Book 1.indb - Journal
Book 1.indb - Journal Book 1.indb - Journal
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Scientifi c Tools<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012<br />
CONTENTS<br />
RGUHS <strong>Journal</strong> of Pharmaceutical Sciences<br />
Vol 2, Issue 1, Jan–Mar, 2012<br />
• Knowing the known to understand the unknown: a systematic approach to reviewing<br />
the scientifi c literature 1<br />
Yamuna V. Kuberappa, Arun H.S. Kumar 10.5530/rjps.2012.1.1<br />
Meeting Report<br />
• Pharmaceutical discovery and development 8<br />
(A report on the symposium held in 63rd annual conference of the Indian Pharmaceutical Congress-2011)<br />
Pitchai Balakumar, Gowraganahalli Jagadeesh 10.5530/rjps.2012.1.2<br />
Review Articles<br />
• Nanobiotechnology: An overview of drug discovery, delivery and development 14<br />
Bhupinder Singh Sekhon 10.5530/rjps.2012.1.3<br />
• Nasal drug delivery–a review 24<br />
Twarita Deshpande, Rajashree Masareddy, Archana Patil 10.5530/rjps.2012.1.4<br />
Research Articles<br />
• Design and optimization of levofl oxacin gastroretentive tablets 38<br />
D. Nagendrakumar, S.B. Shirsand, M.S. Para, A.D. Chauhan 10.5530/rjps.2012.1.5<br />
• Study of the binding properties of hydroxypropyl guar and its utilization in the formulation<br />
and evaluation of metoprolol tartarate tablets 45<br />
Swamy N.G.N., Dharmarajan T.S. Paranjothi K.L.K. 10.5530/rjps.2012.1.6<br />
• Phytochemical investigation of root extract of the plant Carissa spinarum 55<br />
Karunakar Hegde, D. Satyanarayana, Arun B. Joshi 10.5530/rjps.2012.1.7<br />
• Synthesis and antimicrobial activity of 4-hydroxy-1-methyl/phenyl-3- (substituted anilinoacetyl)<br />
quinolin-2(1H)-one 60<br />
Girish Bolakatti, Manjunatha S. Katagi, S.N. Mamledesai, Sujatha M.L., Prakash Dabadi, Narayana Miskin.<br />
10.5530/rjps.2012.1.8<br />
• Evaluation of gastroprotective ability of Amorphophallus paeoniifolius corms against indomethacin<br />
induced gastric ulcers 67<br />
H.N. Nataraj, R.L.N. Murthy, Ramachandra Setty 10.5530/rjps.2012.1.9<br />
• Immunomodulatory activity of methanolic extracts of Pongamia glabra Vent. seeds and bark<br />
in cyclophosphamide induced mice 74<br />
Sanjeev Heroor, Arunkumar Beknal, Nitin Mahurkar 10.5530/rjps.2012.1.10<br />
• Simultaneous fi rst derivative UV spectrophotometric estimation of ramipril and olmesartan 78<br />
Santosh R. Karajgi, C.C. Simpi, Kalyane N.V. 10.5530/rjps.2012.1.11<br />
• Purifi cation and characterization of thermostable amylase from a strain of<br />
thermoactinomyces thalpophilus KSV 17 83<br />
K. Sreenivasa Rao, P. Ellaiah, Karnakumar V. Biradar 10.5530/rjps.2012.1.12
Knowing the known to understand the unknown<br />
A systematic approach to reviewing the scientifi c literature<br />
Yamuna V Kuberappa 1 , Arun HS Kumar 2<br />
1 Freelance MEMS Engineer, Cork, Ireland<br />
2 Department of Medicine (Cardiovascular), University College Cork, College Road, Cork, Ireland<br />
ABSTRACT<br />
Every work must have a starting point, and scientifi c research is no exception. Reviewing literature is an initial<br />
step in undertaking scientifi c research. Reviewing literature, as an ongoing process, provides a critical overview<br />
on the topic of interest and keeps the researcher up-to-date to pursue novel research plans. While reviewing<br />
literature helps early career investigators identify their research niche, established researchers review the literature<br />
to propagate the state of the art progress/opinions in their fi eld of expertise. Most of these reviews often guide<br />
the general research community. Various formats are often adopted in writing a review depending on the context,<br />
audience, depth/complexity of subject and extent of commercial value. The primary goal is to provide a critical<br />
and simplifi ed analysis of the facts. Essentially, every researcher should adapt to these formats in a unique way<br />
and should try to develop their own style of reviewing the literature. In this review, we will describe some of the<br />
essential steps to approaching various formats of reviewing the literature.<br />
Key words: Reviewing literature, scientifi c research, business reviews, book reviews<br />
INTRODUCTION<br />
Reviewing literature is not only a skill but<br />
also an art, wherein scientifi c knowledge<br />
and opinions are disseminated in a critical,<br />
concise and simplifi ed language to scientifi c<br />
and non-scientifi c communities. 1–16 The<br />
purpose of literature review is to provide<br />
critical opinions on established scientifi c<br />
facts and in the process evolve the strengths,<br />
weakness, opportunities and limitations on<br />
the topic of research interests (Figure 1).<br />
Hence, the emphasis is always on providing<br />
critical opinions on a topic rather than<br />
compiling scientifi c facts. 4,10,11,17–19 When one<br />
attempts to critically compile an opinion,<br />
it helps to identify gaps in the existing<br />
literature, and develop research questions<br />
for further investigation. Thus, the seed of<br />
research is sown. A researcher nurtures this<br />
seed over the course of his/her career, to<br />
grow into a fruitful tree. Before starting a<br />
review, clearly defi ne the objectives of the<br />
study and identify the targeted audience.<br />
Based on these two factors, decide the portal<br />
of publication for which the review would<br />
best fi t. The next step is to understand<br />
the procedure for searching and collecting<br />
materials for the intended work. This will<br />
be described in subsequent issues of this<br />
journal. This article focuses on general<br />
steps for systematically reviewing scientifi c<br />
literature.<br />
What necessitates the review<br />
of literature?<br />
Seeking information to gain understanding<br />
of any concept is a fundamental feature of<br />
learning. This is especially true in scientifi c<br />
research, where one needs to understand<br />
an established concept on a relevant topic<br />
to progress further, to evolve innovative<br />
ideas and thoughts, and to avoid any<br />
duplication. 2,6,10,18,22 The famous quote of ‘life<br />
begets life’ in the scientifi c research arena can<br />
Scien fi c Tools<br />
Received Date : 11-02-2012<br />
Revised Date : 29-02-2012<br />
Accepted Date : 03-03-2012<br />
DOI: 10.5530/rjps.2012.1.1<br />
Address for<br />
correspondence<br />
Arun HS Kumar, DVM, PhD.<br />
Senior Scientist and Head<br />
(Functional Biology)<br />
Department of Medicine<br />
(Cardiovascular)<br />
University College Cork<br />
Cork, Ireland<br />
E-mail: a.kumar@ucc.ie<br />
www.rjps.in<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 1
Figure 1: Six major reasons for reviewing the literature.<br />
be represented as ‘research begets research’ and ‘innovations’<br />
i.e., every research work contributes to further research<br />
bringing in scientifi c advancement and innovations.<br />
Therefore, the fundamental reason to proceed with<br />
reviewing the literature is to gain clear understanding on<br />
the topic of interest, so as to set the vision for future<br />
research and to provide the latest source of information<br />
on the topic to a wider scientifi c and non-scientifi c<br />
audience 10,16,25 (Figure 1). The overall objectives of<br />
writing a literature review are summarized in Figure<br />
2. There are several benefi ts of conducting scientifi c<br />
literature review (Box 1).<br />
The following questions are helpful before initiating a<br />
literature review.<br />
Why? Ask yourself, why do you want to review the<br />
literature on a specifi c topic? Come up with rational<br />
thinking on why a review of the literature is required on<br />
the topic at this time.<br />
Why not? This aspect is specifi cally relevant to<br />
young researchers, which emphasizes on curiosity and<br />
inquisitiveness. Hence, start reviewing the literature as<br />
a self-learning step to gain more insight on a specifi c<br />
scientifi c topic and ask yourself if it provokes interests<br />
or motivates you to proceed further.<br />
Box 1: Benefi ts in conducting a methodical literature<br />
review<br />
1. Gives a general and critical overview on the research topic<br />
2. Helps to improve the experimental methods and avoid<br />
duplication of research work<br />
3. Assists in identifying gaps in current research work<br />
4. Helps to build and defi ne research questions and<br />
hypotheses<br />
Yamuna V Kuberappa et al.: Knowing the known to understand the unknown<br />
Why not me? Question yourself on how well you are<br />
suited to reviewing the literature. This is more about<br />
setting the objectives based on strengths and weaknesses<br />
and defi ning targeted audience or scientifi c area of<br />
research.<br />
Why not me now? This question is more relevant to<br />
established researchers, wherein they initiate literature<br />
review to suite the requirement of the scientifi c<br />
community.<br />
The four why’s described above in the context of<br />
literature review might be applicable to any type of<br />
literature review and thus, readers are encouraged to<br />
follow them.<br />
How to start writing a review of literature?<br />
As stated previously, master the skills to search the<br />
literature. A major challenge is how to be effective in<br />
critically judging the published literature? With a recent<br />
surge in the number of scientifi c journals, the number<br />
of scientifi c manuscript/books published on any topic/s<br />
is on an exponential rise. Thus, vast and diversifi ed<br />
resources are available for researchers. It is often diffi cult<br />
to maintain the quality and standards uniform among all<br />
these resources; hence, the researchers should develop<br />
skills to critically evaluate the available literature. 6,9,10,26–28<br />
One of the ways to critique is by avoiding a researcher’s<br />
own inference based on the reported results and rather<br />
looking into the techniques by which the reported<br />
results were derived. Hence, it is emphasised that<br />
while reviewing any literature, one must be critical in<br />
interpretation whether the reported results are based on<br />
rigorous experimental designs or, are merely based on<br />
protocols, which are subjective, controversial and not<br />
rigorous. 10,23,27,28<br />
It is said ‘well begun is half done’; hence, it is important to<br />
start writing with a clear concept and focus by clearly<br />
identifying the objectives of the review. The best way<br />
Figure 2: The basic objectives of any literature review process.<br />
2 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
to do this is, by writing an abstract and clearly outlining<br />
headings and subheadings. 1,4,9,10,16,22,28,29 Once an outline<br />
is framed, start compiling the review by critically<br />
providing an opinion on the topic rather than merely<br />
summarizing the established facts. Attempt to identify<br />
various gaps in existing literature, such as an overworked<br />
area. Based on the existence of such gaps, write research<br />
questions. 10,28,29<br />
It is important to be creative in thoughts and to bring<br />
the researcher’s own signature style of formulating the<br />
literature review. Researchers should not hesitate to<br />
rationally challenge assumptions of existing scientifi c<br />
theory. The review should not be just a free fl ow of<br />
words with stagnant thoughts; rather it should be a<br />
free fl ow of critical thoughts and concepts which are<br />
innovatively constructed/reconstructed. 2,10,27,28,30 An<br />
important factor to innovation is being divergent in<br />
thoughts and attempting to think critically. A detailed<br />
review on critical thinking has recently been published<br />
in this journal, and readers are encouraged to read the<br />
article. 27 In order to draw critical insights from existing<br />
literature, it is necessary to follow an approach of slicing<br />
and dicing of the existing literature to highlight differing<br />
arguments, theories and methodologies. It often<br />
helps to summarize the existing research in the form<br />
of tables, key points on the box and/or fi gures rather<br />
than being extensively textual in literature review. Finally,<br />
it often helps to pre-identify the journal to which you<br />
intend to submit your literature review. This helps you<br />
to structure your review to fi t the aims and scope of<br />
the journal and plan your writing to meet the needs<br />
of specifi c audience.<br />
Defi ning the scope and formulating<br />
the literature review<br />
Composing of literature review begins with clearly<br />
defi ning the objectives based on nature of its intended<br />
audience and the degree of novelty (Figure 3). A wellcomposed<br />
literature review should comprehensively<br />
highlight the current state of the art on the topic,<br />
identify the areas of controversial and/or confl icting<br />
opinions and should hypothesize the topics that need<br />
future research focus. 10,29,30 Since several publications<br />
with either similar and/or contradictory opinions are<br />
being referenced, it is appropriate to use linker words<br />
and phrases such as similarly, in addition to, moreover, likewise,<br />
again (for studies with similar opinions) and albeit, on the<br />
contrary, however, on the other hand, conversely, nevertheless (for<br />
articles with contradictory opinions) while compiling<br />
a literature review. 10,31 It is also essential to include a<br />
summary paragraph at the end to highlight briefl y the<br />
major focus and opinions expressed in a literature review<br />
that often might link to a research hypothesis and future<br />
research directions. 10,22,31<br />
Yamuna V Kuberappa et al.: Knowing the known to understand the unknown<br />
Figure 3: Factors contributing to structuring literature review.<br />
The outline of any literature review is based on nature of audience<br />
and degree of novelty of the contents, which accordingly reflects<br />
its primary goal (creating awareness, educational or scientific<br />
advancement).<br />
Having familiarized with the general formula for<br />
composing a literature review, we now look into the<br />
practical recipes of literature review as it is very much<br />
context dependent. Every scientifi c literature review<br />
begins with the acquaintance of an existing literature and<br />
it is vital to identify type of materials to read. 2,10,18,23,28–30,32<br />
It often helps to categorize the existing literature based<br />
on the degree of novelty and similar versus contradictory<br />
opinions. Although these aspects are more relevant to<br />
established investigators, the novices are often directed<br />
by their guides/supervisors toward a list of introductory<br />
reference materials. While reading these introductory<br />
references, it is important to make notes and write<br />
critical comments as bullet points, which would come<br />
handy at the time of composing the literature review.<br />
The basic elements of a literature review include<br />
an introduction/background section highlighting<br />
the central theme and related context of the review.<br />
This is followed by the body of the literature review<br />
consisting of bulk of the text discussing the origin of<br />
the sources, theories and methods. Finally, the icing on<br />
the cake is the conclusions/recommendations section,<br />
a short paragraph consisting of critical opinion of the<br />
researcher on the subject matter and the scope for further<br />
research. Recently, a ‘Grounded Theory’ based approach<br />
(systematically adopting 5 stage processes) has been<br />
proposed for composing rigorous literature reviews 26,29<br />
(Figure 4), which is a signifi cant advancement of the<br />
matrix-based approach. 33 Although these approaches are<br />
primarily emphasized for information systems, general<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 3
Figure 4: The Grounded theory of composing literature review<br />
involves 5 sequential steps starting from defining to composing the<br />
literature review.<br />
concepts can be adopted for biological/life/medical<br />
sciences as well.<br />
Adopting a language style in composing<br />
literature review<br />
The major purpose of any language style is effective<br />
communication; hence, style is secondary, as long as<br />
writing is formal, clear and concise. 2,9,10,22,28,31 We highly<br />
encourage researchers to develop their own signature<br />
style, which is distinct from others. It is important to<br />
be objective rather than subjective to others opinions<br />
and always use present tense while referring to general<br />
opinions/theories and past tense while addressing specifi c<br />
study results. 10,31 Do not plagiarize any statements. One<br />
of the ways to avoid plagiarism is to make bullet points<br />
and reconstruct sentences to convey your opinion(s) in<br />
simple language. Remember, the essence of science is<br />
to make things as simple as possible. 10,31 Once a draft<br />
is done, it should be read several times and request a<br />
colleague to proofread. Incorporate all corrections and<br />
suggestions in the revised document.<br />
A literature review can take various formats depending<br />
on the purpose for which it is compiled. The following<br />
sections describe each of these formats.<br />
Composing the review of literature<br />
chapter for a thesis<br />
The major purpose of reviewing the literature for a<br />
thesis is to build the context on a research hypothesis<br />
on which the project is based. Thus, the subject matter<br />
should be organized around a pre-defi ned objectives and<br />
research parameters. 10,28,29,34 It often helps to organize<br />
Yamuna V Kuberappa et al.: Knowing the known to understand the unknown<br />
the structure of the literature review by compiling a<br />
list of clearly defi ned research questions/objectives<br />
and selectively reading the relevant literature to fi nd<br />
answers to these questions and in the process identify<br />
the gap in the knowledge base. 10,28,29,34 It is often easy<br />
to get side tracked; hence, keep focused on objectives<br />
and work toward formulating a clear thesis (research<br />
topic) statement and later propose a scientifi cally valid<br />
hypothesis. 7,10,16,20,22,25,26,28,29,34 Box 2 provides a checklist<br />
for compiling a literature review for a thesis.<br />
Organize literature review into a general outline<br />
consisting of introduction, body (major and minor<br />
topics) and conclusion sections. Follow the approach<br />
of composing rather then compiling, and wherever<br />
possible, summarize the data in the form of tables and/or<br />
fi gures. 7,10,16,20,22,25,26,28,29,34 Emphasise on why the intended<br />
work is important for the advancement of scientifi c fi eld<br />
and the likely benefi ts it intended to contribute. 7,10,28,29,34<br />
In doing so, comprehensively cover the existing<br />
literature within and closely related to the scientifi c area<br />
of investigation. Never avoid or ignore any scientifi c<br />
reports that are contradictory or controversial, rather<br />
acknowledge them with a comment why the reported<br />
views are different from those of the researcher. 10,28,34<br />
As noted previously, the review should be proofread<br />
by colleagues and friends who are from outside the<br />
scientifi c domain of the researcher as this clears blind<br />
spots and improves readability. 10,28,34 It is important<br />
that the literature review should lead the reader toward<br />
a conclusion that a rational and realistic research<br />
hypothesis is being proposed.<br />
For most thesis work, the sources are organized in a<br />
chronological pattern. For example, 10 if a researcher<br />
has decided to work on a novel stent-based therapeutics<br />
to achieve myocardial regeneration (MR) following<br />
myocardial infarction, he/she should start with background<br />
information on pathology of myocardial infarction and<br />
epidemiological data on the incidence of myocardial infarction<br />
world-wide and its socio-economic implications. The body of the<br />
review consists of, earlier therapeutic approaches, limitations of<br />
the existing therapeutic approaches and the concept of myocardial<br />
regeneration as a therapeutic option to treat myocardial infarction.<br />
BOX 2: Check list for the compilation of literature<br />
review for a thesis<br />
1. Does the literature review substantiate the research<br />
hypothesis?<br />
2. What is the key message delivered?<br />
3. Do you adequately cover the methodologies and techniques<br />
you intend to adopt?<br />
4. Have you adequately identifi ed the gaps and limitations in<br />
the knowledge base?<br />
5. Have you covered the existing literature up-to-date?<br />
4 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
Follow this with a theory on why stents would be appropriate to<br />
deliver the therapeutics for myocardial regeneration. This should<br />
be followed by a discussion on the developments in design of stents<br />
and the era of introduction of drug eluting stents with its pros<br />
and cons. Furthermore, describe the limitations of current stent<br />
designs, the scope for advancement in the design of stents to deliver<br />
myocardial regenerative therapeutics and its futures perspectives.<br />
Finally, conclude with a statement why the reviewer approach<br />
is better than the other options available. (This paragraph is<br />
reproduced from our previous publication). 10<br />
Instructions for reviewing the literature for<br />
research or review articles<br />
Every research article consists of an introduction with<br />
a brief review of literature, which builds the context<br />
of the research work/hypothesis investigated. The<br />
purpose of literature review in a research article is to<br />
gain support for the scientifi c argument of a researcher.<br />
The purpose in the context of a review article, is to<br />
generate views by composing and synthesizing the<br />
arguments and ideas of others. 1,9,10,18,28,29,35,36 Both<br />
research and review articles have the same elements<br />
of literature but differ in terms of elaboration and<br />
discussion. In research articles, the literature review is<br />
integrated into the introduction and discussion section<br />
and the emphasis is on supporting a researcher’s<br />
argument. On the other hand, in a review article,<br />
the literature review is emphasised on background<br />
information and identifying gaps in knowledge base to<br />
build a critical opinion on the subject topic and future<br />
research directions. 2,9,10,29,36<br />
For example, if one discovers a novel stent design for<br />
treating coronary artery disease (CAD) and report the<br />
same in a research article, the following pattern may<br />
be used.<br />
‘Stents are a widely used medical device for treating CAD…. The<br />
bare metal and drug-eluting stents, which are extensively used,<br />
have serious drawbacks with respect to developing late stenosis<br />
and thrombosis complications … Several studies … report the<br />
complications associated with the currently available stents. Critical<br />
evaluation of the pathology …has revealed that complications are<br />
a consequence of the unique design of the stents. To overcome this<br />
limitation we have designed a novel stent, which we foresee to be<br />
devoid of the side effects observed with the currently available stent<br />
designs,…and tested it for performance in the current study with<br />
the following objectives’. (This paragraph is reproduced from<br />
our previous publication). 10<br />
While all the general guidelines for composing a<br />
literature review are applicable to research as well as<br />
review articles, it is acceptable and essential to include<br />
the reviewer’s opinions in the review articles, especially<br />
on the methodologies adopted and the robustness of<br />
the conclusions reported. Several categories of review<br />
articles exist (comprehensive review, meta-analysis, brief<br />
Yamuna V Kuberappa et al.: Knowing the known to understand the unknown<br />
review, hypothesis-based review, opinion article, stateof-the-art-review,<br />
etc.) depending on the scientifi c insight<br />
and extent of focus on the subject by the reviewer. For<br />
instance, the above paragraph in the context of a review<br />
article can be redesigned as follows.<br />
Start with a brief introduction on CAD, its incidence, its impact<br />
on human health and its socio-economic consequences. Follow this<br />
with a discussion on why are stents the obvious choice for CAD,<br />
what was the approach adopted before the introduction of stents<br />
with its pros and cons, how has the use of stents infl uenced the<br />
CAD events. Include a few paragraphs on the nature/design of<br />
stents used initially and the developments over time on the stent<br />
design and coatings. Follow this with one or two paragraphs on<br />
the types of stents currently in use, their limitations and commonly<br />
encountered side effects (critically evaluate these studies as they<br />
form the basis for further investigations in stent designing and<br />
coatings). Include a paragraph on the reviewer’s opinions for the<br />
limitations encountered with the use of currently available stents<br />
for CAD and the future strategies to overcome these limitations.<br />
Finally, conclude with the room available for future advancement/<br />
development. (This paragraph is reproduced from our<br />
another publication). 10<br />
Art of literature review for books/book chapters<br />
With the growth in digital media and the rising<br />
popularity of tablet devices/computers, the demand<br />
for e<strong>Book</strong>s has surged. <strong>Book</strong>s/book chapters remain a<br />
vital source of established knowledge to both scientifi c<br />
and non-scientifi c audiences. 2,10,18,29 Unlike research<br />
articles, the language used in books must be simple<br />
(avoiding scientifi c jargons wherever possible) and<br />
comprehensible to a wide audience. 10,29 In reviewing a<br />
topic for a book, one should limit to including established<br />
facts and present it with adequate diagrams, tables and<br />
fl ow charts to simplify concepts. It is often essential to<br />
adopt a narrative approach rather than validate scientifi c<br />
facts. Additionally, summarize each subsections, as this<br />
greatly improves readability and comprehension of<br />
the topic. 10,29<br />
Literature review for grant applications and<br />
research proposals<br />
The general outline and focus of the literature review<br />
for grant applications and research proposals are similar<br />
to the one outlined above. However, the emphasis is<br />
more on novelty and applicability aspects of research<br />
proposed and it is important to be precise and to the<br />
point. 10,22,28,29,37 More importantly, the applicant’s own<br />
research to the progress of the proposed project<br />
should be included in the application. This would instill<br />
confi dence in grant reviewers on the abilities and skills<br />
of the researcher to perform the work as described in<br />
the research proposal. 10,28,37 Such confi dence build-up<br />
measure among reviewers helps achieving high rating and<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 5
hence, the success of the grant application. As subject<br />
experts often review the grants/research proposals, it<br />
is necessary to highlight scientifi c advancement rather<br />
than basic information and scientifi c novelty. Needless<br />
to mention that the literature review supporting the<br />
scientifi c advancement must always be tailored to meet<br />
the scope and objectives of the funding agency.<br />
Writing literature review for business proposals<br />
Increasing trends in the academic-industrial partnerships<br />
and academic spin-offs have lead to many academicians<br />
adopting business and entrepreneurship style in running<br />
their research labs. 10,28 Such an integration of corporate<br />
atmosphere in academic environment has progressively<br />
introduced the concept of academic business proposals,<br />
which often involve academic partnerships with<br />
industries to capitalize on the basic/applied science<br />
research to achieve mutual benefi ts. 10,28 Business<br />
proposals by academic researchers should focus on the<br />
following elements.<br />
1. Commercial benefi ts and the economics of the<br />
research work in question.<br />
2. Critical reviews of the applicant’s research work<br />
within the context of commercialization, novelty<br />
and patentability over related research competitors.<br />
Hence, unlike literature reviews for scientifi c manuscripts,<br />
grant proposals, and books, the write-up for business<br />
proposals should highlight on the economic and<br />
commercialization impact of the proposed work with<br />
direct links to novelty and patentability.<br />
SUMMARY<br />
Literature reviews are a valuable knowledge source and a<br />
measure of consolidating the current research progress<br />
in any scientifi c fi eld. There are several categories of<br />
review articles, e.g., comprehensive reviews, metaanalysis,<br />
brief reviews, opinion articles, etc. All literature<br />
reviews adopt the same broad outline/structure, but<br />
vary in detail depending on the objectives. In general,<br />
these objectives identify gaps in existing work, avoid<br />
duplication of work, and propose a scientifi cally valid<br />
hypothesis. Depending on the context in which it is<br />
composed, a literature review has many benefi ts. It may<br />
be a source of self-education or educating others and<br />
publication. In addition, reviews attract successful grant<br />
funding and might build a commercialization case for a<br />
body of research. We emphasise that literature reviewing<br />
is an art of scientifi c communication, which can, if<br />
properly routed, lead to intellectual questions and grants<br />
or business opportunities. Hence, it is necessary to invest<br />
considerable amounts of time, effort and dedication to<br />
this vital scientifi c activity.<br />
Yamuna V Kuberappa et al.: Knowing the known to understand the unknown<br />
CONFLICT OF INTEREST: None.<br />
REFERENCES<br />
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21. Koziol-McLain J, Tanabe P. Reviewing the research literature: you don’t<br />
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22. Price B. Guidance on conducting a literature search and reviewing<br />
mixed literature. Nurs Stand 2009;23:43–49.<br />
23. Sherwin T, Foggin SM, Cartwright VA. Finding information in medical<br />
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S, Gupta YK, Prakash A, editors. Biomedical research: from ideation<br />
to publication. New Delhi: Wolters Kluwer Health, Lippincott Williams &<br />
Wilkins 2010:388–408.<br />
24. Schwartz RB, Russo MC. How to quickly fi nd articles in the top IS journals.<br />
Commun of the ACM 2004;47:98–101.<br />
25. Swanson JM, Easterling P, Costa L, et al. Student-staff collaboration in<br />
identifying nursing problems and reviewing the literature. West J Nurs<br />
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26. McGhee G, Marland GR, Atkinson J. Grounded theory research: literature<br />
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J Pharm Sci 2011;1:97–102.<br />
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gap-spotting or problematization? Organization 2011;18:23–44.<br />
29. Wolfswinkel JF, Furtmueller E, Wilderom CPM. Using grounded theory<br />
as a method for rigorously reviewing literature. Eur J Inform Syst<br />
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/ejis.2011.51;1–11.<br />
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30. Franks H, Hardiker NR, McGrath M, et al. Public health interventions<br />
and behaviour change: Reviewing the grey literature. Public Health<br />
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31. Prakash A. ABC of medical writing: Language and style in scientifi c<br />
communication. In: Jagadeesh G, Murthy S, Gupta YK, Prakash A,<br />
editors. Biomedical research: from ideation to publication. New Delhi:<br />
Wolters Kluwer Health, Lippincott Williams & Wilkins 2010:421–427.<br />
32. Srikumar BN. Reference style and common errors with referencing.<br />
In: Jagadeesh G, Murthy S, Gupta YK, Prakash A, editors. Biomedical<br />
research: from ideation to publication. New Delhi: Wolters Kluwer<br />
Health, Lippincott Williams & Wilkins 2010:479–488.<br />
33. Salipante P, Notz W, Bigelow J. A matrix approach to literature reviews.<br />
Res Organ Behav 1982;4:321–348.<br />
Yamuna V Kuberappa et al.: Knowing the known to understand the unknown<br />
34. Hardy S, Ramjeet J. Refl ections on how to write and organise a research<br />
thesis. Nurse Res 2005;13:27–39.<br />
35. Thomas BH, Ciliska D, Dobbins M, et al. A process for systematically<br />
reviewing the literature: providing the research evidence for public<br />
health nursing interventions. Worldviews Evid Based Nurs 2004;1:<br />
176–184.<br />
36. Humphery SE. What does a great meta-analysis look like? Organ Psychol<br />
Rev 2011;1:99–103.<br />
37. Gulati A. Writing Grant Proposal. In: Jagadeesh G, Murthy S, Gupta<br />
YK, Prakash A, editors. Biomedical research: from ideation to publication.<br />
New Delhi: Wolters Kluwer Health, Lippincott Williams & Wilkins<br />
2010:519–526.<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 7
Pharmaceutical discovery and development<br />
(A report on the symposium held in 63rd annual conference<br />
of the Indian Pharmaceutical Congress-2011)<br />
Pitchai Balakumar 1 , Gowraganahalli Jagadeesh 2,§<br />
1 Department of Pharmacology, Institute of Pharmacy, Rajendra Institute of Technology and Sciences, Sirsa 125055,<br />
Haryana, India<br />
2 Division of Cardiovascular and Renal Products, Center for Drug Evaluation and Research, US Food and Drug Administration,<br />
Silver Spring, MD 20993, USA<br />
ABSTRACT<br />
The 63rd Annual conference of the Indian Pharmaceutical Congress (IPC) held at Bangalore International Exhibition<br />
Centre, Bengaluru, Karnataka, India, from 16 to 18 December, 2011, was organized by ‘Indian Pharmaceutical<br />
Congress Association’ and hosted by ‘The Indian Pharmaceutical Association’, Karnataka State Branch. The<br />
conference included a 3-hour symposium on “Pharmaceutical Discovery and Development”. The objectives of this<br />
symposium were to enlighten the delegates on a few key aspects of drug discovery and development. To that<br />
end, the symposium addressed on drugs and drug targets, early evaluation of pharmacokinetics and metabolism<br />
in drug discovery, toxicity tests in drug development, regulatory review process, and biomarkers.<br />
INTRODUCTION<br />
Drugs are miracles of modern science. They<br />
are used to prevent, treat, or cure diseases<br />
and medical conditions. Disease processes<br />
are often complex and involve a defi ned<br />
sequence of events. Drugs are designed to<br />
intervene in the disease process. They can<br />
simply alleviate symptoms or even control<br />
subsequent development of a chronic<br />
disease condition. Except for antimicrobials<br />
and to some extent anti-infl ammatory and<br />
anti-neoplastic drugs, most other drugs<br />
infrequently cure diseases. Yet, these<br />
drugs improve the quality of life and, in<br />
general, a sense of well-being. Billions of<br />
prescriptions are fi lled worldwide every year.<br />
The process of discovering, developing<br />
and testing new drugs encompasses some<br />
of the most inspiring areas of scientifi c<br />
discovery. 1 Drug development involves<br />
multiple disciplines encompassing a large<br />
number of skilled scientists at different<br />
levels, interdependent science managers<br />
involved in important decision-making,<br />
huge volumes of generated data, and<br />
ultimately the drug regulatory agencies (e.g.,<br />
US FDA, European Medicines Agency,<br />
Japanese Ministry of Health and Welfare). 1<br />
It is a complicated, time-consuming (often<br />
>10 years), and costly process (over US $1<br />
billion) whose end result is seldom known<br />
at the outset. 2<br />
The initial stages of drug discovery<br />
research are characterized by target discovery<br />
(identifi cation and validation) and lead<br />
discovery (generation and optimization).<br />
Integration of combinatorial chemistry,<br />
high throughput screening, and molecular<br />
§ The opinions expressed herein are those of GJ and do not necessarily refl ect those of the US Food and Drug<br />
Administration.<br />
Mee ng Report<br />
Received Date : 15-02-2012<br />
Revised Date : 27-02-2012<br />
Accepted Date : 02-03-2012<br />
DOI: 10.5530/rjps.2012.1.2<br />
Address for<br />
correspondence<br />
Gowraganahalli<br />
Jagadeesh, PhD.<br />
Senior Expert Pharmacologist<br />
Division of Cardiovascular and<br />
Renal Products<br />
Food and Drug<br />
Administration (FDA)<br />
10903 New Hampshire Ave<br />
Bldg 22, Rm 4128<br />
Silver Spring, MD<br />
20993-0002, USA.<br />
Phone: 001-301 796 1093<br />
Fax: 301-796-9838<br />
E-mail: gowra.jagadeesh@fda.<br />
hhs.gov (G Jagadeesh)<br />
pbala2006@gmail.com<br />
(P Balakumar)<br />
www.rjps.in<br />
8 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
modeling generates an abundance of candidate molecules<br />
with drug-like properties. The lead compounds<br />
are selected based on a high degree of selectivity for<br />
the target. Such compounds are also optimized in terms<br />
of potency, physicochemical properties, pharmacokinetics<br />
and drug metabolism, and toxic profi le. 3,4 This<br />
information helps to reduce attrition of compounds<br />
and contributes markedly to success rates in identifying<br />
lead candidates. With only additional minor changes in<br />
structure or other physico-chemical properties of the<br />
lead compound, a potentially viable drug candidate is<br />
launched (Box 1).<br />
The quality of the lead compound is crucial to the<br />
success of the refi nement and development process. The<br />
lead drug candidate that enters the initial developmental<br />
stage (see Box 1) must have a strong chance of meeting<br />
the target profi le in terms of effi cacy, safety, route of<br />
administration, and treatment regimen. An early step of<br />
drug development includes pharmacologic, toxicologic,<br />
pharmacokinetic, and metabolism/biotransformation<br />
studies both in vitro and in animals. These studies<br />
should reveal potentially useful (intended target) and<br />
harmful (off target) properties of the drug candidate<br />
in a qualitative and quantitative manner, thus permitting<br />
an assessment of therapeutic risk. An investigational<br />
new drug (IND in the USA) application is then fi led<br />
with the drug regulatory agency demonstrating that<br />
the CMC (chemistry and manufacturing controls),<br />
nonclinical pharmacologic and toxicologic properties<br />
of the drug have been well characterized. The next<br />
step in drug development (clinical phase) is to<br />
demonstrate that the benefi t-to-risk ratio as observed<br />
in pharmacology and toxicology studies is confi rmed<br />
Box 1: The Discovery to Development Scheme<br />
1. Drug discovery (Early pharmaceutical research)<br />
A. Target discovery (Exploratory phase)<br />
1. Target identifi cation<br />
2. Target validation<br />
B. Hits discovery<br />
1. Hits identifi cation<br />
2. Hits validation (after hits reproducibility and dose effect)<br />
C. Lead discovery<br />
1. Lead generation/identifi cation<br />
2. Lead refi nement/optimization<br />
2. Drug development (Late Pharmaceutical R & D; drug<br />
candidate launched)<br />
A. Drug candidate selection<br />
B. Preclinical development (short and long term studies)<br />
C. IND application fi led<br />
D. Clinical development (Phase 1, 2, 3)<br />
E. NDA fi led<br />
F. Drug regulatory agency approval for marketing<br />
Balakumar, Jagadeesh: Pharmaceutical Discovery and Development<br />
in patients. Standard endpoints in animal safety studies<br />
focus upon morphologic assessments of tissues,<br />
whereas the end points in initial human safety studies<br />
(Phase 1) are largely physiologic, including assessments<br />
of the cardiovascular system, central nervous system,<br />
respiratory, and renal functions. At the completion of<br />
phase 2 and phase 3 trials, if conclusive evidence of the<br />
new molecule’s safety and effectiveness for a specifi c<br />
indication exists, a new drug application (NDA in the<br />
USA) is submitted to the drug regulatory agency. If<br />
benefi ts of a drug for the intended patient population<br />
outweigh the risks, the drug will receive approval from<br />
the regulatory agency and thus, can be marketed (Box 1).<br />
An overview of the symposium: This symposium addressed<br />
selected key topics in drug discovery and development,<br />
such as potential drug targets for therapeutic interventions,<br />
early evaluation of pharmacokinetics and drug<br />
metabolism, preclinical toxicity studies, and regulatory<br />
guidelines in submission of investigative and new<br />
drug applications. It concluded with the role of novel<br />
and traditional biomarkers in monitoring disease or its<br />
treatment during the course of clinical trials. The purpose<br />
of this report is to highlight and disseminate key<br />
information presented at the symposium as a means of<br />
educating the scientifi c community so that they can stay<br />
current with this rapidly developing fi eld.<br />
The symposium had fi ve distinguished speakers affi liated<br />
to drug regulatory affairs, industries and academia<br />
from India and the USA. The symposium was arranged<br />
by Dr. G. Jagadeesh of the US Food and Drug Administration<br />
(FDA), and chaired by Prof. P. Balakumar of the<br />
Institute of Pharmacy, Rajendra Institute of Technology<br />
and Sciences, Sirsa, Haryana, India.<br />
Drug Targets<br />
The symposium began with a welcome address by<br />
Dr. H.G. Shivakumar of J.S.S College of Pharmacy,<br />
Mysore, Karnataka. Prof. P. Balakumar, the chairman<br />
of the symposium, briefl y overviewed the topics for<br />
presentation and profi led the speakers. Dr. G. Jagadeesh<br />
(US FDA) then delivered the fi rst lecture on ‘Drugs and<br />
Drug Targets’. A main element of early pharmaceutical<br />
research is to reveal potential drug targets for therapeutic<br />
intervention in the intended patient population.<br />
Determination of the optimal molecular targets for<br />
drug intervention provides the basis for the discovery<br />
of new medicines. Disease-relevant target identifi cation<br />
is considered an important criterion for determining<br />
the correlation between drug and indication.<br />
An important focus of drug discovery has been on<br />
protein drug targets as a means of treating various<br />
disorders. The advent of genomic sciences has<br />
advanced our knowledge in the development of new<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 9
pharmaceuticals, as we now have a better understanding<br />
of how genes are linked to diseases. It is estimated that<br />
approximately 10% of the 25,000–30,000 known human<br />
genes are linked to diseases. Identifying individual genes<br />
considered potential targets (i.e., drugome) is not an ideal<br />
task, because the functional product of each gene or<br />
proteome is under multiple regulatory controls, including<br />
splice variants and post-translational modifi cations.<br />
Thus, targeting the body’s proteins or proteome is ideal<br />
because druggability, or the ability of a drug to affect<br />
a protein’s function, may be an inherent property of a<br />
given protein. However, this does not constitute a useful<br />
drug target unless the protein is also linked to a disease.<br />
Nearly half of the disease-associated proteins are not<br />
druggable as they lack a hydrophobic binding site for<br />
a potential orally-delivered, cell membrane-permeable<br />
drug molecule. This restricts the number of potential<br />
drug targets to less than 1500. 5 Further, protein drug<br />
targets should be viewed in the context of various<br />
interrelated networks involved in the disease process.<br />
Various factors such as genetic mutations, epigenetic<br />
changes and pathogens can cause perturbations in the<br />
function of networks, disrupting signaling pathways and<br />
resulting in diseases. According to the Online Mendelian<br />
Inheritance in Man, more than 1284 distinct disorders<br />
have been reported. These are classifi ed into 22 disorder<br />
classes based on the physiology of the system and 1777<br />
disease-related genes. 6 Although the majority of disease<br />
genes and the molecular basis for these diseases are not<br />
known, knowledge of the human genome sequence has<br />
increased potential new targets. The effective number of<br />
novel drug targets has been calculated by the intersection<br />
of the number of druggable proteins with the number<br />
of disease-associated proteins, or 600–1500 novel drug<br />
targets (2–5% of the genome). 5,7<br />
Noting the report of Rask-Andersen et al., 8<br />
Dr. Jagadeesh said that for the past thirty years (1982<br />
to 2010) of drug target innovation, on average, about<br />
18 new drugs targeting human proteins have been<br />
approved by the FDA every year, of which about 4<br />
are fi rst-in-class that act on novel target structures that<br />
are encoded by the human genome, and the remaining are<br />
follower or ‘me-too’ drugs. 8 According to these authors,<br />
this translates to 435 drug targets over that time period.<br />
Enzymes are the largest target class (47%) followed by<br />
receptors (G-protein coupled receptors account for 30%,<br />
the second largest), transporters (voltage-gated calcium<br />
channels, the largest) and others. The most common<br />
pharmacologic action of the approved drugs is antihypertensive<br />
followed by anti-cancer, anti-infl ammatory,<br />
sedative and hypnotic, anti-allergic, anti-convulsant, antiarrhythmic,<br />
anti-psychotic, anti-depressant and analgesic.<br />
Most drugs target only a select number of proteins<br />
but some can target as many as 19 (e.g., bromazepam).<br />
Balakumar, Jagadeesh: Pharmaceutical Discovery and Development<br />
The average number of target proteins reported per drug<br />
is 1.8 surpassing the number of single target drugs, 6 and<br />
thus, are called target multipliers (polypharmacology).<br />
Multi-target drugs are useful in cases where a<br />
specifi c disease-associated protein is not druggable.<br />
Dr. Jagadeesh also discussed various interactions, such as<br />
between proteins (interactome), drugs (drug network),<br />
drug and target (drug-target network) and disease and<br />
disease genes (diseasome). It is anticipated that analysis<br />
of these complex networks may also lead to new avenues<br />
for drug target prediction.<br />
Early Pharmacokinetics and Drug Metabolism<br />
After lead molecules are identifi ed, they are optimized<br />
in terms of potency, selectivity, ADME (absorption,<br />
distribution, metabolism, and excretion) and toxicology<br />
before they can become candidates for further drug<br />
development. 3,9 It is important to emphasize the<br />
integral role of drug metabolism and pharmacokinetics<br />
(DMPK) in early phases of drug discovery, so as<br />
to avoid failure due to poor pharmacokinetics and<br />
bioavailability that largely result from suboptimal<br />
physico-chemical properties. 10 DMPK scientists play<br />
a multifaceted role in various functional areas, such as<br />
drug biotransformation, pharmacology and toxicology<br />
that help provide valuable information related to effi cacy<br />
and safety of a new molecule. Dr. Sandhya Mandlekar<br />
(Bristol-Myers Squibb India Ltd., India) spoke on the<br />
topic of ‘Pharmacokinetics and Metabolism in Early<br />
Drug Discovery’. While defi ning the evolving role<br />
of DMPK studies in early pharmaceutical research,<br />
Dr. Mandlekar described traditional ADME properties,<br />
such as characterizing bioavailability and half-life in<br />
animals and man. She also discussed advances in the fi elds<br />
of pharmacogenetics, pharmacogenomics and drug<br />
transporters; the shift in drug metabolism paradigms,<br />
such as enzyme regulation, drug-drug interaction and<br />
inter-individual variability; and predictive biomarkers<br />
from animals to man. She also described the availability<br />
of new technologies and using a tiered approach to<br />
DMPK studies. Her presentation also focused on in vitro<br />
models to predict hepatic clearance, CYP inhibition and<br />
induction assays, Caco-2 permeability determination<br />
assay for poorly soluble compounds, generating<br />
in vivo EC50s by modeling nonclinical PK/PD data, and<br />
investigating unusual metabolic reactions. Dr. Mandlekar<br />
also addressed necessary steps in early pharmaceutical<br />
research ADME studies (Box 2).<br />
Finally, Dr. Mandlekar suggested that the mission of<br />
discovery ADME studies is to ensure no development<br />
candidate fails in the clinic due to unforeseen ADME<br />
properties, and to improve developability characteristics<br />
and risk assessment, in parallel with effi cacy, to get better<br />
success rates during clinical development.<br />
10 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
Box 2: A Focused Application of Discovery ADME<br />
Studies<br />
1. Active involvement earlier in the Discovery Process<br />
2. Timely guidance to Chemistry to select chemotypes with<br />
desirable ADME properties<br />
3. Maximize informed decision making during Lead<br />
Optimization<br />
4. Improved ability to predict human DMPK<br />
5. Stronger partnership with all areas of Drug Discovery and<br />
Development<br />
Toxicology Studies<br />
A remaining critical issue in drug development is the<br />
failure to identify, suffi ciently, toxicological problems<br />
pertaining to new chemical entities early enough in the<br />
drug research and development process. Better success<br />
in developing early toxicologic screens will certainly<br />
enhance the number of successful new chemical entities<br />
entering to the clinical developmental phases and, in<br />
fact, avoid the expense of terminating drugs in latestage<br />
clinical development. Toxicity is a leading cause<br />
of attrition at both clinical and nonclinical stages of<br />
drug development. 4,10 The primary objective of nonclinical<br />
toxicology studies in the drug discovery process<br />
is to evaluate the safety of potential drug candidates for<br />
human use. The ultimate goal is to translate the in vitro<br />
and animal responses into an understanding of the risk<br />
for human subjects under therapeutic conditions. Thus,<br />
non-clinical toxicology testing is utilized at all stages of<br />
the drug discovery and development processes.<br />
Dr. K.S. Rao (Rao Toxicology Foundation, Bengaluru,<br />
India, and Editor-in-Chief of Toxicology International)<br />
spoke at length on ‘Toxicology Issues in Drug<br />
Development’. Dr. Rao addressed various toxicological<br />
issues encountered during the early phases of research<br />
and drug development. The objectives of toxicity<br />
tests, whether conducted in the whole animal or in<br />
cell systems, should be to predict the adverse effects<br />
of the tested compound in human beings both in a<br />
qualitative and quantitative manner. While in silico and<br />
in vitro models will continually be developed and refi ned,<br />
in vivo preclinical safety models remain as the gold<br />
standard for assessing human risk. Various cell culture<br />
systems are an in vitro model of a target tissue in the<br />
human body and mimic the response of human cells<br />
to drug exposure. Several differentiated cell types have<br />
been used in toxicology investigations. Among them,<br />
the more widely used are cells derived from liver, lung,<br />
heart, kidney, muscle, nervous and reticulo-endothelial<br />
systems. Primary cultures or isolated cells are widely<br />
used because the cells retain their specialized functions.<br />
Dr. Rao also discussed the rapid development of<br />
Balakumar, Jagadeesh: Pharmaceutical Discovery and Development<br />
‘Functional genomics’, which includes proteomics and<br />
transcriptomics, as an emerging discipline that represents<br />
a global and systematic approach to identifying biological<br />
pathways and processes in both normal and abnormal<br />
physiological states. Although numerous tests have been<br />
validated in the recent past, they not yet gained wide<br />
spread use in discovery toxicology.<br />
Dr. Rao noted that the fi rst of two stages in new drug<br />
development is comprised of non-clinical (pharmacology<br />
and toxicology) studies. The second stage is initiation<br />
of clinical studies and completion of long-term animal<br />
toxicity studies. The objectives of non-clinical studies<br />
are to demonstrate the biologic activity (effi cacy and<br />
selectivity) against the targeted disease and to provide<br />
data for toxicity and safety evaluation (risk assessment).<br />
Characterization of toxic effects with respect to target<br />
organs, dependence of dose to effect, relationship to<br />
systemic drug exposure, and potential reversibility of<br />
any toxicities are all helpful in human dose escalation<br />
strategies. The toxicity studies are limited at the beginning<br />
of clinical development, but additional long-term toxicity<br />
studies are conducted in tandem with on-going clinical<br />
studies to help support the intended dose and duration<br />
of the clinical trials. Serious adverse effects noted in<br />
ongoing clinical trials may call for additional toxicologic<br />
studies or even result in the clinical trials being halted<br />
(clinical hold).<br />
International regulatory guidelines require that new drugs<br />
be tested for safety and effi cacy before marketing. The<br />
recommendations for the extent of non-clinical safety<br />
studies to support various stages of clinical development<br />
differ only slightly among the regions of Europe, USA<br />
and Japan. International Conference on Harmonization<br />
(ICH) guidelines (ICH-M3) describe the consensus that<br />
exists regarding the scope and duration of non-clinical<br />
safety studies to support the conduct of human clinical<br />
trials for medicinal products. The major toxicology<br />
studies that are conducted to demonstrate the safety of a<br />
Box 3: Major Categories of Toxicology Studies<br />
Conducted During Drug Development (in Tandem<br />
with Clinical Phases)<br />
(http://www.fda.gov/Drugs/GuidanceCompliance<br />
RegulatoryInformation/Guidances/ucm065007.htm)<br />
1. Type, duration, and timing of non-clinical studies in support<br />
of clinical trials (ICH M3(R2))<br />
2. Safety Pharmacology studies (ICH S7A and S7B)<br />
3. Toxicokinetics (ICH S3A)<br />
4. Single and Repeat dose toxicity studies (ICH S4A)<br />
5. Genotoxicity studies (ICH S2(R1))<br />
6. Carcinogenicity studies (ICH S1A, S1B, S1C(R2))<br />
7. Reproductive and development toxicity (ICH S5A, S5B)<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 11
new drug candidate are outlined below (Box 3). Besides<br />
ICH, three other guidelines are available. Each of these<br />
describes methodology and assay criteria for most of the<br />
toxicology studies. They are: i) CFSAN Redbook 2000:<br />
Toxicological Principles for the Safety Assessment of<br />
Food ingredients; 11 ii) OECD guidelines; 12 and iii) EPA<br />
guidelines for toxicity testing. 13<br />
Regulatory Review Process in New Drug<br />
Development<br />
The long road from original concept to fi nal drug approval<br />
has multifaceted phases that include pharmacologic,<br />
pharmacokinetic and drug metabolism, toxicologic and<br />
clinical studies. Dr. Gopi Vudathala (Global Regulatory<br />
Affairs-Interface CMC Sanofi -Aventis, USA) spoke on<br />
‘Overview of the Drug Development Process: The<br />
IND and NDA Process’. His talk focused on the drug<br />
development process describing the regulations and<br />
guidances specifi c to the IND and NDA process. These<br />
included pre-IND, IND application and types of reviews,<br />
various phases of clinical trials, timing and duration of<br />
non-clinical studies necessary to support clinical trials,<br />
NDA registration process and necessary submission<br />
requirements, NDA review process, and fi nally, NDA<br />
actions. Dr. Vudathala integrated many of the issues<br />
with strategic preclinical and clinical drug development<br />
to the regulatory decision involved in licensing the<br />
product for marketing. He also spoke on ‘relevant Laws’<br />
(Federal Food, Drug, and Cosmetic Act; Public Health<br />
Service Act-Part F Licensing of Biological Products and<br />
Clinical Laboratories) and ‘relevant regulations’ (IND<br />
regulations (both drugs and biologics) 21 CFR 312.23;<br />
NDA (drugs) regulations-21 CFR 314.50; Product<br />
licensing (biologics)-21 CFR 601; Protection of human<br />
subjects and informed consent regulations-21 CFR 50;<br />
IRB regulations-21 CFR 56) for INDs and NDAs. In<br />
the presentation, Dr. Vuduthala listed requirements for<br />
a new IND with respect to chemistry/manufacturing/<br />
controls (CMC or Quality) data and pharmacologic and<br />
toxicologic data.<br />
On the subject of clinical trials, Dr. Vuduthala noted that<br />
the key to initiating clinical trials in the US is to ensure<br />
that a good safety and preclinical toxicology package is<br />
available for review in the initial IND stage along with<br />
a good clinical plan to evaluate proof of concept in<br />
healthy volunteers (Phase 1). Subsequently, performing<br />
dose ranging studies and confi rming effi cacy in a small<br />
population of relevant patients will need to be done in<br />
early Phase 2 studies. Based on the results, a detailed<br />
clinical plan is developed for Phase 2b and 3 pivotal<br />
clinical trials in a larger patient population. Throughout<br />
the drug development process, there are a number of<br />
opportunities for the industry to meet with members<br />
of the regulatory agency to discuss preclinical, clinical<br />
Balakumar, Jagadeesh: Pharmaceutical Discovery and Development<br />
and CMC plans and any issues requiring resolution.<br />
A pre-IND meeting, end of phase 2 meeting and pre-<br />
NDA meetings are keys to discuss the path forward<br />
and ensure the sponsor’s development plans are vetted<br />
by the Agency. Development of the product label<br />
(or package insert) occurs in parallel to the above, and<br />
labeling discussions are an important part of the NDA<br />
registration process.<br />
Biomarkers<br />
A fi nal topic discussed, which poses a great challenge<br />
to drug developers, regulators and physicians, is<br />
identifi cation of a ‘biomarker’, a biologic indicator,<br />
parameter or marker that can be used to measure<br />
the disease progress or the effects of treatment.<br />
The parameter can be physical or chemical as well as<br />
biological. A biomarker is defi ned as a characteristic<br />
that is objectively measured and evaluated as an<br />
indicator of normal biologic processes, pathogenic<br />
processes, or biological responses to a therapeutic<br />
intervention. 14 The last speaker of the symposium,<br />
Dr. Samba Reddy (Texas A&M University Health<br />
Science Center, USA) spoke on ‘Biomarkers in<br />
Preclinical and Clinical Drug Development’. Dr. Reddy<br />
focused on current knowledge on biomarkers in drug<br />
development (Box 4).<br />
Dr. Reddy discussed differences between diseaserelated<br />
and drug-related biomarkers. Disease-related<br />
biomarkers give an indication of the disease pathways<br />
or disease mechanism; whereas drug-related biomarkers<br />
indicate whether a drug will be effective in a specifi c<br />
patient and how the patient’s body will process it. The<br />
speaker covered the various aspects of biomarkers,<br />
including ‘Research Breaking in Classifi ed Biomarkers’,<br />
‘Biomarker Discovery Technology’, ‘Biomarker<br />
Quantifi cation and Assay’, ‘Biomarkers in Drug<br />
Discovery and Development’, ‘Disease Biomarkers<br />
and Healthcare’ and ‘Cancer Biomarkers’. The rapid<br />
emergence and evolution of translational medicine have<br />
Box 4: Traits of Biomarkers<br />
1. A biomarker can be a substance that is introduced into an<br />
organism to examine organ function or other aspects of<br />
health<br />
2. Biomarkers can be used in early diagnosis, disease<br />
prevention, drug treatment, drug response, drug target<br />
identifi cation, etc.<br />
3. Biomarkers represent key tools and parameters for the<br />
accelerated new drug discovery and clinical development<br />
4. Biomarkers can be classifi ed into three types: Natural<br />
history markers; Drug activity markers; and Surrogate<br />
markers<br />
5. In clinical trials, the biomarker serves as a surrogate<br />
endpoint for evaluating clinical benefi ts<br />
12 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
pushed biomarkers into the spotlight for successful drug<br />
development. He said that established pharmaceutical<br />
companies are integrating the biomarker strategies into<br />
their research product pipelines. In concluding remarks,<br />
Dr. Reddy said that the path from biomarker discovery<br />
to commercialization is fraught with numerous obstacles.<br />
Novel technologies are needed for biomarker discovery,<br />
and such approaches would be helpful in streamlining<br />
the drug development process by reducing time and<br />
costs, as well as minimizing drug attrition associated<br />
with safety issues.<br />
ACKNOWLEDGEMENTS<br />
We sincerely thank Drs. Sandhya Mandelkar (Head,<br />
Department of Pharmaceutical Candidate Optimization<br />
(PCO), Bristol-Myers Squibb India Ltd., Biocon BMS<br />
R&D Center, Bengaluru, India), K.S. Rao (Chief Scientifi c<br />
Offi cer, Rao Toxicology Foundation, Bengaluru, and<br />
Editor-in-Chief of Toxicology International), Gopi<br />
Vudathala (Associate Vice President, Global Regulatory<br />
Affairs-Interface CMC Sanofi -Aventis, New Jersey, USA)<br />
and Samba Reddy (Associate Professor, & Principal<br />
Investigator, Texas A&M University Health Science<br />
Center, College of Medicine, College Station, Texas,<br />
USA) for providing us their presentation materials<br />
from which this meeting report was prepared. We also<br />
express our gratitude to Dr. B.R. Jagashetty (Drugs<br />
Controller for the State of Karnataka, and Chairman<br />
of the Local Organizing Committee of 63rd IPC),<br />
Dr. N. Udupa (Convener, Scientifi c Services of IPCA),<br />
and Mr. S.M. Mudda (Chairman, Local Scientifi c<br />
Balakumar, Jagadeesh: Pharmaceutical Discovery and Development<br />
Committee of 63rd IPC) for their inspiration and<br />
support for this symposium.<br />
REFERENCES<br />
1. US FDA. Protecting America’s health through human drugs. http://<br />
www.fda.gov/Drugs/ResourcesForYou/Consumers/ucm143455.htm.<br />
Accessed 6 February 2012.<br />
2. Adams CP, Brantner VV. Spending on new drug development. Health<br />
Econ 2010;19:130–41.<br />
3. Terstappen GC, Reggiani A. In silico research in drug discovery. Trends<br />
Pharmacol Sci 2001;22:23–6.<br />
4. Kramer JA, Sagartz JE, Morris DL. The application of discovery toxicology<br />
and pathology towards the design of safer pharmaceutical lead<br />
candidates. Nat Rev Drug Discov 2007;6:636–49.<br />
5. Hopkins AL, Groom CR. The druggable genome. Nature Rev Drug<br />
Discov 2002;1:727–30.<br />
6. Yildirim MA, Goh KI, Cusick ME, Barabasi AL, Vidal M. Drug–target<br />
network. Nat Biotech 2007;25:1119–26.<br />
7. Imming P, Sinning C, Meyer A. Drugs, their targets and the nature and<br />
number of drug targets. Nature Rev Drug Discov 2006;5:821–34.<br />
8. Rask-Andersen M, Almén MS, Schiöth HB. Trends in the exploitation of<br />
novel drug targets. Nat Rev Drug Discov 2011;10:579–90.<br />
9. Lipinski CA. Drug-like properties and the causes of poor solubility and<br />
poor permeability. J Pharmacol Toxicol Method 2000;44:235–49.<br />
10. Kola I, Landis J. Can the pharmaceutical industry reduce attrition rates?<br />
Nat Rev Drug Discov 2004;3:711–5.<br />
11. CFSAN Redbook 2000: Toxicological Principles for the Safety Assessment<br />
of Food Ingredients. Toxicity testing are listed in chapters III and<br />
IV of the book. http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDocuments/FoodIngredientsandPackaging<br />
/Redbook/default.htm. Accessed 6 February 2012.<br />
12. OECD guideline for testing of chemicals, Section 4: e.g., acute toxicity,<br />
repeated dose toxicity, genetic toxicology, reproductive toxicology,<br />
etc. http://www.oecd-ilibrary.org/environment/oecd-guidelines-forthe-testing-of-chemicals-section-4-health-effects_20745788.<br />
(Home page:<br />
http://www.oecd.org/home/.) Accessed 6 February 2012.<br />
13. Environmental Protection Agency (EPA) guidelines for toxicity testing:<br />
http://www.epa.gov/ocspp/pubs/frs/publications/Test_Guidelines/<br />
series870.htm. Accessed 6 February 2012.<br />
14. Biomarkers Defi nitions Working Group. Biomarkers and surrogate<br />
endpoints: Preferred defi nitions and conceptual Framework. Clin<br />
Pharmacol Ther 2001;69:89–95.<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 13
Nanobiotechnology: An overview of drug<br />
discovery, delivery and development<br />
Bhupinder Singh Sekhon<br />
PCTE Institute of Pharmacy, Jhande, Near Baddowal Cantt (Ludhiana)-142 021, Punjab, India<br />
ABSTRACT<br />
Nanobiotechnology is a new technology concerned specifi cally with the functionalism and modifi cation of<br />
chemical-physical structures on a biomolecular scale, and also is the application of nanotechnology to the life<br />
sciences. Nanotechnology for biotechnology and pharmaceutical applications has progressed from the concept<br />
stage to commercialization. Nanobiotechnology represents the future of medicine and healthcare. Various physical,<br />
chemical, electrical tools and methods used to investigate biological nanoobjects include optical tools, nanoforce<br />
and imaging, surface methods, mass spectrometry and microfl udics. Its application has an impact on diagnostics,<br />
drug delivery as well as drug discovery. Nanobiotechnology focuses on various areas such as nanobiotechnology<br />
and cancer, drug discovery and tools, and nanobiotechnology and medicine. Applications are emerging from<br />
all branches of nanobiotechnology in medicine and pharmacy. Several technologies including nanoparticles<br />
and nanodevices such as nanobiosensors and nanobiochips have been used to improve drug discovery and<br />
development. Some nanosubstances such as fullerenes and dendrimers/biodendrimers could be potential drugs<br />
for the future. Moreover, nanobiotechnology has the potential for combining drug design and drug delivery.<br />
However, limitations of the available nanoparticles still to be resolved for their application in the drug-discovery<br />
studies exist. The benefi ts of nanotechnology are enormous and so these benefi ts should be maximized while<br />
efforts are made to reduce the risks.<br />
Keywords: Nanobiotechnology, nanobiomaterials, nanobiostructures, nanocarriers<br />
INTRODUCTION<br />
Nanotechnology is a broader term applicable<br />
to small things in nanometer range (roughly<br />
in the 1−100 nm size regime in at least<br />
one dimension). Nanobiotechnology is a<br />
big word made up of three parts: NANO<br />
is really, really tiny, BIO is living things,<br />
and TECHNOLOGY is about tools.<br />
Nanobiotechnology is an emerging area<br />
of science which is concerned with the<br />
application of tools and processes in order<br />
to assemble devices for the study of objects<br />
in biological systems. Nanobiotechnology<br />
is that branch of nanotechnology that deals<br />
with biological and biochemical applications<br />
or uses. Nanobiotechnology often studies<br />
existing elements of living organisms and<br />
nature to fabricate new nano-devices.<br />
Generally, nanobiotechnology refers to<br />
the use of nanotechnology to further<br />
the goals of biotechnology. Relationship<br />
of nanotechnology, biotechnology and<br />
nanomedicine is shown in Figure 1.<br />
Further, nanobiotechnology (an integration<br />
of physical sciences, molecular engineering,<br />
biology, chemistry and biotechnology) has<br />
yielded healthcare discoveries that have<br />
been used for drug delivery and diagnostic<br />
purposes. 1<br />
In medicine and pharmacology, nanobiotechnology<br />
opens up new perspectives in<br />
analytics and therapy. Medical applications<br />
of nanobiotechnology resulted in appearance<br />
of new fi eld i.e. nanomedicine. An<br />
increasing use of nanobiotechnology by<br />
the pharmaceutical industries includes<br />
(i) drug delivery, and (ii) disease therapy.<br />
The potential topics in nanobitechnology<br />
Review Ar cle<br />
Received Date : 17-11-2011<br />
Revised Date : 04-01-2012<br />
Accepted Date : 17-02-2012<br />
DOI: 10.5530/rjps.2012.1.3<br />
Address for<br />
correspondence<br />
Bhupinder Singh Sekhon<br />
PCTE Institute of Pharmacy<br />
Jhande, Near Baddowal Cantt)<br />
(Ludhiana)-142 021<br />
Punjab, India<br />
E-mail: sekhon224@yahoo.co<br />
Mobile: 91-161-9876242299<br />
www.rjps.in<br />
14 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
Bhupinder Singh Sekhon et al.: Nanobiotechnology: An overview of drug discovery, delivery and development<br />
Figure 1: Relationship of nanotechnology, biotechnology and nanomedicine.<br />
include molecular bioprobes, nanoparticles and nanobiosystems,<br />
nanobiomaterials, biomolecular assemblies<br />
and supra-biomolecules, nanobiosensors and nanobiochips,<br />
BioNEMS and nano-biofl uidics, nanobiophotonics,<br />
single-molecule detection and manipulation<br />
and molecular motors. Experts are of the opinion that<br />
nanobiotechnology has the potential to yield a scientifi<br />
c and industrial revolution, as envisioned by the<br />
numerous programs on nanotechnology/nanobiotechnology<br />
launched over the last decade by councils and<br />
governments worldwide.<br />
The potential uses and benefi ts of nanobiotechnology<br />
are enormous. 2 Nano-systems in biology, the most<br />
complex and highly functional nanoscale materials and<br />
machines have been invented by nature. Proteins and<br />
nucleic acids, and other naturally occurring molecules<br />
(polymers) regulate and control biological systems with<br />
incredible precision. Many nanotechnologists are in fact<br />
drawing inspiration from biology to device new materials<br />
and devices. Moreover, nanotechnology/nanobiotechnology<br />
is expected to rule tomorrow’s world.<br />
NANOBIOSTRUCTURES<br />
The use of biological principles is becoming widespread in<br />
the design of nanomaterials. Representative examples of<br />
nanobiostructures include DNA nanostructure, peptide<br />
structure and biomimetics. 3D-DNA nanostructures<br />
have emerged as promising tools for biology and materials<br />
science. In this regard, DNA cages, nanotubes, dendritic<br />
networks, and crystals with deliberate variation of their<br />
size, shape, persistence length, and porosities exhibited<br />
dynamic character, allowing their selective switching<br />
with external stimuli. Short peptides can spontaneously<br />
associate to form nanotubes, nanospheres, nanofi brils,<br />
nanotapes, and other ordered structures at the nanoscale.<br />
Further, peptides can also form macroscopic assemblies<br />
such as hydrogels with nanoscale order. The theory and<br />
the mechanisms behind peptide self-assembly process<br />
and their bionanotechnology applications have been<br />
reported. 3 In general, nanobiotechnology requires<br />
the organization of atoms and molecules in a two- or<br />
three-dimensional space. Self-assembly properties of<br />
biomolecules have ability to spontaneously organize<br />
into nanostructures, which allows mimicking the living<br />
cell membranes.<br />
Nanoparticle usually forms the core of nanobiomaterial.<br />
Nanobiomaterial is made of nanoparticles and<br />
nanobiomaterials are emerging as the most hopeful<br />
area of research within the area of biological materials<br />
science and engineering. They have an increased number<br />
of atoms and crystal grains at their surfaces and possess<br />
a higher surface area to volume ratio than conventional<br />
microscale biomaterials. These differences in surface<br />
topography alter the corresponding surface energy<br />
for protein adsorption. Nanobiomaterials can be used<br />
for human implant, orthopedics, drug delivery, gene<br />
therapy, antimicrobial treatments, array technologies,<br />
and diagnostics. Moreover, nanobiomaterials help<br />
with targeting, measuring, sensing, and imaging. The<br />
approaches used in constructing nanobiomaterials are<br />
given in Figure 2.<br />
NANOBIOTECHNOLOGY BASED DRUG DELIVERY/<br />
DEVELOPMENT<br />
Nanomedical approaches to drug delivery center<br />
on developing nanoscale particles or molecules to<br />
improve the bioavailability of a drug. Nanomaterials<br />
and nanoparticles are likely to be cornerstone of<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 15
Bhupinder Singh Sekhon et al.: Nanobiotechnology: An overview of drug discovery, delivery and development<br />
Figure 2: Specific configuration of nanobiomaterial when applied to biological or medical applications.<br />
innovative nanomedical devices employed for drug<br />
discovery and delivery, discovery of biomarkers<br />
and molecular diagnostics. 4 Preparation methodsproperties<br />
of nanoscale systems including liposomes,<br />
micelles, emulsions, nanoparticulates, and dendrimer<br />
nanocomposites, and clinical indications are known<br />
for imaging in vivo, sustained and targeted delivery of<br />
drugs, genes, and proteins. The recent development<br />
in drug delivery system has opened up new potential<br />
& possibility for production of pharmaceutical drugs,<br />
oral peptides, gene therapy and nanocosmeceuticals. 5<br />
Nanobiotechnology exploits nanotechnology and<br />
biotechnology to analyze and create nanobiosystems<br />
to meet a wide variety of challenges and develops a<br />
wide range of applications. Biomaterials like DNA and<br />
proteins combined with electronic systems consequently<br />
results in the formation of new devices, sensors, and<br />
systems. Examples include (i) a carbon nanotube<br />
with single-stranded DNA wrapping around it. (ii) a<br />
graphene sheet with duplex DNA molecules. (iii) a<br />
nanoparticle attachment with antibodies, (iv) a nanorod<br />
after enzyme immobilization and (v) Streptavidin<br />
(protein) attachment to nanoparticles along with biotin<br />
(protein). 6 Several technologies, including nanoparticles<br />
and nanodevices such as nanobiosensors, nanobiochips,<br />
nanosubstances were used to improve drug discovery<br />
and development. 7<br />
Gold nano rod-DARPP-32siRNA complexes (nanoplexes)<br />
have been reported suitable for brain-specifi c<br />
delivery of appropriate siRNA for therapy of drug<br />
addiction and other brain diseases. 8 Boron nitride nanotubes<br />
are structural analogues of carbon nanotubes in<br />
nature: alternating B and N atoms entirely substitute for<br />
C atoms in a graphitic like sheet with almost no change<br />
in atomic spacing. By virtue of their magnetic properties,<br />
boron nitride nanotubes could be exploited for<br />
magnetic, physically guided, drug targeting. The interactions<br />
between boron nitride nanotubes and living cells<br />
were reported and the piezoelectric properties of boron<br />
nitride nanotubes make them attractive candidates as<br />
bionanotransducers for cell sensing and stimulation,<br />
a use which still has to be exploited. 9<br />
DENDRIWORMS<br />
Dendriworms (magnetic nanoworm + dendrimer) are<br />
synthetic polymers that can be used to carry a large range<br />
of molecules such as siRNA (made up of magnetic<br />
nanoparticles as well as a fl uorescent nanoparticle)<br />
which allow the nanoworm to be traced as to where<br />
it is. Tremendous progress has been achieved in the<br />
recent years in our understanding of the ability of small<br />
interfering RNAs to silence gene expression in mammalian<br />
cells. This has provided us with a revolutionary new tool<br />
to modulate the expression of disease-causing genes.<br />
Short interfering RNAs (siRNAs) have emerged as a<br />
potent new class of therapeutics, which regulate gene<br />
expression through sequence-specifi c inhibition of<br />
mRNA translation. Lipid-, polymer-, and nanoparticlebased<br />
siRNA delivery vehicles have proven effective<br />
in improving the stability, bioavailability, and target<br />
specifi city of siRNAs following systemic administration<br />
in vivo. Additionally, these methods provided a platform<br />
to modify siRNAs with a variety of contrast agents and<br />
16 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
Bhupinder Singh Sekhon et al.: Nanobiotechnology: An overview of drug discovery, delivery and development<br />
have enabled nuclear and magnetic resonance imaging<br />
of siRNA delivery in preclinical studies. Such imageguided<br />
delivery approaches represent a crucial step in<br />
the transition of siRNA therapeutics to the clinic. 10<br />
Scientists reported the development of dendriworms<br />
as a modular platform for siRNA delivery in vivo.<br />
Researchers have demonstrated that siRNA-carrying<br />
dendriworms can be readily internalized by cells and<br />
enabled endosomal escape across a wide range of loading<br />
doses, whereas dendrimers or nanoworms alone were<br />
ineffi cient. In addition, dendriworms carrying siRNA<br />
against the epidermal growth factor receptor reduced<br />
protein levels of epidermal growth factor receptor in<br />
human glioblastoma cells by 70−80%, 2.5-fold more<br />
effi ciently than commercial cationic lipids. Reported<br />
data established dendriworms as a multimodal platform<br />
that enabled fl uorescent tracking of siRNA delivery<br />
in vivo, cellular entry, endosomal escape, and knockdown<br />
of target proteins. 11<br />
INORGANIC NANOCRYSTALS<br />
Researchers reported the fi rst use of inorganic<br />
fl uorescent lanthanide (europium and terbium) ortho<br />
phosphate [LnPO ·H O, Ln = Eu and Tb] nanorods as<br />
4 2<br />
a novel fl uorescent label in cell biology. These nanorods,<br />
synthesized by the microwave technique, retained their<br />
fl uorescent properties after internalization into human<br />
umbilical vein endothelial cells (HUVEC), 786-O cells,<br />
or renal carcinoma cells (RCC). At concentrations up to<br />
50 μg/ml, the use of [ 3H]-thymidine incorporation assays,<br />
apoptosis assays (TUNEL), and trypan blue exclusion<br />
illustrated the non-toxic nature of these nanorods,<br />
a major advantage over traditional organic dyes. 12<br />
DESIGN OF NEW-AGE DELIVERY SYSTEMS<br />
Scientists have shown a modifi ed version of the bacteriophage<br />
phi29 DNA-packaging motor, when reconstituted<br />
into liposomes and inserted into planar lipid bilayers,<br />
allowed the translocation of double-stranded DNA.<br />
Moreover, this engineered and membrane-adapted<br />
phage connector is expected to have applications in<br />
microelectromechanical sensing, microreactors, gene<br />
delivery, drug loading and DNA sequencing. 13<br />
Aptamers<br />
Aptamers, single stranded DNA or RNA molecules,<br />
generated by a method called SELEX (systematic<br />
evolution of ligands by exponential enrichment) have<br />
been widely used in various biomedical applications.<br />
Technology combination of nanobiotechnology<br />
with aptamers opened the way to more sophisticated<br />
applications in molecular diagnosis. Recent developments<br />
in SELEX technologies and new applications of<br />
aptamers have the ability to discriminate between two<br />
closely related targets, for example a cancerous cell and<br />
an untransformed cell of the same tissue type, makes<br />
aptamers suitable as imaging reagents for non-invasive<br />
diagnostic procedures. 14<br />
Implantable biomedical devices<br />
A bilayer structure comprising a thin gold layer and<br />
a polypyrrole fi lm has been developed as a valve and<br />
holds promise for implantable biomedical devices. 15<br />
Implantable medical devices that comprise a substrate<br />
and a porous layer comprising close packed spherical<br />
pores disposed over the substrate have been reported<br />
and the porous layer may also comprise a therapeutic<br />
agent. 16<br />
Nanoprobes<br />
Tiny nanoprobes have shown to be effective in delivering<br />
cancer drugs more directly to tumor cells - mitigating<br />
the damage to nearby healthy cells - and research has<br />
shown that the nanoprobes are getting the drugs to<br />
right cellular compartments. 17 Carbon nanotubes’<br />
unique properties including low cytotoxicity and good<br />
biocompatibility attract their use as vector system in<br />
target delivery of drugs, proteins and genes. 18<br />
Researchers have focused on the integration of biological<br />
molecules (DNA, antibodies, and enzymes)<br />
into micro- and nanostructures, with state-of-theart<br />
bioelectronic read-out systems, extracting useful<br />
analytical signals with interest for various fi elds. In<br />
this direction, nanotechnology based biosensors are<br />
the product of this integration with great interest for<br />
several applications that aim at a signifi cant improve<br />
of the quality and security of citizen’s life. 19 Rapamune<br />
was the fi rst product based on approved NanoCrystal<br />
Technology. Nanodrugs and nanodevices approved by<br />
the FDA, and emerging nanoproducts that may pose<br />
a challenge for current regulatory schemes both in the<br />
U.S. and internationally, have been reported. 20<br />
Scientists have designed a novel drug delivery vehicle<br />
by hybridizing macrophages with nanoparticles through<br />
cell surface modifi cation. Nanoparticles immobilized<br />
on the cell surface provided numerous new sites for<br />
anticancer drug loading, hence potentially minimizing<br />
the toxic effect of anticancer drugs on the viability and<br />
hypoxia-targeting ability of the macrophage vehicles.<br />
In particular, quantum dots and 5-(aminoacetamido)<br />
fl uoresceinlabeled polyamidoamine dendrimer G4.5,<br />
both of which were coated with amine-derivatized<br />
polyethylene glycol, were immobilized to the sodium<br />
periodate-treated surface of RAW264.7 macrophages<br />
through a transient Schiff base linkage. Further, a<br />
reducing agent, sodium cyanoborohydride, was applied<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 17
Bhupinder Singh Sekhon et al.: Nanobiotechnology: An overview of drug discovery, delivery and development<br />
to reduce Schiff bases to stable secondary amine linkages.<br />
The distribution of nanoparticles on the cell surface was<br />
confi rmed by fl uorescence imaging, and it was found to<br />
be dependent on the stability of the linkages coupling<br />
nanoparticles to the cell surface. 21<br />
Protein therapy<br />
Protein therapy, which delivers proteins into the cell<br />
to replace the dysfunctional protein, is considered the<br />
most direct and safe approach for treating disease and<br />
has been looked into as a possible alternative to gene<br />
therapy. Scientists have demonstrated a general, effective;<br />
low-toxicity intracellular protein delivery system based<br />
on single-protein nanocapsules, and opened a new<br />
direction not only for protein therapy but also for<br />
cellular imaging, tumor tracking, cosmetics and many<br />
other applications. 22 RNAi screen of the protein kinome<br />
identifi ed checkpoint kinase 1 (CHK1) as a therapeutic<br />
target in neuroblastoma. 23<br />
Stabilizing proteins at high concentration is of broad<br />
interest in drug delivery, for treatment of cancer<br />
and many other diseases. Scientists created highly<br />
concentrated antibody dispersions (up to 260 mg/ml)<br />
comprising of dense equilibrium nanoclusters of<br />
protein [monoclonal antibody (mAb) 1B7, polyclonal<br />
sheep Immunoglobulin G (IgG) and bovine serum<br />
albumin (BSA)] molecules, which upon dilution in vitro or<br />
administration in vivo, remained conformationally stable<br />
and biologically active. The nanoclusters are formed by<br />
adding trehalose as a co-solute which strengthened the<br />
short-ranged attraction between protein molecules. The<br />
protein cluster diameter was reversibly tuned from 50 to<br />
300 nm by balancing short-ranged attraction against<br />
long–ranged electrostatic repulsion of weakly charged<br />
protein at a pH near the isoelectric point (pI). 24<br />
Dendrimers/biodendrimers<br />
Dendrimers are nanostructures produced from<br />
macromolecules such as polyamidoamine, polypropyleneimine<br />
and polyaryl ether; and are highly branched with<br />
an inner core. The particle size range is between 1 to<br />
100 nm although their sizes are mostly less than 10 nm.<br />
Biodendrimers are dendrimers composed of repeating<br />
units known to be biocompatible or biodegradable<br />
in vivo to natural metabolites. Medical applications of<br />
dendrimers as multifunctional nanosized containers are<br />
shown in Figure 3.<br />
Dendrimers have successfully proved themselves<br />
as useful additives in different routes of drug<br />
administration to be applied in routes with particular<br />
reference to intravenous, oral, transdermal, and ocular<br />
delivery systems. 25 Dendrimers can act as vectors, in<br />
gene therapy and as an agent for Photodynamic Therapy<br />
of tumorigenic keratinocytes. 26<br />
Figure 3: Some medical applications of dendrimers/biodendrimers.<br />
NANOSTRUCTURED DRUG DELIVERY SYSTEMS<br />
Bionanofabrication<br />
2D crystallization of proteins, especially engineered<br />
proteins, is emerging as a powerful tool for bottom-up<br />
approaches to the nanofabrication of functional<br />
structures. Some aspects of 2D protein crystallization,<br />
including key approaches to growing 2D protein crystals<br />
and their potential applications ranging from biosensors,<br />
diagnostic kits, vaccine applications, and templates for<br />
mineral formation have been reported. 27<br />
Nanostructured lipid carriers (NLC) that can deliver<br />
active pharmaceutical ingredients across the skin have<br />
emerged as novel systems composed of physiological<br />
lipid materials suitable for topical, dermal and<br />
transdermal administration. The design characteristics,<br />
production and composition of semi-solid formulations<br />
containing NLC as API carriers (for example hydrogels)<br />
have been reported. 28 Results of characterization studies<br />
strongly supported the potential application of these<br />
drugs-loaded NLC as prolonged delivery systems for<br />
lipophilic drugs by several administration routes, in<br />
particular for intravenous administration. 29<br />
Fullerenes for Medical Diagnostics<br />
Scientists have created new materials from Fullerenes by<br />
fi lling them with atoms of various metals. An important<br />
example is a fullerene that encases a sensitive contrast<br />
agent (gadolinium) for MRI applications, including as<br />
a diagnostic and therapeutic agent for brain tumors.<br />
Researchers have co-invented a hands-off process for<br />
fi lling fullerenes with radioactive material and these fi nding<br />
could be utilized in medical applications, such as MRIs<br />
and diagnostic and therapeutic agents for brain tumors. 30<br />
NANOBIO TECHNOLOGY AND TISSUE ENGINEERING<br />
Scientists discussed the current applications of nanoscale<br />
materials to bladder tissue engineering. 31 Researchers<br />
18 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
Bhupinder Singh Sekhon et al.: Nanobiotechnology: An overview of drug discovery, delivery and development<br />
explored the bottom-up and top-down nanofabrication<br />
technologies and their use in various drug delivery and<br />
tissue engineering applications. 32<br />
NANOBODIES<br />
The structural properties of Nanobodies and their<br />
possible therapeutic applications were discussed and<br />
data from phase I clinical trials of the novel ‘fi rstin-class’<br />
anti-thrombotic agent ALX-0081 (Ablynx NV)<br />
were reported. 33 Results suggested that the generation<br />
of Nanobody-displaying immune phage libraries and<br />
subsequent in vivo biopanning in appropriate animal<br />
models is a promising approach for the identifi cation of<br />
novel vascular targeting agents. 34<br />
NANOBOTS/NANOROBOTS<br />
Nanorobots could be employed for the diagnostics,<br />
targeted drug dispensation, elimination of xenogenous<br />
particles from the body, and repair of cells and tissues,<br />
e.g. the skin and teeth. 35 Nanorobots could also be<br />
programmed to perform delicate surgeries or remove<br />
obstructions in the circulatory system. 36<br />
BIOMARKERS<br />
Biomarkers are molecules that can be measured in blood,<br />
other body fl uids, and tissues to assess the presence or<br />
state of a disease. They have the potential to help us<br />
detect cancer earlier, determine a tumor’s aggressiveness,<br />
or predict a patient’s response to a particular treatment.<br />
Biomarkers play a role in use of pharmacogenetics,<br />
pharmacogenomics and pharmacoproteomics for<br />
development of personalized medicine. Label-free<br />
nanosensors can detect disease markers to provide<br />
point-of-care diagnosis that is low-cost, rapid, specifi c<br />
and sensitive. Scientists showed specifi c and quantitative<br />
detection of two model cancer antigens from a 10 μl<br />
sample of whole blood in less than 20 min. They used<br />
nanowire sensors to detect and measure concentrations<br />
of two specifi c biomarkers: one for prostate cancer<br />
and the other for breast cancer. This novel device acted<br />
as a fi lter, catching the biomarkers, antigens specifi c<br />
to prostate and breast cancer on a chip while washing<br />
away the rest of the blood. 37 The emerging trends in<br />
the development of biomarkers for early detection and<br />
precise evaluation of cancer disease were reported. 38<br />
This bio-nano-info convergence holds great promise for<br />
molecular diagnosis and individualized therapy of cancer<br />
and other human diseases. Scientists have demonstrated<br />
the feasibility of multiplex detection using the surfaceenhanced<br />
Raman scattering-based molecular sentinel<br />
(MS) technology in a homogeneous solution. Two MS<br />
nanoprobes tagged with different Raman labels were<br />
used to detect the presence of the erbB-2 and ki-67<br />
breast cancer biomarkers. The multiplexing capability of<br />
the MS technique was demonstrated by mixing the two<br />
MS nanoprobes and tested in the presence of single or<br />
multiple DNA targets. 39<br />
NANOBIOTECHNOLOGY IN CANCER<br />
Application of nanotechnology/nanobiotechnology in<br />
biomedicine may contribute to signifi cant advances in<br />
imaging diagnosis and treatment of cancer. Quantum<br />
dots, gold nanoparticles, magnetic nanoparticles,<br />
carbon nanotubes, gold nanowires and many other<br />
materials have been developed over the years, alongside<br />
the discovery of a wide range of biomarkers to lower<br />
the detection limit of cancer biomarkers. Current<br />
developments in cancer detection methods with an<br />
emphasis on nanotechnology were reported. 40 Some<br />
of the recent development in nanotechnologies and<br />
their applications in diagnosing and developing cancer<br />
therapies have been reported. 41<br />
Scientists reported a cooperative nanosystem consisting<br />
of two discrete nanomaterials that work in concert within<br />
the bloodstream to locate, adhere to and kill cancerous<br />
tumors. While one type of nanoparticle improved<br />
detection of the tumor, the other was designed to kill the<br />
tumor. The fi rst component consists of gold nanorod<br />
“activators” that populate the porous tumor vessels and<br />
act as photothermal antennas to specify tumor heating<br />
via remote near-infrared laser irradiation. In addition,<br />
it was found that local tumor heating accelerated the<br />
recruitment of the second component: a targeted<br />
nanoparticle consisting of either magnetic nanoworms<br />
(NW) or doxorubicin-loaded liposomes (LP). Nineamino<br />
acid peptide LyP-1 (Cys-Gly-Asn-Lys-Arg-Thr-<br />
Arg-Gly-Cys) was employed as the targeting species that<br />
binds to the stress-related protein, p32, which was up<br />
regulated on the surface of tumor-associated cells upon<br />
thermal treatment. Mice containing xenografted MDA-<br />
MB-435 tumors that were treated with the combined<br />
gold nanorod /LyP-1- doxorubicin-loaded liposomes<br />
therapeutic system displayed signifi cant reductions in<br />
tumor volume compared with individual nanoparticles<br />
or untargeted cooperative system. 42<br />
A new nanoparticle formulation can be magnetically<br />
guided to deliver and silence genes in cells and tumors<br />
in mice. This formulation, termed LipoMag, consisted<br />
of an oleic acid-coated magnetic nanocrystal core<br />
and a cationic lipid shell. When compared with the<br />
commercially available PolyMag formulation, LipoMag<br />
displayed more effi cient gene silencing in 9 of 13 cell<br />
lines, and better anti-tumour effects when systemically<br />
administered to mice bearing gastric tumours. By<br />
delivering an optimized sequence of a silencing RNA<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 19
Bhupinder Singh Sekhon et al.: Nanobiotechnology: An overview of drug discovery, delivery and development<br />
that targets the epidermal growth factor receptor of<br />
tumor vessels, the intended therapeutic benefi t was<br />
achieved with no evident adverse immune reaction or<br />
untoward side effects. 43<br />
NANOMEDICINE<br />
Nanomedicine has triggered the wind of revolution in<br />
medicine and important areas that nanomedicine covers<br />
include targeted drug delivery in cancer treatment,<br />
biotechnology, disease diagnosis to nanostructure<br />
implants. 44<br />
A variety of recent research combines Heparin (HP) and<br />
nanomaterials for a myriad of applications. HP has been<br />
conjugated to the surface of the nanoparticles, such as<br />
magnetic and metallic nanoparticles, or biodegradable<br />
and nondegradable synthetic polymers. HP has also<br />
been incorporated into the nanoparticles. The different<br />
possibilities of HP-based nanoparticle composites and<br />
their medicinal or biological applications were reported. 45<br />
It was qualitatively demonstrated that enhanced<br />
fl uorescence emission signals occurred from clustered<br />
QDs and deduced that the band 3 membrane proteins<br />
in erythrocytes were clustered. 46<br />
Nanonephrology<br />
Nanonephrology is a branch of nanomedicine and<br />
nanotechnology that deals with (i) the study of kidney<br />
protein structures at the atomic level (ii) nano-imaging<br />
approaches to study cellular processes in kidney cells and<br />
(iii) nanomedical treatments that utilize nanoparticles<br />
and to treat various kidney diseases. Various devices<br />
based on nanotechnology are used for the studying<br />
the different kidney processes and detecting disorders.<br />
Nanotechnological fi lters have the potential to provide<br />
immediate relief for dialysis patients. 47 Scientists have<br />
demonstrated a new artifi cial renal chip by integrating<br />
a high effi cient biocompatible polymeric nanofi bers<br />
membrane with the polydimethylsiloxane(PDMS)<br />
based micro-fl uidic platform via the optimization of<br />
PDMS micro fl uidic channel network and the multiple<br />
packaging of nanofi bers membrane. 48<br />
Nanobiopharmaceutics<br />
Nanobiopharmaceutics aims at the development of<br />
innovative multidisciplinary approaches for the design,<br />
synthesis and evaluation of molecular, nano- and microscale<br />
functionalities for targeted delivery of therapeutic<br />
peptides and proteins (biopharmaceutics). Nanoscale<br />
biomaterials are categorized by metal, non-metal, carbon,<br />
polymer, lipid, virus and miscellaneous nanostructures<br />
as nanobiopharmaceutical carrier systems and their<br />
medical/biological applications as well as toxicological<br />
issues in the fi eld of biomedical nanotechnology<br />
were reported. 49 The basis of technologic application<br />
in biopharmaceutics is Nanoscale Drug Carrying<br />
System and the various units of the system consist of<br />
liposome system which varies between 30 nm to several<br />
micrometers in diameter with their characteristics<br />
governed by their size, surface composition and charge.<br />
Surface modifi ed liposomes carrying doxorubicin and<br />
antisense oligonucleotide system have successfully<br />
targeted multidrug resistance associated protein,<br />
messenger RNA and bcl2 RNA. System after reaching<br />
the cell, delivered the doxorubicin and the antisense<br />
oligonucleotides successfully and inhibited the synthesis<br />
of MRP1 and bc12 RNA and provoked the apoptosis<br />
of carcinomatous cell by arousing the caspase (cysteineaspartic<br />
proteases) dependent pathway. 50<br />
A “nanoviricide” is a fl exible nano-scale material<br />
approximately a few billionths of a meter in size, which<br />
is chemically programmed to specifi cally target and<br />
attack a particular type of virus like a guided missile.<br />
NV-INF-1, the selected candidate drug substance of the<br />
FluCide program, has been shown to be highly effective<br />
in controlling infl uenza viral infection in lethal infection<br />
mouse model. NanoViricides nanotechnology possesses<br />
potent antiviral effi cacy by targeting the mechanisms by<br />
which viruses attach or bind to cells. 51<br />
Nanopharmacology<br />
Nanopharmacology is the use of nanotechnology<br />
for (i) discovery of new pharmacological molecular<br />
entities; (ii) selection of pharmaceuticals for specifi c<br />
individuals to maximize effectiveness and minimize side<br />
effects; and (iii) delivery of pharmaceuticals to targeted<br />
locations or tissues within the body . The potential<br />
applications of biochips, nanosensors, bioreactors,<br />
neural stem cells, immune nanoparticles, biodegradable<br />
polymers, and convection-enhanced drug delivery in the<br />
diagnostics and treatment of diseases were reported.<br />
Numerous novel medicinal forms were reported,<br />
including polymeric nanoparticles, nanotubes, micelles,<br />
liposomes, dendrimers, fullerenes, and hydrogels. In<br />
particular, highly stable glycosphingolipid nanotubes<br />
and nanoliposomes were proposed as drug delivery<br />
systems. For this purpose, the model of stimulation of<br />
skin vasomotor reactions by nitroglycerin application<br />
was developed. The effect of nitroglycerin was found to<br />
increase 1.5 times when used in the form of dispersion<br />
with nanotubes as carriers and almost 2.5 times in the<br />
case of dispersion with nanoliposomes as carriers.<br />
Nanotechnologically manufactured biologically active<br />
substances Apiton-25 (containing apis products) and<br />
Microhydrin (containing SiO nanoparticles, silicon<br />
2<br />
hydrogen bonds Si-H, and free negative charges for free<br />
radical neutralization) results have indicated that both<br />
Apiton-25 and Microhydrin upon peroral administration<br />
20 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
Bhupinder Singh Sekhon et al.: Nanobiotechnology: An overview of drug discovery, delivery and development<br />
enhanced the cyclic trial performance during prolonged<br />
submaximal exercise in endurance-trained cyclists. 52<br />
Scientists have developed a combination drug that<br />
promises a safer, more precise way for medics and fellow<br />
soldiers in battle to give a fallen soldier both morphine<br />
and a drug that limits morphine’s dangerous side effects.<br />
They have used nanotechnology to devise ultra-small<br />
polymer particles capable of carrying the drugs into<br />
the body. The development of the combination drug<br />
makes possible a precise feedback system that can safely<br />
regulate release of the drugs aboard the nanoparticles. 53<br />
NANOBIOTECHNOLOGY AND PERSONALIZED<br />
MEDICINE<br />
Nanobiotechnology will facilitate the integration<br />
of diagnostics with therapeutics and facilitate the<br />
development of personalized medicine. 54 Nanobiotechnology<br />
is being used to refi ne discovery of biomarkers,<br />
molecular diagnostics, drug discovery and<br />
drug delivery, which are important basic components of<br />
personalized medicine. 55 Personalized management of<br />
cancer, facilitated by nanobiotechnology, is expected to<br />
enable early detection of cancer, more effective and less<br />
toxic treatment increasing the chances of cure. 56<br />
Nanobiomaterials/Nanobioanalysis/<br />
Nanobiochips<br />
Novel opportunities and challenges offered by nanobiomaterials<br />
in tissue engineering have been reported. 57<br />
The current state of development of nanodiagnostic<br />
technologies including nanobiochips and nanobiosensors<br />
has been reported. 58 Neuronanobiotechnology<br />
based delivery system are developing rapidly and one<br />
example of current nanobiotechnological research<br />
involved nanospheres coated with fl uorescent polymers.<br />
59 An assay based on gold nanoparticles could<br />
detect recurrences of prostate cancer sooner than is<br />
possible with existing techniques. 60 Carbon nanotubes<br />
have been used to probe the properties of bilayer systems<br />
resembling living cell membranes. 61<br />
A multifunctional one-dimensional nanostructure<br />
incorporating both CdSe quantum dots (QDs) and Fe O 3 4<br />
nanoparticles (NPs) within a SiO -nanotube matrix was<br />
2<br />
successfully synthesized based on the self-assembly of<br />
preformed functional NPs, allowing for control over the<br />
size and amount of NPs contained within the composite<br />
nanostructures. This specifi c nanostructure is distinctive<br />
because both the favorable photoluminescent and<br />
magnetic properties of QD and NP building blocks<br />
were incorporated and retained within the fi nal silicabased<br />
composite, thus rendering it susceptible to both<br />
magnetic guidance and optical tracking. Moreover, the<br />
resulting hydrophilic nanocomposites were found to<br />
easily enter into the interiors of HeLa cells without<br />
damage, thereby highlighting their capability not<br />
only as fl uorescent probes but also as possible drugdelivery<br />
vehicles of interest in nanobiotechnology. 62<br />
A large variety of nanobioanalysis methods have been<br />
reported. 63 Scientists have described the construction<br />
and use of two major classes of nano-bio-chip designs<br />
that serve as cellular and chemical processing units.<br />
These nanobiochips possess capabilities for measuring<br />
such diverse analyte classes as cells, proteins, DNA and<br />
small molecules in the same compact device. Further,<br />
applications such as disease diagnosis and prognosis<br />
for areas including cancer, heart disease and HIV were<br />
reported. 64 One-dimensional nanostructures such as<br />
nanowires are ideal for diagnosis as they can be integrated<br />
into microfl uidic chips that provide a complete sensor<br />
system. 65<br />
DNA nanomachines are synthetic assemblies that<br />
switch between defi ned molecular conformations<br />
upon stimulation by external triggers. Researchers have<br />
reported the construction of a DNA nanomachine called<br />
the I-switch, which is triggered by protons and functions<br />
as a pH sensor based on fl uorescence resonance energy<br />
transfer inside living cells. Moreover, this was found<br />
an effi cient reporter of pH from pH 5.5 to 6.8, with<br />
a high dynamic range between pH 5.8 and 7, thereby,<br />
illustrating the potential of DNA scaffolds responsive<br />
to more complex triggers in sensing, diagnostics and<br />
targeted therapies in living systems. 66<br />
INTEGRATION OF RECOMBINANT TECHNOLOGY<br />
AND NANOSCIENCE<br />
Recombinant technology is the most important<br />
prerequisite for the effective engineering of nanostructured<br />
deoxyribonucleic acid and protein based<br />
materials in nanoscience. This technology allowed the<br />
manipulation of the properties of molecules, including<br />
physico-chemical properties of proteins that control<br />
electron transport and photochemical processes in<br />
the development of molecular electronic devices and<br />
device fabrication. Recombinant molecules, such as<br />
recombinant ovalbumin and recombinant ovalbumin<br />
mutants have provided a powerful means for the study<br />
of their physico-chemical and structural characteristics,<br />
and thereby for their use in nanoscience. The researchers<br />
have provided an overview of the integration of<br />
recombinant technology and nanoscience through<br />
reported studies in areas, including food, environment,<br />
medicine, physics and chemistry. 67<br />
CONCLUSIONS AND PERSPECTIVES<br />
Nanobiotechnology is an emerging fi eld that seeks new<br />
solutions to pressing health and environmental problems<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 21
Bhupinder Singh Sekhon et al.: Nanobiotechnology: An overview of drug discovery, delivery and development<br />
by combining physical sciences and engineering with<br />
life sciences and medicine. This exciting frontier of<br />
discovery is generating new therapies, devices, diagnostic<br />
tools, and a better understanding of the relationship<br />
between cells and disease. Nanobiotechnology also<br />
deals with the investigation and utilization of the newly<br />
conceived nanomaterials, as well as the construction of<br />
functionalized nanobiosystems. Nanopharmaceuticals<br />
offer the ability to detect diseases at much earlier<br />
stages. Drug delivery is the most advanced tool of<br />
nanobiotechnology. Nanomedicine combines nanotechnology<br />
and medicine, and nanotherapeutics is the<br />
use of nanomedicine in therapy. The ultimate goal of<br />
nanotherapeutic is comprehensive monitoring repair<br />
and improvement of all human biologic system.<br />
Nanobiotechnology should provide many of the tools<br />
necessary to enable the components that will one day be<br />
inserted into commercialized products.<br />
Genomics, proteomics, and metabolomics combined<br />
with the power of nanobiotechnology have the potential<br />
to understand the disease in a way that was previously<br />
not possible and it is expected that the disease will<br />
be targeted more effectively and precisely. Experts<br />
support a comprehensive technology assessment of<br />
nanobiotechnologies along with a full-blown risk/benefi t<br />
analysis before their adoption. New biofriendly imaging<br />
probes based on functionalized inorganic nanocrystals are<br />
being developed and are available to facilitate state-of-theart<br />
bioimaging studies. The research on the combination<br />
of chemotherapeutic agents with gene therapy should<br />
be further strengthened to overcome the limitations of<br />
conventional cancer treatment. In this context, a strongly<br />
synergistic antiproliferative effect was observed in colon<br />
cancer cells when E gene expression was combined with<br />
the activity of the 5-Fluorouracil–loaded biodegradable<br />
poly(ε-caprolactone) nanoparticles, thereby indicating<br />
the potential therapeutic value of the combined<br />
therapy. 68 It is hoped that nanobiotechnology will extend<br />
the limits of current molecular diagnostics and enable<br />
point of care diagnostics, integration of diagnostics<br />
with therapeutics, and development of personalized<br />
medicine. Three-dimensional nanobiostructure-based<br />
self-contained devices consisting of a glucose/oxygen<br />
sensor, a biofuel cell and a wireless signal transmitter to<br />
demonstrate wireless monitoring of glucose and oxygen<br />
in biological fl uids, wounds, and cell cultures is under<br />
development. 69 As yet there are no directives to regulate<br />
nanobiotechnology by various regulating bodies but as<br />
products are ready to enter market, these are expected<br />
to be in place. The largest expansion is expected in<br />
coming years. 70 Nanobiotechnology is likely to trigger<br />
advances in the early detection of a variety of diseases<br />
and improvements in biological implants. Experts are of<br />
the opinion that efforts should be made to educate, and<br />
increase awareness about nanobiotechnology through a<br />
transparent public dialogue.<br />
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RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 23
Nasal drug delivery–a review<br />
Twarita Deshpande* 1 , Rajashree Masareddy 2 , Archana Patil 1<br />
1 Rani Chennamma College of Pharmacy, Belgaum<br />
2 College of Pharmacy, K.L.E. University, Belgaum<br />
ABSTRACT<br />
This paper discusses the theory of mucoadhesion along with various approaches to improve nasal absorption by<br />
the use of mucoadhesive polymers and absorption enhancers. An account of various mucoadhesive polymers is<br />
also given. A note on absorption enhancers has been included. Finally the growing market for nasal drug delivery<br />
is discussed.<br />
INTRODUCTION<br />
Nasal drug delivery for systemic effects has<br />
been practiced since ancient times. In modern<br />
pharmaceutics, the nose had been considered<br />
primarily as a route for local drug<br />
delivery. The last two decades heralded a<br />
number of advances in pharmaceutical<br />
biotechnology resulting in possibilities for<br />
large-scale productions of biopharmaceuticals<br />
especially proteins and peptides. The<br />
inability to administer these drugs by routes<br />
other than parenteral injection motivated<br />
scientists to explore other possibilities such<br />
as pulmonary and nasal administration. The<br />
initial enthusiasm was soon confronted<br />
with disappointing in vivo results showing<br />
poor bioavailabilitiy, typically in the order<br />
of 5–10% for large molecules. 1 On the<br />
other hand, very good results were obtained<br />
with small organic molecules, which led<br />
to the successful development of a number<br />
of products currently on the market<br />
list of products that is steadily increasing.<br />
Examination of the causes of failure led<br />
to the conclusion that the short residence<br />
time of the formulation within the nasal<br />
cavity coupled to the low permeability of<br />
the latter did play signifi cant roles. Consequently,<br />
the attention shifted to the evaluation<br />
of mucoadhesive polymers, some<br />
of which would even demonstrate additional<br />
permeation-enhancing capabilities. 2,3<br />
The encouraging results and the desire to<br />
overcome some new challenges stimulated<br />
the development of new generations of<br />
polymers based on pH, thermal responsiveness,<br />
4 modifi ed existing polymers having<br />
improved bioadhesive or permeationenhancing<br />
properties. 5,6,7 Even though a<br />
number of challenges are still to be overcome,<br />
especially with respect to toxicity, the<br />
potential of nasal drug delivery (NDD),<br />
including the ability to target drugs across<br />
the blood–brain barrier (BBB), are very<br />
high and continues to stimulate academic<br />
and industrial research groups so that we<br />
will keep witnessing increasing number of<br />
advanced nasal drug delivery products. To<br />
optimize nasal administration, bioadhesive<br />
hydrogels, Bioadhesive microspheres (dextran,<br />
albumin and degradable starch) and<br />
liposomes have been studied.<br />
ANATOMY<br />
The nasal cavity is divided into two<br />
halves by the nasal septum and extends<br />
posteriorly to the nasopharynx, while the<br />
most anterior part of the nasal cavity, the<br />
nasal vestibule, opens to the face through<br />
the nostril. The atrium is an intermediate<br />
region between the vestibule and the<br />
respiratory region. The respiratory region,<br />
the nasal conchae or turbinates, which<br />
Review Ar cle<br />
Received Date : 08-08-2011<br />
Revised Date : 11-02-2012<br />
Accepted Date : 14-02-2012<br />
DOI: 10.5530/rjps.2012.1.4<br />
Address for<br />
correspondence<br />
Twarita D. Deshpande<br />
Department of Pharmaceutics<br />
Rani Chennamma College of<br />
Pharmacy<br />
Vaibhav Nagar, Belgaum<br />
Pin Code-590010<br />
E-mail: twarita.deshpande@<br />
gmail.com<br />
Mobile: +91 9886181824<br />
www.rjps.in<br />
24 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
occupies the major part of the nasal cavity, possesses<br />
lateral walls dividing it into 3 sections: the superior,<br />
middle and inferior nasal turbinate fi g 1. These folds<br />
provide the nasal cavity with a very high surface area<br />
compared to its small volume.<br />
The epithelial cells in the nasal vestibule are<br />
stratifi ed, squamous and keratinized with sebaceous<br />
glands. Due to its nature, the nasal vestibule is very<br />
resistant to dehydration and can withstand noxious<br />
environmental substances and limits permeation<br />
of substances. The atrium is a transitional epithelial<br />
region with stratifi ed, squamous cells anteriorly<br />
and pseudostratifi ed columnar cells with microvilli<br />
posteriorly. Pseudostratifi ed columnar epithelial cells<br />
interspersed with goblet cells, seromucus ducts, the<br />
openings of sub epithelial seromucus glands cover<br />
the respiratory region (the turbinates). Furthermore,<br />
many of these cells possess actively beating cilia with<br />
microvilli. Each ciliated cell contains about 100 cilia,<br />
while both ciliated and nonciliated cells possess about<br />
300 microvilli each. 8<br />
PHYSIOLOGY<br />
The nasal physiologic functions, such as warming and<br />
humidifi cation, are vital for upper airway function. It has<br />
been estimated that an adult inspires up to 10,000 liters<br />
of air daily.<br />
Figure 1: Anatomy of Nasal Cavity.<br />
Twarita Deshpande et al.: Nasal Drug Delivery-A Review<br />
Nasal breathing is healthy breathing as the air is treated<br />
in many ways by the structures of the nose, paranasal<br />
sinuses and the peculiarities of their lining mucosa.<br />
1. Filtering the air<br />
Filtration of environmental particles occurs fi rst in the<br />
nasal cavity. Nasal mucus traps incoming particulate<br />
matter. The largest particles are fi ltered by nasal hairs<br />
(vibrissae).<br />
2. Moistening the air<br />
Humidifi cation is another important process of nasal<br />
physiology. The nasal cavity is covered with a highly<br />
vascular mucosa that warms and humidifi es incoming<br />
air, increasing the relative humidity to 95% before air<br />
reaches the nasopharynx.<br />
3. Warming the air<br />
Inhaled air must have a temperature between at least<br />
33 and 35 degree Celsius to not cause pathological<br />
reactions at the level of the alveoli. Again, by the<br />
turbulence, the cold air is forced to make contact with<br />
the warm surface of the mucosa and thus heated during<br />
its passage. A number of nasal neurovascular refl exes<br />
occur as well. If needed, underlying capillaries will dilate<br />
and warm up the upper laying mucosa, giving more heat<br />
to the passing air.<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 25
4. The sense of smell<br />
Nasal aerodynamics also contributes to the olfactory<br />
system. In addition, the active process of sniffi ng allows<br />
environmental particles to reach the olfactory system<br />
located at the skull base.<br />
Even the smallest particles are detected by the olfactory<br />
receptors, warning us about danger, food, or any other<br />
biologically meaningful sign detectable through the<br />
sense of smell.<br />
5. The nasal cavity as a sound box<br />
The nose and sinuses serve as contributing factors in<br />
voice modifi cation. Authors have noted that nasal<br />
aerodynamics may have a role in modifying highfrequency<br />
sounds and consonants. The resonance<br />
created within the nasal cavity is characteristic, similar<br />
to a fi nger print and for each person is different (except<br />
in identical twins). Nasal pathologies such as polyps or<br />
rhinitis will directly infl uence the resonance spectrum<br />
and we will”hear“that the person has a cold or something<br />
has changed in his/her voice. 9<br />
MUCOADHESION AS A STRATEGY TO<br />
IMPROVE SYSTEMIC DRUG DELIVERY<br />
VIA THE NASAL ROUTE<br />
Several theories have been put forward to explain the<br />
mechanism of polymer-mucus interactions that lead to<br />
mucoadhesion. To start with, the sequential events that<br />
occur during bioadhesion include an intimate contact<br />
between the bioadhesive polymer and the biological<br />
tissue due to proper wetting of the bioadhesive surface<br />
and swelling of the bioadhesive. Following this is the<br />
penetration of the bioadhesive into the tissue crevices,<br />
interpenetration between the mucoadhesive polymer<br />
chains and those of the mucus. Subsequently low<br />
chemical bonds can become operative. 10, 11 Hydration of<br />
the polymer plays a very important role in bioadhesion.<br />
There is a critical degree of hydration required for<br />
optimum bioadhesion. If there is incomplete hydration,<br />
the active adhesion sites are not completely liberated<br />
and available for interaction. An alternative approach to<br />
the use of chemical enhancers to improve nasal drug<br />
absorption is to increase the duration of formulation<br />
residence within the nasal cavity. This is achieved by<br />
the use of bioadhesive polymers. Apart from these<br />
synthetic and natural polymers, there is now a new class<br />
of promising compounds, the lectins, often referred<br />
to as second-generation mucoadhesive materials.<br />
These are non-immunogenic proteins or glycoproteins<br />
capable of specifi c recognition and reversible binding<br />
to carbohydrate moieties of complex glycoconjugates<br />
without altering the covalent nature of any of the<br />
recognized glycosyl ligands. The use of mucoadhesives<br />
Twarita Deshpande et al.: Nasal Drug Delivery-A Review<br />
can solve a number of problems encountered in<br />
controlled drug delivery. It localizes the formulation at<br />
a particular region in the body, thereby improving the<br />
bioavailability of drugs with low bioavailability. The<br />
increased contact time and localization of the drug<br />
due to the strong interaction between the polymer<br />
and mucus is essential for the modifi cation of tissue<br />
permeability. Furthermore, enzymatic activity can be<br />
locally inhibited to improve the bioavailability of drugs<br />
that are subject to enzymatic degradation. This has<br />
been demonstrated for some mucoadhesive polymers<br />
such as Carbopol 934P and polycarbophil that inhibit<br />
the proteolytic enzyme trypsin, which can thus increase<br />
the stability of co-administered Peptides (Leuben<br />
et al. 1994). Some studies have also demonstrated that<br />
mucoadhesive polymers can also directly interact with the<br />
epithelial tight junctions. 12 The mucoadhesive polymers<br />
reported in the literature are summarized in Table 1.<br />
The theories and mechanisms of bioadhesion are given<br />
Table 2. 13<br />
ADVANTAGES OF MUCOADHESIVE DRUG<br />
DELIVERY SYSTEMS<br />
Mucoadhesive dosage forms have three different advantages<br />
when compared to conventional dosage forms<br />
1. These dosage forms are readily localized in the region<br />
applied to improve and enhance the bioavailability<br />
of drugs. Greater bioavailability of piribedit,<br />
testosterone and its esters, vasopressin, dopamine,<br />
insulin was observed from mucoadhesive dosage<br />
forms when compared to conventional dosage<br />
forms.<br />
Table 1: Mucoadhesive Polymers<br />
Polymer Bioadhesive property<br />
Carboxymethyl cellulose<br />
3<br />
Carbopol 934<br />
3<br />
Polycarbophil<br />
3<br />
Tragacanth<br />
3<br />
Poly(acrylic acid/divinyl benzene)<br />
3<br />
Sodium alginate<br />
3<br />
Hydroxy ethyl cellulose<br />
3<br />
Gum Karaya<br />
3<br />
Gelatin<br />
3<br />
Guar gum<br />
2<br />
Thermally modifi ed starch<br />
2<br />
Pectin<br />
1<br />
Polyvinyl Pyrrolidone<br />
1<br />
Acacia<br />
1<br />
Polyethylene glycol<br />
1<br />
Psyllium<br />
1<br />
Amberlite-200 resin<br />
1<br />
Hydroxy propyl cellulose<br />
1<br />
Chitosan<br />
1<br />
Hydroxy ethyl methacrylate<br />
1<br />
NOTE: 3- Excellent, 2- Fair, 1- Poor.<br />
26 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
2. These dosage forms facilitate intimate contact of the<br />
formulation with the underlying absorption surface.<br />
This allows modifi cation of tissue permeability for<br />
absorption surface. This allows modifi cation of tissue<br />
permeability for absorption of macromolecules, such<br />
as peptides and proteins. Inclusion of penetration<br />
enhancers such as sodium glycholate, sodium<br />
taurocholate and L-lysophosphotidyl choline (LPC)<br />
and protease inhibitors in mucoadhesive dosage<br />
forms resulted in better absorption of peptides and<br />
proteins.<br />
3. Mucoadhesive dosage forms also prolong residence<br />
time of the dosage form at the site of application and<br />
absorption to permit once or twice a day dosing. 14<br />
The absorption enhancement mechanisms can be<br />
grouped into two classes:<br />
NASAL ABSORPTION ENHANCERS<br />
Physicochemical effects: Some enhancers can alter the<br />
physicochemical properties of a drug in the formulation.<br />
This can happen by altering the drug solubility, drug<br />
partition coeffi cient, or by weak ionic interactions with<br />
the drug.<br />
Membrane effects<br />
Many enhancers show their effects by affecting the nasal<br />
mucosa surface. 15 Nasal absorption enhancers involve<br />
two main classes. The most important group involve<br />
microspheres, liposome’s and gels that have been utilized<br />
as drug carriers in the past few years. The second group<br />
Twarita Deshpande et al.: Nasal Drug Delivery-A Review<br />
Table 2: Theories and Mechanisms of Bioadhesion<br />
Theory Mechanism of bioadhesion Comments<br />
Electronic theory Attractive electrostatic forces between<br />
Electrons transfer occurs between the two<br />
glycoprotein mucin network and the<br />
forming a double layer of electric charge at<br />
bioadhesive material.<br />
the surface<br />
Wetting theory Ability of bioadhesive polymer to spread<br />
Spreading coeffi cient of polymers must be<br />
and develop intimate contact with the<br />
positive. Contact angle between polymer and<br />
mucous membrane.<br />
cells must be near to zero.<br />
Adsorption theory Surface force resulting in chemical<br />
Strong primary force: covalent bonds. Weak<br />
bonding.<br />
secondary forces: hydrogen bonds and van<br />
der Waal’s forces<br />
Diffusion theory Physical entanglement of mucin strands<br />
For maximum diffusion and best adhesive<br />
and fl exible polymer chains.<br />
strength, solubility parameters of the<br />
bioadhesive polymer and the mucus<br />
glycoproteins must be similar<br />
Mechanical theory Adhesion arises from an interlocking of<br />
Rough surfaces provide an increased surface<br />
liquid adhesive into irregularities on<br />
area available for interaction along with an<br />
the rough surface.<br />
enhanced viscoelastic and plastic dissipation<br />
of energy during joint failure, which are more<br />
important in the adhesion process than a<br />
mechanical effect.<br />
Fracture theory Analyses the maximum tensile stress<br />
Does not require physical entanglement of<br />
developed during attachment of the<br />
bioadhesive polymer chains and mucous<br />
transmucosal DDS from the mucosal<br />
strands, hence it is appropriate to study the<br />
surface<br />
bioadhesion of hard polymers which lack<br />
fl exible chains<br />
will be discussed under the heading other penetration<br />
enhancers.<br />
Dextran microspheres<br />
Illum et al. introduced well-characterized bioadhesive<br />
microspheres for prolonging the residence time in<br />
the nasal cavity. The slowest clearance was detected<br />
for DEAE-dextran, where 60% of the delivered<br />
dose was still present at the deposition site after 3 h. 16<br />
However, these microspheres were not successful in<br />
promoting insulin absorption in rats. The insulin was<br />
too strongly bound to the DEAE groups to be released<br />
by a solution with an ionic strength corresponding to<br />
physiological conditions. Structural changes due to the<br />
lyophilization process were observed in spheres with<br />
insulin incorporated, which probably further decreased<br />
the release rate. 17<br />
Degradable starch microspheres (DSM)<br />
DSM is the most frequently used microsphere system<br />
for nasal drug delivery and has been shown to improve<br />
the absorption of insulin, gentamicin, human growth<br />
hormone, metoclopramide and desmopressin. Insulin<br />
administered in DSM to rats resulted in a rapid dosedependent<br />
decrease in blood glucose. DSM as a<br />
delivery system for insulin (2 IU.kg_1 ) has also been<br />
tested in sheep. The absolute bioavailability was 4.5%<br />
and the time to reach maximum effect, i.e., a 50%<br />
decrease in plasma glucose, was 60 min. 18 Studies in<br />
rabbits have demonstrated that DSM does not induce<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 27
serious hystopathological changes to the nasal mucosa.<br />
Moreover, the DSM was well tolerated by 15 healthy<br />
volunteers and did not cause signifi cant changes in<br />
mucociliary transport. The effect of starch microspheres<br />
on the absorption enhancing effi ciency of various<br />
enhancer systems with insulin after application in the<br />
nasal cavity of the sheep was investigated. The DSM<br />
was shown to synergistically increase the effect of the<br />
absorption enhancers on the transport of the insulin<br />
across the nasal membrane. 19<br />
Liposomes<br />
Various routes have delivered liposomes. Alpar et al.<br />
studied the potential adjuvant effect of liposomes on<br />
tetanus toxoid, when delivered via the nasal, oral and i.m.<br />
routes compared to delivery in simple solution in relation<br />
to the development of a non-parenteral immunization<br />
procedure, which stimulates a strong systemic immunity.<br />
They found that tetanus toxoid entrapped in DSPC<br />
liposomes is stable and is taken up intact in the gut. The<br />
permeability of liposome entrapping insulin through<br />
the nasal mucosa of rabbit has been studied and<br />
compared with the permeability of insulin solution with<br />
or without pre-treatment by sodium glycocholate (GC).<br />
A comparison of the insulin solution and liposome<br />
suspension showed that the liposome had permeated more<br />
effectively after pre-treatment by GC. The relationship<br />
between the rigidity of the liposomal membrane and<br />
the absorption of insulin after nasal administration of<br />
liposomes modifi ed with an enhancer containing insulin<br />
was investigated in rabbits. The nasal administration<br />
to rabbits showed high fl uidity at 37°C, caused a high<br />
serum glucose reduction, and the reduction effect lasted<br />
for 8 h. The loading and leakage characteristics of the<br />
desmopressin-containing liposomes and the effect<br />
of liposomes on the nasal mucosa permeation were<br />
investigated. The increase of permeability antidiuresis<br />
of desmopressin through the nasal mucosa occured in the<br />
order positively charged liposomes > negatively charged<br />
liposomes > solution. The potential of liposomes as an<br />
intranasal dosage formulation or topical application of<br />
5 (6)-carboxyfl uorescein (CF) was investigated in rats. CF<br />
was rapidly absorbed into the systemic circulation and<br />
no adhesion of CF to the nasal mucosa was observed.<br />
Liposomes suppress drug absorption into the systemic<br />
circulation and concurrently increase drug retention in<br />
the nasal cavity. 20<br />
Gels<br />
Chitin and chitosan have been suggested for use as<br />
vehicles for the sustained release of drugs. Indomethacin<br />
and papaverine hydrochloride were used as model drugs<br />
in gel formulations. It was reported that chitin was able<br />
to control the release of the active agents from gel<br />
Twarita Deshpande et al.: Nasal Drug Delivery-A Review<br />
formulation as compared to the powder formulation.<br />
A similar study done later by Lehr et al. showed that<br />
cationic polymer chitosan was fairly mucoadhesive in<br />
comparison to polycarbophil as a reference substance.<br />
They suggested that a strict distinction should be made<br />
between mucoadhesive of dry polymers on a wet tissue<br />
in air and mucoadhesion of a swollen hydro gel in the<br />
presence of a third liquid phase. 21 Nasal absorption of<br />
nifedipine from gel preparations, PEG 400, aqueous<br />
carbopol gel and carbopol-PEG has been studied in<br />
rats. Nasal administration of nifedipine in PEG resulted<br />
in rapid absorption and high Cmax; however, the<br />
elimination of nifedipine from plasma was very rapid.<br />
The plasma concentration of nifedipine after nasal<br />
administration in aqueous carbopol gel formulation was<br />
very low. The use of PEG 400 in high concentration<br />
in humans should be considered carefully because PEG<br />
400 is known to cause nasal irritation in concentrations<br />
higher than 10%. 22<br />
OTHER PENETRATION ENHANCERS<br />
Cyclodextrin<br />
Several compounds have been investigated for their<br />
nasal absorption enhancement potential using cyclodextrins<br />
as the optimisers. The most studied types are:<br />
cyclodextrins cyclodextrin-cyclodextrin methylcyclodextrin<br />
and Hydroxypropyl-cyclodextrin. Only cyclodextrin<br />
is a compendial substance and is being considered<br />
forGRAS (generally recognized as safe) status. Merkus<br />
et al. reported a study, which investigated the effects of<br />
a dimethyl-cyclodextrin (DM-CD) powder formulation<br />
on intranasal insulin absorption in healthy subjects<br />
and patients with insulin-dependent diabetes mellitus<br />
(IDDM). Mean absolute bioavailabilities of 3.1% and<br />
5.1% were achieved in healthy subjects and diabetics,<br />
respectively. 23<br />
Fusidic acid derivatives<br />
Sodium tauro-24, 25-dihydrofusidate (STDHF) is the<br />
most extensively studied among the derivatives of<br />
fusidic acid. On the basis of its characteristics STDHF<br />
was considered a good candidate for the transnasal<br />
delivery of drugs such as insulin, growth hormone and<br />
octreotide. Lee et al. determined the radioimmunoactive<br />
bioavailability of intranasal salmon calcitonin in<br />
10 healthy human volunteers. The improved nasal<br />
absorption of calcitonin in the presence of STDHF<br />
showed a limited transient irritation of the nasal mucosa<br />
in some subjects. Hedin et al. studied the intranasal<br />
administration of human growth hormone (hGH)<br />
in combination with STDHF at 1% concentration<br />
in patients with hGH defi ciency. They found that in<br />
combination with STDHF, the plasma peak of hGH<br />
28 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
was similar to the endogenous peak. Laursen et al. used a<br />
formulation approach in determining the absorption of<br />
growth hormone in human subjects using didecanoyl-<br />
L-phosphotidylcholine (DDPC) as an enhancer with<br />
different concentrations: 0, 4, 8, and 16%. They<br />
concluded that increasing the relative concentration of<br />
DDPC increases the absorption of nasally administered<br />
hGH. Drejer et al. studied intranasal administration of<br />
insulin with DDPC in healthy human volunteers. They<br />
found that intranasal insulin was absorbed in a dose<br />
dependent manner with slight or no nasal irritation. 24<br />
Phosphatidylcholines (PC)<br />
Phosphatidylcholines are surface-active ampiphilic<br />
compounds produced in biological membranes and<br />
liposomes. Several reports have appeared in the<br />
literature showing that these phospholipids can be used<br />
as enhancers for systemic nasal drug delivery. Newman<br />
et al. investigated the distribution of a nasal insulin<br />
formulation containing DDPC labelled with 99m<br />
Tc-human serum albumin (99m Tc-HAS) in human<br />
volunteers. From the scintigraphic data, the entire dose<br />
from the spray was shown to be deposited in the nasal<br />
cavity with no deposition in the lungs.<br />
The Novo Nordisk study group reported encouraging<br />
results following the nasal administration of an insulin/<br />
DDPC microemulsion formulation in human volunteers.<br />
The study demonstrated good absorption of insulin<br />
whilst preventing or minimizing nasal irritation.<br />
Bile salts and surfactants<br />
Commonly used salts are sodium cholate sodium<br />
deoxy cholate, sodium glycocholate (GC), sodium<br />
taurocholate (TC), sodium taurodeoxycholate (TDC),<br />
and sodiumglycodeoxycholate (GDC). Several studies<br />
indicate that bile salts can be good optimisers in nasal<br />
drug products, there are some reports indicating that<br />
bile salts cause nasal irritation when used above a<br />
concentration of 0.3%. Yokosuka and co-workers<br />
reported a study in which healthy volunteers are dosed<br />
nasally with solution formulation containing insulin<br />
and 1%sodiumglycocholate. Signifi cant decreases in<br />
serum glucose concentrations were observed and<br />
there was a positive correlation between the peak<br />
serum insulin levels and the dose of insulin applied.<br />
Hirata et al. investigated the effi cacy of nasal insulin<br />
formulation containing 1% SGC in healthy volunteers<br />
and diabetic patients. The nasal formulation resulted in<br />
rapid increases in serum insulin levels and decreases in<br />
blood glucose levels in healthy volunteers and diabetics.<br />
Moses and colleagues showed that co administration<br />
of 1% sodium deoxycholate (SDC) enhanced the<br />
intranasal absorption of insulin administered to<br />
human volunteers. High inter-subject variability was<br />
Twarita Deshpande et al.: Nasal Drug Delivery-A Review<br />
observed. Frauman compared the effects of intranasal<br />
and subcutaneous insulin on fasting and post-prandial<br />
blood insulin and glucose concentrations in non-obese<br />
patients with non-insulin-dependent diabetes mellitus<br />
(NIDDM). A nasal solution formulation of insulin<br />
and 1% SGC, administered as a spray, resulted in a<br />
monophasic increase in serum insulin levels. Salzman<br />
et al. investigated the effi cacy of 1% laureth-9 in<br />
enhancing the nasal absorption of insulin in patients<br />
with IDDM and non-diabetic controls. Insulin was<br />
shown to be rapidly absorbed via the nasal route<br />
lowering plasma glucose levels to 50% of basal values<br />
after 45 min in normal subjects compared to 50% in<br />
120 min in diabetics. Paquot et al. investigated the<br />
metabolic and hormonal consequences of an intranasal<br />
insulin formulation administration containing 0.25%<br />
laureth-9 in healthy volunteers. Increase in plasma insulin<br />
levels from 5 to 38 mU.I –1 at 15 min with decreases in<br />
blood glucose concentration from 4.4 to 3.2 mmol.I –1<br />
at 45 min.<br />
DRUG ABSORPTION<br />
The fi rst step in the absorption of drugs from the nasal<br />
cavity is passage through the mucus. Small, uncharged<br />
particles easily pass through this layer. However,<br />
larger or charged particles may fi nd it more diffi cult to<br />
cross. Mucin, the principal protein in the mucus, has<br />
the potential to bind to solutes, hindering diffusion.<br />
Additionally, structural changes in the mucus layer<br />
are possible as a result of environmental changes (i.e.,<br />
pH, temperature, etc) 25 Subsequent to a drug’s passage<br />
through the mucus, there are several mechanisms for<br />
absorption through the mucosa. 26 These include<br />
1. Paracellular Transport Mechanism-It mainly uses<br />
an aqueous mode of transport. Usually the drug<br />
passes through the tight junctions and the open<br />
clefts of the epithelial cells present in the nasal<br />
mucosa. It is a relatively passive mode of transport.<br />
Of Compounds, which are highly hydrophilic in<br />
nature and/or low molecular are most appropriate<br />
for paracellular transport.<br />
2. Transcellular Transport Mechanism-It mainly<br />
encompasses transport via a lipodal route. Small<br />
lipophilc compounds or larger molecules are usually<br />
transported by this route. The transport across nasal<br />
mucosa is mainly a function of lipophilc nature of<br />
the drug. 27<br />
FORMULATION ISSUES<br />
Nasal drug absorption is affected by molecular weight,<br />
particle size, formulation pH, pKa of molecule, and<br />
delivery volume among other formulation characteristics.<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 29
Molecular weight still presents the best correlation to<br />
absorption. 28 The apparent cut-off point for molecular<br />
weight is approximately 1,000, with molecules less than<br />
1,000 having better absorption. Shape is also important.<br />
Linear molecules have lower absorption than cyclicshaped<br />
molecules. Additionally, particles should be larger<br />
than 10 mm, otherwise the drug may be deposited in the<br />
lungs. 29 Hydrophilicity has also been found to decrease<br />
drug bioavailability. 30<br />
Another formulation factor important for absorption<br />
is pH. Both the pH of the nasal cavity and pKa of<br />
a particular drug need to be considered to optimize<br />
systemic absorption. Nasal irritation is minimized<br />
when products are delivered with a pH range of<br />
4.5 to 6.5. Also, volume and concentration are<br />
important to consider. The delivery volume is limited<br />
by the size of the nasal cavity. An upper limit of<br />
25 mg/dose and a volume of 25 to 150mL/nostril has<br />
been suggested. 31<br />
FDA requirements state all nasal drug products sold<br />
in the United States are to be manufactured as sterile<br />
or preserved products. Depending on the drug and/or<br />
formulation characteristics, sterility may be accomplished<br />
via aseptic fi lling processes, terminal sterilization, or both.<br />
Sterility must also be maintained throughout a product’s<br />
shelf life. Unit-dose delivery systems or specialized<br />
multidose delivery systems may be used to ensure<br />
continued product sterility. Alternatively, preservatives<br />
may be added to prevent bacterial growth. The most<br />
commonly used preservative in nasal formulations has<br />
been benzalkonium chloride. 32 However, adverse events,<br />
such as mucosal swelling, irritation, and ciliostastis, are<br />
associated with its use.<br />
Intranasal vaccine administration has also become<br />
a prevalent area of research. One of the major<br />
formulation challenges for nasal vaccination has been<br />
the development of an appropriate adjuvant to stimulate<br />
desirable immune responses. Some examples of<br />
investigational adjuvants include microparticles, genetic<br />
manipulations of Vibrio cholerae and Escherichia<br />
coli toxins, and CpG oligodeoxynucleotides. The<br />
use of adjuvants is especially important for vaccines<br />
composed of recombinant subunits, synthetic peptides,<br />
and plasmid DNA. For instance, when hepatitis B<br />
surface antigen was given alone to mice, little or no<br />
immunogenicity was observed. However, when given<br />
with CpG or cholera toxin, good immunological<br />
responses were achieved. 33<br />
Drugs have been delivered to the nasal cavity in a variety<br />
of formulations, including powders, topical gels, sprays,<br />
drops, and pledgets. As reviewed elsewhere by Behl et al.,<br />
the dosage form should refl ect the intended therapeutic<br />
use, offer easy administration, and provide chemical<br />
stability for the drug. 34<br />
Twarita Deshpande et al.: Nasal Drug Delivery-A Review<br />
With any formulation for intranasal delivery,<br />
interference with normal physiologic processes should<br />
be avoided. The nose is an important defense system<br />
for environmental hazards. Disruption of its normal<br />
physiology or clearance processes may leave a patient<br />
vulnerable to a variety of hosts. Also, formulations<br />
should be designed with patient comfort and acceptance<br />
in mind. Some drugs and excipients have been found<br />
to cause temporary discomfort upon instillation. This<br />
could potentially decrease patient compliance, especially<br />
when chronic administration is indicated. 35–38<br />
NASAL DELIVERY OF VACCINES<br />
Almost all viral, bacterial and parasitic agents causing<br />
common infectious diseases of the intestinal, respiratory<br />
and genital tract enter and infect through the large<br />
surface area made available by mucosal membranes.<br />
The nasal mucosa is an important arm of the mucosal<br />
immune system since it is often the fi rst point of contact<br />
for inhaled antigens and as a consequence, intranasal<br />
immunisation has emerged as possibly the most effective<br />
route for vaccination for both peripheral and mucosal<br />
immunity.<br />
Recently, Partidos has considered the challenges for<br />
intranasal vaccines. He has listed several reasons why the<br />
nose is an attractive route for immunisation.<br />
• Easily accessible.<br />
• Highly vascularized.<br />
• Presence of numerous microvilli covering the nasal<br />
epithelium generates a large absorption<br />
• Surface.<br />
• After intranasal immunisation, both mucosal and<br />
systemic immune responses can be induced.<br />
• Immune response can be induced at distant mucosal<br />
sites owing to the dissemination of effector immune<br />
cells in the common mucosal immune system.<br />
• The nose can be used for the easy immunization of<br />
large population groups.<br />
• Nasal immunisation does not require needles and<br />
syringes, which are potential sources of infection. 39<br />
The utility of intranasal administration for a variety of<br />
clinical applications to include largely the prophylaxis<br />
of disease. Other uses of nasal vaccination have been<br />
reported. For example, Weiner et al. have suggested a<br />
novel mucosal immunlogical approach to Alzheimer’s<br />
disease. Nasal administration of amyloid-beta peptide<br />
decreased cerebral amyloid burden in a mouse model of<br />
the disease. Recently, live attenuated; cold adapted viral<br />
vaccines have been developed as alternatives to inactivated<br />
vaccines. For example, by growing the infl uenza virus<br />
at 25ºC for long periods or using genetic reassortment<br />
methods, it is possible to produce an organism that will<br />
30 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
eplicate effi ciently at 25–28ºC (the temperature of the<br />
nasal passage) but not at 37ºC (the temperature of the<br />
lungs). Clinical studies on live attenuated virus vaccines<br />
have been well reviewed by Wiselka and Keitel and<br />
Piedra. One major goal of intranasal vaccination with<br />
a replicating virus is to induce secretory and systemic<br />
immune responses that more closely resemble those<br />
provided by the natural infection.<br />
BRAIN TARGETING THROUGH NASAL ROUTE<br />
For some time the Blood Brain Barrier has impeded the<br />
development of many potentially interesting CNS drug<br />
candidates due to their poor distribution into the CNS.<br />
The Blood Brain Barrier is a system of layers of cells at<br />
the cerebral capillary endothelium, the choroids plexus<br />
epithelium, and the arachnoid memberanes, which<br />
are connected by tight junctions and which together<br />
separate the brain and the cerebrospinal fl uid from<br />
blood. 40 Owing to the uniqueness of the nose and<br />
the CNS, the intranasal route can deliver therapeutic<br />
agents to the brain bypassing the Blood brain Barrier. 41<br />
Absorption of drug across the olfactory region of the<br />
nose connection provides a unique feature and superior<br />
option to target drugs to brain. When administered<br />
nasally to the rat, some drugs resulted in CSF and<br />
olfactory bulb drug levels considerably higher than<br />
those following intravenous administration. 42 Evidence<br />
of nose to brain transport has been reported by many<br />
scientists. Many previously abandoned potent CNS<br />
drug candidates promise to become successful CNS<br />
therapeutic drugs via intranasal delivery. Recently,<br />
several nasal formulations, such as ergotamine<br />
(Novartis), sumatriptan (GlaxoSmith-Kline), and<br />
zolmitriptan (AstraZeneca) have been marketed to treat<br />
migraine. Scientists have also focused their research<br />
toward intranasal administration for drug delivery to<br />
the brain, especially for the treatment of diseases, such<br />
as, epilepsy, migraine, emesis, depression and erectile<br />
dysfunction. 43,44 Intranasal delivery does not require<br />
any modifi cation of the therapeutic agents and does<br />
not require that drugs be coupled with any carrier.<br />
A wide variety of therapeutic agents, including both<br />
small molecules and macromolecules can be successfully<br />
delivered to the CNS via intranasal method. 45<br />
ADMINISTRATION DEVICE<br />
Drug therapy requires that administration of the dosage<br />
form be accurate and very reproducible, which therefore<br />
places stringent, demands on the device for nasal drug<br />
delivery. The major mechanism of nasal deposition of<br />
particles is by inertial impaction that occurs following a<br />
change in the direction of airfl ow. Other contributory<br />
mechanisms are gravitational sedimentation and<br />
Twarita Deshpande et al.: Nasal Drug Delivery-A Review<br />
Brownian diffusion. Particle deposition by interception<br />
and electrostatic precipitation are of no importance in<br />
nasal deposition (Kublik & Vidgren 1998). Depending<br />
on the type of formulation, a variety of devices have<br />
been used to deliver drugs intranasally. Devices for liquid<br />
formulations include instillation catheter, droppers,<br />
unit-dose containers, squeezed bottle, pump sprays,<br />
airless and preservative-free sprays, compressed air<br />
nebulizers and metered-dose inhalers (MDIs). Devices<br />
for powder dosage forms include insuffl ators, singledose<br />
and multi-dose powder inhalers and pressurized<br />
MDI. Delivery devices are also available for nasal gels<br />
(Kublik & Vidgren 1998). Squeeze bottle delivery<br />
is another option for nasal drug delivery. However<br />
this technique is not able to deliver a measured dose<br />
of drug.<br />
Their angle of insertion into the nostril can infl uence<br />
the part of the nasal cavity that the formulation<br />
comes in contact with initially and as such the overall<br />
deposition pattern. Metered-dose nebulizers and<br />
metered-dose aerosols are superior to other devices in<br />
terms of accuracy and reproducibility (Dondeti 1996).<br />
1985). The particle size of aerosols is very important<br />
with regard to deposition. Particles greater than 10 ml<br />
are deposited within the upper respiratory tract, those<br />
less than 5 ml are inhaled, and those less than 0.5 ml<br />
are exhaled (Sciarra & Cutie 1990; Sanders et al 1997).<br />
A brief description of various intranasal devices is given<br />
as follows Figure 2–8.<br />
1. Caprujet of midazolam–This is a prepackaged<br />
midazolam in 5mg/ml dosing. It is indicated for<br />
treatment of persistent seizure activity. In situations<br />
where the entire volume might not be appropriate,<br />
the syringe can have a indelible mark made by the<br />
clinician or pharmacist at appropriate dosage so the<br />
parent knows how much to give.<br />
2. Direct-Haler powdered Drug Nasal Delivery–This<br />
system enables nasal delivery of fi ne particles for<br />
improved absorption and targeted delivery. Prevents<br />
risk of pulmonary deposition. Prevents risk of<br />
immediate dose swallowing, reduces taste impact<br />
from nasal use.<br />
3. Mucosal Atomization Device(MAD)–Delivers<br />
intranasal medication in a fi ne mist which enhances<br />
absorption and improves bioavailability for fast and<br />
effective drug delivery.<br />
4. Via Nose Electronic Atomizer–This atomizer<br />
creates tiny, Powerful vortices of aerosolized particles,<br />
thus delivering medication more effectively to the<br />
user. This device has a small LCD screen which is<br />
used to specify dose.<br />
5. BDA Accuspray Nasal Spray System–It is a<br />
single use, prefi llable vaccine delivery system which<br />
dispenses a precise dose of fl u vaccine intranasally in<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 31
Figure 2: Nasal Delivery Dosage Forms and Devices.<br />
Figure 3: Caprujet of Midazolam.<br />
the form of a fi ne mist. Recommended for healthy<br />
individuals in the age group of 5–49.<br />
6. Optinose–This device is based on the concept of<br />
a bi-directional nasal drug delivery device. Recently<br />
optinose received positive results from their phase<br />
II clinical studies for chronic rhinosinusitis and<br />
migraine therapies.<br />
Twarita Deshpande et al.: Nasal Drug Delivery-A Review<br />
Regardless of the method chosen to deliver a nasal<br />
medication, several suggestions are worth mentioning:<br />
• Use a highly concentrated form of the medication to<br />
reduce volume and therefore reduce runoff. 1/4 to<br />
1/3 ml per nostril would be preferred. 1/2 to 1 ml per<br />
nostril is tolerable but there will be some loss as the<br />
volume increases. More than one ml per nostril per<br />
dose should likely be split and delivered over several<br />
cycles separated by 10–15 minutes.<br />
• Always use a method that allows the delivery of a<br />
measured dose (syringe or unit dose pump).<br />
• Use a method that fragments the medication into<br />
fi ne particles so the maximal nasal mucosal surface<br />
is covered and minimal volume runs out the nose or<br />
into the throat (atomizer for liquid, powder needs to<br />
be well distributed).<br />
• Utilize both nostrils to double the surface area for<br />
absorption and halve the volume delivered per<br />
nostril.<br />
• Be knowledgeable of the “dead space” within the<br />
delivery device and account for this dead space when<br />
calculating the volume you will deliver to the patient.<br />
Considerations Regarding Device Selection<br />
When selecting a device for nasal administration, it<br />
must be considered, that the administration volume<br />
is comparable low. For liquids, a volume of 100μl is<br />
32 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
Figure 4: Direct-Haler Powdered Drug Nasal Delivery.<br />
Figure 5: Mucosal Atomization Device (MAD).<br />
Twarita Deshpande et al.: Nasal Drug Delivery-A Review<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 33
Figure 6: Via Nase Electronic Atomizer.<br />
Figure 7: BDA Accuspray Nasal Spray System<br />
Twarita Deshpande et al.: Nasal Drug Delivery-A Review<br />
34 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
Figure 8: OptiNose.<br />
optimum per nostrils in a adults, but should be reduced<br />
for children to avoid nasal dripping. Single dose devices<br />
will give best protection for vaccines, but require<br />
highly sophisticated fi lling technology. Multi-dose<br />
spray pumps are option for liquid vaccines, if an in use<br />
microbial contamination of the bottle content can be<br />
prevented. 46<br />
ADVANTAGES OF NASAL DELIVERY<br />
Intranasal delivery is a needle-free, patient-friendly<br />
administration route. Because needles are not involved,<br />
this method of drug delivery is virtually painless. For<br />
patients who fear injections, intranasal administration<br />
offers a more acceptable alternative. Additionally,<br />
the simplicity of nasal delivery would allow for selfadministration<br />
in a home setting. In general, for patients,<br />
the intranasal dosage form provides comfortable, non<br />
threatening, less invasive therapy. This may be especially<br />
important in younger patient populations.<br />
Another major benefi t of intranasal administration, in<br />
contrast to injectables, is that it does not contribute to<br />
biohazardous waste. When the drug has been delivered<br />
intranasally, the administration device may be disposed<br />
of in the normal garbage. There is no need for special<br />
waste containers. Again, this delivery method does not<br />
Twarita Deshpande et al.: Nasal Drug Delivery-A Review<br />
require needles. Hence risk of accidental sticks is not a<br />
concern.<br />
From a pharmacokinetic standpoint, absorption is<br />
rapid, which should provide a faster onset of action<br />
compared to oral and intramuscular administration.<br />
Hepatic fi rst-pass metabolism is also avoided, allowing<br />
increased, reliable bioavailability. In this regard, good<br />
drug candidates for intranasal delivery are those that<br />
undergo extensive fi rst-pass metabolism, display erratic<br />
absorption, or require quick therapeutic onset.<br />
Lastly, patent life of a particular product may be<br />
extended via development of an alternative dosage form,<br />
providing companies the opportunity to maintain their<br />
market share. So from a drug development perspective,<br />
intranasal delivery should stimulate favorable profi t<br />
outcomes. 47<br />
GROWING MARKET FOR NASAL DRUG DELIVERY<br />
Pharmaceutical companies have looked increasingly<br />
towards drug delivery companies for help in lifecycle<br />
management of drugs on the market and with promising<br />
yet hard-to-deliver drugs. The drug delivery market<br />
is currently valued at US$50 billion (or 12.5% of the<br />
global pharmaceutical market) and is expected to reach<br />
US$100 billion by 2005.1 Nasal delivery commands the<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 35
fourth position in market share, with about US$3 billion<br />
in sales, following oral controlled release, pulmonary and<br />
parenteral delivery routes. However, the potential for<br />
growth in this sector is extensive, pending the successful<br />
delivery of proteins and peptides as an alternative to<br />
parenteral delivery. Currently, many nasal drug products<br />
on the market are indicated for the treatment of<br />
local disease such as allergic rhinitis. However, this is<br />
likely to change soon. There are a number of nasally<br />
delivered, systemically acting drugs on the market<br />
in different therapeutic categories, with a growing<br />
number of products in the pipeline. There are many<br />
reasons for this change, including improved patient<br />
compliance (elimination of needles), avoidance of fi rstpass<br />
metabolism and rapid onset of action. Migraine is<br />
a key area where a nasal system (Imitrex ® nasal spray,<br />
GlaxoSmithKline) has provided rapid relief, avoidance<br />
of taking an oral formulation while nauseated, and<br />
pain-free administration circumventing the need for an<br />
injection. Other therapeutic areas where nasal delivery<br />
could provide an alternative to current dosage forms<br />
are crisis situations (seizure and heart attack), erectile<br />
dysfunction, pain management, motion sickness and<br />
psychotropic drugs. Although the market share for nasal<br />
delivery may never take the number one spot enjoyed by<br />
oral controlled release, it remains a drug delivery route<br />
with an enormous potential for growth.<br />
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23. Lee WA, Ennis RD, Longenecker JP, Bengtsson P. The bioavailability<br />
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absorption enhancers. Pharm Res 1997;14:631–7.<br />
27. Vyas Tk, Shahiwala A, Marathe S, Misra A. Intranasal Drug Delivery for<br />
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28. Fisher AN, Brown K, Davis SS, Parr GD, Smith DA. The effect of<br />
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29. Jones NS, Quraishi S, Mason JDT. The nasal delivery of systemic<br />
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30. Corbo D, Liu J, Chien YW. Drug absorption through mucosal membranes:<br />
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31. Behl CR, Pimplaskar HK, Sileno AP, deMeireles J, Romeo VD. Effects<br />
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32. Calcitonin-salmon Nasal spray product information[online]. 2009[cited<br />
2009 Feb]. Available from: URL:http://www.drug.com/pro/calcitoninsalmon-nasal-spray-html<br />
33. Morimoto K, Katsumata H, Yabuta T et al. Evaluation of gelatin<br />
microspheres for nasal and intramuscular administrations of salmon<br />
calcitonin. Eur J Pharm Sci. 2001;13:179–85.<br />
34. Mathiowitz E, Chickering DE, Lehr CM. Bioadhesive drug delivery<br />
systems: Fundamentals, Novel approaches and development. First<br />
edition. New York: Marcel Dekker Inc;1999.<br />
35. Davis SS. Nasal vaccines. Adv Drug Deliv Rev 2001;51:21–42.<br />
36. McCluskie MJ, Davis HJ. Cutting edge: CpG DNA is a potent enhancer<br />
of systemic and mucosal immune responses against hepatitis B<br />
surface antigen with intranasal administration to mice. J Immunol<br />
1998;161:4463–6.<br />
37. Illum L, Davis SS. Intranasal insulin. Clin Pharmacokinet 1992;12:<br />
30–41.<br />
38. van de Donk HJM, Merkus FWHM. Decreases in ciliary beat<br />
frequency due to intranasal administration of propanolol. J Pharm Sci<br />
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39. Mestecky J, Moldoveanu Z, Michalek SM. Current options for vaccine<br />
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36 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
41. Frey WH II. Intranasal delivery: Bypassing the blood- brain to deliver<br />
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cavity to the brain following intranasal cocaine administration in rats.<br />
J Pharm Sci 1999;88:754–8.<br />
44. Sakane T, Akizuki M, Yoshida M. Transport of cephalexin to the<br />
cerebrospinal fl uid directly from nasal cavity. J Pharm Pharmacol<br />
1991;43:449–51.<br />
Twarita Deshpande et al.: Nasal Drug Delivery-A Review<br />
45. Talegaonkar S, Mishra PR. Intranasal Delivery: An approach to bypass<br />
the blood brain barrier. Indian J Pharmacol 2004;36:140–7.<br />
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and Considerations on Device Selection. Indian J Pharm Sci 2009;71:<br />
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47. Wermeling DP, Miller J, Rudy AC. Systematic Intranasal Drug Delivery:<br />
Concepts and Applications. Drug Development & Delivery 2002;2.<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 37
Design and optimization of levofl oxacin<br />
gastroretentive tablets<br />
D. Nagendrakumar 2 , S.B. Shirsand* 1 , M.S. Para 2 , A.D. Chauhan 2<br />
1 Department of Pharmaceutical Technology, H.K.E. Society’s M T R Institute of Pharmaceutical Sciences,<br />
Sedam Road, Gulbarga-585105, India<br />
2Department of Pharmaceutics, S.V.E. Trust’s College of Pharmacy, Kallur Road, Humnabad-585330, India<br />
ABSTRACT<br />
In the present study, gastroretentive fl oating tablets of levofl oxacin hemihydrate were designed with objective<br />
of retention of tablet in acidic pH to improve bioavailability with reduction in dosing frequency. Hydroxypropyl<br />
methyl cellulose of different viscosity grades (K4M and K100LV) was used as polymer and sodium bicarbonate<br />
as gas generating agent to reduce fl oating lag time. Tablets were prepared by direct compression method. The<br />
prepared formulations were evaluated for hardness, friability, weight variation, drug content, swelling index,<br />
in-vitro drug release, short-term stability, fl oating lag time and in-vitro buoyancy. A 32 factorial design was applied<br />
to systematically optimize the drug release profi le.<br />
The proportions of release retardant material HPMC K100LV(X ), sodium bicarbonate (X ) were selected as<br />
1 2<br />
independent variables and t (Y ), and t (Y ) were selected as dependent variables. The promising formulation<br />
50% 1 70% 2<br />
containing levofl oxacin hemihydrate (100 mg), HPMC K100LV (100 mg) and sodium bicarbonate (80 mg) has t50% (5.95 h), t (8.52 h) fl oating lag time was only 10.33 sec and released more than 90% drug in 12 h. This study<br />
70% ,<br />
proved that gastroretentive drug delivery system of levofl oxacin hemihydrate was designed using HPMC K100LV,<br />
which provided excellent gastroretentive property, thus improved the bioavailability of drug.<br />
Key words: Levofl oxacin hemihydrate, Gastroretentive fl oating drug delivery systems, Hydroxypropyl methyl<br />
cellulose, 3² factorial design<br />
INTRODUCTION<br />
The real challenge in the development of an<br />
oral controlled-release drug delivery system<br />
is not just to sustain the drug release, but<br />
also to prolong the presence of the dosage<br />
form within the gastrointestinal tract (GIT)<br />
until the drug is completely released at the<br />
desired period of time. 1 Indeed, gastric<br />
drug retention has received signifi cant<br />
interest in the past few decades. Most of<br />
the conventional oral delivery systems have<br />
shown some limitations related to fast<br />
gastric emptying time. 2<br />
Garg and Gupta3 classifi ed the gastroretentive<br />
dosage forms into four main classes: (i)<br />
fl oating systems4 (ii) expandable systems5 (iii) bioadhesive systems6 and (iv) high<br />
density systems. 7 Floating systems are of<br />
two types: effervescent systems, depending<br />
on the generation of carbon dioxide gas<br />
upon contact with gastric fl uids, and noneffervescent<br />
systems. The non-effervescent<br />
systems can be further divided into four<br />
sub-types, including hydrodynamically balanced<br />
systems, 8 microporous compartment<br />
systems, 9 alginate beads 10 and hollow microspheres/microballons.<br />
11 In addition, superporous<br />
hydrogels 12 and magnetic systems 13<br />
were also described.<br />
As suggested by Singh and Kim, 14 fl oating<br />
drug delivery is of particular interest for the<br />
drugs which: (a) act locally in the stomach;<br />
(b) are primarily absorbed in the stomach;<br />
(c) are poorly soluble at an alkaline pH;<br />
Research Ar cle<br />
Received Date : 11-07-2011<br />
Revised Date : 11-02-2012<br />
Accepted Date : 15-02-2012<br />
DOI: 10.5530/rjps.2012.1.5<br />
Address for<br />
correspondence<br />
S B Shirsand<br />
Department of Pharmaceutical<br />
Technology<br />
H.K.E. Society’s M T R Institute<br />
of Pharmaceutical Sciences<br />
Sedam Road<br />
Gulbarga-585105, India<br />
E-mail: shirsand@rediffmail.<br />
com<br />
www.rjps.in<br />
38 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
D.Nagendrakumar et al.: Design and Optimization of Levofl oxacin Gastroretentive Tablets<br />
(d) have a narrow window of absorption and (e) are<br />
unstable in the intestinal or colonic environment.<br />
Levofl oxacin hemihydrate is a broad spectrum<br />
fl uoroquinolone antibiotic. It is approved for use in<br />
the treatment of Helicobacter pylori infection. H. pylori<br />
was recognized as a major gastric pathogen responsible<br />
for chronic active gastritis, duodenal ulcers and gastric<br />
adeno-carcinoma. 15 The bioavailability of levofl oxacin<br />
hemihydrate is above 99% with a plasma half-life of<br />
6–8 h. It is freely soluble in pH 0.6 to 5.8 range. Above<br />
pH 5.8, the solubility increases rapidly to its maximum<br />
at pH 6.7 and above which the solubility decreases and<br />
reaches a minimum value at a pH of approximately<br />
6.9. 16 Thus solubility of the drug is reduced in intestinal<br />
alkaline pH. Hence, it was selected in the present<br />
investigation as a suitable candidate for the design of<br />
gastric fl oating drug delivery system (GFDDS) with an<br />
improved retention time and bioavailability.<br />
In a full factorial design, two factors were studied at all the<br />
possible combinations, by considering it is most effi cient<br />
in estimating the infl uence of individual variables and<br />
their interactions, using minimum experimentation. In the<br />
present investigation, amount of HPMC K100LV (X 1 )<br />
and amount of sodium bicarbonate (X 2 ) were selected<br />
as independent variables in the 3 2 factorial design. The<br />
time required for drug release of 50% (t 50%, Y 1 ) and 70%<br />
(t 70%, Y 2 ) were selected as dependent variables. A statistical<br />
model incorporating interactive and polynomial<br />
terms was utilized to evaluate the response.<br />
MATERIALS AND METHODS<br />
Levofl oxacin hemihydrate was gifted by Zhejiang Starry<br />
Pharmaceutical Ltd., Mumbai (India), hydroxypropyl<br />
methylcellulose K100LV was gifted by Colorcon<br />
Asia Pvt. Limited, Goa (India). Sodium bicarbonate,<br />
magnesium stearate, talc were purchased from (SD fi ne<br />
chem., Mumbai). All the materials were of analytical or<br />
pharmacopoeial grade and used as received.<br />
PREPARATION OF LEVOFLOXACIN HEMIHYDRATE<br />
FLOATING TABLETS<br />
Preparation<br />
Direct compression method has been employed<br />
to prepare gastroretentive tablets of levofl oxacin<br />
hemihydrate using HPMC K100LV as polymer and<br />
sodium bicarbonate as gas generating agent.<br />
Procedure<br />
All the ingredients including drug, polymer and<br />
excipients were weighed accurately according to the each<br />
tablet formulation (Table 1). The polymer and sodium<br />
bicarbonate were thoroughly mixed on a butter paper<br />
with the help of a stainless steel spatula. The mixture was<br />
mixed with drug and then excipients were added in the<br />
order of ascending weights, the mixture was blended for<br />
10 min in an infl ated polyethylene pouch. The prepared<br />
blend of each formulation was compressed on 10-station<br />
rotary tablet punching machine (Clit, Ahmedabad) to<br />
obtain fl at-faced tablet of 8 mm diameter.<br />
EVALUATION OF GASTRORETENTIVE<br />
FLOATING TABLETS<br />
The prepared formulations were evaluated for hardness,<br />
friability, weight variation, drug content, swelling index,<br />
in-vitro drug release, short-term stability, fl oating lag time<br />
and in- vitro buoyancy.<br />
Hardness test<br />
The crushing strength (kg/cm²) of tablets was<br />
determined by using digital hardness tester 17 (Electro<br />
Lab E01). The mean of three determinations were taken.<br />
Friability test<br />
It was determined by weighing 10 tablets (W ) after<br />
Original<br />
dusting, placing them in the friabilator (Electro lab EF-2)<br />
and rotating the plastic cylinder vertically at 25 rpm for<br />
4 min. 18 After dusting, the total remaining weight of the<br />
tablets (W ) was recorded and the percent friability<br />
Final<br />
was calculated according to Equation 1.<br />
Weight Final − WeightOriginal<br />
Percent friability =<br />
× 100 (1)<br />
Weight<br />
Uniformity of weight<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 39<br />
Original<br />
The weight (mg) of each of 20 individual tablets was<br />
determined using an electronic balance (Shimadzu<br />
BL-220H). The weight data from the tablets were<br />
analyzed for sample mean and percent deviation. 18<br />
Uniformity of drug content<br />
Two tablets were powdered in a glass mortar and the<br />
powder equivalent to 1 mg of drug was placed in a<br />
500 ml conical fl ask. The drug was extracted with<br />
500 ml of pH 1.2 solution with vigorous shaking<br />
on a mechanical gyratory shaker (100 rpm) for 1 h.<br />
Absorbance was measured at 293 nm in Shimadzu<br />
UV-1800 spectrophotometer against blank.<br />
In-vitro fl oating study<br />
Floating time was determined by using USP XXIII tablet<br />
dissolution apparatus (Electro lab TDT-08L) at 50 rpm<br />
using 900 ml of 0.1N HCl at 37 ± 0.5°C temperature.<br />
The fl oating duration was the time during which the<br />
tablet remains buoyant and fl oating lag time was the<br />
time between tablet introduction and its buoyancy.
D.Nagendrakumar et al.: Design and Optimization of Levofl oxacin Gastroretentive Tablets<br />
Table 1: Composition of Factorial Design and Check point Formulations of Levofl oxacin Hemihydrate<br />
Ingredients*<br />
Formulation code<br />
(mg/tablet)<br />
F1 F2 F3 F4 F5 F6 F7 F8 F9 C1 C2 Levofl oxacin hemihydrate 100 100 100 100 100 100 100 100 100 100 100<br />
HPMC K100LV 100 100 100 150 150 150 200 200 200 125 175<br />
Sodium bicarbonate 40 60 80 40 60 80 40 60 80 50 70<br />
Magnesium stearate 4.8 5.2 5.6 5.8 6.2 6.6 6.8 7.2 7.6 5.5 6.9<br />
Talc 2.4 2.6 2.8 2.9 3.1 3.3 3.4 3.6 3.8 2.8 3.5<br />
*Weight expressed as mg per tablet; HPMC-hydroxypropyl methylcellulose; LV-low viscosity; C 1 , C 2 check point formulations.<br />
Duration of fl oating and fl oating lag time was measured<br />
by visual observation.<br />
Swelling index19 The tablet was weighed (W ) and placed in a glass<br />
Wet<br />
beaker, containing 200 mL of 0.1 N HCl, maintained in<br />
a water bath at 37 ± 0.5ºC. At regular time intervals, the<br />
tablet were removed and the excess surface liquid was<br />
carefully removed by a fi lter paper. The swollen tablet<br />
was then reweighed (W ). The swelling index (SI) was<br />
Dry<br />
calculated using the Equation 2.<br />
W − W<br />
Swelling Index =<br />
W<br />
In-vitro dissolution study<br />
wet Dry<br />
Dry<br />
×100 (2)<br />
In-vitro dissolution study of levofl oxacin hemihydrate<br />
fl oating tablets was carried out in USP XXIII tablet<br />
dissolution test apparatus (Electro lab TDT-08L),<br />
employing a paddle stirrer at 50 rpm using 900 ml of<br />
pH 1.2 solution as dissolution medium at 37 ± 0.5ºC.<br />
One tablet was used in each test. At predetermined<br />
time intervals, 5 ml of the samples were withdrawn by<br />
means of a syringe fi tted with pre-fi lter. The volume<br />
withdrawn at each interval was replaced with same<br />
quantity of fresh dissolution medium. The samples were<br />
analyzed for drug release by measuring the absorbance<br />
at 293 nm using Shimadzu UV-1800 spectrophotometer<br />
after suitable dilutions. All the studies were conducted<br />
in triplicate.<br />
Stability study<br />
Short-term stability study was performed at a temperature<br />
of 45° ± 1°C and 75% relative humidity over a period of<br />
thee weeks for the promising GRDF tablet formulation<br />
F . 15 tablets were packed in amber colored, screw-<br />
3<br />
capped bottles and kept in hot air oven maintained at<br />
45° ± 1°C and 75% relative humidity. Samples were taken<br />
at weekly interval for drug content estimation. At the end<br />
of three weeks, dissolution test and in-vitro fl oating study<br />
were performed to determine the drug release profi les,<br />
in-vitro fl oating lag time and fl oating time.<br />
RESULTS AND DISCUSSION<br />
The hardness of the gastroretentive fl oating tablets<br />
of levofl oxacin hemihydrate was found to be in the<br />
range of 6.45 to 6.74 kg/cm² for the factorial design<br />
formulations. The friability of all tablets was from 0.53<br />
to 0.89 % for the factorial design formulations. The<br />
percentage deviation from the mean weights of all the<br />
batches was found to be within the prescribed limits as<br />
per IP. The low values of standard deviation indicated<br />
uniform drug content (Table 2). The swelling index of<br />
the tablets was increased with an increase in the polymer<br />
content and the content of gas generating agent i.e.<br />
sodium bicarbonate (Table 2).<br />
In-vitro fl oating study was performed by placing tablets<br />
in USP XXIII tablet dissolution apparatus containing<br />
900 ml of pH 1.2 maintained at a temperature of<br />
37 ± 0.5ºC. The fl oating lag time and fl oating time were<br />
noted visually. The results were presented in Table 2.<br />
For factorial formulations, lag time was in the range of<br />
10.33 to 64.57 sec. With formulations containing the<br />
same amount of polymer of the same grade, fl oating<br />
lag time was decreased with increase in concentration of<br />
sodium bicarbonate. For formulation F 3 , it was lowest<br />
(10.33 sec) as the drug-polymer (HPMC K100LV) ratio<br />
is 1:1 and sodium bicarbonate is in highest proportion<br />
among the formulations and the tablet remained intact<br />
for 12 h, while for formulation F 7 , lag time was highest<br />
(64.57 sec) as drug-polymer ratio is 1:2 and sodium<br />
bicarbonate is in lowest proportion (40 mg). All the<br />
designed formulations displayed a fl oating time of more<br />
than 24 h.<br />
In-vitro drug release study was performed using USP<br />
XXIII tablet dissolution apparatus From the above data,<br />
it was evident that as the proportion of polymer in the<br />
formulation increased, cumulative percent drug release in<br />
12 h decreased and as the proportion of the gas generating<br />
agent increased, the drug release was increased (Table 3).<br />
Factorial design<br />
Based on the preliminary trial, the levels of independent<br />
variables (X and X ) were fi xed at 100, 150, 200 mg for<br />
1 2<br />
HPMC K100LV (X ) and at 40, 60, 80 mg for sodium<br />
1<br />
bicarbonate (X ) in designing the formulations of 3²<br />
2<br />
40 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
D.Nagendrakumar et al.: Design and Optimization of Levofl oxacin Gastroretentive Tablets<br />
Table 2: Evaluation Results of Factorial Design Formulations of Levofl oxacin Hemihydrate GRDDS<br />
Formulation Code<br />
Mean Hardness<br />
(kg/cm 2 ) Friability (%)<br />
Average<br />
Weight (mg)<br />
Mean Drug Content<br />
Percent* ± SD<br />
Swelling<br />
Index* ± SD<br />
Floating Lag<br />
Time (sec)<br />
Floating<br />
time (h)<br />
F 1 6.61 0.53 248.26 98.60 ± 1.51 4.25 ± 1.35 46.21 >24<br />
F 2 6.55 0.67 268.15 99.64 ± 1.74 13.26 ± 1.87 12.61 >24<br />
F 3 6.45 0.71 289.73 101.48 ± 0.97 49.82 ± 1.79 10.33 >24<br />
F 4 6.69 0.74 300.02 100.05 ± 0.42 07.38 ± 0.06 57.28 >24<br />
F 5 6.62 0.79 319.69 101.87 ± 1.68 23.49 ± 0.48 18.48 >24<br />
F 6 6.51 0.84 342.95 101.62 ± 1.57 53.18 ± 1.42 14.69 >24<br />
F 7 6.74 0.85 352.27 99.74 ± 0.38 10.75 ± 1.29 64.57 >24<br />
F 8 6.67 0.87 369.86 101.85 ± 1.35 28.37 ± 1.15 24.29 >24<br />
F 9 6.59 0.89 393.48 99.39 ± 1.06 56.24 ± 1.45 18.54 >24<br />
*Average of thee determinations, values shown in parenthesis are standard deviations. Formulation F 3 was selected as the best and used for further studies; GRDDSgastroretentive<br />
drug delivery system.<br />
Table 3: In- vitro Cumulative Percent Release of Levofl oxacin Hemihydrate for Factorial Design Batches<br />
Cumulative Percent Drug Release Mean* ± SD<br />
Time (h) F1 F2 F3 F4 F5 F6 F7 F8 F9 01 8.84 ± 0.29 10.37 ± 0.96 13.16 ± 0.65 8.31 ± 0.72 9.51 ± 0.49 09.88 ± 0.34 10.58 ± 0.10 7.51 ± 0.45 6.98 ± 0.17<br />
02 16.96 ± 0.00 18.89 ± 0.90 24.15 ± 0.17 13.24 ± 0.49 13.54 ± 0.89 15.57 ± 0.45 17.40 ± 1.09 14.90 ± 1.44 15.27 ± 1.17<br />
03 24.51 ± 0.50 26.48 ± 0.96 32.33 ± 0.40 20.76 ± 0.79 21.72 ± 1.00 26.13 ± 0.50 24.42 ± 1.17 21.22 ± 1.34 24.29 ± 2.43<br />
04 32.66 ± 2.05 34.06 ± 0.64 40.92 ± 0.72 29.44 ± 1.09 28.88 ± 1.09 36.87 ± 0.47 29.81 ± 2.05 31.91 ± 1.54 34.18 ± 0.61<br />
05 41.65 ± 1.54 42.44 ± 1.15 45.18 ± 0.42 38.36 ± 1.00 35.47 ± 1.12 43.05 ± 0.41 35.70 ± 3.71 39.10 ± 1.20 42.05 ± 0.44<br />
06 48.51 ± 1.41 49.50 ± 0.55 50.05 ± 0.20 46.65 ± 0.80 46.15 ± 0.60 50.13 ± 0.40 44.22 ± 2.75 49.09 ± 1.20 47.15 ± 0.80<br />
07 57.35 ± 1.79 58.25 ± 1.59 63.14 ± 0.56 53.00 ± 1.19 57.67 ± 1.80 54.54 ± 0.54 49.97 ± 1.84 52.58 ± 1.20 56.38 ± 0.61<br />
08 63.41 ± 0.47 65.00 ± 0.74 67.05 ± 1.13 62.39 ± 2.00 60.00 ± 0.37 60.39 ± 0.85 57.16 ± 2.34 56.06 ± 1.20 66.18 ± 0.86<br />
09 71.06 ± 2.42 72.13 ± 0.15 72.18 ± 0.85 66.68 ± 1.34 69.38 ± 1.21 73.51 ± 0.90 64.02 ± 2.05 63.05 ± 1.20 70.05 ± 1.65<br />
10 77.26 ± 2.80 85.61 ± 0.15 88.03 ± 1.02 73.47 ± 2.42 77.36 ± 1.35 79.30 ± 1.95 72.24 ± 2.57 78.54 ± 1.20 72.05 ± 1.14<br />
11 82.58 ± 3.05 91.8 ± 0.37 97.52 ± 0.26 76.78 ± 0.77 80.14 ± 1.43 82.13 ± 0.54 77.26 ± 2.71 81.13 ± 1.32 75.18 ± 1.77<br />
12 87.23 ± 2.67 96.49 ± 0.51 98.91 ± 0.20 83.05 ± 2.50 85.32 ± 1.88 88.91 ± 2.96 81.72 ± 2.22 83.58 ± 1.31 79.13 ± 1.81<br />
*Average of thee determinations, values shown in parenthesis are standard deviations.<br />
Table 4: Dissolution Parameters for 3² Full Factorial Design Batches<br />
Batch Code<br />
Variable Level in<br />
Coded Form<br />
* #<br />
X X 1<br />
2<br />
t 50% (h) t 70% (h)<br />
Cumulative Percent<br />
Drug Release in 12 h<br />
F1 –1 –1 6.20 8.91 87.23<br />
F2 –1 0 6.13 8.71 96.49<br />
F3 –1 1 5.95 8.52 98.91<br />
F4 0 –1 6.68 9.36 83.05<br />
F5 0 0 6.36 9.18 85.32<br />
F6 0 1 6.01 8.74 88.91<br />
F7 1 –1 7.16 9.72 81.72<br />
F8 1 0 6.27 9.47 83.58<br />
F9 1 1 6.32 8.93 79.13<br />
C1 -0.5 +0.5 6.21 8.94 90.98<br />
C2 +0.5 +0.5 6.56 9.23 85.31<br />
C 1 , C 2 check point batches; t 50% , t 70% analyzed by matrix model fi tting using PCP Disso V3 Software. * For HPMCK100LV cps (X 1 ); transformed levels in mg are: –1=100;<br />
‘0’=150, +1=200, –0.5=125, +0.5=175; # For NaHCO 3 (X 2 ), transformed levels in mg are: –1=40; ‘0’=60, +1=80, –0.5=50, +0.5=70; All the batches contained 100 mg of<br />
levofl oxacin hemihydrate,1% talc and 2% magnesium stearate.<br />
full factorial design. The dissolution parameters i.e.,<br />
t 50% , t 70% values were selected as dependent variables.<br />
The formulation codes of the nine batches of factorial<br />
formulations along with dissolution parameter values<br />
(t 50% , t 70% ) and cumulative percent drug released in 12 h<br />
were shown in (Table 4). From the data, it was evident<br />
that formulation F 3 showed highly satisfactory values<br />
for dissolution parameters (t 50% 5.95 h, and t 70% 8.52 h)<br />
and released approximately 98.91% drug in 12 h.<br />
Hence, formulation F 3 was considered as the optimized<br />
promising levofl oxacin hemihydrate gastric fl oating drug<br />
delivery system with improved bioavailability.<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 41
Development of polynomial equations<br />
D.Nagendrakumar et al.: Design and Optimization of Levofl oxacin Gastroretentive Tablets<br />
From the data of dissolution parameters shown in<br />
Table 4 for factorial formulations F 1 to F 9 , polynomial<br />
equations for two dependent variables (t 50% , and t 70% )<br />
have been derived using PCP Disso 2000 V3 software.<br />
The general polynomial equation (Equation 3) for 3² full<br />
factorial design was<br />
2 2 Y = b +b X +b X +b X X +b X +b22X (3)<br />
0 1 1 2 2 12 1 2 11 1 2<br />
Where, Y is dependent variable, b arithmetic mean<br />
0<br />
response of nine batches, and b estimated coeffi cient<br />
1<br />
for factor X . The main effects (X and X ) represented<br />
1 1 2<br />
the average result of changing one factor at a time<br />
from its low to high value. The interaction term<br />
(X X ) showed how the response changes when two<br />
1 2<br />
factors were simultaneously changed. The polynomial<br />
2 2 term (X and X2 ) were included to investigate non-<br />
1<br />
linearity.<br />
The equation derived for t was Y = 6.28 + 0.216X 50% 1 1<br />
– 0.283 X . The negative sign for coeffi cient of X 2 2<br />
indicates that, as the concentration of gas generating<br />
agent (sodium bicarbonate) increases, t value<br />
50%<br />
decreases.<br />
The equation derived for t was Y = 9.02+ 0.31 X –<br />
70% 2 1<br />
0.30 X . 2<br />
The validity of the equations was verifi ed by designing<br />
two check point formulations (C and C ) and studying<br />
1 2<br />
the drug release profi les. The dissolution parameters<br />
predicted from the equations derived and those<br />
observed from experimental results were summarized in<br />
.<br />
Figure 2: Contour plot showing effect of factorial variables on t50% Figure 1: Response surface plot showing effect of factorial vari-<br />
.<br />
ables on t50% Table 5. The closeness of predicted and observed values<br />
for t 50% and t 70% indicated validity of derived equations<br />
for the dependent variables. The computer generated<br />
response surfaces and contour plots for the dependent<br />
variables were shown in Fig. 1–4.<br />
Stability study<br />
Short-term stability study was performed on the<br />
promising formulation F by storing the samples at<br />
3<br />
45 ± 1ºC for 3 weeks. The samples were tested for any<br />
changes in physical appearance and drug content at<br />
weekly intervals. Statistical analysis was performed on<br />
the drug content data and drug release parameters by<br />
using Student t-test. The t value for the drug content<br />
was found to be 3.85. For t and t the t- values<br />
50% 70%,<br />
were found to be 3.46 and 3.00, respectively (Table 6).<br />
These results indicated that there were no signifi cant<br />
changes in drug content and dissolution profi le of<br />
the formulation F during storage at 45ºC for 3 weeks<br />
3<br />
shown no signifi cant effect on physical characteristics<br />
and drug content.<br />
CONCLUSIONS<br />
It was concluded that the gastroretentive fl oating<br />
systems of levofl oxacin hemihydrate with shorter<br />
lag time was prepared by direct compression method<br />
using HPMC K100LV and sodium bicarbonate as<br />
gas generating agent. As the amount of polymer in<br />
the tablet formulation increased, the drug release rate<br />
decreased and as the concentration of gas generating<br />
agent (sodium bicarbonate) increased, the drug release<br />
increased and at the same time fl oating lag time decreased.<br />
42 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
D.Nagendrakumar et al.: Design and Optimization of Levofl oxacin Gastroretentive Tablets<br />
.<br />
Figure 3: Response surface plot showing effect of factorial variables on t70% .<br />
Figure 4: Contour plot showing effect of factorial variables on t70% Table 5: Dissolution Parameters Predicted and Observed<br />
Values for Check point batches<br />
Predicted<br />
Values (h)<br />
Observed<br />
Values (h)<br />
Formulations t50% t70% t50% t70% C1 6.31 9.01 6.20 8.90<br />
C2 6.20 9.02 6.50 9.20<br />
Table.6: Statistical Analysis of Dissolution Parameters<br />
(t 50% , t 70% ) of Stability study for Batch F 3<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 43<br />
Trial<br />
t 50% Values<br />
1st Day<br />
(A)<br />
21st<br />
Day<br />
(B)<br />
A-B<br />
t 70% Values<br />
1st<br />
Day<br />
(A)<br />
21st<br />
Day<br />
(B)<br />
A-B<br />
I 6.01 5.98 0.03 8.69 8.65 0.04<br />
II 5.94 5.93 0.01 8.74 8.73 0.01<br />
III 5.89 5.87 0.02 8.72 8.69 0.03<br />
Mean 5.95 5.92 0.02 8.71 8.69 0.026<br />
SD ± ± 0.060 ± 0.055 ± 0.01 ± 0.025 ± 0.04 ± 0.015<br />
t = 3.46 ;( p
REFERENCES<br />
D.Nagendrakumar et al.: Design and Optimization of Levofl oxacin Gastroretentive Tablets<br />
1. Prajapati ST, Patel LD, Patel DM. Gastric fl oating matrix tablets:<br />
Design and optimization using combination of polymers. Acta Pharm<br />
2008;58:221–9.<br />
2. Sauzet C, Claeys-Bruno M, Nicolas M, Kister J, Piccerelle P, Prinderre<br />
P. An innovative fl oating gastro retentive dosage system: Formulation<br />
and in-vitro evaluation. Int J Pharm 2009;378 (1–2):23–9.<br />
3. Garg R, Gupta GD. Progress in controlled gastroretentive delivery<br />
systems. Trop J Pharm Res 2008;7(3):1055–66.<br />
4. Xiaoqiang X, Minjie S, Feng Z, Yiqiao H. Floating matrix dosage form for<br />
phenoporlamine hydrochloride based on gas forming agent: in-vitro and<br />
in-vivo evaluation in healthy volunteers. Int J Pharm 2006;310:139–45.<br />
5. Deshpande AA, Shah NH, Rhodes CT, Malick W. Development of a<br />
novel controlled release system for gastric retention. Pharm Res 1997;<br />
14:815–9.<br />
6. Chavanpatil MD, Jain P, Chaudhari S, Shear R, Vavia RR. Novel<br />
sustained release, swellable and bioadhesive gastroretentive drug<br />
delivery system for ofl oxacin. Int J Pharm 2006;316 (1–2):86–92.<br />
7. Hwang SJ, Park H, Park K. Gastric retentive drug-delivery systems. Crit<br />
Rev Ther Drug Carrier Syst 1998;15 (3):243–84.<br />
8. Seth PR, Tossounian J. The hydrodynamically balanced system,<br />
a novel drug delivery system for oral use. Drug Dev Ind Pharm<br />
1984;10:313–9.<br />
9. Harrigan RM. Drug delivery device for preventing contact of undissolved<br />
drug with the stomach lining. US patent 4055178. 1977 October 25.<br />
10. Whitehead L, Fell JT, Collett JH. Development of a gastroretentive<br />
dosage form. Eur J Pharm Sci 1996;4(Suppl.):S182.<br />
11. Kawashima Y, Niwa T, Takeuchi H, Hino T, Itoh Y. Hollow microspheres<br />
for use as a fl oating controlled drug delivery system in the stomach.<br />
J Pharm Sci 1992;81:135–40.<br />
12. Chen J, Blevins WE, Park H, Park K. Gastric retention properties of<br />
superporous hydrogel composites. J Control Release 2000;64(1–3):<br />
39–51.<br />
13. Gröning R, Berntgen M, Georgarakis M. Acyclovir serum concentrations<br />
following peroral administration of magnetic depot tablets and the<br />
infl uence of extracoporal magnets to control gastrointestinal transit. Eur<br />
J Pharm Biopharm 1998;46:285–91.<br />
14. Singh BM, Kim KH. Floating drug delivery systems: An approach to<br />
oral controlled drug delivery via gastric retention. J Control Relese<br />
2000;63:235–59.<br />
15. Md. Ismail M, Dehghan MH, Shaikh A, Sahuji T, Chudiwal P. Preparation<br />
of a novel fl oating ring capsule-type dosage form for stomach specifi c<br />
delivery. Soudi Pharm J 2011;19:85–93.<br />
16. [updated 2011 January 6; cited 2011 July 17]. Available from: http://<br />
www.rxlist.com/Levaquin-drug.htm<br />
17. Lachman L, Liberman HA, Kanig JL. The theory and practice of<br />
industrial pharmacy. 3rd ed. Mumbai: Varghese Publishing House;<br />
1991. p. 297–303.<br />
18. Jimenez-Castellanos MR, Hossein Z, Rhodes CT. Design and testing<br />
in vitro of a bioadhesive fl oating drug delivery system for oral application.<br />
Int J Pharm 1994;105:65–70.<br />
19. Tadros IM. Controlled-release effervescent fl oating matrix tablets of<br />
ciprofl oxacin hydrochloride: Development, optimization and in vitro in<br />
vivo evaluation in healthy human volunteers. Eur J Pharm Biopharm<br />
2010;74:332–9.<br />
44 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
Study of the binding properties of hydroxypropyl<br />
guar and its utilization in the formulation and<br />
evaluation of metoprolol tartarate tablets<br />
Swamy N.G.N.*, Dharmarajan T.S., Paranjothi K.L.K<br />
*Department of Pharmaceutics, Government College of Pharmacy, Bangalore – 560 027<br />
ABSTRACT<br />
Derivatization of guar to hydroxypropyl guar enhances the interaction coeffi cient and checks the hydration<br />
rate of the polymer molecule. Hydroxypropyl guar is investigated for its binding property along with sodium<br />
carboxymethylcellulose (1% w/w) as the parallel binding agent. Tablets of metoprolol tartarate were prepared<br />
by wet granulation. Pre-compression parameters such as rate of fl ow, angle of repose, Carr’s index, Hausner’s<br />
ratio, residual moisture content and assay determinations were carried out. Hydroxypropyl guar bound granules<br />
revealed lower Carr’s index value and Hausner’s ratio in comparison to sodium carboxymethylcellulose bound<br />
granules thereby indicating better free fl owing properties. In respect to post compression parameters, tablets<br />
made with hydroxypropyl guar displayed superior hardness, lesser deviation of tablet weight about the mean, a<br />
higher value of (Crushing strength: Friability)/Disintegration time, a faster drug dissolution in comparison to sodium<br />
carboxymethylcellulose bound tablets. While guar with its high intrinsic viscosity restricts its use as a retardant<br />
component in matrix tablets, hydroxypropyl guar with its enhanced interaction coeffi cient and controlled hydration<br />
rate has yielded a promising adjuvant for binding metoprolol tartarate tablets with improved in vitro performance.<br />
Keywords: Carr’s index, crushing strength, disintegration time, friability, hausner’s ratio, hydroxypropyl guar,<br />
interaction coeffi cient, rate of hydration, residual moisture content.<br />
INTRODUCTION<br />
Guar gum1 consists of the endosperm part<br />
of guar plant namely Cyamopsis tetragonolobus<br />
Linn. (Family: Leguminosae). When used in<br />
extemporaneous preparations, guar imparts<br />
colour, which is not desirable. Guar has a<br />
very high intrinsic viscosity but very poor<br />
interaction coeffi cient. 2 Derivatization of<br />
guar to hydroxypropyl guar improves its<br />
interaction coeffi cient. 3 By virtue of its<br />
high intrinsic viscocity, guar gum has been<br />
extensively used as a retarding component<br />
in the formulation of matrix tablets. 4–18<br />
Hydroxypropyl guar with improved<br />
interaction coeffi cient has been investigated<br />
as a gelling agent, 19 as a bodying agent20 in<br />
suppositories, fi lm forming agent21 and<br />
as a suspending agent. 22 Hydroxypropyl<br />
guar with a controlled rate of hydration<br />
is investigated for its solution binding<br />
properties 23 in the formulation of oral<br />
tablets. Sodium carboxymethylcellulose is<br />
tried as the parallel binding agent. 24–28<br />
Metoprolol tartarate 29 (MT), is a selective<br />
β-adrenoreceptor antagonist useful in<br />
the management of hypertension and<br />
ischaemic heart diseases. MT 30 is readily<br />
absorbed after oral administration and<br />
rapidly distributed into body tissues.<br />
The elimination half-life is about 3–7 h.<br />
The drug undergoes extensive fi rstpass<br />
metabolism; only 50% of the orally<br />
administered drug reaches the systemic<br />
circulation. Adult oral dose is 50 mg twice<br />
daily to be optimized subsequently and<br />
not to exceed 400 mg per day. Tablets<br />
containing 50 mg of MT per tablet were<br />
formulated using hydroxypropyl guar<br />
Research Ar cle<br />
Received Date : 26-12-2011<br />
Revised Date : 01-03-2012<br />
Accepted Date : 05-03-2012<br />
DOI: 10.5530/rjps.2012.1.6<br />
Address for<br />
correspondence<br />
Dr. N.G.N. Swamy<br />
Professor<br />
Department of Pharmaceutics<br />
Government College of<br />
Pharmacy,<br />
No. 2, P. Kalinga Rao Road,<br />
Bangalore – 560 027<br />
Mob. No: + 91 9945451772<br />
E-mail: ngnswami@yahoo.co.in<br />
www.rjps.in<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 45
Swamy NGN et al.: Study of the binding properties of hydroxypropyl guar and its utilization in the formulation and evaluation of metoprolol tartarate tablets<br />
(HPG) and sodium carboxymethylcellulose (Na CMC)<br />
as binders.<br />
MATERIALS AND METHODS<br />
MT I.P. was obtained as a gift sample from ASTRA-<br />
IDL ltd. Na CMC (high viscosity grade), Lobachemie<br />
make was used. All other excipients used were of Pharmacopoeial<br />
grade. HPG was obtained by derivatization<br />
of guar, 3 MT tablets weighing around 150 mg each and<br />
containing 50 mg of the drug were made by wet granulation<br />
by employing 1% W/V aqueous dispersions<br />
of HPG and Na CMC respectively as solution binders.<br />
1% W/V HPG dispersion revealed a viscosity of<br />
6400 cps; Na CMC displayed a viscosity of 448 cps.<br />
The aqueous polymer dispersions were stabilized with<br />
0.18% W/V methyl paraben and 0.02% W/V propyl<br />
paraben.<br />
The formulation details of the tablets are shown in<br />
Table 1.<br />
PART-I: PREPARATION OF GRANULES<br />
The quantity of MT as specifi ed in Table 1 was mixed<br />
with lactose IP in the ascending order of weights; the<br />
granulating agent was added little by little so as to get<br />
a damp mass with a pliable consistency. The batter was<br />
allowed to stand for 15 min and passed through sieve<br />
20; the granules so obtained were dried in an oven at 60°<br />
for 1 h duration. The agglomerates were regranulated by<br />
using sieve 20 superimposed on sieve 40. While granules<br />
retained on sieve 20 were used for compression, the<br />
fi nes retained on sieve 40 were used for improvement<br />
of fl ow properties of granules.<br />
PART-II: STUDY OF GRANULE PROPERTIES<br />
(a) Determination of compressibility factor<br />
The untapped and the tapped bulk densities were<br />
obtained by the 10 point method. The 10 cc tared<br />
measuring jar was fi lled with granules up to 1 cc and<br />
the weight was recorded; likewise, the weight of 2 cc,<br />
3 cc ... up to 10 cc was fi lled with granules and the weight<br />
was recorded without tapping. Similarly the procedure<br />
was repeated with the granules fi lled up to 1 cc, tapped<br />
so as to compact the granules, excess granules was added<br />
to record a volume of 1 cc after tapping. The weight<br />
of 2 cc, 3 cc ... up to 10 cc with tapping each time after<br />
the addition of excess granules to adjust the volume<br />
was recorded. A graph of weight values (gm) vs corresponding<br />
volume (cc) was constructed. The bulk density<br />
was arrived from the slope of the linear relationship.<br />
Compressibility factor ‘C’ was calculated using the formula,<br />
c<br />
b a<br />
=<br />
b<br />
− (1)<br />
Table 1: The formulation details of Metoprolol tartarate<br />
tablets<br />
Tablet wt Tablet wt<br />
Ingredient<br />
in gm in gm<br />
Metoprolol tartarate 30 30<br />
Lactose I.P 60 60<br />
Hydroxypropyl guar 1% w/v aqueous<br />
dispersion<br />
Q.S –<br />
Sodium CMC 1% w/v aqueous<br />
dispersion<br />
– Q.S<br />
Talc 2% 2%<br />
Where, b=Tapped bulk density and a = Untapped bulk<br />
density. The graphical representations are shown in<br />
fi gure 1and 2. Compressibility factor multiplied by 100<br />
gives Carr’s index values.<br />
(b) Determination of the optimum amount of<br />
Lactose fi nes to be incorporated to the 20/40<br />
mesh granules to improve the fl ow properties<br />
Since 1% w/v HPG bound granules revealed the lowest<br />
Carr’s index value, 31 1% w/v HPG bound lactose<br />
granules were used for the evaluation.<br />
(i) Determination of angle of repose32 Carried out by employing fi xed bed method using the<br />
formula,<br />
tan θ = ;<br />
h<br />
(2)<br />
r<br />
Where, θ = angle of repose, h = height of the heap<br />
and r = radius of the heap. The exact value of θ was<br />
arrived at by referring to the table on natural tangents.<br />
The advantage of this method being, the dimension of<br />
the base is fi xed, whereas, the height alters as the angle<br />
of repose changes.<br />
(ii) Determination of the rate of fl ow33 Carried out using a glass funnel having an orifi ce and<br />
base diameters of 0.8 cm and 9 cm respectively and<br />
securely clamped to a retort stand. A 50 gm sample was<br />
introduced into the funnel and the powder was allowed<br />
to fall freely under the gravitational force. The fl ow rate<br />
was calculated using the expression:<br />
Flow rate = amount of powder (gm) / time of fl ow (s). (3)<br />
The above studies were conducted on 20/40 # lactose<br />
granules blended with 5% w/w, 10% w/w and 15%<br />
w/w, lactose fi nes. The average value of three such<br />
determinations is recorded in fi gures 3 and 4.<br />
(c) Determination of optimum concentration of<br />
the glidant (Talc) to be added to dummy lactose<br />
granules containing 10% w/w of Lactose fi nes<br />
The θ values and the fl ow rate values were determined<br />
for the above mentioned granules blended with 1%, 2%,<br />
3%, 4% and 5% w/w talc powder. The infl uence of the<br />
46 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
Swamy NGN et al.: Study of the binding properties of hydroxypropyl guar and its utilization in the formulation and evaluation of metoprolol tartarate tablets<br />
Figure 1: Untapped and tapped BD recording for 20/40#MT granules made with 1% w/v of HPG.<br />
Figure 2: Untapped and tapped BD recording for 20/40#MT granules made with 1% w/v of NaCMC.<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 47
Swamy NGN et al.: Study of the binding properties of hydroxypropyl guar and its utilization in the formulation and evaluation of metoprolol tartarate tablets<br />
Figure 3: Influence of percentage lactose fines on the angle of repose for 20/40# lactose granules.<br />
Figure 4: Influence of percentage lactose fines on the rate of flow for 20/40# lactose granules.<br />
talc % w/w on the θ and the fl ow rates is depicted in<br />
fi gure 5 and 6.<br />
From the studies carried out at “b” and “c” above, it<br />
could be concluded that the optimum fi nes concentration<br />
was 10% w/w and optimum glidant concentration was<br />
2% w/w of talc powder.<br />
(d) Determination of Carr’s index for 20/40 mesh<br />
MT granules containing 10% w/w fi nes and<br />
blended with 2% w/w of Talc powder<br />
The graphical representation for granules made with<br />
1% w/v HPG as binder are shown in fi gure 7 and<br />
for granules made with 1% w/v NaCMC is shown<br />
in fi gure 8. Carr’s Index is calculated by using the<br />
formula<br />
CI = (Dt – Db/ Dt) × 100 (4)<br />
(e) Hausner’s ratio<br />
It is an index of ease for powder fl ow. It is calculated by<br />
the following formula,<br />
Hausner s ratio Dt<br />
' = (5)<br />
Db<br />
48 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
Swamy NGN et al.: Study of the binding properties of hydroxypropyl guar and its utilization in the formulation and evaluation of metoprolol tartarate tablets<br />
Figure 5: Influence of % w/w talc added on the angle of repose of 20/40 mesh lactose granules blended with 10% w/w of lactose fines.<br />
Figure 6: Influence of % w/w talc added on the flow rate of 20/40 mesh lactose granules blended with 10% w/w of lactose fines.<br />
Table-2: Infl uence of additives on the angle of repose and the fl ow rate for dummy 20/40 # Lactose granules<br />
Granules composition<br />
20/40 mesh lactose<br />
granules bound with<br />
1% w/v HPG,<br />
20/40 mesh lactose<br />
granules bound with<br />
1%w/v NaCMC,<br />
Angle of<br />
repose for<br />
plain granules<br />
Angle of repose<br />
with the added<br />
additives<br />
Fall in angle<br />
of repose<br />
Flow rate in gm/s<br />
for granules with<br />
additives<br />
Flow rate in<br />
gm/s for plain<br />
granules<br />
Enhancement<br />
in fl ow rate<br />
(gm/s)<br />
40°7’ 38°42’ 1°25’ 3.508 3.385 0.123<br />
39°11’ 37°18’ 1°53’ 3.533 3.496 0.037<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 49
Swamy NGN et al.: Study of the binding properties of hydroxypropyl guar and its utilization in the formulation and evaluation of metoprolol tartarate tablets<br />
Weight (gm)<br />
Figure 7: Untapped and tapped BD recording for 20/40# MT granules made with 1% w/v HPG blended with 10% w/w fines and 2% w/w talc.<br />
Where Dt is the tapped bulk density and Db is the<br />
untapped bulk density.<br />
(f) Determination of angle of repose θ<br />
Angle of repose determination and fl ow time<br />
determination were carried out on 20/40 mesh dummy<br />
granules blended with 10% w/w lactose and 2% W/W<br />
talc powder.<br />
The infl uence of additives on the fl ow properties of<br />
20/40 mesh dummy granules is depicted in Table 2<br />
(g) Determination of residual moisture content<br />
Residual moisture content was determined by drying the<br />
granules at 60° for 15 min. 34<br />
(h) Assay of granules<br />
(i) Carried out to ascertain uniform distribution of drug<br />
in tablets over the entire range of sample (ii) To enable<br />
us to adjust the die volume/ the total weight of the tablet<br />
so as to get tablets having the required drug content. MT<br />
has a λmax at 275 nm in 95% v/v ethanol. It obeys Beer’s<br />
Lamberts law in the concentration range of 10–100<br />
μg/ml. A calibration curve was constructed; the linear<br />
relationship had a slope value of 0.0046 and an intercept<br />
value of 0.0034 which were used in the calculation<br />
of the drug content in the assay of sample. The precompression<br />
parameters are contained in Table 3.<br />
PART-III: COMPRESSION OF GRANULES<br />
INTO TABLETS<br />
The compression was carried out using a 10 station<br />
rotary tablet machine. The capacity of the die cavity was<br />
so adjusted as to accommodate a weight of granules<br />
containing 50 mg of MT. 7mm punches were used and<br />
a compression pressure of 6 Kg was applied on the<br />
rollers for all tablets. The tablets were preserved in tight<br />
screw capped bottles until further evaluation tests were<br />
carried out.<br />
PART-IV: EVALUATION OF TABLETS<br />
The compressed tablets were subjected to the following<br />
evaluation tests.<br />
(i) Hardness test 35<br />
Carried out using Monsonto hardness tester; a mean of<br />
three readings was recorded.<br />
(ii) Determination of disintegration time36 Disintegration time was recorded as per I.P procedure;<br />
6 tablets were subjected to the study.<br />
(iii) Weight variation 36<br />
The test was carried out on 20 tablets. The tablets were<br />
weighed collectively and the average weight was arrived<br />
50 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
Swamy NGN et al.: Study of the binding properties of hydroxypropyl guar and its utilization in the formulation and evaluation of metoprolol tartarate tablets<br />
Table 3: Pre-compression parameters<br />
Granule composition<br />
20/40 mesh MT granules bound with<br />
1% w/v HPG, blended with 10% w/w fi nes,<br />
2% w/w talc.<br />
20/40 mesh MTgranules bound with<br />
1%w/v NaCMC, blended with 10% w/w<br />
fi nes, 2% w/w talc.<br />
RMC* Residual moisture content.<br />
Weight (gm)<br />
Untapped bulk<br />
density (Db) gm/cc<br />
at. The weights of 20 tablets were recorded individually<br />
and the deviation from the mean was calculated. Not<br />
more than 2 tablets to deviate beyond ±7.5% and not<br />
even one to deviate beyond ±15%.<br />
(iv) Friability Test 37<br />
The test was carried out on 20 tablets weighed collectively.<br />
The tablets were loaded on to a Roche friabilator and<br />
Tapped bulk<br />
density (Dt) gm/cc<br />
Carr’s<br />
Index<br />
0.5638 0.5761 2.13<br />
Hausner’s<br />
Ratio<br />
1.0218<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 51<br />
RMC*<br />
Assay Value<br />
(mg)<br />
1.34 47.51<br />
0.5346 0.5728 6.66 1.0714 1.24 48.71<br />
Volume (cc)<br />
Figure 8: Untapped and tapped BD recording for 20/40# MT granules made with 1%w/v NaCMC blended with 10% w/w fines and 2%w/w talc .<br />
Table 4: Post compression parameters<br />
Hardness<br />
in Kg/cm2 Disintegration Friability Assay<br />
time in minutes %<br />
(mg) (CS:FR)/DT* t in min<br />
80%<br />
Tablets made with 1% w/w<br />
HPG as binder<br />
4.5 8.20 0.24 47.86 2.3 20<br />
Tablets made with 1% w/v<br />
of NaCMC as Binder<br />
3.0 7.50 0.18 47.45 2.1 35.45<br />
(CS:FR)/DT*; CS = Crushing strength, FR = Friability and DT = Disintegration time.<br />
rotated at 25 rpm for 4 min; tablets weighed again and<br />
% friability was calculated. It is desirable that the weight<br />
loss is less than 1.0%.<br />
(v) Assay<br />
As mentioned under granules; 5 tablets36 are randomly<br />
powdered; a quantity equivalent to 100 mg of MT was<br />
taken and dissolved in 95% of ethanol; subsequent
Swamy NGN et al.: Study of the binding properties of hydroxypropyl guar and its utilization in the formulation and evaluation of metoprolol tartarate tablets<br />
Figure 9: Dissolution profile of 1% HPG and 1% Na CMC bound Metoprolol Tartarate tablets.<br />
dilutions were made with ethanol to have MT<br />
concentration in the range of 10–100 μg/ml; absorbance<br />
was read at 275 nm; concentration of MT was calculated<br />
by making use of slope and intercept values obtained<br />
from the calibration curve. Post compression parameters<br />
are compiled in Table 4.<br />
(vi) Dissolution profi le<br />
Dissolution testing was carried out in 0.1N Hydrochloric<br />
acid. Amount of medium used was 1000 ml; rpm 50.<br />
Volume withdrawn was 10 ml, samples withdrawn at<br />
the end of 15, 30, 45, 60, 75 and 90 min and analyzed<br />
spectrophometrically at 275 nm. The dissolution profi les<br />
of HPG bound and Na CMC bound tablets are depicted<br />
in fi gure 9.<br />
RESULT AND DISCUSSION<br />
In this study, 1% w/v aqueous dispersion of HPG<br />
has been tried as a binder for MT; 1%w/v aqueous<br />
dispersion of Na CMC is used as the parallel binder.<br />
20/40 mesh lactose granules were made and mixed<br />
with optimum amounts of 10% w/w lactose fi nes<br />
and 2% w/w of talc powder. 20/40 mesh granules as<br />
mentioned afore said were mixed with 10% w/w fi nes<br />
and 2%w/w talc powder and subjected to untapped and<br />
tapped density determination; from this the percentage<br />
compressibility factor i.e. Carr’s index and Hausner’s<br />
ratio were calculated. Granules made with HPG as<br />
binder revealed a Carr’s index value of 2.13 in contrast<br />
to a value of 6.66 for granules made with 1% w/v Na<br />
CMC dispersion. Granules having Carr’s index value in<br />
the range of 5 to 12 reveal excellent fl ow characteristics.<br />
Granules made with HPG revealed a Hausner’s ratio of<br />
1.022 in contrast to a value of 1.071 in case of Na CMC<br />
granules. Lower Hausner’s ratio (1.25). It is a well verifi ed fact<br />
that the more compressible a bed of particles is, the less<br />
fl owable the powder or granules will be. A material having<br />
a compressibility value of less than 16 is considered to<br />
be a free fl owing material; this is in close agreement with<br />
the data compiled in table 3. Repose angle also needs<br />
to be considered along with compressibility; Repose<br />
angle 34 for the experimental samples are in the range of<br />
37–38° which is marginally higher than the optimum<br />
value i.e. 30°. When the angle of repose exceeds 50°,<br />
the fl ow is rarely acceptable for manufacturing process.<br />
Table 5: Flow properties and corresponding angle of<br />
repose<br />
Flow property Angle of repose (Degrees)<br />
Excellent 25–30<br />
Good 31–35<br />
Fair 36–40<br />
Passable 41–45<br />
Poor 46–55<br />
Very poor 56–65<br />
Very, Very Poor >66<br />
52 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
Swamy NGN et al.: Study of the binding properties of hydroxypropyl guar and its utilization in the formulation and evaluation of metoprolol tartarate tablets<br />
The infl uence of angle of repose 38 on fl ow properties<br />
of granules is depicted in Table 5.<br />
The fl ow rate was determined by passing 50 gm of<br />
granules through a glass funnel with orifi ce and base<br />
diameters of 0.8 cm and 9 cm respectively. By and large,<br />
lower the compressibility factor and smaller the repose<br />
angle, the faster is the predicted fl ow rate of granules.<br />
HPG granules have revealed a signifi cant enhancement<br />
in the fl ow rate after the inclusion of additives i.e.<br />
0.123 gm/s in contrast to an enhancement value of<br />
0.037 gm/s incase of granules made with Na CMC.<br />
For trouble free compression, a moisture content of<br />
around 1.5% is desirable. HPG bound granules revealed<br />
a moisture content of 1.34% in contrast to a value of<br />
1.24 in case of granules made with Na CMC as binder.<br />
Moisture content of less than 1% can cause capping and<br />
lamination whereas above 2%, some sticking problem<br />
may develop.<br />
In respect to post compression parameters, from the<br />
weight variation data, it is observed that in case of HPG<br />
bound tablets, the % deviation from the mean was found<br />
to range between (-) 5.86 to (+) 6.73 in contrast to a<br />
value of (-) 7.39 to (+) 8.94 for Na CMC bound tablets.<br />
This observation correlates well with the lower ‘C’<br />
values obtained for HPG bound granules. Tablets made<br />
with HPG as binder revealed a hardness of 4.5 kg/cm 2<br />
in contrast to a value of 3 Kg/cm 2 for tablets made<br />
with Na CMC as binder. The friability value for HPG<br />
bound tablets was 0.24% in contrast to a value of 0.18%<br />
in case of Na CMC bound tablets. Disintegration time<br />
for HPG bound tablets was 8.3 min which was slightly<br />
higher than the Disintegration time value of 7.8 min for<br />
Na CMC bound tablets. Crushing strength – friability<br />
ratio (CS:FR) which is the quotient of the crushing<br />
strength (CS) value divided by the friability (FR) value, is<br />
an index used as a measure of the mechanical strength<br />
of tablets. But the (CS:FR)/DT ratio would be a better<br />
index for measuring tablet quality. 39 It is so because, in<br />
addition to measuring tablet strength (crushing), and<br />
tablet weakness (friability), it does evaluate all negative<br />
effects of these parameters on disintegration time. 40<br />
HPG bound tablets revealed a (CS:FR)/DT ratio of<br />
2.26 in contrast to a value of 2.1 in case of Na CMC<br />
bound tablets. A higher value of (CS:FR)/DT is<br />
indicative of a better balancing act between the cohesive<br />
binding and disintegrating behavior of the tablets. From<br />
the dissolution profi le, a t 80% value of 20 min has been<br />
displayed in case of HPG bound tablets in contrast to a<br />
value of 35.45 min in Case of Na CMC bound tablets.<br />
CONCLUSION<br />
The hydroxypropyl guar with it’s enhanced interaction<br />
coeffi cient and controlled rate of hydration has proved<br />
to be a superior solution binder in achieving suffi ciently<br />
rapid release of the drug from the tablet formulation in<br />
comparison to the conventionally used tablet solution<br />
binder namely sodium carboxymethylcellulose.<br />
ACKNOWLEDGEMENT<br />
The authors wish to thank Shri P. Guruswamy, The<br />
then plant manager, Caryll Pharma, Sarakki main road<br />
Bengaluru, for providing tablet compression facility;<br />
Shri K.R.P Shenoy, Controller, QA and Technical<br />
services, ASTRA-IDL for gifting Metoprolol Tartarate<br />
and Shri Chandrashekhar, Managing Director, Ce-Chem<br />
Pharmaceuticals Pvt Ltd. Peenya, Bengaluru for the gift<br />
Sample of Lactose I.P.<br />
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54 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
Phytochemical investigation of root extract<br />
of the plant Carissa spinarum<br />
Karunakar Hegde 1 , D. Satyanarayana 2 , Arun B. Joshi 3<br />
1 Department of Pharmacology, Srinivas College of Pharmacy, Valachil, Post- Parangepete, Mangalore- 574 143,<br />
Karnataka, India<br />
2Department of Pharmachemistry, N G S M Institute of Pharmaceutical Sciences, Mangalore- 574 160, Karnataka, India<br />
3Department of Pharmacognosy, Goa College of Pharmacy, Panaji- 403 001, Goa, India<br />
ABSTRACT<br />
From the petroleum ether extract of the roots of Carissa spinarum Linn. (Apocynaceae), six compounds namely<br />
stigmasterol, ursolic acid, lupeol, campesterol, 17-hydroxy-11-oxo-nor-β-amyrone and urs-12-ene-3β, 22β-diol-<br />
17-carboxylic acid have been isolated by column chromatography. Their structures were characterized by m.p.,<br />
IR, 1HNMR, 13CNMR and mass spectral data. However, the compounds stigmasterol, campesterol, 17-hydroxy-<br />
11-oxo-nor-β-amyrone and urs-12-ene-3β, 22β-diol-17-carboxylic acid were reported for the fi rst time from the<br />
root of this plant.<br />
Keywords: Carissa spinarum, Root extract, Sterol, Triterpenoids<br />
INTRODUCTION<br />
Carissa spinarum Linn. (Carissa opaca Stapf<br />
ex Haines) is a evergreen shrub with short<br />
stem and strong thorns in pairs, belonging<br />
to family Apocynaceae. The plant is widely<br />
distributed throughout the dry, sandy and<br />
rocky soils of India, Ceylon, Myanmar and<br />
Thailand. 1 In traditional system of medicine<br />
the plant is used as purgative, for the<br />
treatment of rheumatism, cleaning worm<br />
infested wounds of animals and in snake<br />
bite. 1,2 Earlier studies have shown that the<br />
extract of the plant possesses cardiotonic, 3<br />
anticonvulsant, 4 hepatoprotective, 5 antiarthritic,<br />
6 antibacterial, 7 potent antioxidant8 and CNS depressant activitiy. 9 Various<br />
cardiac glycosides, 10 caffeic acid, 11 ursolic<br />
acid, naringin12 germacrane sesquiterpene<br />
and lignans8 were reported from this plant.<br />
Since the chemical investigation of the<br />
roots of Carissa spinarum has not been dealt<br />
in detail, in the present study an effort was<br />
made to establish and report the chemical<br />
investigations, of the petroleum ether<br />
extract of the roots of this plant.<br />
MATERIALS AND METHODS<br />
Chemicals and Instruments<br />
All the chemicals and solvents were<br />
of analytical grade and procured from<br />
Ranbaxy Fine Chemicals Ltd., Mumbai,<br />
India. The melting points were determined<br />
in a Toshniwal melting point apparatus<br />
and were uncorrected. The IR spectra of<br />
the compounds were recorded using KBr<br />
pellet method on Rx-1 Perkin-Elmer FTIR.<br />
1 13 HNMR and CNMR spectra were run on<br />
Bruker Avance II 400 spectrophotometer<br />
using CDCl as a solvent. Mass spectra<br />
3<br />
(FAB-MS) were obtained on a JEOL SX<br />
102/DA-6000 mass spectrometer.<br />
Plant Material<br />
The roots of C. spinarum were collected from<br />
Sirsi, Uttara Kannada District, Karnataka,<br />
Research Ar cle<br />
Received Date : 28-11-2011<br />
Revised Date : 03-02-2012<br />
Accepted Date : 08-02-2012<br />
DOI: 10.5530/rjps.2012.1.7<br />
Address for<br />
correspondence<br />
Karunakar Hegde<br />
Department of Pharmacology<br />
H.K.E. Society’s M T R Institute<br />
Srinivas College of Pharmacy<br />
Valachil, Post- Parangepete<br />
Mangalore- 574 143<br />
Karnataka, India<br />
E-mail: khegde_sh2003@<br />
yahoo.co.in<br />
Phone: +91-824-2274722<br />
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RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 55
Karunakar Hegde et al.: Phytochemical Investigation of Root Extract of the Plant Carissa spinarum<br />
India during May 2007. It was authenticated by<br />
Dr. Gopalakrishna Bhat, Department of Botany, Poorna<br />
Prajna College, Udupi, Karnataka, India. A voucher<br />
specimen no. 105b is deposited in the herbarium of our<br />
institute.<br />
Extraction and Isolation<br />
The shade dried roots of the plant (1000 g) were soaked<br />
in 1.5 L of 95% ethyl alcohol and extracted in the<br />
cold for 4 days with occasional shaking. After 4 days<br />
the ethanol layer was decanted off. The process was<br />
repeated for 4 times. The solvent from the total extract<br />
was fi ltered, the concentrate was evaporated to dryness<br />
under reduced pressure and low temperature (40oC) on a rotary evaporator to give the ethanolic extract<br />
(13% w/w yield), which was stored at 4oC until use.<br />
The ethanol extract (125 g) was suspended in distilled<br />
water and extracted with petroleum ether (60–80oC, 8 ×<br />
500 ml). All the fractions were then washed with distilled<br />
water (30 ml), dried over anhydrous sodium sulphate<br />
and freed of solvent by distillation to give petroleum<br />
ether (60–80ºC) soluble fraction (32 g). Petroleum ether<br />
fraction (25 g) was saponifi ed by refl uxing for 6 h in<br />
500 ml 5% methanolic KOH and allowed to stand at<br />
room temperature for 20 h. The unsaponifi able portion<br />
was extracted with diethyl ether. All the ethereal fractions<br />
Figure 1: Structure of isolated compounds.<br />
were combined and washed with distilled water. The<br />
solvent was evaporated and dried over anhydrous sodium<br />
sulphate to afford a yellowish residue (9 g). 13<br />
The residue (8 g) was dissolved in chloroform (10 ml)<br />
and adsorbed on to silica gel (60–120 mesh, 20 g). After<br />
evaporation of the solvent, it was subjected to column<br />
chromatography over silica gel (150 g) prepared in<br />
n-hexane. The elutions were carried out with n-hexane:<br />
ethyl acetate graded mixture (95:5) afforded compound<br />
I (65 mg) and n-hexane: ethyl acetate graded mixture<br />
(90:10) resulted into compound II (93 mg) and III<br />
(125 mg). Further, the elutions carried out with n-hexane:<br />
ethyl acetate graded mixtures (85:15) resulted into<br />
compound IV (95 mg), V (90 mg) and VI (80 mg)<br />
respectively. The individual compounds were purifi ed<br />
by preparative TLC on silica gel G and further purifi ed<br />
by recrystallization with n-hexane.<br />
RESULTS<br />
Compound I (stigmasterol)<br />
Pearl white crystals (Figure 1); R 0.63 in n-hexane:<br />
f<br />
ethyl acetate (95:05); m.p. 167–170oC; IR v (KBr):<br />
max<br />
3422, 2951, 2851, 1669, 1619, 1465, 1072 cm –1 ; 1HNMR (CDCl , 400 MHz): δ 0.79, 0.78, 0.82, 0.91, 0.94, 1.08<br />
3<br />
(m, 18H), δ 1.13 to δ 2.53 (m, 18H, 9x CH and 8H),<br />
2<br />
56 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
Karunakar Hegde et al.: Phytochemical Investigation of Root Extract of the Plant Carissa spinarum<br />
3.54 (1H, dd, J= 9.1, 6.0 Hz, H-α3), 5.33 ( s, 1H, H-3),<br />
5.11 and 5.15 (2H, br); 13 CNMR (CDCl 3, 400 MHz): δ<br />
33.79 (C-1), 78.98 (C-3), δ 145.83 (C-5), δ 121.81 (C-6), δ<br />
32.50 (C-7), δ 35.35 (C-8), δ 45.99 (C-9), δ 30.02 (C-14),<br />
δ 56.93 (C-17), δ 19.91 (C-19), δ 138.39 (C-22), δ129.35<br />
(C-23), δ 30.35 (C-24), δ 18.89 (C-28); FAB-MS m/z<br />
(rel.int): 412 [M + ] (C 29 H 48 O) (100), 397 (20), 369 (10), 351<br />
(70), 329 (65), 300 (40), 299 (23), 273 (31), 255 (28).<br />
Compound II (ursolic acid)<br />
Pale yellow powder (Figure 1); R 0.51 in n-hexane: ethyl<br />
f<br />
acetate (90:10); m.p. 271–274oC; IR v (KBr): 3455, 2924,<br />
max<br />
2853, 1638, 1460, 1382, 1035 cm –1 ; 1HNMR (CDCl , 400 3<br />
MHz): δ 1.00 to 1.91 (m, 22H), 3.20 (1H, dd, J= 10, 5.7<br />
Hz, H-3), 2.17 (1H, d, J= 4.52 Hz, H-18), 2.28 (m, 1H,<br />
OH), 5.34 (s, 1H, H-12); 13CNMR (CDCl 400 MHz):<br />
3,<br />
δ 37.30 (C-1), 78.49 (C-3), 39.98 (C-4), 41.18 (C-5),<br />
51.87 (C-9), 125.25 (C-12), 137.48 (C-13), 55.39 (C-17),<br />
38.95 (C-18), 32.54 (C-19), 18.45 (C-25), 16.95 (C-26),<br />
23.69 (C-27), 177.48 (C-28), 20.12 (C-29); FAB-MS m/z<br />
(rel.int): 456 [M + ] (C H O ) (71), 428 (50), 289 (49),<br />
30 48 3<br />
277 (35), 248 (100), 149 (27), 85 (11), 83 (44).<br />
Compound III (lupeol)<br />
Pearl white crystals (Figure 1); R 0.56 in n-hexane: ethyl<br />
f<br />
acetate (90:10); m.p. 213–215oC; IR v (KBr): 3435,<br />
max<br />
2928, 2850, 1648, 1455, 1373, 1038, 880 cm –1 ; 1HNMR (CDCl , 400 MHz): δ 0.76 to 1.02 (m, 18H, Me-28,<br />
3<br />
Me-23, Me-24, Me-25, Me-26, Me-27), 1.66 (3H, d,<br />
J= 0.7 Hz, Me-30), 3.21 (1H, dd, J= 9.5, 6.2 Hz, H-α3),<br />
4.57 ( s, 1H, H-3); 13CNMR (CDCl 400 MHz): δ 38.55<br />
3,<br />
(C-1), 78.49 (C-3), 38.98 (C-4), 54.34 (C-5), 18.44 (C-6),<br />
39.68 (C-8), 49.87 (C-9), 42.28 (C-14), 42.98 (C-17),<br />
48.24 (C-18), 47.42 (C-19), 150.48 (C-20), 28.25 (C-21),<br />
16.51 (C-25), 15.96 (C-26), 109.29 (C-29); FAB-MS m/z<br />
(rel.int): 426 [M + ] (C H O) (50), 409 (10), 395 (13), 318<br />
30 50<br />
(9), 218 (74), 203 (65), 187 (46), 175 (30), 161 (39), 147<br />
(64), 125 (71), 121 (70), 93 (100).<br />
Compound IV (campesterol)<br />
Pearl white crystals (Figure 1); R 0.79 in n-hexane:<br />
f<br />
ethyl acetate (85:15); m.p. 157–159oC; IR v (KBr):<br />
max<br />
3425, 2935, 2843, 1642, 1474, 1032, 601 cm –1 ; 1HNMR (CDCl , 400 MHz): δ 0.76, to 1.08 (m, 18H, Me-18,<br />
3<br />
Me-21, Me-26, Me-27, Me-24’, Me-19), δ 1.12 to δ 1.26<br />
(m, 16H, 8xCH ), δ 1.39 to δ 1.86 (m, 9H), 3.22 (m, 1H,<br />
2<br />
H-3a), 5.36 (1H, t, J= 3.4 Hz, H-12); 13CNMR (CDCl3, 400 MHz): δ 32.96 (C-1), δ 31.92 (C-2), δ 72.98 (C-3),<br />
δ 150.96 (C-5), δ 124.43 (C-6), δ 50.46 (C-9), δ 40.85<br />
(C-12), δ 43.01 (C-13), δ 29.98 (C-14), δ 56.32 (C-17),<br />
δ 12.66 (C-18), δ 19.80 (C-19), δ 19.39 (C -21), δ 33.86<br />
C-23), δ 31.18 (C-26), δ 21.09 (C-24’); FAB-MS m/z<br />
(rel.int): 400 [M + ] (C H O) (70), 394 (21), 351 (35), 300<br />
28 48<br />
(12), 271 (65), 255 (54), 213 (28), 159 (23), 145 (9), 133<br />
(15), 105 (34), 83 (16), 55 (100 %).<br />
Compound V (17-hydroxy-11-oxo-nor-β-amyrone)<br />
Semisolid compound (Figure 1); R f 0.59 in n-hexane:<br />
ethyl acetate (85:15); m.p. 85–87 o C; IR v max (KBr): 3445,<br />
2931, 1789, 1649, 1454, 1376, 1082, 882 cm –1 ; 1 HNMR<br />
(CDCl 3 , 400 MHz): δ 0.79 (s, 3H, 1xCH 3 , H-27), δ 0.87<br />
(s, 6H, 2xCH 3 , H-25 & H-26), δ 0.91 (s, 6H, 2xCH 3 , H-23<br />
& H-24), δ 0.97 (s, 6H, 2xCH 3 , H-28 & H-29), δ 1.13 to<br />
1.61 (m, 18H, 9xCH 2 ), δ 1.68 to δ 2.31 (s, 3H, 3xCH), δ<br />
3.61 (s, 1H, OH), δ 5.18; 13 CNMR (CDCl 3, 400 MHz): δ<br />
38.94 (C-1), δ 206.82 (C-3), δ 55.34 (C-5), δ 40.02 (C-8),<br />
δ 47.72 (C-9), δ 198.36 (C-11), δ 124.45 (C-12), δ 139.61<br />
(C-13), δ 75.68 (C-17), δ 59.11 (C-18), δ 39.88 (C-19),<br />
δ 39.39 (C-20), δ 41.87 (C-22), δ 28.10 (C-28), δ 15.67<br />
(C-29); FAB-MS m/z (rel.int): 440 [M+H] + (C 29 H 44 O 3 )<br />
(52), 425 (19), 409 (100), 391 (78), 353 (26), 325 (45).<br />
Compound VI (urs-12-ene-3β, 22β-diol-<br />
17-carboxylic acid)<br />
White crystalline powder (Figure 1); R 0.54 in n-hexane:<br />
f<br />
ethyl acetate (85:15); m.p. 211–214oC; IR v (KBr):<br />
max<br />
3455, 2948, 2850, 1642, 1462, 1374, 1055 cm –1 ; 1HNMR (CDCl , 400 MHz): δ 1.00 to 1.91 (m, 23H), δ 2.36 (1H,<br />
3<br />
d, J=7.3 Hz, H-18), δ 3.38 (dd, 1H, OH), δ 3.61 (dd,<br />
1H, OH), δ 5.28 (s, 1H); 13CNMR (CDCl 400 MHz): δ<br />
3,<br />
38.30 (C-1), δ 78.98 (C-3), δ 54.34 (C-5), δ 33.02 (C-7),<br />
δ 39.38 (C-8), δ 46.72 (C-9), δ 123.25 (C-12), δ 139.35<br />
(C-13), δ 41.37 (C-14), δ 48.35 (C-17), δ 38.89 (C-19),<br />
δ 70.68 (C-22), δ 14.99 (C-23), δ 179.33 (C-28), δ 16.61<br />
(C-30); FAB-MS m/z (rel.int): 472 [M] + (C H O ) (100),<br />
30 48 4<br />
456 (45), 428 (12), 289 (30), 277 (35), 241 (70), 234 (25),<br />
149 (9), 85 (42), 83 (20).<br />
DISCUSSION<br />
Compound I was obtained as pearl white crystals<br />
and gave characteristic color reaction for sterol. The<br />
FAB-MS spectrum showed a molecular ion peak at m/z<br />
412 corresponding to molecular formula C H O. The<br />
29 48<br />
IR spectrum exhibited strong absorptions at 3422 cm –1<br />
(hydroxyl group), 2951 and 1669 cm –1 . The 1HNMR spectrum exhibited six tertiary methyl groups at δ 0.79,<br />
0.78, 0.82, 0.91, 0.94, and 1.08, one vinylic proton at δ 5.33<br />
and two olefi nic protons at δ 5.11 and 5.15 respectively.<br />
In 13CNMR spectrum the most down fi eld signals at δ<br />
145.83 was accommodated for sp2 (olefi nic) carbon at<br />
C-5 and the next downfi eld signal at δ 138.39 ppm and<br />
δ 129.35 ppm to C-22 and C-23. The downfi eld signal at<br />
δ 121.81 is to C-6. The oxygenated carbon at C-3 gave<br />
a downfi eld signal at δ 78.98 ppm. The next downfi eld<br />
signal at δ 56.93 ppm was accommodated for C-17.<br />
Other carbon atoms of the steroidal skeleton except<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 57
Karunakar Hegde et al.: Phytochemical Investigation of Root Extract of the Plant Carissa spinarum<br />
that in the side chain appeared in the range δ 45.99 to<br />
δ 30.02 ppm. The angular methyl groups and the side<br />
chain methyl carbons gave signals in the region δ 19.91<br />
to δ 18.89 ppm. 13 CNMR spectral data matched exactly<br />
with that of Stigmasterol 14 . Its identity as stigmasterol<br />
was confi rmed by m.p., IR, 1 HNMR, 13 CNMR and<br />
mass spectral data and by co-chromatography with an<br />
authentic sample.<br />
Compound II was obtained as pale yellow powder and<br />
it gave characteristic color reaction for triterpenoids..<br />
The FAB-MS showed a molecular ion peak at m/z 456<br />
corresponding to a molecular formula C 30 H 48 O 3 . It was<br />
unambiguously identifi ed as ursolic acid on the basis<br />
of its m.p., IR, 1 HNMR, 13 CNMR and mass spectral<br />
data 15 .<br />
Compound III was obtained as pearl white crystals and<br />
it gave characteristic color reaction for triterpenoids.<br />
The FAB-MS spectrum showed a molecular ion peak at<br />
m/z 426 corresponding to molecular formula C 30 H 50 O.<br />
The IR spectrum exhibited strong absorptions at<br />
3435 cm –1 (hydroxyl group) and 2928, 1648, 880 cm –1<br />
(exomethylene group). The 1 HNMR spectrum exhibited<br />
six tertiary methyl groups at δ 0.76, 0.78, 0.82, 0.91,<br />
0.94 and 1.02, a methane group at δ 1.66, a secondary<br />
carbinol group at δ 3.21 and an exomethylene group at<br />
δ 4.56 and 4.68 implies a typical pentacyclic triterpenoid<br />
of the lupeol. Based on the m.p., IR, 1 HNMR, 13 CNMR<br />
and mass spectral data the compound was identifi ed as<br />
lupeol. 16,17<br />
Compound IV obtained as pearl white crystals and it<br />
gave characteristic color reaction for sterol. The peak at<br />
1642.47 cm –1 in IR spectra indicated C=C streching, while<br />
the peaks at 1474.27 cm –1 and 1032.65 cm –1 indicated the<br />
C-H deformation in gem dimethyl and C-O streching of<br />
secondary alcohol respectively. The peak at 3425.25 cm –1<br />
indicated the presence of OH group. The proton NMR<br />
signal at δ 2.17 indicated OH at C–3 position. The signal<br />
at δ 5.36 gave singlet which indicated one vinylic proton<br />
at C-6 position, while the signal at δ 5.12 gave doublet<br />
which indicated two allylic protons at C–7 position. The<br />
13 CNMR spectrum revealed 28 signals which were duly<br />
assigned as CH 3 , CH 2 , CH and -C- group represent in the<br />
compound. The FAB-MS spectrum showed a molecular<br />
ion peak at m/z 400 corresponding to molecular formula<br />
C 28 H 48 O. The mass fragmentation was typical to that of<br />
campesterol. Based on the m.p., IR, 1 HNMR, 13 CNMR<br />
and mass spectral data the compound was identifi ed as<br />
campesterol.<br />
Compound V obtained as semisolid mass and gave<br />
characteristic color reaction for triterpenoids. The peak at<br />
1789.68 cm –1 in IR spectra indicated C=O streching. The<br />
peak at 882.12 cm –1 indicated =C-H out plane bending.<br />
The peak at 1649.64 cm –1 indicated C=C streching and<br />
peak at 1376.69 cm –1 indicated the C-H deformation<br />
in gem dimethyl. The peak at 3445.42 cm –1 indicated<br />
the presence of OH group. The proton NMR signals<br />
at δ 5.18 indicates vinylic protons and another singlet<br />
at δ 3.61 indicated OH at C-17 position. The 13 CNMR<br />
spectrum revealed 29 signals which were duly assigned<br />
as CH 3 , CH 2 , CH, -C- and -C=C- group represent in the<br />
compound. The FAB-MS spectrum showed a molecular<br />
ion peak at m/z 400 corresponding to molecular formula<br />
C 29 H 44 O 3 . The mass fragmentation was typical to that<br />
of β-amyrone skeleton. From the above evidences the<br />
compound was identifi ed as 17-Hydroxy-11-oxo-norβ-amyrone<br />
18 .<br />
Compound VI obtained as white crystals and gave a<br />
characteristic color reaction for triterpenoids. The FAB-<br />
MASS spectrum displayed the characteristic retro-Diels-<br />
Alder fragment peak at m/z 234 indicating a C-12 /<br />
C-13 double bond which suggested an ursane structure 19<br />
substituted by two hydroxyl groups (-OH), one located<br />
at A/B rings and one on D/E rings. These results<br />
are in good agreement with the IR spectrum, which<br />
showed absorption bands at 3455.84 cm –1 assigned to<br />
the hydroxyl groups and 1642.89 cm –1 corresponds to<br />
carbonyl group of the carboxylic acid. The 1 HNMR<br />
spectrum displayed seven methyl signals. In addition,<br />
the presence of the olefi nic triplet at δ 5.28 and two<br />
hydroxyl bearing methine protons exhibiting two double<br />
doublets at δ 3.38 and δ 3.61. On the basis of the<br />
1 HNMR spectrum of the compound, a β-confi guration<br />
of the two hydroxyl groups was assigned according to<br />
the fact that their respective geminal protons appeared<br />
as dd thus confi rming axial orientation. 20 Further the<br />
13 CNMR spectrum revealed 30 signals which were duly<br />
assigned as CH 3 , CH 2 , CH and -C- group represent in the<br />
compound. Therefore the structure of compound VI is<br />
identifi ed as Urs-12-ene-3β, 22β-diol-17-carboxylic acid,<br />
whose mass value obtained as 472 by FAB-MASS which<br />
corresponds to the C 30 H 48 O 4 ; the molecular formula of<br />
the compound.<br />
CONCLUSION<br />
From the spectral data, the compounds I-VI was analyzed<br />
as stigmasterol, ursolic acid, lupeol, campesterol,<br />
17-hydroxy-11-oxo-nor-β-amyrone and urs-12-ene-3β,<br />
22β-diol-17-carboxylic acid respectively. However, the<br />
compounds stigmasterol, campesterol, 17-hydroxy-11oxo-nor-β-amyrone<br />
and urs-12-ene-3β, 22β-diol-17carboxylic<br />
acid were reported for the fi rst time from the<br />
root of this plant.<br />
REFERENCES<br />
1. Kirtikar KR, Basu BD. Indian Medicinal Plants, Vol. II, Lalit Mohan Basu,<br />
Allahabad, 2003,1548–9.<br />
2. Chopra RN, Nayar SL, Chopra IC. Glossary of Indian Medicinal Plants,<br />
(CSIR, New Delhi), 1956;52.<br />
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3. Vohra MM, De NN. Comparative cardiotonic activity of Carissa carandas<br />
and Carissa spinarum. Indian J Med Res 1963;51:937–40.<br />
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5. Karunakar Hegde, Arun B Joshi. Hepatoprotective and antioxidant effect<br />
of Carissa spinarum root extract against CCl 4 and paracetamol induced<br />
hepatic damage in rats. Bangladesh J Pharmacol 2010;5:73–6.<br />
6. Karunakar Hegde, Cijo Issac, Arun B Joshi. Antiarthritic activity of<br />
Carissa spinarum root extract in Freund’s adjuvant induced polyarthritis<br />
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new germacrane sesquiterpene from Carissa spinarum. Nat Prod Res<br />
2005;19:763–9.<br />
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Screening of Indian plants for biological activity: Part III. Indian J Exptl<br />
Biol 1971;9:91–102.<br />
10. Rastogi RC, Kulshreshtha DK, Rastogi RP. Cardioactive constituents<br />
from Carissa spinarum Linn. Indian J Chem 1969;7:1102–4.<br />
11. Raina MK, Bhatnaga JK, Atal CK. Isolation of caffeic acid from the roots<br />
of Carissa spinarum Linn. The Indian J Pharm 1971;33:76–7.<br />
12. Mathuram V, Brahmadhayalaselvam A, Hussain AJ, Rao RB, Patra<br />
A. Chemical constituents of Carissa spinarum and their antibacterial<br />
activity. J Indian Chem Soc 1998;75:262–4.<br />
13. Pearlman WH, Emily C. The isolation of C 21 steroids from human<br />
placenta. J Biol Chem 1951;8:807–14.<br />
14. Sright JLC, McInnes AG, Shimizu S, et al. Identifi cation of C-24<br />
alkyl epimers of marine sterols by 13C nuclear magnetic resonance<br />
spectroscopy. Can J Chem 1978;56:1898–1903.<br />
15. Saeidnia S, Gohari AR, Uchiyama N, Ito M, Honda G, Kiuchi F. Tow<br />
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RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 59
Synthesis and antimicrobial activity of<br />
4-hydroxy-1-methyl/phenyl-3- (substituted<br />
anilinoacetyl) quinolin-2(1H)-one<br />
Girish Bolakatti 1 , Manjunatha S. Katagi* 1 , S.N. Mamledesai 2 , Sujatha. M.L. 3,<br />
Prakash Dabadi 1 , Narayana Miskin. 1<br />
1 Bapuji Pharmacy College, Davangere 577 004, Karnataka, India<br />
2 P.E.S’s College of Pharmacy education and Research, Ponda 403 401, Goa, India<br />
3 Bapuji Institute of Engineering and Technology, Davangere 577 004, Karnataka, India<br />
ABSTRACT<br />
A series of new 2-quinolone derivatives were synthesized, purifi ed and characterized on the basis of IR, 1H NMR, 13C NMR and Mass spectral studies. Same compounds were evaluated for antimicrobial activity against<br />
Staphylococcus aureus, Bacillus substilis, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and<br />
Asparagillus niger. Among eighteen synthesized novel compounds, in which fi ve compounds (3a, 3b, 3d, 4a, 4c)<br />
shows promising antibacterial activity as compared to Ciprofl oxacin (100 μg/ml), however most of them (3a, 3b,<br />
3d, 3e, 4a, 4b, 4d, 4e) showed potent antifungal activity as compared to Fluconazole (100 μg/ml).<br />
Key words: Mass spectral, 2-quinolone, antibacterial and antifungal<br />
INTRODUCTION<br />
Although considerable advances have been<br />
achieved over recent decades in the research<br />
and development of new structural prototypes<br />
as effective antimicrobials, current antimicrobial<br />
chemotherapy still suffers from<br />
two major limitations. The fi rst is the lack<br />
of selectivity of conventional antimicrobial<br />
agents, which in turn brings about unwanted<br />
side effects. The second is acquisitions by<br />
the microorganism of multi drug resistance.<br />
The design of new agents, active against<br />
resistant organism is of critical importance.<br />
In the fi eld of quinolone antimicrobial<br />
agents, the new generation of quinolone<br />
has achieved signifi cant improvements in<br />
terms of potency, spectrum and pharmacokinetic<br />
properties. But these agents faced<br />
a rapid increase of resistance from grampositive<br />
organisms. Therefore, enhancing<br />
the potency of quinolone especially against<br />
gram-positive organism has become most<br />
urgent. 1<br />
The resistance to antimicrobial drugs is<br />
wide spread, the development of new<br />
antimicrobial agents and understanding<br />
their mechanisms of action are becoming<br />
vital nowadays. Nitrogen containing heterocyclic<br />
compounds is an indispensable<br />
structural unit for both the chemist and<br />
the biochemist. Among the antimicrobial<br />
agents discovered in recent years the various<br />
2-quinolone as potent and selective antimicrobial<br />
agent has stimulated remarkable<br />
interest in the synthesis of 2-quinolones<br />
bearing heterocycles. 2 Numerous biological<br />
activities of 2-quinolone have been<br />
described; antimicrobial, 3–5 antioxidant and<br />
anti-infl ammatory, 6 antitumor, 7 Fornesyl<br />
transferase inhibitor 8 , antiangiogenic 9 and<br />
anti-tuberculosis. 10–13<br />
EXPERIMENTAL PROCEDURE<br />
All the chemicals and solvents were<br />
supplied by Merck, S.D Fine-Chem.<br />
Research Ar cle<br />
Received Date : 19-11-2011<br />
Revised Date : 02-03-2012<br />
Accepted Date : 05-03-2012<br />
DOI: 10.5530/rjps.2012.1.8<br />
Address for<br />
correspondence<br />
Manjunatha S. Katagi<br />
Bapuji Pharmacy College<br />
S.S. Layout<br />
Davangere-577 004<br />
Karnataka, India<br />
Phone: 08192-221459<br />
Mobile: +919886499160<br />
Fax: 08192-222561<br />
Email: manju_mpharm@<br />
rediffmail.com<br />
www.rjps.in<br />
60 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
Girish Bolakatti et al.: Synthesis and antimicrobial activity of 4-Hydroxy-1-methyl/phenyl-3- (Substituted anilinoacetyl) quinolin-2(1H)-one<br />
Limited, Mumbai. All the solvents were distilled before<br />
use and chemicals were purifi ed by either distillation or<br />
recrystallisation before use. The melting points were<br />
taken on the Veego (VMP–MP) melting point apparatus<br />
and are uncorrected. The IR spectra of the compounds<br />
were recorded using KBr on Jasco FTIR spectrometer<br />
(model-4100). The 1 H NMR and CHN elemental<br />
analysis of the synthesized compounds was recorded on<br />
Bruker avance II 400 NMR spectrometer (with TMS as<br />
internal references) and Perkin Elmer 2400 respectively<br />
at Sophisticated Analytical and Instrumentation Facility<br />
(SAIF), Punjab University (Chandigarh). Mass spectra<br />
were recorded on Shimadzu LC MS-2010A at Quest<br />
Research and Training Institute (Pvt) Ltd, Bangalore.<br />
4-Hydroxy-6-methyl/phenyl-2H-pyrano [3,2-c] quinoline-<br />
2,5(6H)-dione, were synthesized according to literature<br />
procedure and subjected to hydrolysis to yield 1a and<br />
1b, 14 further 1a and 1b subjected to bromination to yield<br />
3-Bromoacetyl-4-hydroxy-1-methyl/phenyl quinolin-<br />
2(1H)-one 2a and 2b. The synthesis of title compounds<br />
3a-h and 4a-h were accomplished by condensing with<br />
different substituted aniline. The compounds thus<br />
obtained were characterized by IR, 1 HNMR and mass<br />
spectral data. The physicochemical properties of titled<br />
compounds were given in Table 1 and Table 2.<br />
General procedure for synthesis of<br />
3-(Bromoacetyl)-4-hydroxy-1-methyl/<br />
phenylquinolin-2(1H )-one 2a, 2b<br />
A solution of 3-Acetyl-4-hydroxy-1-methyl/phenyl<br />
quinolin-2(1H )-one (0.065 mol) in glacial acetic acid<br />
(50 ml) was heated (80–100°)( follow the instruction<br />
provided on page no 5 of instruction to authors) with<br />
constant stirring, then bromine (0.065 mol) in glacial<br />
acetic acid (20 ml) was added drop wise over a period<br />
of 1 h and heated the solution until slight change in the<br />
color of the solution was seen. The reaction mixture was<br />
then cooled to room temperature. The product obtained<br />
as yellow crystals and recrystallized from ethanol.<br />
3-(Bromoacetyl)-4-hydroxy-1-methylquinolin-<br />
2(1H )-one 2a<br />
This was prepared and purifi ed as per the above<br />
mentioned procedure: yield 10.26 g (73%), mp 174–176<br />
°C, IR (KBr, v, cm –1 ): 756.14 cm –1 and 1190.18 (CH 2 -Br),<br />
1615.47 cm –1 (– C = O amide), 3046.24cm –1 (aromatic<br />
–C–H stre). 1 H NMR (400MHz, CDCl 3 ) δ (ppm): δ<br />
3.78 (s, 3H, N–CH 3 ), 4.98 (s, 2H, CH 2 –Br), 7.72–8.35<br />
(m, 4H, Ar–H), 15.82 (s, 1H, OH).<br />
3-(Bromoacetyl)-4-hydroxy-1-phenylquinolin-<br />
2(1H )-one 2b<br />
This was prepared and purifi ed as per the above<br />
mentioned procedure: yield 12.24 g (67.43%), mp<br />
192–194°, IR (KBr, v, cm –1 ): 786.11 cm –1 and 1210.06<br />
(CH 2 -Br), 1620.31 cm –1 (– C = O amide), 3058.11cm –1<br />
(aromatic –C – H stre). 1 H NMR (400MHz, CDCl 3 ) δ<br />
(ppm): δ 5.23 (s, 2H, CH 2 -Br), 7.65–8.85 (m, 9H, Ar–H),<br />
15.91 (s, 1H, OH).<br />
General procedure for synthesis of 4-Hydroxy-<br />
1-methyl/phenyl-3-(Substituted anilinoacetyl)<br />
quinolin-2(1H )-one 3a-h and 4a-h<br />
A solution of 3-(Bromoacetyl)-4-hydroxy-1-methyl/<br />
phenylquinolin-2(1H)-one 2a, 2b (0.004 mol) in acetone<br />
Table 1: Characterization data of 4-Hydroxy-1-methyl -3-(Substituted anilinoacetyl)-quinolin-2(1H)-one 3a-h<br />
Analysis( % )Found (Calcd) in % Yield<br />
Comp R Mol formula M.P. °C * Rf C H N<br />
3a -2,5-Dichloro C H Cl N O 18 14 2 2 3 210–212 0.56 57.31 (57.16) 3.74 (3.61) 7.43 (7.37)<br />
3b -2,4-Dichloro C H Cl N O 18 14 2 2 3 206–208 0.53 57.31 (57.19) 3.74 (3.64) 7.43 (7.39)<br />
3c -p-Nitro C H N O 18 15 3 5 198–200 0.47 61.19 (61.08) 4.28 (4.21) 11.89 (11.79)<br />
3d -p-Fluro C H FN O 18 15 2 3 204–206 0.41 66.25 (66.18) 4.63 (4.51) 8.58 (8.49)<br />
3e p-Chloro C H ClN O 18 15 2 3 211–213 0.42 63.07 (63.01) 4.41 (4.35) 8.17 (8.11)<br />
3f -3-Chloro-4-fl uro C H ClFN O 18 14 2 3 220–222 0.77 59.93 (59.85) 3.91 (3.86) 7.76 (7.71)<br />
3g -2,4-Dimethyl C H N O 20 20 2 3 225–227 0.51 71.41 (71.37) 5.99 (5.91) 8.33 (8.24)<br />
3h -3,4-Dimethyl C H N O 20 20 2 3 222–224 0.53 71.41 (71.35) 5.99 (5.92) 8.33 (8.25)<br />
*TLC Solvent system: Chloroform: Methanol: Strong ammonia-(10:5:3) .<br />
OH O<br />
N<br />
CH 3<br />
3a-h<br />
O<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 61<br />
NH<br />
R
Girish Bolakatti et al.: Synthesis and antimicrobial activity of 4-Hydroxy-1-methyl/phenyl-3- (Substituted anilinoacetyl) quinolin-2(1H)-one<br />
Table 2: Characterization data of 4-Hydroxy-1-phenyl -3-(Substituted anilinoacetyl) quinolin-2(1H)-one 4a-h<br />
(50 ml) was cooled at 0º, primary amine (0.004 mol)<br />
dissolved in acetone (25 ml) was added with stirring<br />
at 0–5° followed by drop wise addition of sodium<br />
hydroxide (0.004 mol) in water (25 ml). Contents were<br />
stirred for 3 hour and poured in to ice water acidifi ed<br />
with dil. HCl, fi ltered, washed, dried and purifi ed by<br />
crystallization from a ethanol + water (9:1).<br />
3-{2-[(2,5-dichlorophenyl)amino]acetyl}-4hydroxy-1-methylquinolin-2(1H)-one<br />
3a<br />
This was prepared and purified as per the above<br />
mentioned procedure: yield 0.75 g (49.73%); IR<br />
(KBr, v, cm –1 ): 1617.61 cm –1 (– C = O amide), 3156.20<br />
cm –1 (– CH stre), 3266.33 cm –1 (– NH), 3363.09 cm –1<br />
(– OH); 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm):<br />
2.85 (s, 3H, N- CH 3 ), 3.87 (s, 1H, NH), 4.81 (s, 2H,<br />
COCH 2 ), 6.45–7.5 (m, 7H, Ar–H), 15.24 (s, 1H,<br />
OH); 13 C NMR (400MHz, DMSO-d 6 ) δ (ppm): 31.5<br />
(N-CH 3 ), 59.8 (CH 2 ), 108.01 115.12, 115.65, 119.32,<br />
121.23, 121.93, 124.99, 126.11, 128.84, 132.36,<br />
134.24, 135.85, 145.40 (Aromatic carbons), 164.53<br />
(exocyclic ketone), 166.32 (Hydroxyl carbon), 196.85<br />
(Carbonyl carbon); LCMS: C 18 H 14 Cl 2 N 2 O 3 (M + ) m/z<br />
377.29; calcd. 377.22.<br />
Analysis( % )Found (Calcd) in % Yield<br />
Comp R Mol formula M.P. 0C * Rf C H N<br />
4a -2,5-Dichloro C H Cl N O 23 16 2 2 3 240–242 0.58 62.88 (62.81) 3.67 (3.60) 6.38 (6.32)<br />
4b -2,4-Dichloro C H Cl N O 23 16 2 2 3 236–238 0.55 62.88 (62.83) 3.67 (3.62) 6.38 (6.34)<br />
4c -p-Nitro C 23 H 17 N 3 O 5 245–247 0.45 66.50 (66.41) 4.12 (4.01) 10.12 (10.01)<br />
4d -p-Fluro C 23 H 17 FN 2 O 3 251–253 0.43 71.13 (71.02) 4.41 (4.32) 7.21 (7.16)<br />
4e p-Chloro C 23 H 17 ClN 2 O 3 211–213 0.45 68.23 (68.17) 4.23 (4.14) 6.92 (6.87)<br />
4f -3-Chloro-4-fl uro C 23 H 16 ClFN 2 O 3 208–210 0.73 65.33 (65.23) 3.81 (3.74) 6.63 (6.52)<br />
4g -2,4-Dimethyl C H N O 25 22 2 3 246–248 0.47 75.36 (75.23) 5.57 (5.48) 7.03 (6.97)<br />
4h -3,4-Dimethyl C H N O 25 22 2 3 251–253 0.49 75.36 (75.28) 5.57 (5.45) 7.03 (6.98)<br />
* TLC Solvent system: Chloroform: Methanol: Strong ammonia-(10:5:3).<br />
OH O<br />
N<br />
3-{2-[(2,4-dichlorophenyl)amino]acetyl}-4hydroxy-1-methylquinolin-2(1H)-one<br />
3b<br />
This was prepared and purifi ed as per the above<br />
mentioned procedure: yield 0.71 g (47.08%); IR (KBr,<br />
v, cm –1 ): 1620.54 cm –1 (– C = O amide), 3158.51 cm –1<br />
(– CH stre), 3271.31 cm –1 (– NH), 3359.05 cm –1 (– OH);<br />
1 H NMR (400MHz, DMSO-d6 ) δ (ppm): 2.82 (s, 3H,<br />
N–CH 3 ), 3.85 (s, 1H, NH), 4.75 (s, 2H, COCH 2 ), 6.35–<br />
7.58 (m, 7H, Ar–H), 15.04 (s, 1H, OH).<br />
3-[2-(4-Nitrophenylamino)acetyl]-4-hydroxy-<br />
1-methylquinolin-2(1H)-one 3c<br />
This was prepared and purifi ed as per the above<br />
mentioned procedure: yield 0.86 g (60.90%); IR (KBr,<br />
v, cm –1 ): 1623.11 cm –1 (– C = O amide), 3186.22 cm –1<br />
(– CH stre), 3252.38 cm –1 (– NH), 3345.23 cm –1 (– OH);<br />
1 H NMR (400MHz, DMSO-d6 ) δ (ppm): 2.91 (s, 3H, N-<br />
CH 3 ), 4.06 (s, 1H, NH), 4.96 (s, 2H, COCH 2 ), 6.86–7.92<br />
(m, 8H, Ar–H), 14.52 (s, 1H, OH); 13 C NMR (400MHz,<br />
DMSO-d 6 ) δ (ppm): 32.5 (N-CH 3 ), 60.2 (CH 2 ), 107.25,<br />
114.16, 115.21, 121.76, 121.99, 124.65, 126.11, 128.84,<br />
135.05, 136.56, 153.65 (Aromatic carbons), 164.36<br />
(exocyclic ketone), 166.32 (Hydroxyl carbon), 196.12<br />
62 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012<br />
O<br />
NH<br />
4a-h<br />
R
Girish Bolakatti et al.: Synthesis and antimicrobial activity of 4-Hydroxy-1-methyl/phenyl-3- (Substituted anilinoacetyl) quinolin-2(1H)-one<br />
(Carbonyl carbon) ; LCMS: C 18 H 15 N 3 O 5 (M + ) m/z<br />
353.33; calcd. 353.32.<br />
3-[2-(4-Fluorophenylamino)acetyl]-4-hydroxy-<br />
1-methylquinolin-2(1H)-one 3d.<br />
This was prepared and purifi ed as per the above<br />
mentioned procedure: yield 0.56 g (42.94%); IR (KBr,<br />
v, cm –1 ): 1613.16 cm –1 (– C = O amide), 3180.01 cm –1<br />
(– CH stre), 3245.02 cm –1 (– NH), 3305.03 cm –1 (– OH);<br />
1 H NMR (400MHz, DMSO-d6 ) δ (ppm): 2.75 (s, 3H, N-<br />
CH 3 ), 3.89 (s, 1H, NH), 4.53 (s, 2H, COCH 2 ), 6.45–7.53<br />
(m, 8H, Ar–H), 14.96 (s, 1H, OH).<br />
3-[2-(4-Chlorophenylamino)acetyl]-4-hydroxy-<br />
1-methylquinolin-2(1H)-one 3e<br />
This was prepared and purifi ed as per the above<br />
mentioned procedure: yield 0.68 g (49.63%); IR (KBr,<br />
v, cm –1 ): 1613.51 cm –1 (– C = O amide), 3123.26 cm –1<br />
(– CH stre), 3243.36 cm –1 (– NH), 3345.12 cm –1 (– OH);<br />
1 H NMR (400MHz, DMSO-d6 ) δ (ppm): 2.63 (s, 3H, N-<br />
CH 3 ), 3.91 (s, 1H, NH), 4.56 (s, 2H, COCH 2 ), 6.49–7.62<br />
(m, 8H, Ar–H), 14.99 (s, 1H, OH).<br />
3-[2-(3-Chloro-4-fl uorophenylamino)acetyl]-<br />
4-hydroxy-1-methylquinolin-2(1H)-one 3f<br />
This was prepared and purifi ed as per the above<br />
mentioned procedure: yield 0.78 g (54.09%); IR (KBr,<br />
v, cm –1 ): 1613.05 cm –1 (– C = O amide), 3123.12 cm –1<br />
(– CH stre), 3243.56 cm –1 (– NH), 3355.10 cm –1 (– OH);<br />
1 H NMR (400MHz, DMSO-d6 ) δ (ppm): 2.83 (s, 3H, N-<br />
CH 3 ), 3.99 (s, 1H, NH), 4.63 (s, 2H, COCH 2 ), 6.49–7.51<br />
(m, 7H, Ar–H), 14.82 (s, 1H, OH).<br />
3-[2-(2,4-dimethylphenylamino)acetyl]-4-hydroxy-<br />
1-methylquinolin-2(1H)-one 3g<br />
This was prepared and purifi ed as per the above<br />
mentioned procedure: yield 0.81 g (60.26%); IR (KBr, v,<br />
cm –1 ): 1632.05 cm –1 (– C = O amide), 3178.28 cm –1 (– CH<br />
stre), 3288.88 cm –1 (– NH), 3355.51 cm –1 (– OH); 1 H NMR<br />
(400MHz, DMSO-d 6 ) δ (ppm): 2.32 (s, 6H, CH 3 ), 2.42 (s,<br />
3H, N- CH 3 ), 3.75 (s, 1H, NH), 4.85 (s, 2H, COCH 2 ),<br />
6.38–7.53 (m, 7H, Ar–H), 15.23 (s, 1H, OH); 13 C NMR<br />
(400MHz, DMSO-d 6 ) δ (ppm): 21.9, 24.6 (2 CH 3 ), 31.5<br />
(N–CH 3 ), 60.2 (CH 2 ), 107.25, 113.16, 115.21, 121.5,<br />
121.76, 124.99, 126.76, 126.96, 128.84, 131.75, 135.05,<br />
143.65 (Aromatic carbons), 164.10 (exocyclic ketone),<br />
166.82 (Hydroxyl carbon), 196.65 (Carbonyl carbon) ;<br />
LCMS: C 20 H 20 N 2 O 3 (M + ) m/z 336.32; calcd. 336.38.<br />
3-[2-(3,4-dimethylphenylamino)acetyl]-4-hydroxy-<br />
1-methylquinolin-2(1H)-one 3h<br />
This was prepared and purifi ed as per the above<br />
mentioned procedure: yield 0.96 g (71.42%); IR (KBr,<br />
v, cm –1 ): 1632.00 cm –1 (– C = O amide), 3178.28 cm –1<br />
(– CH stre), 3288.88 cm –1 (– NH), 3355.51 cm –1 (– OH);<br />
1 H NMR (400MHz, DMSO-d6 ) δ (ppm): 2.15 (s, 6H,<br />
CH 3 ), 2.42 (s, 3H, N- CH 3 ), 3.75 (s, 1H, NH), 4.85 (s, 2H,<br />
COCH 2 ), 6.38–7.53 (m, 7H, Ar–H), 15.23 (s, 1H, OH).<br />
3-{2-[(2,5-dichlorophenyl)amino]acetyl}-4hydroxy-1-phenylquinolin-2(1H)-one<br />
4a<br />
This was prepared and purifi ed as per the above<br />
mentioned procedure: yield 0.95 g (54.10%); IR (KBr, v,<br />
cm –1 ): 1625.61 cm –1 (– C = O amide), 3166.00 cm –1 (– CH<br />
stre), 3260.83 cm –1 (– NH), 3392.08 cm –1 (– OH); 1 H<br />
NMR (400MHz, DMSO-d 6 ) δ (ppm): 3.89 (s, 1H, NH),<br />
4.76 (s, 2H, COCH 2 ), 6.60–7.64 (m, 12H, Ar–H), 15.20<br />
(s, 1H, OH); 13 C NMR (400MHz, DMSO-d 6 ) δ (ppm):<br />
59.91 (CH 2 ), 107.5, 113.01, 115.22, 118.21, 118.32,<br />
118.93, 119.21, 119.31, 120.23, 127.84, 128.36, 129.7,<br />
131.1, 133.25, 137.2, 140.7, 145.4 (Aromatic carbons),<br />
159.53 (exocyclic ketone), 166.30 (Hydroxyl carbon),<br />
196.85 (Carbonyl carbon) ; LCMS: C 23 H 16 Cl 2 N 2 O 3 (M + )<br />
m/z 439.23; calcd. 439.29.<br />
3-{2-[(2,4-dichlorophenyl)amino]acetyl}-<br />
4-hydroxy-1-phenylquinolin-2(1H)-one 4b<br />
This was prepared and purifi ed as per the above<br />
mentioned procedure: yield 0.92 g (52.39%); IR (KBr,<br />
v, cm –1 ): 1620.54 cm –1 (– C = O amide), 3158.51 cm –1<br />
(– CH stre), 3271.31 cm –1 (– NH), 3359.05 cm –1 (– OH);<br />
1 H NMR (400MHz, DMSO-d6 ) δ (ppm): 3.93 (s, 1H,<br />
NH), 4.81 (s, 2H, COCH 2 ), 6.63–7.69 (m, 12H, Ar–H),<br />
15.05 (s, 1H, OH).<br />
3-[2-(4-Nitrophenylamino)acetyl]-4-hydroxy-<br />
1-phenylquinolin-2(1H)-one 4c<br />
This was prepared and purifi ed as per the above<br />
mentioned procedure: yield 1.06 g (63.85%); IR (KBr,<br />
v, cm –1 ): 1628.65 cm –1 (– C = O amide), 3145.06 cm –1<br />
(– CH stre), 3245.38 cm –1 (– NH), 3354.23 cm –1 (– OH);<br />
1 H NMR (400MHz, DMSO-d6 ) δ (ppm): 4.06 (s, 1H,<br />
NH), 4.82 (s, 2H, COCH 2 ), 6.86–7.92 (m, 13H, Ar–H),<br />
14.82 (s, 1H, OH).<br />
3-[2-(4-Fluorophenylamino)acetyl]-4-hydroxy-<br />
1-phenylquinolin-2(1H)-one 4d<br />
This was prepared and purifi ed as per the above<br />
mentioned procedure: yield 0.49 g (31.57%); IR (KBr,<br />
v, cm –1 ): 1619.13 cm –1 (– C = O amide), 3188.05 cm –1<br />
(– CH stre), 3251.02 cm –1 (– NH), 3389.03 cm –1 (– OH);<br />
1 H NMR (400MHz, DMSO-d6 ) δ (ppm): 4.15 (s, 1H,<br />
NH), 4.91 (s, 2H, COCH 2 ), 7.12–8.13 (m, 13H, Ar–H),<br />
14.92 (s, 1H, OH).<br />
3-[2-(4-Chlorophenylamino)acetyl]-4-hydroxy-<br />
1-phenylquinolin-2(1H)-one 4e.<br />
This was prepared and purifi ed as per the above<br />
mentioned procedure: yield 0.56 g (34.61%); IR<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 63
Girish Bolakatti et al.: Synthesis and antimicrobial activity of 4-Hydroxy-1-methyl/phenyl-3- (Substituted anilinoacetyl) quinolin-2(1H)-one<br />
(KBr, v, cm –1 ): 1619.51 cm –1 (– C = O amide), 3187.26<br />
cm –1 (– CH stre), 3256.31 cm –1 (– NH), 3385.18 cm –1<br />
(– OH); 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 4.11<br />
(s, 1H, NH), 4.67 (s, 2H, COCH 2 ), 7.05–8.11 (m, 13H,<br />
Ar–H), 14.91 (s, 1H, OH).<br />
3-[2-(3-Chloro-4-fl uorophenylamino)acetyl]-<br />
4-hydroxy-1-phenylquinolin-2(1H)-one 4f<br />
This was prepared and purifi ed as per the above<br />
mentioned procedure: yield 0.88 g (52.07%); IR (KBr,<br />
v, cm –1 ): 1610.05 cm –1 (– C = O amide), 3142.02 cm –1<br />
(– CH stre), 3251.16 cm –1 (– NH), 3420.15 cm –1 (– OH);<br />
1 H NMR (400MHz, DMSO-d6 ) δ (ppm): 3.85 (s, 1H,<br />
NH), 4.68 (s, 2H, COCH 2 ), 6.84–8.11 (m, 12H, Ar–H),<br />
13.92 (s, 1H, OH).<br />
3-[2-(2,4-dimethylphenylamino)acetyl]-<br />
4-hydroxy-1-phenylquinolin-2(1H)-one 4g<br />
This was prepared and purifi ed as per the above<br />
mentioned procedure: yield 0.83 g (52.13%); IR (KBr,<br />
v, cm –1 ): 1653.12 cm –1 (– C = O amide), 3251.06 cm –1<br />
(– CH stre), 3296.80 cm –1 (– NH), 3395.22 cm –1 (– OH).<br />
3-[2-(3,4-dimethylphenylamino)acetyl]-<br />
4-hydroxy-1-phenylquinolin-2(1H)-one 4h<br />
This was prepared and purifi ed as per the above<br />
mentioned procedure: yield 0.96 g (60.30%); IR (KBr,<br />
v, cm –1 ): 1612.00 cm –1 (– C = O amide), 3178.33 cm –1<br />
(– CH stre), 3269.28 cm –1 (– NH), 3358.01 cm –1 (– OH);<br />
1 H NMR (400MHz, DMSO-d6 ) δ (ppm): 2.13 (s, 6H,<br />
CH 3 ), 3.78 (s, 1H, NH), 4.96 (s, 2H, COCH 2 ), 6.98–8.35<br />
(m, 12H, Ar–H), 14.01 (s, 1H, OH).<br />
ANTIMICROBIAL SCREENING15–18 All the synthesized compounds 3a-h and 4a-h have<br />
been screened in vitro for their antibacterial activity<br />
against gram-negative bacteria Escherichia coli (ATCC<br />
10536), Pseudomonas aeruginosa (ATCC 10145) and grampositive<br />
bacteria Staphylococcus aureus (ATCC11632),<br />
Bacillus substilis (ATCC 60511), while antifungal activity<br />
against Candida albicans (ATCC 2501) and Asparagillus<br />
niger (ATCC 1781) at 100 μg/ml concentration by cupplate<br />
agar diffusion method using dimethylsulfoxide<br />
as solvent. After 24 and 48h of incubation at 37° ±1,<br />
the antibacterial and antifungal activity respectively<br />
was determined by measuring the zones of inhibition<br />
in mm. Standard antibacterial ciprofl oxacin and<br />
fungicide fl uconazole were used under similar condition<br />
for comparison. Control test with solvent were<br />
performed for every assay but showed no inhibition<br />
of microbial growth. The observed zone of inhibition<br />
for antibacterial and antifungal activity is presented in<br />
Table 3<br />
Table 3: In vitro antimicrobial activities of the 2-quinolone<br />
derivatives at a concentration of 100 µg/ml (zone of<br />
inhibition in mm)<br />
Zone of inhibition<br />
Compounds Sa Bs Ec Pa Ca An<br />
3a 19 22 29 28 28 33<br />
3b 18 23 28 26 30 34<br />
3c 10 12 13 NA 16 18<br />
3d 20 23 25 27 31 34<br />
3e 09 07 10 08 27 33<br />
3f 07 08 NA 10 15 16<br />
3g 08 10 13 15 17 12<br />
3h 11 13 NA 17 13 19<br />
4a 19 24 31 29 28 31<br />
4b 11 12 11 13 27 30<br />
4c 21 25 32 30 11 16<br />
4d 12 16 19 21 29 28<br />
4e 09 07 10 11 32 34<br />
4f 12 11 13 NA 13 15<br />
4g 09 07 10 11 NA NA<br />
4h 08 NA NA NA 10 NA<br />
Ciprofl oxacin 23 26 34 31 ND ND<br />
Fluconazole ND ND ND ND 33 36<br />
Sa = Staphylococcus aureus, Bs = Bacillus substilis, Ec = Escherichia coli,<br />
Pa = Pseudomonas aeruginosa, Ca = Candida albicans, An = Asparagillus niger, NA = No<br />
activity, ND = Not determined.<br />
RESULTS AND DISCUSSION<br />
4-Hydroxy-6-methyl/phenyl-2H-pyrano[3,2-c]<br />
quinoline-2,5(6H)-dione, were synthesized according to<br />
literature procedure14 and subjected to hydrolysis to yield<br />
1a and 1b, further 1a and 1b subjected to bromination<br />
to yield 3-Bromoacetyl-4-hydroxy-1-methyl/phenyl<br />
quinolin-2(1H)-one 2a and 2b. The synthesis of title<br />
compounds 3a-h and 4a-h were accomplished by<br />
condensing with different substituted aniline.<br />
The structures of newly synthesized compounds were<br />
confi rmed by their spectral data. The characteristic<br />
IR absorption peaks 1617.61 cm –1 was due to amide<br />
carbonyl, 3156.20 cm –1 mainly because aromatic –C – H<br />
stretching, 3266.33 cm –1 may be due to free amine group<br />
of acetamido and 3363.09 cm –1 of hydroxyl group<br />
indicates the completion of the reaction. Their structure<br />
was further supported by their 1H NMR spectral data<br />
that exhibited three proton of N-methyl signal found<br />
at δ value 2.85, where as aromatic proton signal shows<br />
the δ value 6.45–7.5. Subsequent purifi cation yielded<br />
fi nal compounds in moderate to higher yields. Physical<br />
data of the synthesized compounds are listed in Table<br />
1 and Table 2. Some of these compounds have shown<br />
antibacterial and potent antifungal agents.<br />
64 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
CONCLUSION<br />
Girish Bolakatti et al.: Synthesis and antimicrobial activity of 4-Hydroxy-1-methyl/phenyl-3- (Substituted anilinoacetyl) quinolin-2(1H)-one<br />
4-Hydroxy-1-methyl/phenyl-3-(Substituted<br />
anilinoacetyl)quinolin-2(1H)-one compounds were<br />
synthesized and evaluated for antimicrobial activity<br />
against Staphylococcus aureus, Bacillus substilis Escherichia coli,<br />
Pseudomonas aeruginosa, Candida albicans and Asparagillus<br />
niger. Among synthesized novel compounds 3a, 3b, 3d,<br />
4a, 4c shows promising antibacterial activity as compared<br />
to Ciprofl oxacin (100 μg/ml), however most of them<br />
3a, 3b, 3d, 3e, 4a, 4b, 4d, 4e showed potent antifungal<br />
activity as compared to Fluconazole (100 μg/ml).<br />
Suitable molecular modifi cation of these compounds<br />
may generate potent antimicrobial agents in future.<br />
REFERENCE<br />
1. Alireza F, Saeed E, Masood M, Mohammad HM, Abbas S. Synthesis<br />
and antibacterial activity of N-[2-(5-bromothiophen-2-yl)-2-oxoethyl] and<br />
N-[2-(5-bromothiophen-2-yl)-2-oximinoethyl] derivatives of piperazinyl<br />
quinolones. Bioorg Med Chem Lett 2005; 15: 4536–9.<br />
2. Thomas K. The pyrano route to 4-hydroxy-2-quinolone and 4-hydroxy-<br />
2-pyridones. IL farmaco 1999; 54:309–15.<br />
3. Jayashree BS, Seeja T, Yogendra N. Design and synthesis of<br />
2-quinolones as antioxidants and antimicrobials: a rational approach.<br />
Med Chem Res 2010;19:193–209.<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 65
Girish Bolakatti et al.: Synthesis and antimicrobial activity of 4-Hydroxy-1-methyl/phenyl-3- (Substituted anilinoacetyl) quinolin-2(1H)-one<br />
4. El-Dine SA, El-Khawass SM. Synthesis of 4-[(2-alkylaryl or arylalkyl)<br />
amino-1,3,4-thiadiazol-5-yl]-2-synthesized quinolines and compounds<br />
reasonable antifungal and antimicrobial activity. Pharmazie 1979;<br />
34:537–8.<br />
5. Joseph P, Charles B, Gladys D, Trevor V. Refomatsky reactions<br />
with N-arylpyrrolidine-2-thiones: Synthesis of tricyclic analogues of<br />
quinolone antibacterial agents. Tetrahedron 2001;57:9635–48.<br />
6. Anastasia D, Dionysia B, Kyriakos CP, Maria K, Giorgos A, Georgia<br />
M. Design and synthesis of novel quinolinone-3-aminoamides and<br />
their α-lipoic acid adducts as antioxidant and anti-infl ammatory agents.<br />
J Med Chem 2007;50:2450–8.<br />
7. Joseph S, Francis D. Aryl-2-quinolone derivatives having in vivo and<br />
in vitro antitumour activity. J Med Chem 2002;45:2543.<br />
8. Qun Li, Keith WW, Weibo W, et al. Design, synthesis, and activity of<br />
achiral analogs of 2-quinolones and indoles as non-thiol farnesyltransferase<br />
inhibitors. Bioorg Med Chem Lett 2005;15:2033–9.<br />
9. Gisela CM, Mariela B, Ana MB, Silvia EA. Evaluation of antiparasitic,<br />
antituberculosis and antiangiogenic activities of 3-aminoquinolin-2-one<br />
derivatives. J Chil Chem Soc 2006; 51:859–63.<br />
10. Anquetin G, Greiner J, Mahmoudi N, et al. Design, synthesis and activity<br />
against Toxoplasma gondii, Plasmodium spp., and Mycobacterium<br />
tuberculosis of new 6-fl uoroquinolones. Eur J Med Chem 2006;41:<br />
1478–93.<br />
11. Alexandra A, Veziris N, Cambau E, Truffot-Pernot C, Jarlier V, Fisher LM.<br />
Novel Gyrase Mutations in Quinolone-Resistant and Hyper susceptible<br />
Clinical Isolates of Mycobacterium tuberculosis: Functional Analysis of<br />
Mutant Enzymes. Antimicrob Agents Chemother 2006;50:04–112.<br />
12. Cheng AF, Yew WW, Chan EW, Chin ML, Hui MM, Chan RC. Multiplex<br />
PCR amplimer conformation analysis for rapid detection of gyrA<br />
mutations in fl uoroquinolone-resistant Mycobacterium tuberculosis<br />
clinical isolates. Antimicrob Agents Chemother 2004; 48:596.<br />
13. Ginsburg AS, Grosset JH, Bishai WR. Fluoroquinolones, tuberculosis,<br />
and resistance. Lancet Infect Dis 2003;3:432–42.<br />
14. Roschger P, Fiala W, Sradlabauer W. Nucleophilic substitution and<br />
ringclosure reactions of 4-chloro-3-nitro-2-quinolones. J Heterocycl<br />
Chem 1992;29:225–31.<br />
15. Fairbrother RW, Martyn G. The Disc Technique for Determining<br />
Sensitivity to the Antibiotics. J Clin Pathol 1951;4:374–77.<br />
16. Gould JC, Bowie JH. The determination of bacterial sensitivity to<br />
antibiotics. Edinb med J 1952;59:178–99.<br />
17. Handan A, Oznur A, Seher B, Gulten O, Melten U, Dilek S. Synthesis<br />
of mannich bases of some 2,5-disubstituted-4-thiazolidinones and<br />
evaluation of their antimicrobial activities. Turk J Chem 2005;29:425–35.<br />
18. Wolfson J, Hooper DC. Fluoroquinolone Antimicrobial agents. Clini<br />
Microbiol Rev 1989;2(4): 378–24.<br />
66 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
Evaluation of gastroprotective ability of<br />
Amorphophallus paeoniifolius corms against<br />
indomethacin induced gastric ulcers<br />
H.N. Nataraj 1 , R.L.N. Murthy 1 , Ramachandra Setty 2<br />
1 Dept. of Pharmacognosy, T.V.M. College of Pharmacy, Bellary<br />
2 Dept. of Pharmacology, Govt. College of Pharmacy, Bangalore<br />
ABSTRACT:<br />
Peptic ulcer is regarded as a multifactorial gastrointestinal disorder in its pathophysiology including free radical<br />
generations. Thereby free radical scavengers can play an important role in such diseases. The methanolic extract<br />
of Corms of Amorphophallus paeoniifolius exhibited remarkable anti-oxidant activity in various In-vitro models.<br />
Further, preliminary phytochemical screening revealed the presence of polyphenolic compounds; fl avonoids and<br />
tannins which are known to possess anti-ulcer activity. In light of these fi nding, it was under taken to investigate the<br />
gastroprotective activity of methanolic extract gainst NSAID–Indomethacin (30 mg/kg p.o.) induced gastotoxicity<br />
in Wistar albino rats wherein the animals were orally administered with two different doses of test extract<br />
(250 and 500 mg/kg b.w.) or with reference drug Lansoprozole (8 mg/kg p.o.). Animals were analyzed for Ulcer<br />
score, and in vitro estimation of GSH and LPO. Extract showed signifi cant (p
H.N Nataraj et al.: Evaluation of gastroprotective ability of Amorphophallus paeoniifolius corms against indomethacin induced gastric ulcers<br />
antisecretory ctivity can prove effectively in PUD<br />
(Peptic Ulcer Disease). Acid neutralization is being recognized<br />
as effective treatment for many centuries ago,<br />
but with the understanding of pathogeneses of PUD<br />
the treatment has become more effective. A number<br />
of antiulcer drugs by various mechanisms like gastric<br />
anti-secretory drugs, H 2 -receptors antagonists, antimuscarine<br />
drugs, proton pomp inhibitors and mucosal protective<br />
agents are in most common usage as a remedy<br />
for peptic ulcer.<br />
In ancient system of medicine, herbal preparations are<br />
being used for treating duodenal ulcers. Presently, a large<br />
section of the world’s population relies on traditional<br />
and medicinal herbs due to their less cost and easy<br />
access. 3 In the Indian Pharmaceutical Industry, antacids<br />
and antiulcer share 6.2 million rupees and occupy 4.3%<br />
of the market share. 4 Some of the phytoconstituents<br />
known to possess antiulcer activity include fl avonoids,<br />
saponins, tannins, gum and mucilages.<br />
Amophophallus paeoniifolius (Dennst.) Nicolson (Family:<br />
Araceae) is a perennial herb with underground tuber<br />
commonly known as ‘Suran’ in Hindi. The plant is widely<br />
distributed in India, Bangladesh, and Africa. 5–6 These<br />
tuberous roots of the plant have been used traditionally<br />
for the treatment of piles, abdominal pain, tumours,<br />
enlargement of spleen, asthma and rheumatism. 5,6 The<br />
tuberous roots have been reported to possess tonic,<br />
stomachic and appetizer properties. 6,7<br />
It was revealed that the corms are reported in the<br />
management of Haemorroids, 8 Obesity; 9 evaluated<br />
for Antiprotease, 10 Analgesic, 11 Immunomodulatory, 12<br />
Anthelmintic, 13 Hepatoprotective 14 and Cytotoxic<br />
activity. 15<br />
Previously we have reported that the methanolic extract<br />
of corms Amophophallus paeoniifolius (MECAP) found to<br />
be containing appreciable quantities of phenolics and<br />
fl avonoids as upon their quantitative estimations, 16 based<br />
on which the possible free radical scavenging potential<br />
by various models of MECAP was anticipated. 17<br />
The present investigation was aimed to evaluate the<br />
gastroprotective potential of Amophophallus paeoniifolius<br />
corms against indomethacin-induced gastric ulcer in<br />
albino rats in pursuit of newer gastro protectant.<br />
MATERIALS AND METHODS<br />
Plant material and preparation of extract<br />
The corms of Amorphophallas paeoniifolius were collected<br />
from cultivated lands from Hassan district of Karnataka<br />
and authenticated by Dr. Kotresh Botany department,<br />
Karnataka University, Dharwad. The voucher specimens<br />
of these plants and tubers were preserved in the<br />
herbarium of the pharmacognosy department of this<br />
institution.<br />
The air dried powdered plant material-corm was<br />
extracted with methanol in soxhelt extraction apparatus<br />
as methanol is one of best solvents for phenolics and<br />
it gave more quantitative colour reaction for phenolics<br />
than hydroalcoholic extract. The methanolic extract was<br />
fi ltered and evaporated to dryness in vacuum (at 35ºC &<br />
0.8Mpa) in a Buchi evaporator, R-114. The dry extract<br />
(MECAP-Methanolic Extract Corms of Amorphophallas<br />
paeoniifolius) was kept in vacuum desiccators until use<br />
and the preliminary phytochemical analysis revealed the<br />
presence of mainly polyphenols, tannins, fl avonoids,<br />
coumarins and triterpenoids.<br />
Animals<br />
Healthy adult Wistar albino rats weighing 200–250 g<br />
were used for the present investigation. They were<br />
housed in clean polyacrylic cages with not more than<br />
four animals per cage and maintained under standard<br />
laboratory conditions (temperature 25 ± 2°C, relative<br />
humidity 55–65%, with dark/light cycle 12/12 h).<br />
They were allowed free access to standard pellet diet<br />
and water ad libitum. The animals were acclimated to<br />
laboratory condition for one week prior to experiment.<br />
All the procedures were performed in accordance with<br />
the institutional Animal Ethics Committee (IAEC)<br />
constituted as per the direction of the Committee<br />
for the Purpose of Control and Supervision of<br />
Experiments of Animals (CPCSEA), under Ministry<br />
of Animal Welfare Division, Govt. of India,<br />
New Delhi. The experiment protocol was approved<br />
by IAEC S.C.S. College of Pharmacy, Harapanahalli,<br />
Karnataka (SCSCP/753/2009-10/Sl No.15).<br />
EXPERIMENTAL<br />
Acute Oral toxicity Studies and selection of dose<br />
Fixed dose method of Organization of Economic<br />
Co-operation and Development (OECD) Guideline<br />
No.423 (Annex 2d) given by CPCSEA18 was adopted for<br />
toxicity studies and it was concluded that the extract was<br />
unclassifi ed w.r.t. Globally Hormonized Classifi cation<br />
system and their LD was infi nite i.e. more than<br />
50<br />
5000 mg/kg. Hence, the doses 500 mg/kg and 250 mg/<br />
kg (1/10th & 1/20th of 5000 mg/kg) were selected for<br />
pharmacological screening of the MECAP.<br />
Antiulcer Activity Studies<br />
Antiulcer activity was evaluated in indomethacin<br />
induced gastric ulcer model according to the method<br />
described by Datta GK et al. 19 In this method, animals<br />
were divided into four groups (each group contains<br />
six rats) and animals were fasted for 36 h prior to the<br />
experiment, but allowed free access to water.<br />
68 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
H.N Nataraj et al.: Evaluation of gastroprotective ability of Amorphophallus paeoniifolius corms against indomethacin induced gastric ulcers<br />
Grouping and Treatment of Animals<br />
The different groups were treated as follows;<br />
Group-I : + ve control water, 10 ml/kg,<br />
P.O. + Indomethacin, 30 mg/kg, P.O.<br />
Group-II : Standard Lansoprazole, 8 mg/kg,<br />
P.O. + Indomethacin, 30 mg/kg, P.O.<br />
Group-III : Methanolic extract 250 mg/kg,<br />
P.O. + Indomethacin, 30 mg/kg, P.O.<br />
Group-IV : Methanolic extract 500 mg/kg<br />
P.O.+ Indomethacin, 30 mg/kg, P.O.<br />
Groups of 6 animals each were pretreated with water<br />
or standard drug or methanolic extract of corm (250,<br />
500 mg/kg P.O.) 30 minutes before the administration<br />
of indomethacin at dose 30 mg/kg orally.<br />
Determination of gastric ulcer index<br />
Four hours later the albino rats were sacrifi ced by an<br />
overdose of anesthetic ether. Subsequently, stomachs<br />
were removed and cut open along the greater curvature<br />
and pinned on a soft board. The number of ulcers in the<br />
glandular portion per stomach were noted and severity<br />
of gastric mucosal lesions scored microscopically with<br />
the help of hand lens (10x) and scoring was done as<br />
fallows. 20<br />
0 = Normal stomach 0.5 = Red coloration<br />
1 = Spot ulcers 1.5 = Haemorrhagic streaks<br />
2 = Ulcer > 3 mm < 5mm 3 = Ulcers > 5mm<br />
Mean ulcer score for each animal was expressed<br />
as ulcer index. The percentage protection was<br />
calculated by using the formula; Percentage protection<br />
= 1–U t /U c × 100 ; Where, U t = Ulcer index of treated<br />
group; U c = Ulcer index of control group (Table 1).<br />
GSH estimation in Indomethacin induced<br />
gastric ulceration<br />
Tissue samples of stomach were homogenized in ice<br />
cold Trichloroacetic acid (1 gm tissue plus 10 ml 10%<br />
TCA) in an ultra turrax tissue homogenizer. Glutathione<br />
measurements were performed using the modifi cation<br />
of the Ellamn procedure. 21 Briefl y, after centrifugation<br />
at 3000 rpm for 10 minutes, 0.5 ml supernatant was<br />
added to 2 ml of 0.3 M disodium hydrogen phosphate<br />
solution. A 0.2 ml solution of dithiobisnitrobenzoate<br />
(0.4 mg/ml in 1% sodium citrate) was added and the<br />
absorbance at 412 nm was measured immediately<br />
after mixing. Percentage increase in OD was directly<br />
proportional to the increase in the levels of Glutathione.<br />
Hence, Percentage increase in OD was calculated by the<br />
formula: Percentage increase = { (Test OD - Control<br />
OD) / Control OD } x 100 (Figure 1).<br />
In vivo lipid peroxidation estimation in gastric<br />
ulceration<br />
The degree of lipid peroxide formation was assayed by<br />
monitoring thiobarbituric reactive substance formation. 22<br />
Combined 1.0 ml of tissue samples of stomach<br />
biological sample (0.1–2.0 mg of membrane protein or<br />
0.10.2μ mol of lipid phosphate) with 2.0 ml of TCA-<br />
TBA-HCl (15% w/v trichloroacetic acid; 0.375% w/v<br />
thiobarbituric acid; 0.25N hydrochloric acid) and mixed<br />
thoroughly. The solution was heated for 1 hr in a boiling<br />
water bath. After cooling, the fl occulent precipitate was<br />
removed by centrifugation at 1000 rpm for 10 mins. The<br />
absorbance of the sample was determined at 535 nm<br />
against a blank that contains all the reagents minus the<br />
lipid. The malondialdehyde concentration of the sample<br />
was calculated by using an extinction coeffi cient of<br />
1.56 × 105 M –1 cm –1 [Percentage inhibition = { (Control<br />
– Test) / Control OD } × 100] (Figure 2).<br />
Statistical analysis<br />
All the results were expressed as mean ± SEM, n = 6.<br />
Statistical analysis was performed with one way analysis<br />
of variance (ANOVA) followed by Tukey-Kramer<br />
Multiple Comparisons Test by using Graph Pad Instat<br />
3.06 Software P value less than < 0.05 was considered<br />
to be statistically signifi cant. * P < 0.05, **
H.N Nataraj et al.: Evaluation of gastroprotective ability of Amorphophallus paeoniifolius corms against indomethacin induced gastric ulcers<br />
Figure 1: Effect of MECAP on tissue GSH levels.<br />
Figure 2: Effect of MECAP on tissue GSH levels.<br />
of ethyl acetate were spotted in the form of bands on<br />
precoated and preactivated aluminium silica gel 60GF 254<br />
HPTLC plates (10 cm × 10 cm width, 0.2 mm thickness-<br />
E-Merk) by means of Linomat IV automatized spray<br />
on band applicator equipped with a 100 μL syringe. The<br />
linear ascending development was carried out in Camag<br />
HPTLC twin trough chamber saturated with mobile<br />
phase consisting of chloroform : ethyl acetate : formic<br />
acid (5:4:1). Subsequent to the development the plates<br />
were dried in a current of air with the help of an air dryer.<br />
Densitometric scanning was performed on Camag TLC<br />
scanner III in absorbance mode at 254 nm (Figure 3).<br />
For references, HPTLC fi nger print analysis of standards<br />
markers viz., Gallic acid and Quercetin were developed<br />
70 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
H.N Nataraj et al.: Evaluation of gastroprotective ability of Amorphophallus paeoniifolius corms against indomethacin induced gastric ulcers<br />
Figure 3: HPTLC Finger print profile after densitometric scan of Sample (EtOAc fraction of MeOH extract) under uv 254 nm (Chloroform:<br />
Ethyl acetate: Formic acid - 5:4:1).<br />
with same solvent system. Densitometric scanning was<br />
performed and R f values were recorded (Table 2).<br />
RESULTS<br />
Effect of MECAP on tissue GSH & LPO levels,<br />
Gastric ulceration<br />
In pylorus ligation induced gastric ulcer there was<br />
marked reduction in the tissue GSH levels in control<br />
group. Treatment with MECAP increased GSH levels<br />
by both the doses 250 and 500 mg/kg to 47.01% &<br />
73.09% respectively and inhibition of lipid peroxidation<br />
generation up to 29.27% and 36.81% respectively in a<br />
dose dependant manner. Besides, MECAP at both<br />
concentrations showed signifi cant (p
H.N Nataraj et al.: Evaluation of gastroprotective ability of Amorphophallus paeoniifolius corms against indomethacin induced gastric ulcers<br />
role in gastric ulceration induced by several types of<br />
stress. ROS also decrease the levels of endogenous<br />
antioxidants, such as GSH, α-tocophenol and ascorbate<br />
and make more prone to oxidative damage. 26 Most of<br />
the world’s population relies on traditional remedies<br />
for its treatment. It is recommended that natural drugs<br />
containing antiulcer, antioxidant and antisecretory<br />
activity can exhibit effectiveness in peptic ulceration.<br />
The well known Phytoconstituents possessing antiulcer<br />
activity includes polyphenols, tannins, fl avonoids, gums<br />
and mucilages, saponins etc.<br />
In the present study indomethacin induced gastric ulcer<br />
model experiment, ulcer index parameter was used<br />
for the evaluation of gastroprotective activity. Oral<br />
administration of indomethacin (30 mg/kg) has induced<br />
ulcers as indicated by higher ulcer index values. Further<br />
it also reduced the gastric tissue glutathione levels<br />
and enhanced the lipid peroxidation. Indomethacin<br />
induced gastric damage particularly due to inhibition of<br />
cycloxygenase pathway of arachidonic acid metabolism<br />
which has a consequence diversion of pathway resulting<br />
in over production of leukotrienes 27,28 and these<br />
leukotrienes have been attributed an important role<br />
in gastrointestinal ulceration. 29 Apart from this, these<br />
agents break the mucosal barrier, provoke an increase in<br />
the gastric mucosal permeability to H + and Na + ions and<br />
drop in the transmucosal potential differences inducing<br />
the formation of erosions and ulcer and in addition<br />
indomethacin markedly decrease gastric mucosal PGE 2<br />
levels causing ulceration. 30<br />
Whereas, the Methanolic extract of corms of<br />
Amorhophallus paeoniifolius (MECAP) signifi cantly reduced<br />
the ulcer score in dose dependent manner compared to<br />
control group. Along with this, it also lowered destructive<br />
lipid peroxidation (LPO) levels and restored back the<br />
protective GSH levels.<br />
Preliminary phytochemical screening of MECAP indicated<br />
the presence of the phytoantioxidants viz., Tannins,<br />
Flavonoids, Sterols, Polyphenolics and Coumarins<br />
which are well known powerful natural antioxidants due<br />
to their electron donating property which either scavenge<br />
the principal propagation free radicals or halt the<br />
radical chain. The HPTLC analysis revealed the presence<br />
of nine phenolics including Gallic acid and Quercetin<br />
in the tested extract.<br />
Besides, various mechanisms have been thought to be<br />
involved in the ulcer production; hence it is not possible<br />
to propose a single mechanism for anti-ulcer effect to a<br />
particular drug or especially for an extract. It has been<br />
demonstrated that the antioxidant compounds could<br />
be active in producing antiulcerogenic effect as these<br />
substances are capable of settling on the membrane<br />
and counteracting lipid peroxidation. 31 Along with<br />
this the fl avonoids present in the test extract not only<br />
exhibit antioxidant activity 32 but also have effects in<br />
gastrointestinal tract, including mucosal protection in<br />
rat colitis. 33 In addition to this, it is being reported that<br />
fl avonoids are able to protect the gastric mucosa against<br />
a variety of ulcerogenic agents via several mechanisms<br />
of action, mainly free radical scavenging and antioxidant<br />
properties, increased mucus production. 34 Thus the<br />
antiulcerogenic effect observed with oral administration<br />
of MECAP could be related to the presence of fl avonoids<br />
and other phytoantioxidants detected in test extract.<br />
However, further studies are required to establish the<br />
exact mode of action and the active principles involved<br />
in its gastroprotection.<br />
CONCLUSION<br />
In conclusion, from the results of the present study, it is<br />
clear that Amorphophallus paeoniifolius corms methanolic<br />
extract has shown signifi cant gastroprotective<br />
activity in indomethacin induced gastric ulceration<br />
animal model. The MECAP at both doses exhibited<br />
signifi cant gastoprotection in dose dependent manner<br />
in comparison to standard Lansoprazole. In addition to<br />
tannins, sterols, fl avonoids, coumarins and nine phenolic<br />
compounds including quercetin and gallic acid (resolved<br />
in HPTLC analysis) present in extract and some other<br />
compounds might have contributed to the protection<br />
offered. Further, evaluation of the said compounds for<br />
gastroprotection is under study to ascertain the claim.<br />
ACKNOWLWDGMENTS<br />
The authors are grateful to principal and management,<br />
T.V.M. College of Pharmacy, Bellary and authorities<br />
of SCS College Pharmacy, Harapanahalli for providing<br />
necessary facilities. We wish to extend our thanks to<br />
Dr. Kotresh also for authentication of plant.<br />
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9. Li B, Xia J, Wang Y, Xie B, ‘Antiobesity activity of corm of Amophophallus<br />
paeoniifolius. J. Agri Food. Chem., 2002;53:740–5.<br />
10. Pratibha, S, Nambison B and Leelamma, ‘Enzyme inhibiters in tubers<br />
crops’, Plant Food Hum Nutr., 1995;48:247.<br />
11. Shilpi JA, Ray PK, Sarder MM, and Uddin SJ ‘Analgesic activity of<br />
Amorphophallus companulatus tuber’, Fitoteropia, 2005, 367–9.<br />
72 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
H.N Nataraj et al.: Evaluation of gastroprotective ability of Amorphophallus paeoniifolius corms against indomethacin induced gastric ulcers<br />
12. Shastry RA, Mahaden KM, Habbu PV, Patil JP. Immunomodulatory<br />
activity of corm extracts of Amophophallus paeoniifolius (Dennst),<br />
Pharmakine, 2009;1(4):26–33.<br />
13. Ramalingam J, Hima Bindu K, Madhavi B, Ravinder Nath A, David Banji,<br />
‘Phytochemical and Anthelmintic eavaluation of corm Amorphophallus<br />
companulatus’, International <strong>Journal</strong> of Pharma and Bioscinces, 2010,<br />
VI(2), 1–9.<br />
14. Jain S, Dixit VK and Malviya N, ‘Antioxidant and hepatoprotective activity<br />
of Ethanolic and Aqueous extracts of Amorphophallus campanulatus’ ,<br />
Acta poloniac Pharmaceutica, 2009;66(4):423–428.<br />
15. Angayarkanni J, Ramkumar KM, Poornima T, Priyadarshini U, Cytotoxic<br />
activity of Amorphophallus paeoniifolius tuber extract, American-<br />
Euresian J Agri Environ Sci, 2007;2:395–8.<br />
16. Nataraj HN, Murthy RLN, Ramachandra setty S, In vitro quantifi cation<br />
of Flavonoids And Phenolic content of–Suran, Int. J. ChemTech Res,<br />
2009;1(4):1063–1067.<br />
17. Nataraj HN, Murthy RLN, Ramachandra setty S, ‘In vitro antioxidant<br />
and free radical scavenging potential of Amorphophallus paeoniifolius’,<br />
Oriental journal of chemistry, 2008;24(3):895–902.<br />
18. Mrs. Prema Veeraraghavan. Expert consultant, CPCSEA, OECD<br />
guideline No. 423, Oct. 2000.<br />
19. Datta GK, Sairam K, Priyambada S, Debnath PK, Goel RK.<br />
Antiulerogenic activity of Satavaru mandur- An ayurvedic herbomineral<br />
Preparation. Indian J Expl Biol 2002;40:1173–7.<br />
20. Kulkarni SK. Hand book of experimental pharmacology, New Delhi,<br />
Vallabhah prakashan ; 1999, 128–131.<br />
21. Aykae G, Vysal M, Yalein AS, Kocak-Toker N, Sivas A, Oz H. The effect<br />
of chronic ethanol ingestion on hepatic lipid peroxide, Glutathione,<br />
glutathione peroxidase and glutathione transferase in rats. Toxicology<br />
1985;36:71–6.<br />
22. John Buege A, Steven Aust D, Microsomal lipid peroxidation. London:<br />
Moury Kleiman Co., 1978, 302.<br />
23. Repetto MG and Liesuy SF, Antioxidant properties of natural compounds<br />
used in popular medicine for gastric ulcers, Brazil J Med Biol Res, 2002;<br />
35(5):523–524.<br />
24. Pihan G, Rogers C, Szabo S, Free radicals and lipid peroxidation in<br />
ethanol and aspirin induced gastric mucosal injury, Digestive Diseases<br />
Sci, 1987;32(12):1395–1399.<br />
25. Vaananann PM, Medding JB and Wallace JL, Role of oxygen derived<br />
free radicals in indomethacin-induced gastric injury, Amer J Physiol,<br />
1991;261:470–475.<br />
26. Phull PS, Green CJ, Jaycna MRA, A radical review of the stomach; the<br />
role of oxygen derived free radicals and antioxidants in gastroduodenal<br />
diseases, Eur J Gastroenterol Hepatol, 1995;7:265–271.<br />
27. Pihan G, Rogers C, Szabo S., Vascular injury in acute gastric mucosal<br />
damage, mediatory role of leukotrienes. Dig. Dis. Sci 1988;33:<br />
625–632.<br />
28. Rainsford KD. Gastric ulcerogenicity of non-steroidal anti-infl ammatory<br />
drugs in mice with mucosa sensitized by anolinmimetic treatment.<br />
J. Pharmacol 1978;39:669–672.<br />
29. Wallace JL, McKnight, G.W, Keenan CM., Byles, NI, MacNaughton WK.<br />
Effects of leukotrienes on susceptibility of the rat stomach to damage<br />
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1990;98:1178–1186.<br />
30. Whittle BJ. Temporal relationship between cyclooxygenase inhibition,<br />
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damage induced by indomethacin in rat. Gastroenterology 1981;80(1):<br />
94–98.<br />
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1996;16:33–50.<br />
32. Magnani L, Gaydou EM, Hubaud JC. Spectrophotometric, measurement<br />
of antioxidant properties of fl avones and fl avonols agaisnt superoxide<br />
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33. Gálvez J, Cruz T, Crespo E, Ocete MA, Iorente MD, Sánchez de Medina<br />
F, Zarzuelo, A. Rutoside as mucosal protective in acetic acid-induced<br />
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simple method for the uniform production of gastric ulceration in the rat.<br />
Gastroenterology 1945;5:43–61.<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 73
Immunomodulatory activity of methanolic<br />
extracts of Pongamia glabra Vent. seeds and<br />
bark in cyclophosphamide induced mice<br />
Sanjeev Heroor 1 , Arunkumar Beknal 1 , Nitin Mahurkar 2<br />
1 Dept. of Pharmacognosy and Phytochemistry, HKES’s MTR institute of Pharmaceutical Sciences, Gulbarga, Karnataka, India<br />
2 Dept. of Pharmacology, HKES’s MTR institute of Pharmaceutical Sciences, Gulbarga, Karnataka, India<br />
ABSTRACT<br />
Immune activation is an effective as well as a protective and novel approach against emerging infectious<br />
diseases. Traditionally Pongamia glabra Vent claimed to cure infectious diseases needs scientifi c validation as<br />
immunomodulatory agent. Methanolic extracts of seeds and barks at the doses of 250 mg/kg and 500 mg/kg<br />
(per oral) of Pongamia glabra Vent. were studied for the assessment of immunomodulatory activity on<br />
cyclophosphamide induced immunosuppression in mice. The activity was assessed by determining the RBC,<br />
Hb%, platelet, total WBC and differential counts. Methanolic extracts of seeds and barks of Pongamia glabra<br />
Vent. showed dose dependent highly signifi cant counteracting effect (p
Sanjeev Heroor et al.: Immunomodulatory activity of methanolic extracts of Pongamia glabra Vent. seeds and bark in cyclophosphamide induced mice<br />
Karanja, a tree found all over India bearing imparipinnate<br />
leaves and pinkish white colored fl owers. 8 The seeds of<br />
the plant reported to contain fi xed oil and traces of<br />
essential oil. Bark contains a bitter alkaloid, resin and<br />
mucilage. 9 The ancient folklore claims the uses of the<br />
seeds to treat rheumatic joints and anti-paracytic. Bark is<br />
astringent and powdered seeds are used as febrifuge and<br />
tonic. 10 The present study was aimed at screening of seeds<br />
and bark methanolic extracts of Pongamia glabra Vent.<br />
for immunomodulatory activity in cyclophosphamide<br />
induced immunosuppressed albino mice.<br />
MATERIALS AND METHODS<br />
Plant material<br />
Pongamia glabra Vent. seeds and bark were collected from<br />
local areas of North Karnataka and a voucher specimen<br />
has been deposited at the departmental herbarium. The<br />
plant was authenticated by Dr. Srinathrao, Prof. and<br />
HOD, Dept. of Botany, Gulbarga University, Gulbarga<br />
and Ref. No. GUG/BOT/Herbarium/2008–09/09. The<br />
mentioned parts of the plant were dried and pulverized<br />
to particle size (#) 40 and then were fi rst defatted with<br />
petroleum ether (40–60°C) and extracted with methanol<br />
by continuous hot percolation method using Soxhlet<br />
apparatus at 40°C for 48 h to obtain methanolic extracts<br />
of seeds and bark of the plant respectively. The fi ltrates<br />
of the extracts were concentrated to dryness at 40°C<br />
under reduced pressure in a rota fl ash evaporator. The<br />
yields of the methanolic extracts of seeds and barks were<br />
43.16 gm (30.59%w/w) and 37.76 gm (22.19%w/w)<br />
respectively.<br />
Preliminary Phytochemical Studies<br />
The methanolic extracts of seeds and bark were<br />
subjected for preliminary qualitative chemical tests<br />
and the presence of major phytoconstituents were<br />
confi rmed by Thin Layer Chromatography (TLC)<br />
studies11,12 (Table 1).<br />
Thin Layer Chromatography studies<br />
• TLC profi le for fl avonoids<br />
Solvent system: ethyl acetate: formic acid: glacial<br />
acetic acid: water<br />
(10:1.1:1.1:2.6).<br />
Detection: UV 365 (blue fl uorescent spots).<br />
• TLC profi le for alkaloids<br />
Solvent system: toluene: ethyl acetate: diethylamine<br />
(7:2:1)<br />
Detection: wagner’s reagent (brown colored spots).<br />
• TLC profi le for steroids<br />
Solvent system: petroleum ether: acetone (7:3).<br />
Detection: anisaldehyde: sulphuric acid reagent (pink<br />
to red colored spots)<br />
• TLC profi le for saponins<br />
Solvent system: chloroform: glacial acetic acid:<br />
methanol: water (6.4:3.2:1.2:0.8).<br />
Detection: anisaldehyde: sulphuric acid reagent (pink<br />
colored spots).<br />
Animals<br />
Swiss albino mice of either sex, weighing 25–30 g<br />
housed in standard conditions of temperature, humidity<br />
and light were used. They were fed with standard rodent<br />
diet and water ad libitum. The study was approved by<br />
Institutional Animal Ethical Committee, Ref. No.<br />
HKECOP/IAEC/45/2011–12.<br />
Acute Toxicity Studies<br />
Acute toxicity studies were conducted as per OECD<br />
guideline by 425 method. 13 The animals did not show<br />
any mortality at the dose of 5000 mg/kg and hence its<br />
1/10th dose i.e. 500 mg/kg and 1/20th dose i.e. 250 mg/<br />
kg were used as the therapeutic doses for the methanolic<br />
extracts of the study.<br />
Test Samples<br />
Weighed quantities of test extracts were suspended<br />
in 1% sodium carboxy methyl cellulose to prepare a<br />
suitable dosage form. 14 The control animals were given<br />
an equivalent volume of sodium CMC vehicle.<br />
Drugs<br />
Cyclophosphamide was used as a standard immunosuppressant,<br />
Cycloxan ® (Biochem–pharmaceutical<br />
indus tries Ltd., Mumbai) containing 200mg – cyclosphosphamide<br />
, was procured from the market and dilutions<br />
were made using sterile water for injection as mentioned<br />
on the label of the marketed product.<br />
Cyclophosphamide Induced Myelosuppression15 Animals were divided into six groups of six animals<br />
each. Group I served as control group and received<br />
Table 1: Thin Layer Chromatography (TLC) results of Pongamia glabra Vent. seeds and bark methanolic extracts<br />
Flavonoids Alkaloids Steroids Saponins<br />
S.No. Extracts No. of spots Rf value No. of spots Rf value No. of Spots Rf Value No.of spots Rf value<br />
1 Seed Ext. 2 0.59, 0.63 2 0.89, 0.80 2 0.61, 0.76 2 0.58, 0.65<br />
2 Bark Ext. 2 0.62, 0.71 2 0.82, 0.53 4 0.64,0.72, 0.78, 0.88 2 0.72, 0.87<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 75
Sanjeev Heroor et al.: Immunomodulatory activity of methanolic extracts of Pongamia glabra Vent. seeds and bark in cyclophosphamide induced mice<br />
the vehicle (1% sodium CMC) for 13 days. Group<br />
II (Cyclophosphamide group) received the vehicle<br />
(1% sodium CMC) for a period of 13 days and on 11th,<br />
12th and 13th days was injected with cyclophosphamide<br />
(30 mg/kg intraperitonial). Groups III and IV were<br />
administered methanolic extracts of seeds of the plant<br />
at the doses of 250 mg/kg and 500 mg/kg (per oral)<br />
daily for 13 days respectively. Similarly Groups V and<br />
VI were administered methanolic extracts of bark of<br />
the plant at the doses of 250mg/kg and 500mg/kg (per<br />
oral) daily for 13 days respectively. The groups III, IV, V<br />
and VI were injected with cyclophosphamide (30 mg/kg<br />
intraperitonial) on the 11th, 12th and 13th days, 1 hour<br />
after the administration of the Respective oral treatment.<br />
Blood samples were collected on 14th day of the<br />
experiment by retro orbital puncture and hematological<br />
parameters were studied for RBC, Hb%, platelets, total<br />
WBC counts and differential leucocytes counts (DLC).<br />
Statistical Analysis<br />
Data were expressed as mean ± SEM and differences<br />
between the groups were statistically determined by<br />
analysis of variance (ANOVA) followed by Dunnet’s<br />
test. p-values
Sanjeev Heroor et al.: Immunomodulatory activity of methanolic extracts of Pongamia glabra Vent. seeds and bark in cyclophosphamide induced mice<br />
Immune activation is an effective as well as protective<br />
approach against emerging infectious diseases. 17<br />
Immunomodulatory activity of methanolic extract of<br />
seeds and bark of Pongamia glabra Vent. was explored<br />
by evaluating their effects on cyclophosphamide<br />
induced myelosuppression in mice at 2 dose levels of<br />
250 mg/kg and 500 mg/kg (per oral) Results of the<br />
study revealed the dose dependent counteracting effect<br />
of the extracts to the cyclophosphamide induced bone<br />
marrow activity suppression i.e. myelosuppression, as<br />
indicated by increase in RBC, total WBC platelet counts,<br />
Hb% and DLC in the extract treated groups (Group III,<br />
IV, V and VI), when compared to cyclophosphamide<br />
treated group (Group II). However the counteracting<br />
activity to reduction in blood cell counts of the seeds<br />
extract was more than that of the bark extract at the<br />
mentioned doses, but both extracts showed similar level<br />
of signifi cance of activity. The potentiated activity of<br />
seed extract may be due to presence of excess number<br />
of fl avonoids and steroids in the seed extract. The<br />
results indicate modulation of bone marrow activity,<br />
viz., – suppression when used cyclophosphamide alone<br />
and stimulation to counteract the cyclophosphamide<br />
induced myelosuppression in pretreated methanolic<br />
extract groups of seeds and barks of Pongamia<br />
glabra Vent.<br />
CONCLUSION<br />
From the phytochemical investigation, it was found<br />
that the major chemical constituents of the methanolic<br />
extracts of seeds and barks were steroids, saponins,<br />
tannins, fl avonoids, alkaloids, proteins and carbohydrates.<br />
Saponins are either triterpenoid or steroidal glycosides<br />
proven as important phytoconstituent with different<br />
pharmacological activities such as antiallergic,<br />
cytotoxic, antitumour, antiviral, immunomodulating,<br />
antihepatotoxic, and antifungal activities. Recently three<br />
diosgenyl saponins isolated from Paris polyphylla were<br />
reported for immunostimulating activity. 18 Tannins are<br />
also known to possess immunostimulating activites. The<br />
well known ayurvedic formulation, Triphala contains<br />
Terminalia chebula, Terminalia belerica and Emblica offi cinalis,<br />
which are rich in tannins reported for immunostimulating<br />
activity. 19 Hence the collective presence of steroids,<br />
saponins, tannins and fl avonoids in the methanolic<br />
extracts would be attributed for immunostimulating<br />
activity. However this is a preliminary research work<br />
and the precise mechanism(s) of immunomodulatory<br />
action infl uenced by potent bio-active constituents of<br />
methanolic extracts of seeds and bark of Pongamia glabra<br />
Vent. against cyclophosphamide induced immunesuppression<br />
needs to be investigated.<br />
ACKNOWLEDGMENT<br />
Authors are thankful to authorities of HKE Society and<br />
MTR Institute of Pharmaceutical sciences, Gulbarga,<br />
Karnataka India, for providing necessary facilities to<br />
carry out the study.<br />
REFERENCES<br />
1. Charak Samhita (Trans.). Shree Gulab Kunvera Ayurvedic Society,<br />
Jamnagar, India. 1949; 249–50.<br />
2. Patwardhan B, Kalbag D, Patki P. S, Nagasampagi B.A. Search of<br />
Immunomodulatory agents: a review. Indian Drugs 1990;28(2):56–63.<br />
3. Wagner H. Immunomodulatory agents. Proceedings of the Alfred<br />
Benzon Symposium. 1983:20;559.<br />
4. Makare N, Bodhankar S, Rangari V. Immunomodulatory activity of<br />
alcoholic extracts of Mangifera indica Linn in mice. J. Ethnopharmacol.<br />
2001;78:133–7.<br />
5. Baumann F, Preiss R. Cyclophosphamide and related anticancer drugs,<br />
B. chrmoatogr. B. Biomed Sci. Appl.2001;764:173–92.<br />
6. Goodman J. W. In : Stites D. P., Terr. A.I., Parslow T. G. (Eds.), The<br />
Immune response in Basic and Clinical Immunology. 8th Edn. NJ:<br />
Prentice-Hall, Engle wood Cliffs; 1994; 40–7.<br />
7. Angulo I., Jimenez M.B., Garcia Bustos J.F, Gargallo D. Candida albicans<br />
infection enhances immunosuppression induced by cyclophosphamide<br />
by selective priming of suppressive myeloid progenitors. cell immunol<br />
2002:218;46–58.<br />
8. Kirtikar K. R, Basu B.D. Indian Medicinal Plants, Oriental Enterprises.<br />
Dehradun, 2nd Ed. 1984;I:830–32.<br />
9. Nadkarni K. M. Indian Materia Medica, Popular Prakashan.1996;<br />
I: 1001–04.<br />
10. Hartwell J. L. Plants used against cancer. A Survey, Lloydia.1967–1971;<br />
30–34.<br />
11. Khandelwal K.R. Practical Pharmocognosy Techniques and<br />
Experiments. 10th Ed. Pune: Nirali Prakashan; 2003; 149–58.<br />
12. Hildebert Wagner, Sabine Bladt. Plant Drug Analysis-A Thin layer<br />
Chomoatography Atlas, Springer-verlag. 2nd Ed. New York: Berlin<br />
Heidelberg; 2001; 1–3, 195–197, 305–206.<br />
13. Committee for the purpose of control and supervision of Experimental<br />
Animals (CPCSEA), OECD Guidelines for the testing of Chemicals,<br />
revised draft guidelines 425: Acute oral toxicity-Acute toxic class method,<br />
revised document. India: Ministry of Social Justice and Empowerment;<br />
2008, No.26.<br />
14. Satpute K. L, Jadhav M. M, Karodi R. S, Patil M.J. Immunomodulatory<br />
activity of fruits of Randia dumetorum Lamk. J. Pharmocog. and<br />
Phytother. 2009;1:1–5.<br />
15. Manjarekar P. N, Jolly C.L, Narayan S. Comparative studies of<br />
immunomodulatory activity of Tinospora cordifolia and Tinopora<br />
sinensis. Fitoterpia 2001;71:254–7.<br />
16. Pelczar M.J, Chan E.C.S, Krieg N. R. Microbiology, 5th Ed. New Delhi:<br />
Tata Mcgraw Hill; 1990; 703–15.<br />
17. Hackett C J. Allergy. Clin. Immunol. 2003;112:686–94.<br />
18. Xiu-feng Z, Yan C, Jiajun H. Ya-Zhou Z, Zhou N. Ya-Lin T, Yang L.<br />
Immunostimulating properties of diosgenyl saponins isolated from Paris<br />
polyphylla. Bioorganic and Med. Chem. Letters 2007;7:255–9.<br />
19. R Srikumar, Parthasarathy N.J, Sheeladevi R. Immunomodulatory<br />
activity of Triphala on Neutrophil Functions. Biol. Pharm. Bull. 2005;<br />
28(8):1398–1403.<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 77
Simultaneous fi rst derivative UV<br />
spectrophotometric estimation of<br />
ramipril and olmesartan<br />
Santosh R. Karajgi, Simpi C.C., Kalyane N.V.<br />
Department of Pharmaceutical Chemistry, BLDEA’s College of Pharmacy, BLDEA University Campus, Solapur Road,<br />
Bijapur, Karnataka, India<br />
ABSTRACT<br />
A simple, precise and economical procedure for the simultaneous estimation of Olmesartan Medoxomil and<br />
Ramipril in tablet formulation has been developed. Olmesartan and Ramipril are antihypertensive agents belonging<br />
to category of angiotensin-converting enzyme inhibitor. The present study involves the simultaneous estimation<br />
done by fi rst derivative UV Spectrophotometric method using Shimadzu 1700 spectrophotometer. Olmesartan<br />
has zero crossing point at 240 nm in methanol and Ramipril has zero crossing point at 246 nm in methanol.<br />
Both these drugs obey Beer’s law in the concentration range employed for the present method. The result<br />
of analysis has been validated statistically by recovery studies. The slope and intercept for Olmesartan were<br />
0.0364 and 0.0078 and for Ramipril were 0.0010 and –0.0001 respectively as determined by the method of<br />
least squares.<br />
INTRODUCTION<br />
Ramipril is a prodrug belonging to the<br />
class of ACE inhibitors used to treat<br />
hypertension and congestive heart failure<br />
where as Olmesartan is an ARB used<br />
to treat high blood pressure. Both ACE<br />
inhibitors and ARBs are widely used in<br />
renal failure patients in the treatment of<br />
hypertension, left ventricular dysfunction,<br />
and diabetic nephropathy. Their effi cacy<br />
in these conditions is well established, and<br />
generally both classes of drugs are well<br />
tolerated, with a low incidence of side<br />
effects. 1 Combination of ACE inhibitors and<br />
ARBs is proved to be a useful combination<br />
therapy for the treatment of ischemic heart<br />
diseases. 2<br />
Review of literature revealed that there<br />
are very few methods reported for the<br />
estimation of Ramipril and Olmesartan<br />
individually and in combined dosage<br />
forms, 3–26 no derivative spectrophotometric<br />
method has been so far reported for<br />
simultaneous estimation of these drugs in<br />
combined dosage forms. Therefore, the<br />
aim and objective of present study were<br />
to develop a fi rst derivative UV-Visible<br />
spectrophotometric analytical method for<br />
the estimation of Ramipril and Olmesartan<br />
in bulk and formulated dosage form in<br />
combination without prior separation and<br />
to establish a simple, sensitive, standard,<br />
reproducible method for the quality control<br />
of Ramipril and Olmesartan.<br />
MATERIALS AND METHODS<br />
Materials<br />
Ramipril and Olmesartan were gift samples<br />
from Ajantha Pharmaceuticals, Aurangabad.<br />
The commercial formulations were<br />
purchased from local market. Methanol<br />
A.R. Grade (Qualigens, Fine chemicals)<br />
was used as the solvent.<br />
Research Ar cle<br />
Received Date : 27-08-2011<br />
Revised Date : 06-02-2012<br />
Accepted Date : 08-02-2012<br />
DOI: 10.5530/rjps.2012.1.11<br />
Address for<br />
correspondence<br />
Santosh R. Karajgi<br />
Department of Pharmaceutical<br />
Chemistry, BLDEA’s College of<br />
Pharmacy, BLDEA<br />
University Campus,<br />
Solapur Road, Bijapur,<br />
Karnataka, India<br />
Email: santosh.karajgi@gmail.<br />
com<br />
Mobile: +919739619395<br />
www.rjps.in<br />
78 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
Equipments<br />
Santosh R. Karajgi et al.: Simultaneous First Derivative UV Spectrophotometric Estimation of Ramipril and Olmesartan<br />
Shimadzu spectronic 1700 double beam UV-visible<br />
spectrophotometer with 1 cm matched quartz cells was<br />
used for all the absorbance measurements.<br />
Method<br />
The solutions of Ramipril and Olmesatan were<br />
prepared separately in methanol at a concentration<br />
range of 10 μg/ml. Both these solutions were scanned<br />
in the wavelength range of 200 nm to 300 nm. Data<br />
were recorded at the wavelength interval of 1 nm in the<br />
derivative mode to obtain fi rst order derivative spectra<br />
at N=2. After examining the overlain fi rst derivative<br />
spectra, wavelengths for analysis were selected where<br />
one drug showed zero crossing and the other drug<br />
showed substantial absorbance. The wavelength selected<br />
for Ramipril analysis was 240 nm where Olmesartan<br />
has zero absorbance and the wavelength selected for<br />
Olmesartan analysis was 246 nm where the absorbance<br />
of Ramipril is zero. The overlain derivative spectra of<br />
the drugs are given in the Figure 1.<br />
Linearity<br />
Standard stock solutions of Ramipril and Olmesartan<br />
were prepared by dissolving 100 mg each of standard<br />
drug samples in 100 ml volumetric fl asks separately<br />
and the volume was made up with methanol to<br />
get a concentration of 5 – 50 μg/ml for each drug.<br />
Figure 1: Zero crossing points of Ramipril and Olmesartan.<br />
The absorbances of derivative spectra were measured<br />
at 240 nm and 246 nm for Ramipril and Olmesartan<br />
respectively. Five replicate analyses were carried out.<br />
Absorbance against concentrations were plotted to<br />
obtain the calibration graph. Both the drugs obeyed<br />
Beer’s law with the above concentration range with<br />
the R 2 value of 0.9994 and 0.9996 for Ramipril and<br />
Olmesartan respectively (Figure 2 and 3).<br />
Analysis of marketed formulation<br />
Twenty tablets were weighed accurately and powdered.<br />
An accurately weighed quantity of powder equivalent<br />
to 20 mg of Ramipril and 20 mg of Olmesartan was<br />
taken in a 50 ml volumetric fl ask and dissolved in<br />
methanol. The volume was made up with methanol so<br />
that the theoretical concentrations of both drugs were<br />
10μg/ml and the concentrations were determined at<br />
240 nm for Ramipril and 246 nm for Olmesartan by<br />
using calibration graph. (Table 1). The overlain spectra<br />
of synthetic mixture, Ramipril bulk drug, Olmesartan<br />
bulk drug and combined tablet formulation of Ramipril<br />
and Olmesartan is given in the Figure 4.<br />
Recovery Studies<br />
To determine the accuracy of the method, recovery<br />
study was performed using the method of standard<br />
addition. To the pre- analyzed marketed formulation<br />
powder of combined Ramipril and Olmesartan, diluted<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 79
Santosh R. Karajgi et al.: Simultaneous First Derivative UV Spectrophotometric Estimation of Ramipril and Olmesartan<br />
Table 1: Analysis of marketed formulation<br />
Label claim (mg) Amount found (mg) Label claim %<br />
Sample Ramipril Olmesartan Ramipril Olmesartan Ramipril Olmesartan<br />
1 10 10 10.08 10.18 100.8 101.8<br />
2 10 10 10.11 10.17 101.1 101.7<br />
3 10 10 9.99 10.11 99.9 101.1<br />
4 10 10 10.04 10.18 100.4 101.8<br />
5 10 10 9.98 9.88 99.8 98.8<br />
Mean 10.04 10.10 100.4 101.0<br />
S.D. 0.0561 0.1285 0.561 1.285<br />
Figure 2: Calibration graph of Ramipril at 240 nm.<br />
Figure 3: Calibration graph of Olmesartan at 246 nm.<br />
Figure 4: First derivative spectra of Ramipril, Olmesartan, Synthetic mixture and Tablet formulation.<br />
80 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
Santosh R. Karajgi et al.: Simultaneous First Derivative UV Spectrophotometric Estimation of Ramipril and Olmesartan<br />
to 10 μg/ml with methanol, weighed quantities of bulk<br />
drugs were added at four levels, i.e. 50 %, 75 %, 100 %<br />
and 125 % for both drugs and the total drug contents<br />
were determined as described for formulation. The<br />
percentage recovery was determined (Table 2).<br />
RESULTS AND DISCUSSION<br />
Simultaneous analysis of Ramipril and Olmesartan by zero<br />
order UV-Visible spectrophotometric method appears to<br />
be quite impossible because of the total overlap of bands.<br />
(Figure 5). But derivative spectrophotometric technique<br />
involves the differentiation of the normal spectrum with<br />
respect to the wavelength. The fi rst derivative spectra of<br />
the Olmesartan shows an absorbance at 246 nm, where<br />
the absorption for Ramipril is zero and Olmesartan can<br />
be specifi cally measured at that wavelength. Ramipril<br />
on the other hand, has absorbance at 240 nm where<br />
absorbance for Olmesartan is zero; hence Ramipril is<br />
specifi cally measured at that wavelength.<br />
Figure 5: Overtain zero order spectra of Ramipril and Olmesartan.<br />
Table 2: Recovery study of marketed formulation<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 81<br />
Drug<br />
Amount present in<br />
formulation (μg/ml)<br />
Amount<br />
added (%)<br />
Mean<br />
Recovery (%)<br />
Ramipril 10.11 50 99.62<br />
75 100.93<br />
100 98.89<br />
125 99.85<br />
Olmesartan 10.04 50 100.43<br />
75 99.93<br />
100 100.18<br />
125 99.75<br />
The percentage RSD values in precision shows that<br />
proposed method provides acceptable variation of<br />
Ramipril and Olmesartan which were found to be less<br />
than 2% shows its capacity to remain unaffected by<br />
small variations in method parameters and provides<br />
an indication of its reliability during normal usage.<br />
The good recovery values indicate the accuracy of the<br />
Table 3: Validation of the proposed method<br />
Sl. No. Parameters<br />
Ramipril<br />
Experimental value<br />
Olmesartan<br />
ICH Limits<br />
1 Beer’s Range 5–50 μg/ml 5–50 μg/ml –<br />
2 Linearity (R2 ) 0.9994 0.9996 0.9990<br />
3 Precision (% RSD) 0.1754 0.0349 2.0000<br />
4 Accuracy (% Recovery) 99.62–100.93 % 99.75–100.43 % 97–103 %<br />
5 Intercept -0.0001 +0.0078 –<br />
6 Slope +0.0010 +0.0364 –
Santosh R. Karajgi et al.: Simultaneous First Derivative UV Spectrophotometric Estimation of Ramipril and Olmesartan<br />
method (Table 3). Method validation was carried out as<br />
per ICH guidelines. 27<br />
CONCLUSION<br />
The described method gives accurate and precise results<br />
for determination of Ramipril and Olmesartan mixtures<br />
in tablets without prior separation and are easily applied<br />
for routine analysis. This method also provides simple<br />
and reproducible quantitative analysis without any<br />
interference from the excipients.<br />
REFERENCES<br />
1. Macdougall I.C. The role of ACE inhibitors and angiotensin II receptor<br />
blockers in the response to epoetin. Nephrol. Dial. Transplant<br />
1999;14(8):1836–41.<br />
2. Mohamed Saleem T.S., Bharani K., Gauthaman K. Angiotensin II<br />
receptor antagonists: A useful combination therapy for ischemic heart<br />
disease. Emergency Medicine 2010;2:51–9.<br />
3. Hassan Y., Aboul enein, Thiffault C. Determination of Ramipril<br />
and Its Precursors by Reverse Phase High Performance Liquid<br />
Chromatography. Analytical Letters 1991;24(12):2217–24.<br />
4. Al-Majed A.A., Belal F., Abadi A., et al. The voltammetric study and<br />
determination of Ramipril in dosage forms and biological fl uids. Farmaco<br />
2000;55(3):233–8.<br />
5. Zhu Z., Vachareau A., Neirinck L. Liquid chromatography–mass<br />
spectrometry method for determination of Ramipril and its<br />
active metabolite Ramiprilat in human plasma J. Chromatogr. B<br />
2002;779(2):297–306.<br />
6. Ayad M.M., Abdalla A.S., Hisham E., et al. Spectrophotometric and<br />
AAS determination of Ramipril and enalapril through ternary complex<br />
formation. J. Pharm. Biomed. Anal. 2002;28(2):311–21.<br />
7. Rahman N., Ahmad Y., Azmi S.N.H. Kinetic Spectrophotometric Method<br />
for the Determination of Ramipril in Pharmaceutical Formulations.<br />
AAPS Pharm. Sci. Tech. 2005;6(3): E543-E551.<br />
8. Baing M.M., Vaidya V.V., Sane R.T., et al. Simultaneous RP-LC<br />
Determination of Losartan Potassium, Ramipril, and Hydrochlorothiazide<br />
in Pharmaceutical Preparations. Chromatographia 2006;64:293–6.<br />
9. Gowda K.V., Mandal U., Selvan P.S., et al. Liquid chromatography<br />
tandem mass spectrometry method for simultaneous determination of<br />
metoprolol tartrate and Ramipril in human plasma. J. Chromatogr. B.<br />
2007;858:13–21.<br />
10. Patel C.V., Khandhar A.P., Captain A.D., et al. Validated absorption<br />
factor spectrophotometric and Reversed phase High performance<br />
liquid chromatographic methods for the determination of Ramipril and<br />
Olmesartan medoxomil in pharmaceutical formulations. Eurasian J.<br />
Anal. Chem. 2007;2(3):159–71.<br />
11. Liu D., Hu P., Matsushima N., et al. Quantitative determination of<br />
Olmesartan in human plasma and urine by liquid chromatography<br />
coupled to tandem mass spectrometry. J.Chromatogr. 2007;856<br />
(1–2):190–7.<br />
12. Shah N.J., Suhagia B.N., Shah R.R., et al. Development and validation<br />
of a simultaneous HPTLC method for the estimation of Olmesartan<br />
Medoxomil and hydrochlorothiazide in tablet dosage form. Ind. J.<br />
Pharm. Sci. 2007;69(6):834–6.<br />
13. Lincy J., George M., Ranga Rao V.B. Simultaneous estimation of<br />
atorvastatin & Ramipril by RP-HPLC & Spectroscopy. J. Pharm. Sci.<br />
2008;21(3):282–4.<br />
14. Yuan B., Wang X., Zhang F., et al. Simultaneous Determination of<br />
Ramipril and Its Active Metabolite Ramiprilat in Human Plasma by LC–<br />
MS–MS. Chromatographia 2008;68:533–9.<br />
15. Kadukara S.S., Ranjanea P.N., Ranhera S.S., et al. Spectrophotometric<br />
Methods for Determination of Olmesartan Medoxomil and<br />
Hydrochlorothiazide in Tablet Dosage Form. The Pharma Review<br />
2008.<br />
16. Garg G., Saraf S., Saraf S.S. Simultaneous estimation of atorvastatin<br />
& Ramipril by RP-HPLC & Spectroscopy. J. Ind. Chem. Soc.<br />
2008;85(7):769–72.<br />
17. Thamake S.L., Jadhav S.D., Pishawikar S.A. Development and<br />
Validation of Method for Simultaneous Estimation of Atorvastatin<br />
Calcium and Ramipril from Capsule Dosage Form by First Order<br />
Derivative Spectroscopy Asian J. Res. Chem. 2009;2(1):52–3.<br />
18. Patil P.R., Rakesh S.U., Dhabale P.N., et al. Simultaneous Estimation of<br />
Ramipril and Amlodipine by UV Spectrophotometric Method. Reseach.<br />
J. Pharm. and Tech 2009;2 (2):304–7.<br />
19. Karajgi S.R., Simpi C.C., Zambare Y.B. Simultaneous Estimation<br />
of Atorvastatin and Ramipril by First Derivative Spectrophotometric<br />
method. J. Pharm. Res. 2009;2(5):874–7.<br />
20. Kurade K. B., Pai V.P., Gude M.G. RP-HPLC estimation of Ramipril and<br />
telmisartan in tablets. Ind. J. Pharm. Sci. 2009;71(2):148–51.<br />
21. Gupta Y. isocratic RP-HPLC method development & validation for the<br />
simultaneous estimation of Ramipril and Telmisartan in tablet dosage<br />
form. Asian J. Pharm. Clin. Res. 2009;2(4):2441.<br />
22. Bankey S., Tapadiya G.G., Saboo S.S., et al. Simultaneous<br />
Determination of Ramipril, Hydrochlorothizide and Telmisartan by<br />
Spectrophotometry. Int. J. ChemTech Res. 2009;2:183–8.<br />
23. Panchal H.J., Suhagia B.N., Patel N.J., et al. Simultaneous Estimation<br />
of Atorvastatin Calcium, Ramipril and Aspirin in Capsule Dosage Form<br />
by RP-LC. J.Chromatogr. 2009;69:1–2.<br />
24. Qutab S.S., Razzak S.N., Ashfaq M. Simultaneous quantitation of<br />
Olmesartan Medoxomil and Amlodipine besylate in combined Tablets<br />
using HPLC. J. Chili. Chem. Soc. 2009;54 (3):234–7.<br />
25. Rote A.R., Bari P.D. Ratio Spectra Derivative and Zero-Crossing<br />
Difference Spectrophotometric Determination of Olmesartan Medoxomil<br />
and Hydrochlorothiazide. AAPS Pharm. Sci. Tech. 2009;10(4):1200.<br />
26. Wankhede S.B., Wadkar S.B., Raka K.C., et al. Simultaneous estimation<br />
of amlodipine besilate and Olmesartan Medoxomil in pharmaceutical<br />
dosage form. Ind. J. Pharm. Sci. 2009;71 (5):563–7.<br />
27. ICH Harmonized Tripartite Guideline. Text on Validation of Analytical<br />
Procedures. Q2A and Q2B 1995:1–5 and 1–7.<br />
82 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
Purifi cation and characterization of thermostable<br />
amylase from a strain of thermoactinomyces<br />
thalpophilus KSV 17<br />
K. Sreenivasa Rao 1 , P. Ellaiah 2 , Karnakumar V. Biradar 3<br />
1 Department of Pharmaceutics, RRKS College of Pharmacy, Bidar-585402, India<br />
2 Department of Biotechnology, College of Pharmaceutical Sciences, Visakhapatnam, Andhra University, Andra Pradesh, India<br />
3 Department of Pharmacology, RRKS College of Pharmacy, Bidar-585402, India<br />
ABSTRACT<br />
This research reported the Purifi cation and Characterization of Thermostable Amylase from a strain of<br />
T. Thalpophilus KSV 17. The result showed that the purifi ed enzyme specifi c activity of 145.80 U mg –1 , this was<br />
an increase of 21 fold than the crude enzyme extract. The analysis of SDS- polyacrylamide gel electrophoresis<br />
showed that the molecular weight of the enzyme was 52 kDa. The Optimum pH of the purifi ed enzyme showed<br />
maximum activity at pH 7.0, but the enzyme was stable in the pH range of 5.5–7.0. The optimum temperature<br />
of the purifi ed enzyme was 85°C in absence of 10 mM CaCl while 90°C in presence of 10 mM CaCl K and<br />
2 2., m<br />
V values for the purifi ed enzyme were calculated as 5.2 mg ml max –1 , 0.45 mg ml –1 /minute respectively. The thermal<br />
stability of the purifi ed enzyme at 80°C in absence of CaCl and 85°C in presence of CaCl . The purifi ed enzyme<br />
2. 2<br />
mostly inhibited by diethyl pyrocarbonate and N-bromosuccinimide and at 5 mM conc. Ca2+ , Na + and Mg2+ showed stimulatory effect while Cu2+ , Zn2+ , Hg2+ and Mn2+ shown inhibitory effect. The purifi ed enzyme showed<br />
good stability and compatibility on commercial detergents and on being stored for 60 minutes at 85°C.<br />
Key words: Purifi cation, Characterization, α-Amylase, Sephadex G-200, SDS-PAGE<br />
INTRODUCTION<br />
Microbial enzymes and coenzymes are<br />
widely used in several industries, notably<br />
in detergent, food processing, brewing<br />
and pharmaceuticals. They are also used<br />
for diagnostic, scientifi c and analytical<br />
purposes. Since ancient times they have<br />
been used in the preparation of fermented<br />
foods, especially in oriental countries. 1<br />
The activity of an enzyme is determined<br />
by the enzyme concentration, substrate<br />
concentration and its availability,<br />
concentration of co-factors, allosteric<br />
effectors, the concentration and type of<br />
inhibitors, ionic strength, pH, temperature<br />
and time since start of the reaction. The<br />
way in which each of these parameters<br />
affects enzyme activity can be determined<br />
by studying the enzyme kinetics, many assay<br />
procedures for determining the enzyme’s<br />
activity are available. Some of them are<br />
typically dependent on measurement of<br />
chemical changes Eg: Biuret Method,<br />
Lowry Method, Bradford Method etc.<br />
and others are based on physical changes.<br />
Some of these assays are also designed<br />
to investigate particular properties of the<br />
enzyme preparations and physical changes<br />
Terms, which are frequently encountered,<br />
especially in investigations of enzyme<br />
purifi cation and use are, specifi c activity and<br />
molecular activity per milligram of enzyme.<br />
Where the enzyme preparation is not pure,<br />
specifi c activity is frequently expressed in<br />
units per milligram of protein or in units<br />
per milligram of enzyme preparation. Due<br />
to wide range of application of α-Amylase,<br />
in order to get not only high quality and but<br />
Research Ar cle<br />
Received Date : 26-12-2011<br />
Revised Date : 04-03-2012<br />
Accepted Date : 06-03-2012<br />
DOI: 10.5530/rjps.2012.1.12<br />
Address for<br />
correspondence<br />
K.Sreenivasa Rao<br />
R.RK.S. College of Pharmacy<br />
Naubad, Bidar-585401,<br />
Karnataka, India<br />
Email: bidarkaran@gmail.com<br />
www.rjps.in<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 83
K. Sreenivasa Rao et al.: Purifi cation and Characterization of Thermostable Amylase from a Strain of Thermoactinomyces thalpophilus KSV 17<br />
also high grade α-Amylase we made objectives behind<br />
the sudy.<br />
In this research, α-amylase was purifi ed from a strain of<br />
T. Thalpophilus KSV 17 in three steps of purifi cation<br />
processes, namely precipitation with ammonium sulfate,<br />
precipitation with zink sulfate and Sephadex G-200<br />
gel fi ltration chromatography. The enzyme was then<br />
characterized. The enzyme activity was determined<br />
based on Bernifi eld method, 2 while protein content<br />
was determined by Lowry method. 3<br />
MATERIAL AND METHODS<br />
Chemicals and Microorganism Used<br />
Agarose, acrylamide, bis-acrylamide, sodium dodecyl<br />
sulfate (SDS), TEMED, ammonium per sulfate and<br />
other chemicals for polyacrylamide gel electrophoresis<br />
(SDS-PAGE) were purchased from Sigma Chemical Co.,<br />
U.S.A. A strain of T. thalpophilus mutant KSV 17 was<br />
used for the study. The research phases done were as<br />
follows: Production, Purifi cation and Characterization<br />
of purifi ed enzyme.<br />
Production of α-Amylase<br />
The amylase production was carried out in a 2 liter<br />
fermentor, containing 1.5 L modifi ed production<br />
medium containing Starch 2%, Yeast extract 0.05%, Mg<br />
SO 4 0.01 and salt solution (KH 2 PO 4 0.15%, K 2 HPO 4<br />
0.22% CaCl 2 .2H 2 0 0.0025% and FeSO 4 . 7H 2 0 0.00025%).<br />
A 10% (v/v) level of inoculum was added and the<br />
fermentor was run at 55°C for 48h. Whole fermentation<br />
broth was centrifuged at 10,000 g at 4°C and the clear<br />
supernatant was separated. The supernatant (crude<br />
enzyme) was subjected to recovery and purifi cation<br />
processes.<br />
Purifi cation of α-Amylase<br />
The purifi cation of enzyme was done by following steps,<br />
Ammonium Sulfate Precipitation<br />
Ammonium sulfate was added at different<br />
concentrations ranging from 40 to 80 % saturation.<br />
The precipitates so obtained were suspended in cold<br />
saline solution (2ml) and tested for amylase activity and<br />
total protein content. The salting out concentration of<br />
crude enzyme was established to be 60% on the basis<br />
of enzyme activity. To obtain complete precipitation<br />
of the crude enzyme, the remaining harvest fl uid was<br />
subjected to ammonium sulfate precipitation at 60%<br />
saturation. For this purpose, solid ammonium sulfate<br />
(195g) was added gradually with mechanical stirring<br />
to 500ml of harvest fl uid at 4˚C to a saturation of<br />
60%. The precipitate so formed was separated by<br />
centrifugation (8000g) for 15 min., again suspended in<br />
cold saline solution (100ml) and dialyzed in cold against<br />
1 L of 0.05M Phosphate buffer (pH 7) for 20 h. After<br />
dialysis, the solution was centrifuged and supernatant<br />
obtained was designated as fraction-I. It was used for<br />
zinc sulfate precipitation.<br />
Zinc Sulfate Precipitation<br />
About 100ml of the pooled supernatant (fraction-I)<br />
with activity of 20 U/mg protein was diluted with<br />
1.4 L of 0.05M Phosphate buffer (pH 7) to obtain 2<br />
units of absorbance at 280 nm. To this solution 75 ml<br />
of 0.1M zinc sulfate was added drop wise with stirring<br />
at 0˚C in an ice bath. The precipitate thus formed was<br />
separated by centrifugation (8000g) for 15 min. and<br />
re-suspended in 50ml of 0.4M sodium citrate. This<br />
solution was dialyzed in cold (4ºC) against 240 ml of<br />
0.05M Phosphate buffer, pH 7. The resulting solution<br />
was designated as fraction-II, and subjected to gel<br />
fi ltration chromatography.<br />
Sephadex G-200 Gel Filtration Chromatography<br />
The dialyzed enzyme (fraction-II) was centrifuged at<br />
8000rpm for 15 min. and supernatant was chromatographed<br />
on a column of Sephadex G-200. The sample<br />
(fraction-II) was loaded onto a column of Sephadex<br />
G-200 (1.5cm × 24cm) equilibrated with 0.05M phosphate<br />
buffer (pH 7). The column was eluted at a fl ow<br />
rate of 4.0ml /h with a discontinuous gradient from<br />
0.1 M to 0.8 M phosphte buffrer in the same buffer.<br />
A total of 25 fractions were collected. Fractions (15–18)<br />
with high amylase activity were pooled together, dialyzed,<br />
concentrated by lyophilization and used for further<br />
studies. It was labeled as fraction-III.<br />
Sodium Dodecyl Sulfate Polyacrylamide<br />
Gel Electrophoresis (SDS-PAGE)<br />
After Sephadex G-200 column chromatography, the<br />
fractions (19–23) showing the highest specifi c activity<br />
were pooled, dialyzed, lyophilized and then subjected to<br />
SDS-PAGE. The SDS-PAGE was performed according<br />
to Laemmli (1970) 4 using 12% acrylamide.<br />
Characterization of Purifi ed Enzyme<br />
The Characterization of purifi ed enzyme includes:<br />
Determination of optimum pH, Temperature, effects<br />
of metal ions, effects of inhibitors and chelators and<br />
stability towards temperature, pH, and detergents, kinetic<br />
data, Activity test of α-amylase and protein content.<br />
Determination of Optimum pH of Purifi ed Enzyme<br />
The pH was adjusted using the following buffers: 0.05 M<br />
phosphate (pH 5.0–7.0), Tris-HCl (pH 8.0) and glycine-<br />
NaOH (pH 9.0–12.0). Reaction mixtures were incubated<br />
at 55ºC for 30 min. and the activity of the enzyme<br />
84 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
K. Sreenivasa Rao et al.: Purifi cation and Characterization of Thermostable Amylase from a Strain of Thermoactinomyces thalpophilus KSV 17<br />
was measured. For stability study, the purifi ed enzyme<br />
was diluted in different relevant buffers (pH 6.0–12.0)<br />
in two sets. One set incubated for 2h and another set for<br />
20 h at 55ºC. The relative activity at each exposure was<br />
measured as per assay procedure.<br />
Determination of Optimum Temperature<br />
of Purifi ed Enzyme<br />
The activity of the purifi ed enzyme was determined by<br />
incubating the reaction mixture at different temperatures<br />
ranging from 30 to 95°C for 30 min. in the absence and<br />
presence of 10 mM CaCl 2 . Thermostability of the purifi ed<br />
enzyme was determined in the presence and absence<br />
of CaCl 2 (10mM) by pre-incubating enzyme samples in<br />
phosphate buffer for 30 minutes at temperatures ranging<br />
from 30 to 95ºC.<br />
Determination of Amylase Activity on<br />
Various Metal Ions<br />
Metal ions viz. Ca2+ , Mg2+ , Hg2+ , Fe3+ , Na + , Zn2+ , Mn2+ and Cu2+ (10 mM) were investigated by adding them<br />
into the reaction mixture. The mixture was incubated<br />
for 30 min. at 55°C and the relative amylase activities<br />
were measured.<br />
Determination of Amylase Inhibitors and<br />
Chelators on Purifi ed Enzyme<br />
The effect of various amylase inhibitors (at 5mM), such<br />
as Diethyl pyrocarbonate (DEPC), N-bromo succinimide<br />
(N-BS) cysteine-inhibitors p-chloromercuric benzoate<br />
(pCMB) and β-mercaptoethanol (β-MCE), and a<br />
chelator of divalent cations [Ethylene diamine tetra<br />
acetic acid (EDTA)] were determined by preincubation<br />
with the enzyme solution for 30 min at 55°C before the<br />
addition of substrate. The relative amylase activity was<br />
measured.<br />
Determination of Enzyme Stability in<br />
Presence of Detergents<br />
The compatibility of KSV-17 amylase with local<br />
laundry detergents was investigated in the presence of<br />
10 mM CaCl . The detergents were diluted in distilled<br />
2<br />
water (0.7% w/v), incubated with 0.1ml of enzyme<br />
(500U/ml) for 4 h at 55°C and the residual activity was<br />
determined.<br />
Determination of Enzyme Kinetic Data<br />
of Purifi ed Enzyme<br />
Initial rates of soluble starch hydrolysis was determined<br />
with various concentrations at various substrate<br />
concentrations ( g/100ml: 0.10, 0.20, 0.30, 0.40, 0.50,<br />
0.75, 1.00, 1.25, 1.50, 1.75 and 2.00). The kinetic<br />
constants K m and V max were determined by the method<br />
of Lineweaver-Burk.<br />
Activity Test of α-Amylase and Protein<br />
Content Determination<br />
The α- amylase activity was done using 3, 5-dinitro<br />
salicylic acid as a coupling agent by following mandels<br />
(1976) method. The content of protein was determined<br />
based on method of lowry et al. (1951). 3<br />
RESULTS AND DISCUSSION<br />
Table 1: Summary of purifi cation steps of amylase from T. Thalpophilus KSV 17<br />
Purifi cation step<br />
Total enzyme<br />
activity (U)<br />
Total<br />
protein (mg)<br />
Purifi cation of Amylase of Thermoactinomyces<br />
Thalpophilus KSV 17<br />
The enzyme production was carried in a 2 L fermentor<br />
and the extraction of growth media that contain the<br />
extracellular enzyme with cold centrifugation to get<br />
the raw extract enzyme which further precipitated<br />
by salt of ammonium sulfate at 60% of saturation<br />
grade, and obtained supernatant precipitated with zinc<br />
sulfate, resulting solution was subjected for gel fi ltration<br />
column chromatography using Sephadex G-200, then<br />
subjected for SDS-PAGE was performed according<br />
to Laemmli et al.(1970) 4 using 12% acrylamide fi naly<br />
α- amylase activity was done using 3,5-dinitro salicyclic<br />
acid as a coupling agent according to Mande l (1976)<br />
method. Purifi cation steps were showed in Table1.<br />
Gel fi ltration Chromatography using<br />
Sephadex G-200<br />
The purifi cation of enzyme with Gel fi ltration<br />
chromatography using Sephadex G-200, used phosphate<br />
buffer 0.05M, pH 7 and column eluted with 0.1 M to<br />
0.8 M NaCl as elution buffer. The protein pattern (A ) 280<br />
and activity of α-amylase obtained from gel fi ltration<br />
chromatography using Sephadex G-200. From the<br />
fi gure 1 showed only one peak showed α-amylase activity<br />
observed at 0.4M NaCl concentration that was fraction<br />
(15th–18th), the highest activity was fraction 18th as<br />
showed in fi gure 1.<br />
Specifi c<br />
activity (U/mg)<br />
Purifi cation<br />
fold % Recovery<br />
Crude enzyme 175000 26800 6.52 1.0 100<br />
(NH4)2SO4 precipitation 131000 7890 16.60 2.5 74.85<br />
Sephadex G-200 13560 93 145.80 22.3 7.74<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 85
K. Sreenivasa Rao et al.: Purifi cation and Characterization of Thermostable Amylase from a Strain of Thermoactinomyces thalpophilus KSV 17<br />
Figure 1: Chromatography of α-amylase from Thermoactinomyces thalpophilus KSV 17 at Gel filtration column chromatography using<br />
Sephadex G-200, elution solution of buffer phosphate 0.05M, pH 7.0, column size 1.5 × 24 cm and flow rate 1.0 ml/h.<br />
Homogenity test of Purifi ed Enzyme by SDS-<br />
Polyacrylamide gel Electrophoresis<br />
The result of electrophoresis SDS-polyacrylamide of<br />
purifi ed enzyme can be seen in electrophoregram as<br />
shown in Figure 2. This fi gure showed that the molecular<br />
weight of purifi ed enzyme was 52 kDa.<br />
Characterization of Purifi ed Enzyme<br />
Determination of Optimum pH of Purifi ed Enzyme<br />
The activity (%) of purifi ed enzyme and stability can<br />
be seen in fi gure 3. It showed optimum pH of purifi ed<br />
enzyme is 6.0 and stable in the pH range of 4–7.5 in<br />
case of 2h. preincubation and at 20h. preincubation the<br />
enzyme was stable in the pH range 5.5–7.0 (Figure 3).<br />
Determination of Optimum Temperature<br />
of Purifi ed Enzyme<br />
The activity (%) of purifi ed enzyme at various<br />
temperatures in presence and absence of CaCl 2 can be<br />
seen in fi gure 4. The recorded optimum temp of purifi ed<br />
enzyme was 85ºC in the absence of CaCl 2 , while 90ºC in<br />
the presence of CaCl 2 (Figure 4).<br />
Determination of Inhibitors and Chelators<br />
effect on Purifi ed Enzyme<br />
The inhibitors tested (at 5 mM conc.), diethyl<br />
pyrocarbonate (DEPC) was able to inhibit the almost<br />
completely, N-bromo succinimide (N-BS) exhibited<br />
89% inhibition. Results are similar to Bolton et al.,<br />
(1997) 5 study. This indicated that it is an alpha amylase<br />
and, 51% inhibition was observed with EDTA and slight<br />
stimulation effect by β -mercaptoethanol (fi gure 5).<br />
Determination of Compatibility with<br />
Detergents on Purifi ed Enzyme:<br />
Besides pH, a good detergent amylase is expected to<br />
be stable in presence of commercial detergents. The<br />
amylase from KSV 17 showed excellent stability and<br />
compatibility in the presence of locally available detergents<br />
(Nirma, Wheel, Henko, Surf excel, Ariel and Rin).<br />
Determination of Metal ions Effect on<br />
Purifi ed Enzyme<br />
The data indicates that Ca 2+ , Mg 2+ and Na + have slight<br />
stimulatory effect activation of the enzyme may be<br />
due to activation of metal ions while other ions have<br />
slight inhibitory effect on the enzyme. These results<br />
suggest, the metal ions apparently protected the enzyme<br />
against thermal denaturation and played a vital role in<br />
maintaining the active confi rmation of the enzyme at high<br />
temperatures (Donaghy and McKay (1993) 6 (Figure 6).<br />
Determination of Kinetic data of<br />
Purifi ed Enzyme<br />
The graph of Determination of K and V values<br />
m max<br />
of the purifi ed enzyme can be seen in fi gure 7. From<br />
the graph, the K (Michaelis constant) and V m max<br />
were calculated as 5.2 mg/ml and 0.45 mg/ml/min<br />
respectively. (Figure7). Aguilar et al., (2000) reported<br />
a K of 3.44 mg/ml and V of 0.45 mg/ml/min for<br />
m max<br />
alpha amylase from Lactobacillus. A K of 0.90 g/l was<br />
m<br />
reported for a thertmostable alpha amylase from Bacillus<br />
licheniformis by Ivanova et al., (1993). 7 Krishnan and<br />
Chandra, (1983) 8 reported a K of 1.274 mg/ml and<br />
m<br />
V of 0.738 mg/ml/min for alpha amylase obtained<br />
max<br />
from Bacillus licheniformis CUMC 305.<br />
86 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
K. Sreenivasa Rao et al.: Purifi cation and Characterization of Thermostable Amylase from a Strain of Thermoactinomyces thalpophilus KSV 17<br />
Figure 2: SDS-PAGE of Amylase from T. Thalpophilus KSV 17. Lane A, crude enzyme; Lane B, purified enzyme; Lane C, molecular mass<br />
markers. The molecular weight of standard protein markers used are: BSA (67 kDa), Ovalbumin (45 kDa), Carbonic anhydrase (30 kDa),<br />
Trypsinogen (24 kDa) and α-lactalbumin (14 kDa).<br />
Figure 3: The optimum pH of purified enzyme 6.0, same figure also showed that the purified enzyme was stable in the pH range of 4–7.5<br />
in case of 2h. preincubation group. But in the case of 20 h. preincubation group, the enzyme was stable between pH 5.5 and pH 7.<br />
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 87
K. Sreenivasa Rao et al.: Purifi cation and Characterization of Thermostable Amylase from a Strain of Thermoactinomyces thalpophilus KSV 17<br />
Figure 4: For the study temperature ranging from 30 to 90 o C in the absence and presence of 10 mM CaCl 2 . The result indicates purified<br />
enzyme is stable at 80 o C and showed high activity at 85 o C. The optimum temperature recorded was 85 o C for purified enzyme in the<br />
absence of CaCl 2 , while 90 o C in the presence of CaCl 2 .<br />
Figure 5:: The inhibitors like DEPC, N-BS exhibited 89% inhibition., 51% inhibition was observed with EDTA and slight stimulation effect<br />
by α-mercaptoethanol.<br />
CONCLUSION<br />
Based on above results and discussion, the following<br />
conclusions are made: The analysis of SDS- PAGE<br />
indicates the molecular weight of α-Amylase is 52 kDa<br />
depending on the relative mobility. The purifi ed enzyme<br />
specifi c activity was an increase of 21 fold than the crude<br />
enzyme extract. The purifi ed enzyme has optimum<br />
pH 7.0. The enzyme was stable between pH 6 and<br />
88 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
K. Sreenivasa Rao et al.: Purifi cation and Characterization of Thermostable Amylase from a Strain of Thermoactinomyces thalpophilus KSV 17<br />
Figure 6: The metal ions Ca 2+ , Mg 2+ and Na + have slight stimulatory effect while other slight inhibitory effect on the enzyme.<br />
Figure 7: Amylase activity done according to Bernfield (1955), using 3,5- dinitro salicylic acid as a coupling agent.<br />
pH 7 even after 20 h. preincubation. The optimum<br />
temperature for purifi ed enzyme activity was 85°C<br />
in absence of CaCl 2 while 90ºC in the presence of<br />
CaCl 2 . Amylase inhibitors like DEPC, N-BS and<br />
pCMB showed exhibitory however Ca 2+ , Mg 2+ and<br />
Na + ions have slight stimulatory effect. This purifi ed<br />
enzyme showed good compatibility and stability with<br />
commercial detergents.<br />
REFERENCES<br />
1. Reed G. Enzymes in Food Processing, 2nd ed, Academic Press,<br />
Orlando, 1975, 256–60.<br />
2. Bernfeld P. Amylases, α and β, Methods in enzyme, Vol. I, Academic<br />
Press, New York, 1955, 149–58.<br />
3. Lowry OH. Rosebrough N J, Farr AL, Randall RJ. Adenyl cyclase<br />
activity in human platelets. J Biol Chem1951;193:265–71.<br />
4. Laemmli UK. Purifi cation and Biochemical Charectirisation of Ricin fom<br />
Castor. Nature1970;227:680–7.<br />
5. Bolton DJ, Kelly CT, Fogarty WM. Purifi cation and charecterization of<br />
Amylase of Bacillus. Enz Microb Technol 1997;20:340–3.<br />
6. Ivanova VN, Dobreva EP, Emanuilova EI. Efect of temparature on some<br />
charecteristics thermostable Amylase enzyme. J Biotechnol 1993;28:<br />
277–89.<br />
7. Krishnan T, Chandra AK. Effect of oilseed cakes on alpha-amylase<br />
production by Bacillus licheniformis CUMC305. Appl Environ Microbiol<br />
1982;44:270–4.<br />
8. Domingues CM, Peralta RM. Production of amylase by soil fungi and<br />
partial biochemical characterization of amylase of a selected strain<br />
(Aspergillus fumigatus Fresenius). Can. J Microbiol 1993;39:681–5.<br />
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