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African Journal of
Pharmacy and
Pharmacology
Volume 5 Number 17 8 November, 2011
ISSN 1996-081

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Himanshu Gupta
University of Colorado- Anschutz Medical Campus,
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Molecular Cardiovascular Research Program
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Arizona Health Sciences Center
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Editorial Board
Prof. Fen Jicai
School of life science, Xinjiang University,
China.
Dr. Ana Laura Nicoletti Carvalho
Av. Dr. Arnaldo, 455, São Paulo, SP.
Brazil.
Dr. Ming-hui Zhao
Professor of Medicine
Director of Renal Division, Department of Medicine
Peking University First Hospital
Beijing 100034
PR. China.
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Guangdong Cardiovascular Institute, Guangdong General
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China.
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Faculty of Engineering and Applied Science,
Memorial University of Newfoundland,
Canada.
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Medical Intensive Care Unit
University hospital Ibn Sina, Univesity Mohamed V
Souissi, Rabat,
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Department of Gynaecology and Obstetrics, the 1st
hospital, NanFang University,
China.
Prof. Ma Hui
School of Medicine, Lanzhou University,
China.
Prof. Gu HuiJun
School of Medicine, Taizhou university,
China.
Dr. Chan Kim Wei
Research Officer
Laboratory of Molecular Biomedicine,
Institute of Bioscience, Universiti Putra,
Malaysia.
Dr. Fen Cun
Professor, Department of Pharmacology, Xinjiang
University,
China.
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Department of Chemical Engineering, Annamalai
University,
Annamalai Nagar, Tamilnadu,
India.
Prof. Ehab S. EL Desoky
Professor of pharmacology, Faculty of Medicine
Assiut University, Assiut,
Egypt.
Dr. Yakisich, J. Sebastian
Assistant Professor, Department of Clinical Neuroscience
R54
Karolinska University Hospital, Huddinge
141 86 Stockholm ,
Sweden.
Prof. Dr. Andrei N. Tchernitchin
Head, Laboratory of Experimental Endocrinology and
Environmental Pathology LEEPA
University of Chile Medical School,
Chile.
Dr. Sirajunnisa Razack
Department of Chemical Engineering,
Annamalai University, Annamalai Nagar, Tamilnadu,
India.
Dr. Yasar Tatar
Marmara Unıversıty,
Turkey.
Dr Nafisa Hassan Ali
Assistant Professor, Dow institude of medical technology
Dow University of Health Sciences,Chand bbi Road, Karachi,
Pakistan.
Dr. Krishnan Namboori P. K.
Computational Chemistry Group, Computational
Engineering and Networking,
Amrita Vishwa Vidyapeetham, Amritanagar, Coimbatore-
641 112
India.
Prof. Osman Ghani
University of Sargodha,
Pakistan.
Dr. Liu Xiaoji
School of Medicine, Shihezi University,
China.

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African Journal of Pharmacy and Pharmacology
International Journal of Medicine and Medical Sciences
Table of Contents: Volume 5 Number 17 8 November, 2011
ences
Research Articles
ARTICLES
A review on Hyssopus officinalis L.: Composition and biological activities 1959
Fatemeh Fathiazad and Sanaz Hamedeyazdan
Identification of phenolic compounds and assessment of in
vitroantioxidants activity of 30% ethanolic extracts derived from two
Phyllanthus species indigenous to Malaysia 1967
Elrashid Saleh Mahdi, Azmin Mohd Noor, Mohamed Hameem Sakeena,
Ghassan Z. Abdullah, Muthanna Abdulkarim and Munavvar Abdul Sattar
Cultural importance and antibacterial activity of Ziziphus mucronata
(Willd.) in the Umlazi community in Durban 1979
R. M. Coopoosamy, K. K. Naidoo and N. J. Ndlazi
Altitude-related changes in activities of carbon metabolism enzymes
and secondary plant products-menthoforon an active pharmaceutical
constituents yield in pippermint (Mentha piperita L. Var. Kukarail) 1983
A. Misra and N. K. Srivastava
Simultaneous determination of gatifloxacin and dexamethasone sodium
phosphate in bulk and pharmaceutical formulations by HPLC 1990
K. R. Sireesha and K. Prakash
Anti-inflammatory and wound healing activity of Fagonia schweinfurthii
alcoholic extract herbal gel on albino rats 1996
Saleh I. Alqasoumi, Hasan S. Yusufoglu and Aftab Alam

Table of Contents: Volume 5 Number 17 8 November, 2011
ences
ARTICLES
The effects of maximal aerobic exercise on cortisol and thyroid hormones
in male field hockey players 2002
Malik BEYLEROGLU
Cationic liposomes as gene delivery system 2007
Mohsen M. Mady
Nitrous oxide versus pethidine with promethasine for reducing labor
pain 2013
Batool Teimoori, Nahid Sakhavar, Masoome Mirteimoori and
Behzad Narouie
Differential analysis of human kidney stone samples using electrospray
ionization mass spectrometry 2018
Zhiquan Zhou, Yongzhong Ouyang, Xiangtai Zeng, Tingting Zhang, Bin Jia,
Xinglei Zhang, Huanwen Chen and Jianhua Ding
Radical scavenging and antioxidant activity of Hibiscus rosasinensis
extract 2027
Rajesh Mandade, S. A. Sreenivas, D. M. Sakarkar and Avijit Choudhury
Therapeutic monitoring of isoniazid, rifampicin, ethambutol and
pyrazinamide serum levels in the treatment of active pulmonary
tuberculosis and determinants of their serum concentrations 1713
Servet Kayhan and Alper Akgüneş

African Journal of Pharmacy and Pharmacology Vol. 5(17), pp. 1959-1966, 8 November, 2011
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP11.527
ISSN 1996-0816 © 2011 Academic Journals
Review
A review on Hyssopus officinalis L.: Composition and
biological activities
Fatemeh Fathiazad* and Sanaz Hamedeyazdan
Department of Pharmacognosy, Faculty of Pharmacy, Tabriz University of Medical Sciences, Iran.
Accepted 5 October, 2011
Hyssopus officinalis L. (Hyssop) is one of the most popular herbal preparations, mainly distributed in
the East Mediterranean to central Asia. The plant has been used traditionally for medicinal purposes;
generally, these therapeutic uses and health benefits of hyssop are largely based on folklore rather
than on scientific substantiation, making it a good candidate to gather documentations, including the
phytochemical content, in vitro experiments, animal models and human studies available in the recent
scientific studies. A literature review on the chemical and biological aspects of the plant indicates that
the main constituents of H. officinalis include several polyphenolic compounds, primarily the
flavonoids apigenin, quercetin, diosmin, luteolin and their glucosides followed by other phenolic
compounds chlorogenic, protocatechuic, ferulic, syringic, p-hydroxybenzoic and caffeic acids. Reports
on the essential oils extracted from aerial parts of H. officinalis revealed several principal components,
including terpenoids pinocamphone, isopinocamphone and β-pinene. Hyssop has moderate antioxidant
and antimicrobial activity against Gram positive and negative bacteria activities together with antifungal
and insecticidal antiviral properties in vitro. Animal model studies indicate myorelaxant, antiplatelet and
α-glucosidase inhibitory activities for this plant. However, human studies, adverse reactions and
clinical trials examining the reported properties of hyssop are absent and needs more attention to
determine whether biological differences in findings of the studies reflect the different isolation
procedures, different types of plant material used, collection time, locations or different chemotypes.
Key words: Hyssopus officinalis L., phenolic compounds, essential oil, extract.
INTRODUCTION
One of the most frequently consumed herbal remedies
available today is the hyssop preparations prepared from
Hyssopus officinalis (L) which is gaining increased
importance as a minty flavor, condiment and spices in
food industries as well (Dragland et al., 2003; Jung et al.,
2004; Lugasi et al., 2006). Not surprisingly, like many
other herbal preparations used in traditional medicinal
cultures, the therapeutic uses and health benefits of
hyssop are largely based on folklore rather than on
scientific substantiation. Regardless of the wide range of
literatures suggesting health benefits of herbal remedies
associated with hyssop, evidence-based information
regarding the effects of hyssop is quite limited.
Developing an efficient herbal remedy is reliant to a
*Corresponding author. E-mail: fathiazad@tbzmed.ac.ir. Tel:
+98 411 6692405. Fax: +98 411 3344798.
better understanding of the relationship between
chemical constituents and biological properties of the
natural product. In view of these aspects, natural
products, particularly higher plant species, continue to be
important sources of medicine and supplementary health
products which represent a challenge to science due to
their various properties, including chemical diversity,
synergism to biological activity and variable
compositions. Herein, we tried to gather detailed
documentations of the available scientific papers related
to the bioactivity and potential health benefits of hyssop
ensuring a high quality herbal medicine to meet the ever
more demands of the public.
NOMENCLATURE
The genus Hyssopus comprises aromatic perennial herbs
or subshrubs that are mainly cultivated, but can also be

1960 Afr. J. Pharm. Pharmacol.
found in the wild. The inflorescence is 20 to 25 cm long,
false spikelike, composed of 4 to 10 flowered
pseudoverticils in the terminal. The root of H. officinalis is
a strongly branching, multi-headed tap root. The stems
are 0.5 to 0.7 m in height, erect or decumbent dividing
into many woody stems. The leaves are opposite, shiny
dark-green, entire-edged and lanceolate or oblong,
obtuse to acuminate that are 2 to 4 cm long and 0.5 to 1
cm wide. Hyssopus L. comprises of about 10 to 12
species distributed mainly in the East Mediterranean to
central Asia. H. officinalis L. (Family: Lamiaceae alt.
Labiatae) has a long history of medicinal use as
carminative, tonic, antiseptic, expectorant and cough
reliever. Despite having a slightly bitter taste, H. officinalis
is often used as a minty flavor and condiment in food
industries. The merit of the traditional use of H. officinalis
has been supported by some prior studies from the
genus Hyssopus, providing several biologically active
constituents especially main compounds from essential
oils. Although, a great body of papers refers to the
composition of H. officinalis oil, far too little attention has
been paid to the chemical constituent structures present
in the plant. Herein, we offer documentations including
the phytochemical content, in vitro experiments, animal
models and human studies available in the recent
scientific literatures.
PHYTOCHEMICAL CONSTITUENTS
The phytochemical study of the aerial parts of H.
officinalis cultivated in Xinjiang, China, revealed isolation
of two new flavonoid glycosides and nine other known
flavonoids from the ethanolic extract of the plant. The
new compounds were identified as; quercetin 7-O-b-Dapiofuranosyl-(1→2)-b-dxylopyranoside
(1) and quercetin
7-O-b-D-apiofuranosyl-(1→2)-b-D-xylopyranoside 30-Ob-D-glucopyranoside
(2), together with nine known
flavonoids apigenin (3), apigenin 7-O-b-Dglucopyranoside
(4), apigenin 7-O-b-Dglucuronopyranoside
methyl ester (5), luteolin (6),
apigenin 7-O-b-D-glucuronide (7), apigenin 7-O-b-Dglucuronopyranoside
butyl ester (8), luteolin 7-O-b-Dglucopyranosid
(9), diosmin (10) and acacetin 7-O-a-Lrhamnopyranosyl-(1→6)-b-D-glucopyranoside
(11). The
free radical scavenging activity of the compounds 1 to 11
was also determined using 2,2-diphenyl-1-picrylhydrazyl
(DPPH). The isolated compounds were found to possess
noble radical scavenging activity. Out of the isolated
compounds, 1, 2, 6 and 9 with IC50 values in the range
of 2.81 to 10.41 mmol/L exhibited stronger scavenging
activity on DPPH assay than butylated hydroxytoluene
and L-ascorbic acid as standards (Wang and Yang,
2010).
Mario et al. (1997) revealed the presence of the most
widespread class of secondary metabolites, flavonoids, in
H. officinalis L. using high-performance liquid
chromatography and magnetic-resonance imaging (NMR)
spectroscopy. The major flavone, diosmin, was present in
the plant with 51 and 40.5% in sepals and leaves,
respectively that were identified as the total content of
diosmin in whole plant. Nonetheless, there were changes
in diosmin levels during the development of hyssop
leaves, stems and roots. The other identified compound
in the plant was considered to be isoferulyl D-glucose
ester (Marin et al., 1998). Previously, Hilal et al. (1979)
reported isolation of seven glycosides of flavanone type
from H. officinalis where the aglycon of the glycosides
were determined as 5,4'-dihydroxy-7,3'-dimethoxy
flavanone.
The content of free phenolic acids (PhAs) for ten
popular medicinal plants used in Polish phytotherapy
including H. officinalis belonging to the family Lamiaceae
were determined by a rapid, selective and accurate
extraction method combining solid-phase extraction and
high-performance liquid chromatography. Considering the
findings of the study, methanolic extract of the H.
officinalis was shown to be rich in phenolic compounds,
especially high in chlorogenic, protocatechuic, ferulic,
syringic, p-hydroxybenzoic and caffeic acids followed by
vanillic, p-coumaric, rosmarinic and gentisic acids
(Murakami et al., 1998; Varga et al., 1998a; Kochan et
al., 1999; Zgorka and Głowniak, 2001). Elsewhere, the
presence of caffeic acid and its derivatives in the roots of
H. officinalis L. cultivated in Romania with a content of
1.69% was reported. Additionally, rosmarinic acid, ferulic
acid and phenylpropanic compounds were also identified
in the plant by chromatography and spectrophotometric
analyses (Benedec et al., 2002). Later, Proestos et al.
(2005) employed reversed phase high-performance liquid
chromatography with UV detection for the identification
and quantification of the phenolic compounds for some
plant extracts including H. officinalis. The most abundant
phenolic acids in H. officinalis were considered to be
ferulic acid (13.2 mg/100 g of dry sample) and caffeic
acid (6.5 mg/100 g of dry sample). Moreover, syringic,
gentisic and p-hydroxybenzoic acids along with two
flavonoids (+)-catechin and apigenin were also detected
in the genus H. officinalis (Proestos et al., 2005).
ESSENTIAL OIL
Garg et al. (1999) reported on the characteristics of the
oil of H. officinalis L. ssp. officinalis cultivated in the North
Indian plains as an annual crop. The GC and GC-MS
analysis of the colourless essential oil led to the
identification of 21 compounds representing 95.6% of the
oil, comprising seven monoterpene hydrocarbons
(32.3%), five oxygenated monoterpenes (60.5%), one
phenol (0.2%) and six sesquiterpene hydrocarbons
(0.35%). The major constituents of the camphorous
predominant monoterpenes of the oil were
pinocamphone (49.1%) >β-pinene (18.4%)

isopinocamphone (9.7%) (Shah et al., 1986; Garg et al.,
1999). Myrtenol methyl ether, myrtenic acid, methyl
myrtenate, pinic acid, cis-pinic acid, (+)-2hydroxyisopinocamphone,
pinonic acid and cis-pinonic
acid were identified for the first time in H. officinalis oil by
Joulain (Joulain, 1976; Joulain and Ragault, 1976). The
analysis of the composition of two essential oils from H.
officinalis L. ssp. officinalis grown in two different
localities near Urbino (Marche, Italy) revealed major
essential oil components as pinocamphone (34 and
18.5%), isopinocamphone (3.2 and 29%) and β-pinene
(10.5 and 10.8%). However, they showed detectable
differences in the ratio of
pinocamphone/isopinocamphone and in the percentage
of linalool (0.2 and 7.9%) and camphor (0.3 and 5.3%).
All the same, the essential oils exhibited antifungal
activity against 13 strains of phytopathogenic fungi; the
essential oil of the plants grown at 1000 m above sea
level was superior (Daniele et al., 2004). Another study
performed with the H. officinalis from U.P. Himalaya
explained the presence of isopinocamphone 38.1%,
pinocarvone 20.3%, 1-8-cineole 12.2% and β-pinene
10.2% as the main compounds and the total 47 chemical
constituents represented 98.56% of the total oil (Shah,
1991). Salma et al. (2002) identified H. officinalis as a
new source of essential oil in Egypt that was
characterized by high content of β-pinene (19.60%),
pinocamphone (19.20%) and camphor (16.3%). The
highest yield of oil production was determined at the
flowering stage of growth, in July (Salma et al., 2002).
Bulgarian and Italian essential oils of H. officinalis L. were
analyzed and the main difference between these two
kinds of hyssop oils was in the higher quantity of
terpenoids in Bulgarian oil. Isopinocamphone and its
biogenetic precursor β-pinene, camphor, 1,8-cineole,
cubenene and germacrene B were detected in the
Bulgarian oil, whereas in the case of Italian hyssop oil, βpinene
was the minor component and phenyl propanoids,
safrole and benzyl benzoates, were the predominant
constituents of the oil (Manitto et al., 2004). Garcia-
Vallejo et al. (1995) examined the volatile oil of H.
officinalis grown in Spain by gas chromatography (GC)
and gas chromatography/mass spectrometry (GC/MS)
and reported a high content of 1,8-cineole (52.89%) and
β-pinene (16.82%) as the main components of the oil. In
another study, Özer et al. (2005) analyzed the essential
oil of Hyssopus officinalis L. subsp. angustifolius (Bieb.)
Arcangeli wild-growing in the Eastern Anatolian region of
Turkey. The essential oil of this plant demonstrated the
presence of many monoterpenes that were identified by
gas chromatography; about thirty-four components were
characterized, representing 91.0% of the total
components detected. The main components were
identified as pinocarvone (36.3%), pinocamphone
(19.6%), β-pinene (10.6%), 1,8-cineole (7.2%) and
isopinocamphone (5.3%) (Hold and Sirisoma, 2002; Ozer
et al., 2005). Salvatore et al. (1997) performed detailed
Fathiazad and Hamedeyazdan 1961
examination of the essential oil of H. officinalis L. var.
decumbens from the High-Provence Alps in Banon,
France. Linalool (49.6%), 1,8-cineole (13.3%), limonene
(5.4%), β-caryophyllene (2.8%), β-pinene (3.0%) and αpinene
(2.4%) were identified as the major components
of the essential oil, while iso-pinocamphone and
pinocamphone were present at a lower content level
suggesting the existence of different chemotypes in that
province (Salvatore et al., 1997). Analysis of the essential
oils of H. officinalis L. var. decumbens (HOD) from
France (Banon) and H. officinalis (HO) from Italy by GC
and GC/MS exhibited notable differences in the amounts
of components. The bicyclic monoterpene ketones,
pinocamphone and isopinocamphone, were present in
HO, but their percentages were very low in HOD, where
instead linalool (49.6%), 1,8-cineole (13.3%) and
limonene (5.4%) were predominant (Salvatore et al.,
1998). Chemical analysis for three essential oils of
endemic H. officinalis cultivated in Yugoslavia: f. albus
Alef., f. cyaneus Alef. and f. ruber Mill. showed that
components mainly composed of cis- and transpinocamphone
and pinocarvone, together with lesser
amounts of germacrene D, bicyclogermacrene, elemol
and spathulenol (Chalchat et al., 2001). Furthermore, the
presence of aliphatic fatty acids, such as palmitic acid
15.60%, stearic acid 10.73%, linolenic acid 63.98%,
arachidic acid 2.64% and eicosadienoic acid 0.68% in the
Romanian hyssop oil was determined (Benedec et al.,
2002). In our previous study for the essential oil from
Iran, the main constituents were myrtenyl acetate
(74.08%), camphor (6.76%), germacrene (3.39%),
spathulenol (2.14%), caryophyllen oxide (2.13%) and βcaryophyllene
(2.10%) with lesser amounts of cis-sabinol
(1.75%), β- bourbonene (1.47%) and bornyl acetate
(1.42%) (Fathiazad et al., 2011).
Kerrola et al. (1994) investigated the volatile
compounds of the four phenotypes of H. officinalis L.
differentiated by the color of the corolla, by Soxhlet
extraction and Supercritical Fluid Extraction (SFE). The
main components of all extracts were identified as
pinocamphone, isopinocamphone, and pinocarvone.
However, differences in the quantity of the constituents
were worth mentioning; the lower amount of
monoterpene hydrocarbons and a higher amount of
oxygenated hydrocarbons were obtained in the SFE
(Kerrola et al., 1994). Detailed examination of the SFE of
the hyssop oil was undertaken by Kazazi et al. (2007) at
various pressures, temperatures, extraction (dynamic and
static), times and modifier (methanol) concentrations.
Considering the impacts of different factors during the
extraction, it was shown that the composition of the
extracted oils was significantly influenced by the
operating conditions. Major components of the extracts
under different SFE conditions were sabinene (4.2 to
17.1%, w/w), iso-pinocamphene (0.9 to 16.5%) and
pinocamphene (0.7 to 13.6%). Consequently, SFE
offered more choices with parameters for the extraction

1962 Afr. J. Pharm. Pharmacol.
of different components of the hyssop oil (Kazazi et al.,
2007). Kazazi and Rezaei (2007) in another study
evaluated effects of various parameters on the selective
extraction of compounds from hyssop using SFE and
hydrodistillation. Sabinene, pinocamphene and isopinocamphene
were the major compounds applying SFE
with different operational conditions. The optimized
conditions of SFE for the highest extraction selectivity of
pinocamphene and iso-pinocamphene were achieved at
100 atmosphere, 45°C temperature, with 4.5 µl (0.14%,
w/w) methanol, dynamic extraction time of 20 min and
static extraction time of 25 min. Nonetheless, the results
of the study suggested that the hyssop collected from
Iran could be a special chemotype with a high sabinene
concentration (11.04%) (Kazazi and Rezaei, 2009). More
recently, Langa et al. (2009) studied the effects of
pressure, temperature and flow rate of CO2, as well as
the particle size of the vegetable material, on the yield
and composition of the SFE of essential oil from H.
officinalis in comparison with HD extraction. The major
compounds for both techniques were 1,8-cineol
(eucalyptol) (60 to 75%) followed by terpinen-4-ol (4 to
10%), pinocarvone (2 to 6%) and β-pinene (1 to 6%). In
spite of the major similar compounds with comparable oil
yields for both SFE and HD methods, heavier compounds
were detected for the oil obtained from SFE technique
(Toth et al., 1989; Langa et al., 2009).
On the whole, the essential oil content may vary
considerably within a single species from one growth
season to another, affected by extraction method,
climatic parameters and agrotechnical factors
(Benhammou et al., 2008; Ghalem and Mohamed, 2009;
Xu et al., 2011). Additionally, many plants have various
chemotypes that differ in their both quantitative and
qualitative diversity in the composition of essential oils
obtained (Varga et al., 1998b). Further studies are
mandatory to determine the origin of the differences
observed during examinations.
IN VITRO STUDIES
Antimicrobial and antioxidant activities
Mazzanti et al. (1998) published a paper in which they
reported that essential oil of both H. officinalis L. and H.
officinalis L. var decumbens possessed strong
antimicrobial activity in vitro. The findings of the study
showed that all yeasts including seven strains of Candida
albicans, Candida krusei and Candida tropicalis were
strongly inhibited by both species. In liquid medium the
minimal inhibitory concentration (MIC) of H. officinalis L.
was 41.2% v/v for bacteria and between 0.6 and 1.2% v/v
for yeasts, while the MIC of var. decumbens was
between 0.15 and 0.6% v/v for the Gram positive
bacteria, 0.3 and 1.2% v/v for the Gram negative bacteria
and 0.15 and 0.3% v/v for the yeasts. Regarding the
contribution of pure components to the antimicrobial
activity of the oils, pinocamphone and isopinocamphone
were present in H. officinalis L. (4.4 and 43.3%,
respectively), and instead linalol (51.7%), 1,8-cineole
(12.3%) and limonene (5.1%) were predominant in var.
decumbens representing the special microbiological
properties of the essential oils. On the whole, the effect of
var. decumbens was generally bactericidal. Linalol and
1,8-cineole, may contribute to the greater antimicrobial
activity of var. decumbens compared to H. officinalis L.,
while limonene may be responsible for the antimycotic
action observed in both oils (Mazzanti et al., 1998).
Marino et al. (2001) evaluated three groups of essential
oils including hyssop oil for their inhibitory effects against
nine strains of Gram negative bacteria and six strains of
Gram positive bacteria. On the contrary to the previously
published paper by Mazzanti et al. (1998) the findings of
the study exhibited that the hyssop oil in general was less
inhibitory against different strains of bacteria, suggesting
variation in the composition of the essential oils according
to the environmental conditions and plant chemotypes
(Marino et al., 2001).
Recently, Kizil et al. (2010) evaluated antimicrobial and
antioxidant activities of the essential oil of H. officinalis
(L.) collected from wild in the Southeast Anatolian,
Turkey. Isopinocamphone (57.27%), (-)-β-pinene
(7.23%), (-)-terpinen-4-ol (7.13%), pinocarvone (6.49%),
carvacrol (3.02%), p-cymene (2.81%) and myrtenal
(2.32%) were determined as the major components of the
hydrodistilled essential oil by GC-MS analysis. The
essential oil with 5 and 10 µl concentrations was carried
out for anti-microbial disc diffusion tests. The results of
the study were indicative of the oils strong antimicrobial
activities against Staphylococcus pyogenes,
Staphylococcus aureus, C. albicans and Escherichia coli,
but not against Pseudomonas aeruginosa. The
antioxidant activity of H. officinalis essential oil was lower
as compared to butylated hydroxytoluene and ascorbic
acid. Generally, hyssop essential oil showed relatively
low antioxidant activity and good antimicrobial activity
against some test organisms (Kizil et al., 2010). In
addition to the all cited papers indicating the importance
of this genus for its antibacterial activity, publication of a
patented product identified as KR 2005073080 of 2005-
07-13 is an extra confirmation of the fact. The invention
comprises of an anti-acne composition which exhibits
excellent anti-bacterial activity to propionbacterium acnes
as causative bacteria of acne, while no adverse reaction
to human body by comprising essential oil extracted from
plants including H. officinalis as effective ingredient.
Besides, another patent product has also been reported
as JP 2004262861 of 2004-09-24 with the aim of
cosmetics skin-conditioning and antiwrinkle topical
formulations containing 0.01% H. officinalis and 50%
ethanolic extract (Handa, 2004; In Hong et al., 2005).
The essential oil of H. officinalis L. subsp. angustifolius
and methanolic extract of the plant were examined for

their in vitro antimicrobial and antioxidant activities.
Although, the methanol extract in the DPPH assay
provided 50% inhibition at a concentration of 117.0 µg/ml
and 40% inhibition at the concentration of 2 g/L in linoleic
acid test system, it showed no effective activities in the
antimicrobial assays, whereas, the essential oil exhibited
activity against eight bacteria, ten fungi and yeast, C.
albicans, with MIC values ranging from 15.625 to 250
µl/ml; no distinctive anti oxidant properties were achieved
for the essential oil (Ozer et al., 2006).
Ebrahimzadeh et al. (2010) employed six different in
vitro methods for evaluating antioxidant and free radical
scavenging activities of methanolic extract of the aerial
parts of H. officinalis L. var. angustifolius along with three
other plants. They showed that it showed potent to
modrate antioxidant activities in reducing powers and
DPPH radical-scavenging as well as Fe2+ chelating
ability assays, respectively. Although, in the case of nitric
oxide and hydrogen peroxide scavenging and ferric
thiocyanate methods, the results for the anti oxidant
activities of H. officinalis L. extracts were very low and
weak (Ebrahimzadeh et al., 2010). SFE extraction of
antioxidant fractions from certain Lamiaceae herbs with
their antioxidant capacity was evaluated (Babovic et al.,
2010). Antioxidant activity of the obtained extracts,
including H. officinalis were determined by measuring
their ability to scavenge stable DPPH free radical and
reactive hydroxyl radical during the Fenton reaction
trapped by 5,5-dimethyl-1-pyroline-N-oxide, using
electron spin resonance spectroscopy. According to the
results of the study, hyssop extract showed much weaker
antioxidant activity as compared to the rosemary, sage,
and thyme extracts in different methods of antioxidant
evaluations (Dragland et al., 2003; Fernandez-Lopez et
al., 2003; Babovic et al., 2010). Ludmila and Viera (2005)
assessed the antiradical activity and the reduction power
of H. officinalis extracts in another study. All the extracts
showed high activities by both evaluation criteria.
Besides, among the phenolic acids, gallic acid was found
to be the most active component in scavenging free
radicals and caffeic acid had the highest reducing power.
In a study conducted by Glamoålija et al. (2005),
essential oil of the H. officinalis L. was evaluated for its
antifungal activity against Mycogone perniciosa (Mang),
one of the major pathogenic diseases of the cultivated
mushroom Agaricus bisporus (Lange) Imbach in Serbia.
The findings of the study revealed its positional antifungal
activity with minimal inhibitory quantity of 5 µl/ml and a
minimal fungicidal quantity of 15 to 20 µl/ml. These kinds
of studies have been placed in the focus of medical and
aromatic plants investigations for their antifungal
properties since relative biological control systems are
not much used in mushroom cultivation (Ghfir et al.,
1994; Ghfir et al., 1997; Glamoålija et al., 2005; Raila et
al., 2009). Twelve essential oils from Mediterranean
aromatic plants were tested at different doses against
four fungi: Botrytis cinerea, Penicillium italicum,
Fathiazad and Hamedeyazdan 1963
Phytophthora citrophthora, and Rhizopus stolonifer. The
findings of the study revealed weak to moderate fungicide
activities in the case of hyssop oil; however, these
essential oils together with hyssop oil could be
considered as natural preservatives for food products
(Camele et al., 2010). Motiejunaite and Kalediene (2003)
carried out an antifungal screening for essential oils of
some Lamiacae plants using agar-diffusion method. In
most cases including H. officinalis L. a complete inhibition
of Aspergillus niger growth was observed at 0.5 to 1.5%
v/v concentrations.
Pavela (2004) investigated insecticidal activities of
eight medicinal plants including H. officinalis in third instar
larvae of Egyptian cottonworm (Spodoptera littoralis).
Methanolic extract of H. officinalis at the concentration of
10 % (w/v) significantly affected the growth indexes which
showed a certain degree of larval toxicity with 1.78 LC50
and a range of 1.66 and 1.82 confidence interval of 95%
(Pavela, 2004).
Anti viral activity
Crude extracts of the dried leaves of H. officinalis were
also tested for its effectiveness on inhibition of human
immunodeficiency virus (HIV) replication. Not only a safe
non-toxic activity was determined for the uninfected Molt-
3 cells, but also, a strong anti-HIV activity was revealed
as measured by inhibition of syncytia formation, HIV
reverse transcriptase (RT) and p17 and p24 antigen
expression. In the experiment, either extracts from direct
extraction, after removal of tannins or from the residue
after dialysis of the crude extract, also showed good
antiviral activity. Eventually, Kreis et al. (1990) concluded
that the hyssop extracts contained caffeic acid,
unidentified tannins, and possibly a third class of
unidentified higher molecular weight compounds which
exhibited strong anti-HIV activity, and might be useful in
the treatment of patients with AIDS (Kreis et al., 1990).
In another study conducted by Gollapudi et al. (1995), an
isolated polysaccharide (MAR-10) from the aqueous
extract of H. officinalis was examined for its activity
against HIV-1 (SF strain) in HUT78 T cell line and
primary cultures of peripheral blood mononuclear cells.
They demonstrated that the MAR-10 inhibited HIV-1
replication in a concentration-dependent manner with no
substantial direct toxicity or effect on lymphocyte
functions or CD4+ and CD8+ T cell counts (Gollapudi et
al., 1995).
Other activities
Methanolic and hexane extracts of twelve plants including
H. officinalis that are used in traditional European
medicine to treat different central nervous system
disorders were tested for the symptomatic treatment of

1964 Afr. J. Pharm. Pharmacol.
Alzheimer’s disease using Ellman’s colorimetric method.
Since the therapy of early and moderate stages of
Alzheimer’s disease is mainly based on the choline
esterase inhibitors; effects of the plant extracts on
acetylcholinesterase (AChE) and butyrylcholinesterase
(BuChE) inhibitors were investigated (Wszelaki et al.,
2010). Ultimately, H. officinalis revealed no significant
inhibitory activity, as the methanolic and hexane extracts
showed 5.2 ± 8.2 and 29.6 ± 2.3 AChE inhibition (%) and
11.5 ± 0.5 and 23.2 ± 2.0 BuChE inhibitions (%) at the
concentrations of 100 mg/ml -1 , respectively (Wszelaki et
al., 2010).
Animal model studies
Matsuura et al. (2004) evaluated aqueous methanol
extracts of dried H. officinalis leaves for their αglucosidase
inhibitory activity. The active principles
against α-glucosidase, prepared from rat small intestine
acetone powders, were isolated and the amount of
glucose derived from sucrose in the reaction mixture was
measured. The extract showed inhibitory activity which
led to the further isolation and identification of the
responsible compounds for the α-glucosidase inhibitory
activity. The structures of the two isolated compounds
were determined to be (7S,8S)-syringoylglycerol-9-O-(60-
O-cinnamoyl)-β-d-glucopyranoside (1) and (7S,8S)syringoylglycerol
9-O-β-d-glucopyranoside (2) by analysis
of physical and spectroscopic data together with chemical
syntheses that exhibited 53 and 54% inhibitory activity at
the concentration of 3 × 10 -3 M for the compounds 1 and
2, respectively (Matsuura et al., 2004). In another study,
Miyazaki et al. (2003) evaluated the α-glucosidase
inhibitory effects of the hyssop extracts on
hyperglycemia, intestinal carbohydrate absorption by
examining the inhibitory effects on intestinal carbohydrate
absorption in rat everted gut sac and carbohydrateloaded
hyperglycemia in mice. According to the results, in
the presence of 0.5 and 1.0 mg/ml hyssop extracts, the
carbohydrate-loaded excessive increase in blood glucose
was inhibited within 120 min, suggesting that hyssop
might be a useful supplemental food for inhibiting of
postprandial hyperglycemia (Miyazaki et al., 2003).
Confirming this idea, they had provided a patented
product identified as JP 2004256467 of 2003-50400 from
the derivatives of this plant as α-glucosidase inhibitors
(Miyazaki et al., 2004). Elsewhere, the inhibitory activities
of some plants used in Lebanon traditional medicine
containing H. officinalis extracts against angiotensin
converting enzyme (ACE) and digestive enzymes related
to diabetes were investigated (Loizzo et al., 2008). They
demonstrated that the H. officinalis chloroform extract
was active only on the α-glucosidase enzyme, with IC50
values ranging from 127.3 to 908.4 µg/ml. At the same
time the n-hexane extract of the H. officinalis exhibited a
strong inhibitory potency against ACE (IC50 values of
52.0 µg/ml) (Loizzo et al., 2008). Churl et al. (2005)
carried out an experiment to study the simulative or
sedative effects of some inhaling essential oils by using a
forced swimming test with mice. Inhalation of the hyssop
oil (P < 0.01) increased the immobile state in mice that
were artificially over agitated by an intra parental injection
of caffeine (a psycho-stimulant). Accordingly, the authors
stated that the inhaling fragrant hyssop oil possessed
sedative effects (Churl et al., 2005).
Essential oils extracted from different plants including
H. officinalis were tested for their antiplatelet activity and
inhibition of clot retraction in guinea pig and rat plasma.
As Tognolini et al. (2006) mentioned in their study,
phenylpropanoid moiety is a favorable chemical feature
for the inhibition of platelet aggregation and lack of this
moiety in the hyssop oil is responsible for the oil to be
inactive.
The myorelaxant effect of the hyssop essential oil on
isolated preparations of guinea-pig and rabbit intestinal
musculature was determined (Lu et al., 2002).
Isopinocamphone, the major component of the essential
oil, was considered to be responsible of the relaxing
effect. Accordingly, essential oil and isopinocamphone
inhibited the acetylcholine- and BaCl2-induced
contractions in guinea-pig ileum in a concentrationdependent
manner (IC50 42.4 and 61.9 µg/ml to
acetylcholine; 48.3 and 70.4 µg/ml to BaCl2), whereas
limonene or β-pinene left tissue contraction was
unchanged. Nonetheless, synergic actions among the
other several components of the essential oil could not be
excluded. They believed that the myorelaxant activity
induced by the hyssop oil could originate from its
interaction with the plasma membrane and subsequent
alteration of the ionic channels. Considering the inactivity
of the β-pinene and limonene, it had been suggested that
the interaction not only depends on the lipophilicity of the
essential oil and its components, but also on the chemical
structure of the components of the essential oil (Lu et al.,
2002). Mazzanti et al. (1998) reported the spasmolytic
activity of the essential oil from H. officinalis L. var.
decumbens. The essential oil and its major pure
components, linalool, 1,8-cineole and limonene inhibited
the acetylcholine- and BaCl2-induced contractions on
isolated guinea-pig ileum with IC50 values of 37, 60, 10
and 51 µg/ml, correspondingly. Generally, it has been
suggested that the hyssop oil would provide us with
valuable feature of myorelaxant activity in antispasmodic
remedies (Mazzanti et al., 1998).
Cytotoxicity of the essential oils of H. officinalis L. (HO)
and H. officinalis var. decumbens (HOD) was evaluated
using the brine shrimp (Artemia salina Leach) test. The
percent of nauphii dead within 24 h was reported to
determine the cytotoxic activity of the essential oils.
Accordingly, HOD with LC50 of 156.03 µg/ml lower than
HO 191.06 mg/ml revealed stronger cytotoxic activity
probably in support of linalool rich in HOD (Renzini et al.,
1999).

CONCLUSION
Concisely, these issues furnish the background for the
experiments on the associated basic studies for the H.
officinalis (L.) which is an important source of bioactive
substances of medicinal interest. Nevertheless, several
experimental studies are required to confirm the
therapeutic potential of this plant and determine whether
biological differences reflect the different isolation
procedures, different types of plant material used,
collection time, locations or different chemotypes.
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African Journal of Pharmacy and Pharmacology Vol. 5(17), pp. 1967-1978, 8 November, 2011
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP11.034
ISSN 1996-0816 © 2011 Academic Journals
Full Length Research Paper
Identification of phenolic compounds and
assessment of in vitro antioxidants activity of 30%
ethanolic extracts derived from two Phyllanthus
species indigenous to Malaysia
Elrashid Saleh Mahdi 1 *, Azmin Mohd Noor 1 , Mohamed Hameem Sakeena 1 , Ghassan Z.
Abdullah 1 , Muthanna Abdulkarim 1 and Munavvar Abdul Sattar 2
1 Department of Pharmaceutical Technology, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden
11800 Pulau Pinang, Malaysia.
2 Department of Physiology, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden 11800 Pulau Pinang,
Malaysia.
Accepted 21 July, 2011
Phenolic compounds were identified in 30% ethanolic extracts derived from Phyllanthus niruri (P. niruri)
and Phyllanthus urinaria (P. urinaria) using high performance liquid chromatography (HPLC) assay. In
vitro antioxidants activity of the extracts was studied based on total phenolic contents (TPC) using
Folin-Ciocalteu reagent and their scavenging activity towards radical 2,2-diphenyl-1-picrylhydrazyl
(DPPH). The HPLC results show that gallic acid (GA), corilagin (Cor) and ellagic acid (EA) were the
major components of the extracts and their quantifications in P. niruri were 11.867 � 0.130, 89.579 �
0.602 and 37.309 � 0.033 mg/g of extract respectively and in P. urinaria were 8.710 � 0.091, 56.382 �
0.364 and 27.880 � 0.263 mg/g of extract. The TPC of P. niruri and P. urinaria were 262.10 � 1.04 and
277.98 � 1.04 mg of gallic acid equivalent per gram of extract respectively and their scavenging based
on IC50 was 32.64 and 25.00 �g mass of the extract compared to the IC50 of references standards GA
(3.28 �g) and EA (2.99 �g). The results revealed the extracts as a potential source of natural
antioxidants that can be utilized in cosmetics as skin antiaging, sun-blocking and whitening agents.
Key words: Corilagin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), ellagic acid, Folin-Ciocalteu, gallic acid.
INTRODUCTION
Reactive oxygen species (ROS) and free radicals (FR)
are small molecules naturally generated as by-product of
cellular metabolism. Exposure to environmental hazards
such as radiations, chemicals and gases increases their
production in the body to toxicity levels (Govindarajan et
al., 2005). Endogenous antioxidants in the body, such as
glutathione and α-tocopherol can maintain and counteract
the produced FR and ROS when they are within the
physiological limit (Halliwell, 1996). Improper balance
between the oxidants (FR and ROS) and antioxidants in
*Corresponding author. E-mail: elrashidm@yahoo.com.
favour of the oxidants, is potentially leading to oxidative
stress state (Sies, 1997). Oxidative stress is a fragmental
state of DNA and cellular membrane damage, thus can
ultimately lead to cells and tissues death due to proteins
denaturation and lipids peroxidation (Ratnam et al.,
2006). Consequently, make way for various human
generative diseases like myocardial infarction, heart
failure, hypertension, atherosclerosis, Parkinson‟s
disease, Alzheimer‟s disease, muscular dystrophy,
multiple sclerosis, diabetes, rheumatoid arthritis, chronic
inflammatory diseases, sickle cell anaemia, acute renal
failure, cancers and premature aging (Ferrari et al., 2004;
Halliwell, 1987; Lefer and Granger, 2000; Nath and
Norby, 2000; Pham-Huy et al., 2008; Praticò and Delanty,

1968 Afr. J. Pharm. Pharmacol.
2000; Ratnam et al., 2006). Antioxidants are compounds
capable of scavenging FR/ROS by terminating oxidative
reaction chain in the biological tissues and hence can
prevent cellular damage and oxidative stress associated
with free radical induced generative diseases (Ratnam et
al., 2006). Recognized dietary antioxidants such as
ascorbic acid (vitamin C) and �-tocopherols (Vitamin E)
can pick up and neutralize FR and ROS, prevent and
reversed age related disorder and diseases (Halliwell,
1996). Plants secondary metabolites such as flavonoids
and polyphenols compounds exhibited important
commercial and biological role due to their antioxidants
activity (Agati et al., 2007; Bendini et al., 2007; Di
Mambro and Fonseca, 2005; Rice-Evans et al., 1997).
They are good electron donors and having potentials
redox pattern that can scavenge FR and ROS and
prevent their harmful effects (Pietta et al., 1998). They
are relatively stable due to resonance, delocalization and
formation of side conjugated system with the hydroxyl
group attached to the aromatic ring (Srinivasan et al.,
2007). Therefore, they have been the major research
issues for the last two decades (Gourine et al., 2010).
Consequently, their commercial application as food
supplements, food preservatives in nutraceuticals and
skin anti-aging, sun-blocking and whitening agents in
cosmeceuticals is highly increased (Peschel et al., 2006).
Phyllanthus urinaria and Phyllanthus niruri belong to
widely distributed genus “Phyllanthus”, family
Phyllanthaceae (Samuel et al., 2005). The genus is found
all over the world in the tropical and subtropical countries.
More than 750 species of genus Phylanthus have been
described (Calixto et al., 1998; Wehtje et al., 1992). The
genus “Phyllanthus” has been traditionally used internally
to treat a broad spectrum of diseases such as diarrhoea,
hepatitis, diabetes, abdominal pain, and kidney disorder
(Chularojmontri et al., 2005; Mellinger et al., 2005). It is
also used topically as a poultice to treat skin ulcers,
sores, itching and wounds healing. The phytochemical
compounds of many of Phyllanthus species such as
tannins, ellagitannins flavonoids have been isolated and
characterized (Ahmeda, 2005; Chang et al., 2003; Fang
et al., 2008; Liu et al., 1999; Murugaiyah and Chan,
2007). The potential pharmacological effects of the many
of these isolated compounds have been assessed
(Ambali et al., 2010; Calixto et al., 1998; Krithika et al.,
2009). Several studies have shown the antioxidants
activity of various Phyllanthus species using different
solvents and methods of extractions (Chularojmontri et
al., 2005; Fang et al., 2008; Harish and Shivanandappa,
2006). Various phenolic compounds with antioxidants
effect have been identified in P. niruri and P. urinaria
(Harish and Shivanandappa, 2006; Markom et al., 2007;
Murugaiyah and Chan, 2007). P. urinaria and P. niruri,
being investigated in this study are found in Malaysia.
The two species are closely-related in appearance and
traditionally uses and locally known as “Dukung anak”
which means carry baby; because the plants carry the
fruits on their backs and underneath the feathered-like
leaves (Ahmeda, 2005; Markom et al., 2007; Ong and
Norzalina, 1999). Our aim of this study was to identify
and quantify the major components of the extracts
chromatographically using high performance liquid
chromatography (HPLC) and to assess an in vitro
antioxidants activity in terms of total phenolic content and
scavenging activity toward the radical 2, 2-Diphenyl-1-
Picrylhydrazyl (DPPH) and to compare it to the
scavenging activity of gallic and ellagic acids as control
positive reference standards.
MATERIALS AND METHODS
Reagents
Folin-Denis‟ reagent, sodium carbonate 99%, 2,2-Diphenyl-1picrylhydrazyl
(DPPH 95%), gallic acid (GA, 99%) and ellagic acid
(EA, 95%) were purchased from Sigma–Aldrich (St. Louis, MO.,
USA). Methanol was purchased from J. T. Baker (Philipsburg,
USA), formic acid 98 to 100% assay from Merck (Darmstadt,
Germany). Ethanol (99.7%) was purchased from Brightchem Sdn
Bhd (Malaysia), sodium hydroxide from R & M Marketing (Essex,
UK) and corilagin in-house prepared working standard was
obtained as a gift from Nova Laboratories Sdn Bhd (Malaysia).
Extracts
The extracts of P. urinaria and P. niruri were prepared by Nova
Laboratories Sdn. Bhd. (Malaysia) by dissolving 50 g of dried
powdered materials from the aerial part of each plant (P. niruri and
P. urinaria) in 500 ml of 30% ethanol at 60°C water bath for 1 h.
The 30% ethanol liquid extract was filtered using Whatman No. 1
filter paper. The residue was re-extracted with another 500 ml of
30% ethanol. The two filtrates were combined and dried using
rotatory evaporator at 60°C.
Identification of major phenolic compounds
GA external reference standard was prepared by dissolving 10 mg
into 100 ml of distilled water. 10 mg EA of external reference
standard was transferred into 100 ml volumetric flask and dissolved
into 10 ml of sodium hydroxide 0.1 M and the volume was
completed to 100 ml with water. Serial dilutions from the two
references standards solutions of GA and EA were prepared in the
range of concentration from 0.5.0 to 16 µg/ml. Corilagin (Cor)
working standard was prepared in concentration of 250 µg /ml
methanol. The solutions were ultra sonicated at ambient
temperature for 10 min and filtered through nylon membrane filters
47 mm 0.45 µm (Whatman, UK) before HPLC analysis was
performed. The calibration curves were plotted with six
concentrations each of the standard solution of GA and EA versus
the areas under the peaks. The GA and EA standard curves
equations were used to quantify in the extracts. Since corilagin is
commercially unavailable and the small quantity available was
enough only for identification purposes and triplicate runs was used
to quantify corilagin in the extracts.
Samples solutions
10 mg of each extracts were weighed and transferred into 10 ml
volumetric flasks. Each sample of P. niruri and P. urinaria was

Table 1. Liquid chromatography (LC) time programme of binary gradient pumps modules.
Time (min)
Module action
Pump A (% concentration) Pump B (% concentration)
0.00 0 100
2.00 5 95
5.00 30 70
8.00 34 66
11.00 45 55
14.00 45 55
17.00 0 100
20.00 0 100
dissolved in formic acid 0.2%, vortexed for 20 s and then ultra
sonicated at ambient temperature for 10 min at room temperature.
The resulting samples solutions were filtered through nylon
membrane filter 47 mm 0.45 µm diameter (Whatman, UK) before
HPLC analysis were performed.
Detection and quantification limits (DL and QL)
The DL and QL were estimated based on the standard deviation of
the response and slope (Guideline, 2005). The standard deviation
(�) of the responses of the lowest concentration in calibration curve
of six runs (n = 6) and the slope of the calibration curves of GA and
EA (S) were used to calculate DL and QL using Equations 1 and 2,
respectively (Guideline, 2005). The sensitivity of the method was
evaluated by the relative standard deviation (RSD %) of mean area
under the peaks of the reference standards. The selectivity of the
method and suitability of system were evaluated by comparing the
retention time of the standards peaks to the samples peaks. The
means of retention time of the GA and EA in the samples solutions
were compared to external reference standards GA and EA mean
retention time and were analyzed for null hypothesis using student‟s
paired t-test.
Chromatographic condition
The HPLC analysis was performed using LC 20 AD Class LCsolution
software, connected to SPD-20A UV/VIS detector, binary
pump and temperature controlled column oven, (Shimadzu, Japan).
Thermo Hypersil Gold TM (250 × 4.6 mm i.d., 5 µm) reversed phase
column was used for all separations. The column oven temperature
was set at 40°C and the external reference standards solutions and
extracts were eluted with a binary gradient mode at a UV
wavelength of 270 nm and 20 �l volume of injection (Rangkadilok et
al., 2005). Methanol (solvent A) and formic acid 0.2% (solvent B)
were used as mobile phase at flow rate of 1 ml/min. The liquid
chromatography (LC) time programme of modules of the binary
gradient pumps was set as in Table 1 and the retention time of the
peaks was specified by band of 0.2 min.
(1)
(2)
Determination of total phenolic content (TPC)
Mahdi et al. 1969
The total phenolic content (TPC) of the extracts was studied
spectrophotometrically by folin–ciocalteu reagent assay with slight
modification (Bajpai et al., 2009). GA standard curve was plotted in
the concentrations range of 50, 100, 150, 200, 250 and 500 mg/L in
ethanol:water (10/90) solution and their corresponding
absorbencies were measured at 765 nm visible wavelength. 10 mg
of each extracts material were transferred in 25 ml volumetric flask
and dissolved into ethanol:water (30:70). 100 µl from the samples
solutions were placed in screw-capped test tubes, 500 µl of the
Folin–Ciocalteu reagent and 1500 µl of distilled water (1/15 dilution)
were added to the samples solutions respectively. The test tubes
were properly shaken before incubated at room temperature for 1
min. After 1 min, 1000 µl of 20% sodium carbonate (Na2CO3)
aqueous solution was added. The final mixture was vortexed for 10
s and then incubated for 2 h at room temperature. After 2 h, the
absorbance was measured at 765 nm UV wavelength using Hitachi
U-2000 spectrophotometer, Perkin Elmer Lambda, USA. The
procedure was carried out in triplicate manner (n = 3) and the TPC
the extracts was calculated using Equation 3. The results were
expressed as milligram gallic acid equivalent per gram dry extract
weight (mg GAE/g DW).
The experimental TPC were compared to the predicted TPC based
on the quantified composes of the extracts. GA contains three
hydroxyl group attached to aromatic ring as was used; the
reference standard is equivalent to unity. The number of GA
equivalent to each composes in the extracts was obtained by
dividing the number of hydroxyl group in the composes by the
number of hydroxyl group of GA (GAE). The predicted TPC were
evaluated by summation of the total quantitative amount of each
composes equivalent to GA multiplied by its quantified amount in
the extract as shown by expression 4. In which, PTPC was the
predicted TPC, COC was the content of composes in the extract
and GAE was the GA equivalent of the composes.
Determination of radical scavenging activity
The powdered extracts and EA reference standard were dissolved
in 99.7% ethanol in the concentration range of 0.1 to 2.0 mg/ml.
(3)
(4)

1970 Afr. J. Pharm. Pharmacol.
0.0 2.5
5.0 5.0 7.5 10.0 12.5 15.0 17.5 min
12.5 15.0
min
Figure 1. P. niruri chromatogram showing the peaks of Compound 1 gallic acid at a retention time 8.522 min, Compound 2, corilagin at
12.196 min, Compound 3 expected to be geraniin at 12.406 min and compound 5, ellagic acid at 16.963 min.
While GA reference standard was dissolved in distilled water in the
range of 0.2 to 2.0 mg/ml. The extracts and the reference standards
solutions of GA and EA were allowed to react with 2,2-Diphenyl-1picrylhydrazyl
(DPPH) (Fang et al., 2008; Krithika et al., 2009). 0.3
ml of 0.394 mg/ml DPPH and 2.4 ml of 99.7% ethanol were mixed
in screw-capped test tubes. 0.1 ml of the extracts and the reference
standards were added to a separate reaction mixture in the test
tubes and allowed to stand for 30 min in the dark. The scavenging
activity of the mixtures was measured at 517 nm visible wavelength.
The experiment was carried in triplicates (n = 3) for each
concentration of the reference standard and the extracts and the
percentages of scavenging activity of the extracts were calculated
using Equation 5, where Sc. A is the scavenging activity, Ao is the
absorbance of the blank mixture (the absorbance of reaction
mixture without extract or the reference standards), Am was the
absorbance of the reaction mixture with the extract or the reference
standards. The results of scavenging activity of the extracts and the
reference standards were plotted against their dose of the extracts
and the standards in the mixtures (µg). The dose that inhibits 50%
of DPPH radical activity (IC50) were calculated from the equation of
the scavenging activity plotted curves. The scavenging activities of
the extracts were compared with GA and EA scavenging activity as
positive control standards. The relative scavenging activity of the
extracts to the reference standards GA and EA scavenging activity
were analyzed for null hypothesis using student‟s t-test.
Statistical analysis
Results were expressed as means�standard deviation (SD) of
triplicates measurements. Student‟s t-test was used to analyse the
(5)
data and p-value

Mahdi et al. 1971
Figure 2. P. urinaria chromatogram shows the peaks of Compound 1, gallic acid at a retention time 8.436 min, Compound 2
corilagin at 12.144 min, Compound 3 expected to be geraniin at 12.353 min and Compound 5 ellagic acid at 16.868 min.
0.0
0.0
2.5 5.0 7.5 10.0 12.5 15.0 17.5 min
2.5 5.0 7.5 10.0 12.5 15.0 17.5 min
Figure 3. The chromatogram of external references standards mixture of gallic acid Compound 1, corilagin Compound 2 and ellagic A
Compound 5 peaks at 8.477, 12.183 and 16.961 min, respectively.
min, respectively in P. urinaria chromatogram (Figure 2).
The chromatograms of GA, Cor and EA reference
standards mixture shows peaks of retention‟s time at
8.477, 12.183 and 16.961 min, respectively (Figure 3).
Figure 4 shows the chemical structure of the phenolic
compounds identified in the extracts and geraniin which
is structurally closed to corilagin, its peak could be much
closed to the corilagin peak such as Compound 3 in the
chromatograms (Figures 1 and 2). The expectation was
compared to previously identification of geraniin closed to
corilagin in previous identification of phenolic compounds
described previously (Thitilertdecha et al., 2010). It was
cleared that these compounds are large molecules, have
high molecular and their solubility are varied from water
soluble compound such as GA to lower water soluble EA.
It was also cleared from the chromatogram (Figure 1) and

1972 Afr. J. Pharm. Pharmacol.
HO
HO
O
HO
OH
O
O
HO
OH
Compound 1 Gallic Acid
Figure 4. Chemical structures of the major compounds identified in the extracts.
that corilagin was the major compound in the extracts
followed by Compound 3 which was expected to be
geraniin, then EA and lastly GA. The important of
polyphenols compounds is that they have amphiphilic
properties which facilitate their antioxidants mechanism in
both water and lipid phase (Sies and Stahl, 2004).
Therefore, their application in cosmeceuticals will have
important role in scavenging the ROS and FR results
from excessive exposures to solar UV radiations. As it
was known that the antioxidants properties of the
polyphenolic compounds is because of the presence of
hydroxyl group attached to aromatic ring as electron
donating group (Ng et al., 2000). The more electron
donating group available in the antioxidants compound,
the more potent and stronger scavenging activity
(Srinivasan et al., 2007). Based on this, geraniin which
possess eleven hydroxyl groups attached to aromatic will
be more potent compared to corilagin which has nine
OH
OH HO
O
O
OH
OH
O
O
O
O
O
O
OH
OH
OH
O
O
O
OH
OH
OH
OH
OH
OH
Compound 2 Corilagin
Compound 3 expected to be Geraniin Compound 5 ellagic acid
HO
HO
O
OH
HO
O
O O
O
O
O
OH
O
OH
O
OH
O O
hydroxyl groups and consequently, corilagin is a strong
antioxidant compared to EA and GA which has four and
three hydroxyl groups, respectively Figure 4.
Furthermore, carboxylic acid group in GA can provide
additional attack sites for free radicals and thus prevent
them from attacking cell membrane and denaturation of
protein (Srinivasan et al., 2007). Similarly, EA which
contains two lactones‟ groups have contribution to its
antioxidant activity.(Barch et al., 1996). Hence, the
variation in content of these active ingredients in the
extracts will affect the antioxidant activity. The
quantification of major compounds in extracts was shown
in Table 2. These compounds were also found in other
plant extracts with content almost similar the P. niruri and
P. urinaria and their uses as antioxidants material were
explored (Rangkadilok et al., 2007). These polyphenolic
compounds have been shown as antioxidants able to
scavenge the free radicals reactive oxygen species (Fang
OH
OH
OH
OH
OH
OH

Table 2. Quantitative results of major active ingredients in the extracts (n = 3).
Dry weight of the extract (mg/g) P. niruri P. urinaria
Gallic acid (GA) 11.867�0.130 8.709�0.091
Corilagin (Cor) 89.579�0.602 56.382� 0.364
Ellagic acid (EA) 37.309�0.033 27.880�0.263
Mahdi et al. 1973
Table 3. Selectivity of the HPLC method based on the retention time of the external reference standards GA and EA to their retention in the
extracts.
Retention time
(min)
GA standard (n
= 6)
EA standard (n
= 6)
P. niruri (n = 3) P. urinaria (n = 3)
GA EA GA EA
Mean (min) 8.398 16.897 8.526 16.964 8.406 16.861
SD 0.013 0.042 0.033 0.031 0.012 0.010
RSD (%) 0.154 0.249 0.387 0.183 0.143 0.059
P-value - - 0.530 0.127 0.420 0.324
et al., 2008; Lin et al., 2008). They also can absorb the
UV radiations and act as sun-blocking and whitening
agents. It can penetrate the skin, pick up and neutralized
the excessively generated ROS and FR due to the
exposure to UV radiation and act as skin antiaging. To
utilized the extracts as skin cosmetics, they were
subjected to further treatment such as identification of
TPC and scavenging activity towards stable radical
DPPH.
Detection and quantification limits (DL and QL)
The HPLC analysis results in the present study show the
linearity of the method by references standards at six
levels of concentrations in the range of 0.5 to 16 �g/ml by
the equations of the calibration curves of reference
standards GA and EA. The calibration curve of reference
standard GA equation was y = 4978.79 + 60900.7 × x, R 2
= 0.9994. While the EA equation was y = 54933.8 +
36348.1 × x with regression factor R 2 = 0.9981. The DL of
the two external reference standards GA and EA were
0.021 and 0.076 �g/ml, respectively. While the QL of the
two external reference standards GA and EA were O.070
and 0.252 �g/ml, respectively. The lower values of DL
and QL reflect the sensitivity of the method. Furthermore,
the sensitivity of the method was confirmed by the result
of the relative standard deviation (RSD%) of the mean
area under peak of both GA and EA reference standards
1.34 and 1.63%, respectively. It was also cleared that the
DL and QL of GA is lower than EA. Hence the method
was more sensitive to GA compared to EA.
Selectivity of the HPLC method
The selectivity of the HPLC method was evaluated by
comparing the retentions time of the external references
standards GA and EA with retentions time of GA and EA
active ingredients in the extracts Table 3. The small value
of the relative standard deviation (RSD %) show the
selectivity of the method. The SD of the means of the
retention time is less than the retention time band set in
the instrument which was 0.2 min. In addition, the
statistical result also shows that the paired t-test tested
satisfies the null hypothesis for two-sided with p-value >
0.05 and 95% confidence interval of the difference
between the external reference standards retention time
and the sample solution (Table 3). The proposed HPLC
method was efficient, simple, rapid, cost effect and safe
for the column on long uses since small amount of formic
acid was utilized. Since the proposed HPLC method
identified and quantified high content of phenolic
compounds in the extracts, the extracts were subjected
for further study to evaluate their antioxidants activities
with respect to TPC and scavenging activity towards
DPPH.
Total phenolic content
The quantification of the total phenolic content of P.
urinaria and P. niruri were expressed as means ± SD and
was found to be 277.98 � 1.04 and 262.10 � 1.04 mg of
GAE/g dry weight of plant extract, respectively. The
present study also revealed that P. urinaria and P. niruri
extracts possessed high TPC compared to previous
studies of extract from P. niruri (Harish and
Shivanandappa, 2006; Kumaran and Joel, 2007). The
TPC of the P. niruri was lower compared to the predict
TPC based on the quantity of the major active ingredients
in the extract; while the TPC of P. urinaria was higher
compared to the predicted TPC which based on the

1974 Afr. J. Pharm. Pharmacol.
Table 4. Predicted TPC of the extracts based on the polyphenolic contents
compared to the experimental TPC.
Compose P. niruri (GAE/g DW) P. urinaria (GAE/g DW)
GA 11.87�0.13 8.71�0.09
Corilagin 268.74�1.81 169.15�1.09
EA 48.50�0.04 36.24�0.34
Predicted TPC 329.11�1.98 214.10�1.53
Experimental TPC 262.10� 1.04 277.98�1.04
GA has 3 OH groups is equivalent to 1 GAE/g DW, corilagin has 9 OH groups. Therefore
= 3GAE/g DW equivalent and EG has 4 OH groups = 1.33 GAE/g DW.
number of hydroxyl groups in each of the phenolic
compounds to the number of hydroxyl groups in GA and
its content in the extracts (Table 4). The variations in the
predicted and experimental TPC of P. urinaria could be
due to presence of much major and high content of
unidentified compounds. Such compounds were 6, 7 and
8 which were found only in P. urinaria and Compound 4
which was expected to be geraniin with highest peak in
P. urinaria chromatogram (Figure 2) compared to P. niruri
chromatogram (Figure 1). Therefore, its high content in P.
urinaria certainly will increase its TPC. This also could be
reason of P. urinaria has high phenolic content despite it
contain less amount of GA, Cor and EA compared to P.
niruri. In case of P. niruri, the predicted and experimental
TPC results (Table 4) were closely relevant since only
Compound 4 was not quantified (Figure 1). The predicted
TPC gives quick estimation to the TPC of the extracts
despite that it required more study and broad application
before been normalize as a measure.
Scavenging activity
The scavenging activity of P. niruri towards the stable
radical DPPH was calculated as IC50 from the plotted
graph equation, Y = -1684.82/X + 101.618, with
correlation coefficient factor R 2 = 0.9938 (Figure 5). The
IC50 is the dose in mass of the antioxidants materials
(extract or reference standard) necessary to inhibit the
initial DPPH radical activity by 50%. The smaller value of
IC50 means high scavenging activity and potent
antioxidants compound. The IC50 of P. niruri was 32.64 �g
mass of the extract and its inhibition capacity to DPPH
was 91.57% at dose of 200 �g. The IC50 of P. urinaria
was 25.00 �g mass of the extract and was inhibited
93.81% of DPPH scavenging activity at a dose of 200 �g
based on the curve equation Y = -1288.4/X + 101.528
with regression coefficient R 2 = 0.9982 (Figure 6) . The
results of scavenging activities of P. niruri and P. urinaria
were compared to the scavenging activity of the control
positive reference standard GA and EA. The IC50 of GA
and EA were 3.28 μg and 2.99 �g mass of the reference
standard, respectively. The maximum scavenging
capacity of the positive control external references
standards GA and EA against DPPH was 95.95% and
94.01% at a dose of 200 μg and 100 �g respectively
(Figures 7 and 8). The scavenging activity curves of the
extracts and the reference standards were non-linear with
increasing mass of the extract or the reference standards
in the form Y = A/X + B. These curves show the
maximum scavenging activity of the extracts and the
control reference standards and behave as biological
curves. The inhibition capacity was the maximum
percentage of scavenging activity of the extract/reference
standard towards DPPH. The maximum inhibition
capacity never reaches 100% due to the plateau feature
of the scavenging activity profile of both the extracts and
the control positive standards which is the characteristic
of biological activity. The profiles were typical biological
activity curve and closely resemble enzyme kinetic curve.
The results of scavenging activity of the extracts and the
references standards reported in Table 5 show that the
scavenging activity of both extracts were much lower
compared to the control reference standards compare to
the TPC of the extracts. According to the TPC of the
extracts, they must have lower IC50 than actually
obtained. This might be because the high TPC phenolic
content of the extracts was due to high molecular weight
and bulky molecule corilagin and geraniin compared to
the planar GA and EA used as reference standards. The
bulky molecules, corilagin and geraniin might have some
steric hindrance to donate the phenolic hydrogen of the
hydroxyl group to DPPH compared to the relatively small
molecules GA and EA (Figure 4). Hence corilagin and
geraniin might be slower in scavenger DPPH. While in
case of GA and EA which were small planar compounds
geometry enable them to participate easily in the reaction
with DPPH. Therefore, the scavenging activity of the
extracts was mainly due to GA and EA in the extracts.
This can be predicted also from the relative scavenging
activity of the extracts to the reference standards. The
relative scavenging activity (RScA) was the ratio of the
scavenging activities of the extracts to the scavenging
activity of the control positive standard. From the relative
scavenging activity (RScA) of the extract to GA and EA.

Table 5. Antioxidants activity based on IC50 (�g) and the capacity of inhibition
to DPPH (%) of the extracts compared to the reference standards.
Extract/standard IC50
Capacity of DPPH inhibition
(%)
P. niruri 32.64 91.57
P. urinaria 25.00 93.81
Gallic acid 3.28 95.95
Ellagic acid 2.99 94.01
p-value - 0.00
Table 6. Relative scavenging activity (RScA) of extracts to GA and
EA scavenging activity.
RScA P. niruri P. urinaria
GA 9.96 7.63
EA 10.90 8.35
Mean�SD 10.43�0.67 7.99�0.51
P-value of RScA 0.029 0.029
Figure 5. P. niruri scavenging activity profile against 2, 2-diphenyl-1-picrylhydrazyl radical
(DPPH), the curve equation Y = -1684.82/X + 101.618, correlation coefficient R 2 = 0.9938 and
IC50 = 32.64 �g.
standards (Table 6), the RScA of P. urinaria was 7.630 GA
and contained 8.709 �g GA /mg dry weight of the extract.
While in P. niruri, the RScA was 9.960 and the content of
GA was 11.867�g /mg dry weight of P. niruri. The
percentage ratio of the RScA of GA to its contents in P.
niruri and P. urinaria extracts were approximately 83.9
and 87.6%, respectively. This result shows that the
scavenging activity of the extracts was mainly due to GA.
Similarly, the contribution of EA in the scavenging activity
Phyllanthus niruri extract (in �g)
Mahdi et al. 1975
of the P. niruri and P. urinaria extracts was 22.5 and
23.1%, respectively. Therefore, the scavenging activity of
both extracts towards DPPH was due to the GA and EA
in the extracts. Hence DPPH was not suitable indicator
for scavenging activity of the extracts contains such bulky
molecules like corilagin and geraniin. The inhibition
capacity and the relative scavenging activity were
significantly different and the null hypothesis was rejected
for both p-value

1976 Afr. J. Pharm. Pharmacol.
Phyllanthus urinaria extract (in �g)
Figure 6. P. urinaria scavenging activity profile against 2, 2-diphenyl-1-picrylhydrazyl radical
(DPPH), the curve equation Y = -1288.4/X + 101.528, correlation coefficient R 2 = 0.9982 and IC50
= 25.00 �g.
Gallic acid extract (in �g)
Figure 7. Gallic acid scavenging activity profile against 2, 2, diphenyl-1-picrylhydrazyl radical
(DPPH), the curve equation Y = -138.615/X + 92.3058, correlation coefficient R 2 = 0.9395 and
IC50 = 3.28 �g.
Ellegic acid extract (in �g)
Figure 8. Ellagic acid scavenging activity profile against 2, 2-diphenyl-1-picrylhydrazyl radical
(DPPH), the curve equation Y = -126.56/X + 92.27, correlation coefficient R 2 = 0.9394 and
IC50 = 2.99 �g.

The results of scavenging activity (Table 5) consequently
show that P. urinaria was more potent compared to P.
niruri based on IC50 value and capacity of scavenging
activity. The result was consistent with the TPC finding
which showed P. urinaria with high TPC compared to P.
niruri. Therefore P. urinaria was strong inhibitor to DPPH
activity compared to P. niruri as well as it has high total
phenolic content (TPC). This result was also consistent
with the previous finding which is, the higher the TPC, the
stronger scavenging to DPPH (Tawaha et al., 2007;
Zheng and Wang, 2001). Similarly, it was also noticed
that EA IC50 was lower compared to GA IC50, hence it
was more potent than GA. EA also retuned high
scavenging capacity towards DPPH since it inhibited
94.01% of DPPH activity at a dose of 100 �g compare to
GA which inhibited 95.95% of DPPH activity but at a dose
of 200 �g (Figure 7 and 8). This might be because of
differences in the number of hydroxyl group attached to
the aromatic ring in GA and EA chemical structures and
hence more availability of donating the phenolic hydrogen
of hydroxyl group as scavenger in the reaction (Figure 4).
The results of scavenging activity of GA and EA from this
study and the scavenging activity of P. niruri and P.
urinaria were comparable to the previous study of plants
material of similar content of our extracts (Rangkadilok et
al., 2007). The scavenging capacity of the extracts from
this study was higher at higher dose as compared to
previous (Kumaran and Joel, 2007).
Conclusions
The proposed HPLC method successfully identified and
quantified the phenolic compounds of the extracts. The
method was quite simple, rapid, sensitive, selective, cost
effective procedure, friendly and safe to the column and
instrument on long uses. The HPLC analysis, TPC and
the scavenging activities results show that the extracts
contain high phenolic materials comparable to the
references standards gallic acid and ellagic acid. DPPH
was not a good indicator of scavenging activity of the
extract because it mainly composes bulky molecules
such as corilagin and geraniin. The result of antioxidants
evaluation is revealed to the extracts as a potential
natural source of antioxidants. The high antioxidants
properties of the extracts might make them versatile in
various fields of nutraceuticals, pharmaceuticals and
cosmeceuticals applications. Specifically, our interest in
this time is formulation of these extracts as skin
antiaging, sun-blocking and whitening agents.
ACKNOWLEDGEMENTS
The authors thank Biotechnology Directorate Malaysia for
allocation of the research grant, Universiti Sains Malaysia
(USM) for financial support for Elrashid and Nova
Mahdi et al. 1977
Laboratories Sdn.Bhd. (Malaysia) for supplying the
extracts materials and corilagin working standard.
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African Journal of Pharmacy and Pharmacology Vol. 5(17), pp. 1979-1982, 8 November, 2011
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP11.045
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Cultural importance and antibacterial activity of
Ziziphus mucronata (Willd.) in the Umlazi community in
Durban
R. M. Coopoosamy 1 *, K. K. Naidoo 1 and N. J. Ndlazi 2
1 Department of Nature Conservation, Mangosuthu University of Technology, P. O. Box 12363, Jacobs, 4026, Durban,
KwaZulu-Natal, South Africa.
2 Faculty of Natural Science, Mangosuthu University of Technology, P. O. Box 12363, Jacobs, 4026, Durban, KwaZulu-
Natal, South Africa.
Accepted 21 October, 2011
The Zulu community of Umlazi, Durban makes extensive use of Ziziphus mucronata (Buffalo thorn) for
predominantly cultural purposes. There are many superstitions and beliefs associated with the tree. A
validation of its antimicrobial properties from leaf, bark and root extracts were attempted against three
Gram positive and three Gram negative bacteria. In addition, an antifungal assay was attempted, using
ethanol, aqueous and boiled aqueous extracts on six fungal species. Acetone and ethyl acetate
samples showed greater inhibition of mostly Gram positive bacteria. Extracts from the leaves showed
the greatest inhibition, while root extracts showed the least inhibition. Ethanol extracts were found to
possess greater antifungal activity than aqueous and boiled aqueous extracts. Extracts were most
effective against Aspergillus flavus and Aspergillus glaucus with little effect on Candida albicans and
Candida tropicalis. It was evident that the bark and leaves of Z. mucronata possess greater
antimicrobial properties than the root and can be used as a substitute for other extensively harvested
species demonstrating similar properties.
Key words: Ziziphus mucronata, antimicrobial, cultural importance.
INTRODUCTION
In recent years, the interest of traditional medicine from
different cultures has increased significantly in
industrialized countries, with prescription drugs worldwide
originating from tropical flora (Nelson-Harrison et al.,
2002). Traditional medicine plays an important role in
many areas of South Africa, where communities do not
have access to proper healthcare facilities. In Southern
Africa, it is estimated that 70% of the black population
consults traditional healers for health hazard problems
and utilize traditional medicines, most of which is derived
from plant species indigenous to the region (Jager et al.,
1995). One of such community, living in Umlazi, Durban
makes extensive use of buffalo thorn for predominantly
cultural purposes.
*Corresponding author. E-mail: rogercoopoosamy@gmail.com.
Tel: +27 82 200 3342. Fax: +27 31 907 7665.
Africans have many beliefs and superstitions attached to
this tree. Zulus use the buffalo thorn in connection with
burial rites. It was once customary that when a Zulu chief
died, the tree was planted on his grave as a reminder or
symbol of where the chief lies, hence, the name
umLahlankosi (that which buries the chief, Pers. Comm:
Fakazi). A twig from the tree was and is still used to
attract and carry the spirit of the deceased from the place
of death to the new resting place. When a stock owner
died and was buried, according to custom, within the
cattle or goat kraal, some branches were placed on the
grave so that the animals nibbled on leaves and twigs,
and so understood that their master had died (Palmer
and Pitman, 1972). In other parts, Africans drag a branch
round the village to protect it from evil spirits, as it is
believed to keep evil spirits away (Pers. Comm: Fakazi).
In Botswana as well as most parts of South Africa, the
residents believed the buffalo thorn to be immune against
lightning; anyone standing under one in a storm would be

1980 Afr. J. Pharm. Pharmacol.
safe. It is also believed that if it is felled in summer, a
drought, hail or lightning will certainly follow (Pers.
Comm: Fakazi). A decoction of the glutinous roots is
commonly administered as a sedative for all sorts of
pains as well as dysentery (Nadembega et al., 2011). A
concoction of the bark and the leaves is used for
respiratory ailments and other septic swellings of the
skin. Pastes of the root and leaves can be applied to treat
boils, swollen glands, wounds and sores (Hutchings et
al., 1996). Steam baths from the bark are used to purify
and improve complexion (Palmer and Pitman, 1972). A
study using root extracts from buffalo thorn showed
activity against dermatophytes causing skin diseases
(Adamu et al., 2006). Furthermore, aqueous and
methanol extracts of the stem bark showed antifungal
activity against Candida albicans (Gundidza, 1986). In
East Africa, roots are used for treating snake bites
(Hutchings et al., 1996). It has been reported that rural
communities in Burkina Faso use a decoction of the root
to prevent obesity and stave off hunger pangs
(Nadembega et al., 2011).
Programmes for the screening of plant remedies are
important for validating the traditional use of herbal
remedies and for providing leads in the discovery of new
active principles. It is evident that buffalo thorn is used
sparingly by traditional healers in Umlazi, possibly
because of its scarcity in the area as well as the fact that
there is an abundance of other species exhibiting high
medicinal value. Although, some work has shown
antifungal properties of the plant, not much focus has
been placed on the antibacterial properties. Validation of
the antibacterial and antifungal properties of buffalo thorn
may thus ease existing pressure on other intensively
harvested medicinal plant species and intensify efforts to
re-introduce this species in previously occupied habitats
where it has become almost extinct due to
overharvesting.
METHODOLOGY
Plants species were located with the assistance of Mr Fakazi and
samples were collected from Umlazi, Durban, South Africa.
Antibacterial assay
One kilogram of dried material (leaf, root and bark) was crushed
and placed in a 2 L conical flask containing one of the three
mediums, that is, water, ethyl acetate and acetone, for extraction
based on varying polarity of solvents. The media were left for 72 h
in an orbital shaker at 20 shakes per minute. After 72 h, the extracts
were filtered. The extracts were then used for further tests.
The plant extract was then tested for antibacterial properties
against three strains of Gram positive (Bacillus subtilis, Micrococcus
kristinae and Staphylococcus aureus) and three strains of Gram
negative bacteria (Escherichia coli, Proteus vulgaris and
Enterobacter aerogenes) for antibacterial activity using the cupplate
method. Each organism was prepared by diluting in 24 h old
broth cultures with sterile nutrient broth. The cultures were then
diluted 100 fold to give approximately 10 6 bacteria ml -1 . Cultures
were incubated for 72 h at 60°C. Each treatment was done in
triplicate and the mean values were calculated.
Antifungal assay
Buffalo thorn leaf, bark and root samples (approximately 1 kg of
each) were cut into small pieces and crushed in a homogenizer.
The plant materials were soaked in ethanol (95% v/v) and in
distilled water in 2 L conical flasks for 3 weeks. The extracts (water
and ethanol) obtained were evaporated at reduced pressure (45°C)
to a residue. Extracts for testing ethanol and aqueous extract were
prepared in three different concentrations. The stock solutions were
prepared by dissolving 100 mg of dry extract in 1 ml of ethanol and
water separately in order to obtain a concentration of 100 mg/ml
dilutions (1:10, 1:100 and 1:500). These stock solutions were then
used in phosphate buffer at pH 6.0 to evaluate the antifungal
activity (Champion et al., 1992). The solutions were then tested for
antifungal activity using the following fungal cultures: Aspergillus
flavus, Aspergillus glaucus, Candida albicans, Candida tropicalis,
Trichophyton mentagrophytes, and Trichophyton rubrum. Plates
containing potato dextrose agar served as controls. All tests were
done in triplicate and the mean values were calculated.
RESULTS AND DISCUSSION
Water did not serve as a good extraction medium with
only bark extract samples inhibiting B. subtilus and M.
kristinae (Table 1). Acetone and ethyl acetate samples
showed greater inhibition of mostly Gram positive
bacteria (Tables 2 and 3). Acetone extracts from leaves
showed the greatest inhibition while extracts from roots
showed the least inhibition (Table 2). All Gram positive
bacteria and one Gram negative bacteria, that is, E. coli
were inhibited by acetone extracts obtained from the
leaves. Staphylococcus aureus was the only Gram
positive bacteria not inhibited by acetone extracts from
bark (Table 2).
All Gram positive bacteria were inhibited by ethyl
acetate extracts from leaves and bark while extracts from
roots inhibited only S. aureus (Table 3). In contrast, none
of the Gram negative bacteria were inhibited by ethyl
acetate extracts of the different plant parts (Table 3). The
results of the present study are similar to findings
obtained by other researchers who showed that
antibacterial activity was more prevalent in Gram positive
strains (Coopoosamy and Magwa, 2007; Grierson and
Afolayan, 1999).
The mixed extracts of buffalo thorn showed similar
activities to that of the leaves (Tables 1, 2 and 3).
However, the mixed extract was less effective when
compared with the leaf extract. This can be explained by
the fact that these samples contained extracts from
leaves, bark and roots; extracts from bark and root being
less effective.
The antifungal activities (Table 4) of the ethanol
extracts were found to be more effective than aqueous
and boiled aqueous extracts. Extracts were most
effective against A. flavus and A. glaucus with little effect
on C. albicans and C. tropicalis (Table 4). No effects

Table 1. Minimal inhibitory concentration (MIC) of Z. mucronata in water extract.
Bacteria
Gra
m
+/-
Leave
Stem bark
Plant part
Roots
Coopoosamy et al. 1981
Mixture of leaves,
bark and roots
B. subtilis + Na Na Na 4.0 5.0 Na Na Na Na 6.0 4.0 Na
M. kristinae + Na Na Na 4.0 4.0 Na Na Na Na Na Na Na
S. aureus + Na Na Na Na Na Na Na Na Na Na 6.0 Na
E. coli - Na Na Na Na Na Na Na Na Na Na Na Na
P. vulgaris - Na Na Na Na Na Na Na Na Na Na Na Na
E. aerogenes - Na Na Na Na Na Na Na Na Na Na Na Na
Na = No activity; all activities were done in triplicate.
Table 2. Minimal inhibitory concentration (MIC) of Z. mucronata in acetone extract.
Bacteria
Gra
m +/-
Leaves
Stem bark
Plant part
Roots
Mixture of leaves,
bark and roots
B. subtilis + 3.0 4.0 3.0 2.0 3.0 Na 5.0 Na Na 3.0 4.0 Na
M. kristinae + 4.0 3.0 5.0 3.0 3.0 4.0 Na Na Na 4.0 Na Na
S. aureus + 2.0 4.0 4.0 Na Na Na Na Na Na 4.0 5.0 Na
E. coli - 3.0 3.0 3.0 Na Na Na Na Na Na Na Na Na
P. vulgaris - Na Na Na Na Na Na Na Na Na Na Na Na
E. aerogenes - Na Na Na Na Na Na Na Na Na Na Na Na
Na= No activity; all activities were done in triplicate.
Table 3. Minimal inhibitory concentration (MIC) of Z. mucronata in ethyl-acetate extract.
Bacteria
Gram
+/-
Leaves
Stem bark
Plant part
Roots
Mixture of leaves,
bark and roots
B. subtilis + 4.0 3.0 3.0 2.0 3.0 Na 5.0 Na Na 4.0 4.0 2.0
M. kristinae + 5.0 4.0 5.0 4.0 4.0 5.0 Na Na Na 4.0 Na 3.0
S. aureus + 3.0 Na 4.0 3.0 3.0 4.0 5.0 3.0 3.0 3.0 2.0 2.0
E. coli - Na Na Na Na Na Na Na Na Na Na Na Na
P. vulgaris - Na Na Na Na Na Na Na Na Na Na Na Na
E. aerogenes - Na Na Na Na Na Na Na Na Na Na Na Na
Na = No activity; all activities were done in triplicate.
were recorded for T. mentagrophtes and T. rubrum
(Table 4).
This contradicts a study by Adamu et al. (2006) where
methanol extracts of root samples showed antifungal
activity against T. rubrum, T. mentagrophytes, Aspergillus
fumigatus and Microsporum canis. Unfortunately, those
researchers did not test leaf nor bark samples based on
their results on modifications of existing methods.
Growth inhibition (zone of inhibition) was recorded as
very high (++++), high (+++), medium (++) and low
(+),which indicated zones of inhibition between 41 to 50,
31 to 40, 21 to 30 and 11 to 20 mm, respectively.
These zones of inhibition were determined from the
central point to the point where growth inhibition has
occurred and measured, respectively. The ethanol extract
and boiled aqueous extract of the leaves were noted to
have more antimicrobial effects as compared to the
aqueous extracts.
The high zones of inhibition noted in the ethanol
extracts (using a 1:10 concentration) suggest further
explanation of the possibility of using this plant against
certain ailments caused by the aforementioned fungal

1982 Afr. J. Pharm. Pharmacol.
organisms.
Conclusion
Table 4. Effect of ethanol, aqueous extracts and boiled aqueous extracts obtained from Z. mucronata on different fungal
species (Tests were done in triplicate).
Fungal species
Ethanol extract
1:10 1:100 1:500
Aqueous extract
1:10 1:100 1:500
Boiled aqueous extract
1:10 1:100 1:500
A. flavus ++ ++ ++ ++ - - +++ + -
A. glaucus +++ + - ++ + - +++ - -
C. albicans + - - - - - ++ - -
C. tropicalis + - - - - - - - -
T. mentagrophytes - - - - - - - - -
T. rubrum - - - - - - - - -
- = Negative antifungal activity; + = Positive antifungal activity (low inhibition); ++ = Positive antifungal activity (medium
inhibition); +++ = Positive antifungal activity (high inhibition); ++++ = Positive antifungal activity (very high inhibition). Plates
containing potato dextrose agar served as controls. Controls did not show any inhibition of any of the test fungal species.
It was evident that Ziziphus mucronata does possess
antimicrobial properties and can be used as a substitute
for other extensively harvested species demonstrating
similar properties. Although, the roots of the plant did not
yield positive results, the study showed that leaves and
bark possesses greater antimicrobial properties.
Therefore, this study was important as it may help to
sustain remnants of the existing population in Umlazi.
However, further investigations are needed, including
purification and identification of the active compounds
present in the leaves and bark.
ACKNOWLEDGEMENTS
The researchers are greatly appreciative for the help from
Mr Bheki Fakazi for the site identification and traditional
information pertaining to Ziziphus mucronata in Umlazi,
Durban. Further acknowledgement goes to the Research
Directorate of Mangosuthu University of Technology for
providing fund towards this investigation.
REFERENCES
Adamu HM, Abayeh OJ, Ibok NU, Kafu SE (2006). Antifungal activity of
extracts of some Cassia, Datarium and Ziziphus species against
dermatophytes. Nat. Prod. Rad., 5: 357-360.
Champion RH, Burton J L, Ebling FJG (1992). Textbook of Dermatology
(5 th edition). London, Blackwell., 3: 1130-1175.
Coopoosamy RM, Magwa ML (2007). Traditional use, antibacterial
activity and antifungal activity of crude extract of Aloe excelsa. Afr. J.
Biotech., 6: 2406-2410.
Grierson DS, Afolayan AJ (1999). An Ethnobotanical study of plants
used in treatment of wounds in the Eastern Cape, South Africa. J.
Ethnopharmacol., 67: 327-332.
Gundidza M (1986). Screening of extracts from Zimbabwean higher
plants. Part 2: Antifungal properties. Fitoterapia, 57: 11-113.
Hutchings A, Scott AH, Lewis G, Cunningham AB (1996). Zulu
Medicinal plants: An inventory. University of Natal Press. pp. 53-54.
Jager AK, Hutchings A, Van Staden J (1995). Screening of Zulu
medicinal plants for prostaglandin-synthesis inhibitors. J.
Ethnopharmacol., 52: 95-100.
Nadembega P, Boussim JI, Nikiema JB, Poli F, Antognoni F (2011).
Medicinal plants in Baskoure, Kourittenga Province, Burkina Faso: An
ethnobotanical study. J. Ethnopharmacol., 133: 378-395.
Nelson-Harrison ST, King SR, Limbach C, Jackson C, Galiwango A,
Kato SK, Kanyerezi BR (2002). Ethnobotanical research into the 21 st
century. In: Iwu MM, Woottron JC (Eds.), Ethnomedicine and Drug
Discovery. Elsevier, Amsterdam, pp. 283-307
Palmer E, Pitman N (1972). Trees of Southern Africa covering all known
indigenous species in the Republic of South Africa, South-West
Africa, Botswana, Lesotho and Swaziland. Volume 1. A.A. Balkema,
Cape Town.
Pers. Comm. Mr Fakazi BT (Principal): Kwa Mathanda High School,
Durban.

African Journal of Pharmacy and Pharmacology Vol. 5(17), pp. 1983-1989, 8 November, 2011
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP11.118
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Altitude-related changes in activities of carbon
metabolism enzymes and secondary plant productsmenthoforon
an active pharmaceutical constituents
yield in pippermint (Mentha piperita L. Var. Kukarail)
A. Misra* and N. K. Srivastava
Central Institute of Medicinal and Aromatic Plants, P. O. CIMAP, Lucknow-226015, India.
Accepted 15 September, 2011
Activities of some enzymes related to carbon metabolism were studied in different ecotypes of
pippermint (Mentha piperita L. Var. Kukarail), growing at 950 and 1,250 m above mean sea level.
Activities of peroxidase and superoxide dismutase (SOD) and geranyl pyro phosphate (GPP) are
significantly higher in higher altitude cultivated cultivars. The GPP which converts to geranyl geranyl
pyro phosphate (GGPP) comprises of two subunits, the smaller one and the large subunits.
Electrophoretic bands of PCR showed the gene specific primers. In higher altitude, the smaller and
large subunits of the key enzyme are present where as in the plain cultivated plants, the larger subunits
are absent and the only smaller subunits are more conspicuously synthesized. The plain cultivated
plants at 0.05 ppm Zn showed the higher carbon dioxide exchange rate (0.65 µg(CO2)m- 2 s -1 ) and sacride
formation 0.470 µg(CH2O)m -2 s -1 ). The higher altitude pippermints plants at 0.1 Zn mg/L grown plants,
showed higher total amounts of monoterpne oil(s) (0.67%) and the increased was 26% over control, with
higher menthol, menthyl actate and of medicinal uses menthofuron contents in comparison to plain
cultivations.xide-dismutase (SOD) and peroxidise iso-enzymes with altitude were studied, where as
activities of SOD did not show a significant difference with change in altitude. RFPD of the two altitude
grown cultivars showed the different lines of conspicuous lane bandings. The key enzymes are from
geranyl pyro phosphate (GPP).
Key words: Peroxidase, superoxide dismutase, peroxidise iso-enzymes, gernul geranul pyruphosphate,
phosphoenolpyruvate carboxylase; ribulose-1,5-bisphosphate, carboxylase/oxygenase.
INTRODUCTION
Peppermint (Mentha piperita L) was mostly affected by
the leaf blight disease of Alternaria alternata. Therefore, a
protocol has been established by tissue culture
techniques, to get an improved and resistant variety of
Mentha piperita L. var. Kukarail. Further, an adaptation to
environment refers to the capacity of organisms or cells
to alter their phenotype in response to changes. Plants
display enormous plasticity to survive under changing
environmental variables with altitude (Clements et al.,
1950; Billings et al., 1961; Tranquillini, 1964). This
plasticity in variety is according to altitude cropping
response to environmental variables, This may involve
*Corresponding author. E-mail: amisracimap@yahoo.co.in.
the changes at anatomical, morphological, physiological
and biochemical levels to enable plants to combat ‘harsh’
climatic conditions at high altitude and maintain a
reasonably efficient carbon harvesting system to
compensate for the relatively short growing period
(Tranquillini 1964, Larcher, 1995; Purohit, 2003).
Plasticity in each of these responses could be of special
significance under specific environment. Morphological
plasticity is related to high competition in productive
environments, whereas species acclimate through
physiological plasticity in unproductive environments
(Cordell et al., 1998). Mountain plants evolved in
response to their particular altitude environment differ in
physiological response to the respective ecotypes from
lowland areas (Billings et al., 1961; Hiesey et al., 1971;

1984 Afr. J. Pharm. Pharmacol.
Mächler et al., 1977; Körner and Diemer, 1987; Cordell et
al., 1998; Hovenden and Schimanski, 2000; Hovenden
and Schoor, 2003; Kumar et al., 2005, 2006; Vats and
Kumar, 2006). However, there is little information on
altitude related changes in enzymatic activities related to
carbon metabolism except for enzymes such as
peroxidase , superoxide-dismutase(SOD) and peroxidise
iso-enzymes and geranyl geranyl pyro phosphate
(GGPP), geranyl geranyl pyro phosphate (GGPP),
comprises of two subunits, the smaller one and the large
subunits. Further apart from the monoterpenes
biosynthesis, the primary carbon metabolism, that is, the
ribulose-1, 5-bisphosphate carboxylase/oxygenase
(RuBPCO) plays an important role in carbon
sequestration and carbon capturing for carbon balancing.
The higher RuBPCO activity was reported in high altitude
ecotypes of Selinum vaginatum (Pandey et al., 1984).
Lower activation state of RuBPCO but higher total
carboxylase activity in barley, pea, and wheat at high
elevation suggested responsiveness of the enzyme to
low partial pressures of CO2 at high altitude (Kumar et al.,
2004). While low temperature could stimulate RuBPCO
activity in C4 plant Atriplex (Osmond et al., 1982), neither
high altitude nor chilling could enhance RuBPCO capacity
in C4 plants Bouteloua gracilis and Muhlenbergia
montanum (Pittermann and Sage, 2000, 2001).
Increased phosphoenolpyruvate carboxylase (PEPC)
activity was reported in C3 species Glycine soja with
increase in elevation from about 500 to 3 650 m, though
the implication of this fact was not discussed (Pandey
and Purohit, 1980). Earlier, we reported that crop plants
like barley and wheat when grown at high altitude
significantly increased carboxylase and oxygenase
activities of RuBPCO and activities of PEPC, aspartate
aminotransferase (AspAT), and glutamine synthetase
(GS) as compared to those grown at low altitude (Kumar
et al., 2006). The objective of the present study was to
find the response of some carbon capturing enzymes for
secondary plant products production in altitudinal
ecotypes of wild species, using Rumex nepalensis as the
target plant that has its natural distribution spread along a
wide altitudinal gradient in Himalaya (Bahar, 2002).
Keeping in view the facts that a study has been made to
raise the disease free and efficient genotype of M.
piperita L. Var. Kukarail at high altitude at Purera and in
plains to see the carbon sequestration.
MATERIALS AND METHODS
Plant
Plant tips (5 to 6 inches) with of efficient and disease free M.
piperita L. Var. Kukarail genotype were obtained from the the tissue
culture techniques as described previously (Saxena et al., 2008) in
CIMAP, Lucknow, India. Uniform cuttings were initially planted in
10000 cm 3 earthen pots filled with purified silica sand (Agarwala
and Sharma, 1961), for the development of roots. After 15 days,
rooted cuttings were transferred to 2500 cm 3 pots. The salts used
in nutrient solution culture were purified for Zn (Hewitt, 1952).
Hoagland and Arnon’s (1952) nutrient solution was used in the
experiment except Fe which was supplied as Fe-EDTA. Three pots
each of Zn treatments ranging from 0.0 to 1.0 g Zn ml -1 were
maintained in controlled glass house condition at ambient
temperature (30±5°C) and irradiance (between 800 and 1000 �mol
m -2 s -1 ). The nutrient solution in each treatment was added at
alternate days. With onset of deficiency and toxicity (after 20 days)
growth observation and detailed physiological and biochemical data
with growth attributes were performed.
CO2 exchange rate (PN)
CO2 exchange rate was measured using a computerized portable
photosynthesis system (Srivastava and Misra, 1991) (Model LiCOR
6000, LiCOR, USA).
Chlorophyll (Chl) content
A known mass of leaf tissue (3 rd leaf) was extracted with 80%
acetone and observance was recorded on spectrophotometer (Pye
Unicham PU8610, USA) for determination of Chl a and b according
to Arnon (1949)., and carotenoids were calculated as described by
Deming-Adams(1992).
Growth attributes and Zn analysis
Leaf fresh and shoot dry mass and area (area meter LICOR Li-
3000) were recorded. For tissue elemental analysis 1.g. dried leaf
samples were digested with I N HCl at 60°C for 24 h. Aliquot
samples of the clear digest were diluted with water (10 cm 3 ) and
analyzed for Zn by atomic absorption spectrophotometer (Pye
Unicam SP 2800) (Misra and Sharma, 1991).
For antioxidant reactive peroxidase enzyme assays of peroxidise,
Iso-enzymes of peroxidase, SOD and GGPP enzymes, frozen leaf
samples were ground with a mortar and pestle in extraction buffer
containing 5 ml 0.1 M phosphate buffer (pH 6.8), as described
previously (Shanon et al., 1960) and peroxidise iso-enzymes by
polyacrylamide gel electrophoresis (PAGE). Leaf samples of
Mentha piperita were collected for enzymatic studies from three
different altitudinal locations, including Purera (1,250 m,
32°17'41"N, 7°10'76"E), and of plains ( 950 m, 32°20'47''N,
77°13'17"E). Fully developed young leaves were harvested
between 09:00 and 10:00 h on a clear sunny day and stored in
liquid nitrogen for further use. All the assays were performed in the
Institute’s laboratory at Lucknow. The range for photosynthetic
photon flux density varied from 1500 to 1700 and 2200 to 2500
μmol m -2 s -1 for Purera, and Plains, respectively. Mean monthly day
temperatures during the month of data recording at these localities
were 9.2±2.2 and 22.2±3.2°C, respectively. Further, using 2 g of
fresh chopped leaves at 3rd position, were homogenized with 5 ml
of 0.1 M phosphate buffer (pH 6.8). Each treatment was replicated
3 times and assayed on SDS page electrophoresis. Superoxide
dismutase (SOD) activity was assayed by the method of Henary
et.al. (1976). Geranyl pyrophosphate synthtase (GPP) assayed was
as described previously (Misra and Sharma, 1991). RFPD through
PCR and cDNA analysis were performed as described by Saxena
et al. (2008).
Estimation of essential monoterpene oil(s)
Geranium oil estimation was done by steam distillation of 100 g
freshly plucked leaves in a clevenger’s apparatus (Clevenger,

Figure 1. Disease free plants produced through plant tissue
culture technique in the culture tubes.
(a)
(b)
Figure 2. The plants with full growth in the controlled conditions at
glass hous. (a) At hills cultivation; (b) At plains cultivation.
1928). The oil constituents mainly geraniol, citronellol and other
associated oil contents were determined by gas liquid
chromatography (Perkin –Elemer model 3920 B). The stainless
steel column was packed with 10% carbowax (20 mesh) on
Misra and Srivastava 1985
Figure 3. Effect of peroxidise activity in peppermint cultivated at
hiher altitude- Purera and plains: Series#1 to 4 at hills, and
series#5 to 8 are at plains cultivation.
chromosorb WNAW. Injector and detector temperature was
maintained at 200°C. The flow of H2 was 0.47 cm S -1 data
processing for area % was done on a Hewllet Packard integrator
model HP-33%.
Statistical analysis
The results were statistically analyzed for the least significant
differences (LSD) using the layout of a complete randomized
design (CRD). Further, the results were analyzed for the correlation
coefficient to determine the relationship among the characters
studied, using the relationship Y= a+b x.
RESULTS AND DISCUSSION
Disease free somaclonal variants of Mentha piperita Var.
Kukarail were obtained from tissue culture techniques invitro
from agar medium at controlled condition (Figure 1),
and further planted in plains and at higher altitudes of
CIMAP Resource Center at Purera.
Peroxidase, isoenzymes of peroxidises, increased with
increase in altitude and was higher by 60%, at Purera,
than in plants grown at Plains (Figure 1). Similar trend
was shown by and an antioxidant enzyme - SoD and
activities of GPP (Figure 2). Activity also showed similar
trend with almost double activity c-DNA at Purera as
compared to plains (Figure 3). Three out of four probable
enzymes of antioxidants activities in monoterpene
synthesis during primary plant products and secondary
plant products- the monoterpenes metabolism, including
Peroxidise, iso-enzymes of peroxidises (Figure 5) and
SOD exhibited lower activities at plains, compared to
Purera (Figure 2). The fourth probable enzyme of GPP of
involved in Carbon metabolism monoterpene metabolism,
showed higher activity by 43% at Purera, compared to
plains (Figure 3).
GPP activity, which is associated with the capacity of

1986 Afr. J. Pharm. Pharmacol.
Figure 4. RNA isolated from leaves and shoeing the bands in
electrophoresis.
Figure 5. Native polyacrylamide gel electrophoresis (PAGE): Zn treatment 0.00 to 1.0 Ug/ml. Showed the
peroxidase isoenzyme band profiles in Peppermints cultivation at plains (#1 to 4) and at higher altitudes
of Purera (#5 to 7b and unnumbered the last band).
carbon fixation, may increase with altitude (Chabot et al.,
1972). Temperature drops consistently with altitude and
may impart a major impetus to shape leaf’s
photosynthetic response at high elevation. Response of
GPP to temperature is largely explained by the function
of its activating enzyme, GPP activase which has a low
temperature optimum (Robinson and Portis, 1989; Crafts-
Brandner et al., 1997). RuBPCO activase is instrumental
in maintaining high GPP activity at low temperatures
(Pearcy, 1977). GPP was transformed into GGPP
through GPP synthase enzyme. GPP synthase is
composed of two subunits, one larger subunits and
another of smaller subunits. Here in our studies the plains
cultivated peppermint is composed of only smaller
subunits which showed the lesser activity of GPP of plain
crops (Figure 4). The same trend was also obtained in

Figure 6. Electrophoretic bands after PCR with gene specific primer.
cDNA analysis with RFPD lesser bands then the
cultivations of higher altitudes of Purera (Figure 6). Again
the total photosynthesis, saccarides formation, and total
oil production was 2 folds much higher than the plain
cultivations (Table 1). The same production of
menthofuron was obtained in the higher cultivation of
peppermints. Further, CD values indicated significant
differences at p

1988 Afr. J. Pharm. Pharmacol.
Table 1. Effect of M. pipeperita cultivation on growth parameters.
Harvesting
Growth attribute
Plane side Hill side LSD LSD
1 st Harvest 2 nd Harvest 3 rd Harvest 4 th Harvest 5 th Harvest 6 th Harvest At 5% At 1%
Plant height (cm) 57.0 58.0 61.0* 63.4** 64.1** 59.0 2.5 4.1
No. of branches 9 10* 13** 10* 10* 8 1.1 3.2
Fresh mass (g plant -1 ) 218.8 238.6* 224.8 282.5** 215.5** 196.2 11.1 16.3
Dry mass (g plant -1 ) 14.11 16.33* 16.81* 19.36** 18.46** 15.85 2.10 3.30
Leaf area (cm 2 ) 8.2 12.1* 25.2** 40.3** 37.2** 11.2 3.5 6.2
Chl a (g kg -1 (FM)) 0.68 0.79* 0.94** 1.48** 1.01** 0.82* 0.11 0.15
Chl b (g kg -1 (FM)) 0.50 0.56 0.61* 0.79** 0.40 0.29 0.08 0.12
Chl a/b 1.36 1.41 1.54 1.87 2.53 2.83 - -
PN (μg(CO2) m -2 s -1 ) 0.15 0.19* 0.75** 0.82** 0.71** 0.42** 0.03 0.06
Saccharides (μg (CH2O)
m -2 s -1 )
0.102 0.129 0.510 0.558 0.483 0.286 - -
Oil % 0.35 0.36 0.47* 0.56** 0.46 0.47 0.02 0.04
Menthone % of total oil 21 27** 27** 25** 38** 37** 0.01
Menthol % of total oil 0.59 59 67** 67** 69** 69** 0.01
Menthofuron % of total oil 5 10** 9.** 19** 18** 17** 0.04
Fe (mg kg -1 ) 98 112 142** 537** 419** 312** 21
Mn (mg kg -1 ) 26 37** 41** 98** 62** 53** 9
Zn (mg kg -1 ) 12 19* 34** 64** 41** 36** 7
Cu (mg kg -1 ) 7 9 11** 12** 7 5 3
Chl, Chlorophyll; PN, net photosynthetic rate; oil amounts in % of total oil. * and **, Values are significant at P=0.05 and P=0.01 levels, respectively.
possible source. Further, these enzymatic alterations
could provide adaptive advantage to plant in order to
conserve carbon at high elevation. The high total oil
contents, menthofuron at higher altitude cropping of
peppermints at Purera leads to value addition. This
higher menthofurn enrich plants at higher altitude than
the plains, leads to control of dangerous smoking and as
a good reliever to smoker if added into the candies.
Further, it is highly available in candies of states, which
have menthofuron as an additive in candies.
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(Nothofagaceae). New Phytol., 161: 585-594.
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and stomatal morphology of Southern seech Nothofagus
cunninghamii, exposed to depleted CO2 concentrations. Aust. J.
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temperature and carbon dioxide in herbaceous plants from low and
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primary products of photosynthesis and the associated enzymes in
barley and wheat. Photosynth. Res. 88: 63-71.
Kumar N, Kumar S, Ahuja PS (2004). Differences in the activation state
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African Journal of Pharmacy and Pharmacology Vol. 5(17), pp. 1990-1995, 8 November, 2011
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP11.172
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Simultaneous determination of gatifloxacin and
dexamethasone sodium phosphate in bulk and
pharmaceutical formulations by HPLC
K. R. Sireesha 1 * and K. Prakash 2
1 S. N. Vanitha Pharmacy Mahavidyalaya, Exhibition grounds, Hyderabad (AP), India.
2 Nirmala College of pharmacy, Mangalagiri, Guntur (AP), India.
Accepted 20 September, 2011
A liquid chromatography method was developed and validated for the simultaneous determination of
gatifloxacin and dexamethasone sodium phosphate in bulk and pharmaceutical formulations. Optimum
separation was achieved in less than 5 min using a C18 column (250 × 4.6 mm i.d, 5 µ particle size) by
isocratic elution. UV detection was carried out at 254 nm. Developed method was economical in terms
of the time taken and amount of solvent consumed for each analysis. It was also validated with respect
to linearity, limit of detection, limit of quantification, precision, accuracy, specificity, robustness and
system suitability. The limits of detection for gatifloxacin and dexamethasone sodium phosphate were
0.397 and 0.11 µg/ml, respectively. Limits of quantification were found to be 1.203 and 0.342 µg/ml for
gatifloxacin and dexamethasone sodium phosphate, respectively. The developed method was
successfully applied to the simultaneous determination of gatifloxacin and dexamethasone sodium
phosphate in bulk and pharmaceutical formulations.
Key words: Gatifloxacin, dexamethasone sodium phosphate, high performance liquid chromatography (HPLC),
isocratic elution.
INTRODUCTION
Dexamethasone sodium phosphate (DSP) is a highly
selective glucocorticoid which is widely used in ocular
inflammatory diseases. It’s chemical name is 9- fluoro-
11b, 17, 21-trihydroxy-16α- methylpregna-1,4- diene-
3,20-dione 21-(dihydrogen phosphate) disodium salt
(Indian Pharmacoepia, 2007; Balaji et al., 2008).
Gatifloxacin (GFN) is a fourth generation fluoroquinolone
antibiotic used in bacterial infections. It is chemically 1cyclopropyl-6-fluoro-
1,4- dihydro-8-methoxy-7-(3methylpiperazin-1-yl)-
4-oxo-3-quinoline carboxylic acid
(USP, 1995; Sayed et al., 2011). Dexamethasone in
combination with Gatifloxacin is used in several antiinfective
eye preparations to treat acute and sub acute
conjunctivitis caused by susceptible strains of the
following aerobic gram positive and negative bacteria
such as Staphyloccocus aureus, Staphyloccocus
*Corresponding author. E-mail: rupak1@rediffmail.com. Tel:
08686654459.
epidermidis, Streptococcus pneumonia and Haemophilus
influenza.
In the literature, methods were described for the
individual estimation of fluoroquinolones and
dexamethasone in aqueous samples and biological fluids
by liquid chromatography (Chen et al., 2006; Hyung and
Donald, 1995) liquid chromatography-fluorescence
detection (Joana et al., 2011). A few methods were also
given for the simultaneous determination of
Dexamethasone with other drugs such as
Chloremphenicol (Iqbal et al., 2006), ciprofloxacin (Rele
et al., 2010) and ofloxacin (Ali et al., 2002).But simulta-
neous determination of these drugs in pharmaceutical
formulations has not been reported in the literature. So
an attempt was made to develop a HPLC method for the
estimation of these drugs available as eye drops.
The purpose of the present study was to develop a
simple, sensitive, precise and accurate HPLC method for
simultaneous determination of GFN and DSP in bulk and
pharmaceutical formulations. The developed method has
been validated (USP, 1995; ICH Q2B 2003) by evaluation

of the system suitability, specificity, linearity, limit of
detection and quantitation, precision, accuracy and
recovery. The validated method was applied to the
commercially available pharmaceutical formulations
containing both the drugs.
MATERIALS AND METHODS
DSP and GFN were obtained as gift samples from Ajanta Pharma
Ltd, Mumbai. HPLC grade acetonitrile was purchased from SD
Fine-chemicals, India. Triple distilled water was used during the
study. The pharmaceutical formulations containing 3 mg/ml of GFN
and 1 mg/ml DSP (ZIGAT-D eye drops, FDC proxima, India.) were
purchased from local market.
Instrumentation
A high performance liquid chromatograph (Shimadzu-10 AT VP)
equipped with two pumps (Model-10AT VP) and Shimadzu UV-
Visible detector (SPD-10AT VP), ultrasonic bath (Spincotech Pvt.
Ltd, India).
Chromatographic conditions
For chromatographic analysis, a Hypersil C18, (250 × 4.6 mm i.d, 5
µ particle size) was used. Separation was carried out by isocratic
elution. The mobile phase consisting of a mixture of mixed
phosphate buffer (pH 6.8) and acetonitrile (ACN) in the ratio of
60:40 was filtered under vacuum from 0.45 membrane filter and
degassed in ultrasonic bath for 30 min before passing through the
instrument. The injection volume was 20 µl and the flow rate was 1
ml/min. UV detection was carried out at 254 nm.
Preparation of standard solution
Stock standard solutions of GFN and DSP were prepared in the
mobile phase at a concentration of 1200 and 400 µg/ml. The
working standard solutions were prepared by serial dilution of stock
solutions with the mobile phase.
Sample preparation
Sample solutions of GFN and DSP were prepared at a
concentration of 1200 and 400 µg/ml by diluting 10 ml of the
ophthalmic solution to 25 ml with the mobile phase. From this, 1 ml
was taken and diluted to 10 ml to get a concentration of 120 and 40
µg/ml of GFN and DSP.
Validation
The developed analytical method was validated as per International
Conference on Harmonization (ICH) and United States
Pharmacopeia (USP) guidelines for the parameters like linearity,
limit of detection (LOD), limit of quantification (LOQ), precision,
specificity, accuracy, robustness and system suitability.
Linearity
Six working standard solutions of each analyte in the concentration
range of 24 to 144 µg/ml for GFN and 8 to 48 µg/ml for DSP was
prepared in triplicate and injected. Calibration curves were
constructed by plotting concentration versus mean peak area.
Limits of detection and quantification
Sireesha and Prakash 1991
Limits of detection (LOD) and quantification (LOQ) were calculated
based on the standard deviation of the response and slope of the
calibration curve. LOD and LOQ are calculated from the formulae
3.3 and 10 σ/s, respectively, where σ is the standard deviation of yintercepts
of the regression line and s is the slope of the calibration
curve.
Precision
Method precision
The precision of the method was evaluated in terms of intermediate
precision, that is, intra-day and inter-day precision and by different
analysts. For intra-day precision, three different concentrations of
GFN and DSP in the linearity range were prepared in triplicate and
were analyzed during the same day. For inter-day precision the
same concentrations were analyzed on three consecutive days.
The relative standard deviations (RSD) values for GFN and DSP
showed that the precision of the method was satisfactory.
System precision
System precision was analyzed by injection repeatability. This was
examined by analyzing six injections of the mixture containing GFN
and DSP at 120 and 40 µg/ml, respectively. The RSD were
calculated from the peak areas and retention times of GFN and
DSP.
Accuracy
Accuracy of the method was determined by recovery studies. These
studies were carried out by addition of known amounts of GFN and
DSP to a sample solution of known concentration and comparing
calculated and measured concentrations. A sample solution
containing GFN and DSP (1.2 and 0.4 mg/ml, respectively) was
prepared by diluting 10 of the ophthalmic solution to 25 ml in
volumetric flask and make-up of the solution with the mobile phase,
samples (0.4 ml) of the filtered solution were transferred to 10 ml
volumetric flasks containing 0.4, 0.6 and 0.8 ml of GFN and DSP
standard solution, dilutions were made and analyzed. The
percentage recovery and the percentage RSD was calculated and
found to be within the limits.
Specificity
Specificity of an analytical method may be defined as the ability of
the method to measure accurately and specifically the analyte in
presence of additional components, such as matrix, impurities,
degradation products and other related substances.
Robustness
Robustness of the method is a measure of capacity of the method
to remain unaffected by small but deliberate variations in method
parameters and provides an indication of the variability during
normal usage. Robustness of the method was evaluated by varying
method parameters, such as detection wavelength and flow rate.
Detection wavelength was changed from 254 nm to 254 ± 2 nm and
flow rate was changed from 1 ml/min to 1 ± 0.1 ml/min. Effect of
these changed parameters was studied by injecting the sample into
the system.

1992 Afr. J. Pharm. Pharmacol.
System suitability
Voltage (mV)
50
Figure 1. Chromatogram for blank.
System suitability was established in order to determine the adequate
resolution and reproducibility of the proposed method.
Suitability parameters including retention factor, resolution,
asymmetry factor and plate number were investigated.
Assay of the marketed formulation
The developed method was applied to the simultaneous
determination of GFN and DSP in pharmaceutical formulations.
Sample was analyzed by performing six independent
determinations and each series was injected in triplicate.
RESULTS AND DISCUSSION
Mobile phase optimization
Chromatographic parameters were optimized to develop
a HPLC method for simultaneous determination of GFN
and DSP with short analysis time (< 5 min) and
acceptable resolution (RS > 2). Various compositions of
mobile phases like methanol: buffer and ACN: buffer in
different ratios were tried.
But with mixed phosphate buffer (pH 6.8) and ACN in
the ratio of 60:40 at a flow rate of 1 ml/min symmetrical
peaks with good resolution were obtained.
Chromatogram for the mobile phase (blank
chromatogram) is shown in (Figure 1) and shows no
interference with the drug peaks. The optimum
wavelength for detection was set at 254 nm at which
Time (min)
better detector response for both drugs was obtained.
The retention times were 2.42 and 4.81 min for GFN and
DSP, respectively (Figure 2).
Validation
Calibration graphs were constructed by plotting the peak
area versus their corresponding concentrations. Good
linearity was obtained in the range of 24 to 144 µg/ml and
8 to 48 µg/ml for GFN and DSP. The results are shown in
Table 1. LOD and LOQ were calculated from the slope
and standard deviation y-intercepts of the regression line
of the calibration curve. For GFN it was found to be 0.397
and 1.203 µg/ml and for DSP 0.11 and 0.342 µg/ml,
respectively. The precision of the method and instrument
precision was evaluated and relative standard deviation
(RSD) values were calculated. The RSD values for GFN
and DSP showed that the precision of the method was
satisfactory. The results are shown in Table 2. The
accuracy of the method was determined by recovery
studies. The recoveries were close to 100% for GFN and
DSP; the results are as shown in Table 3. Developed
method was found to be robust when the detection
wavelength and flow rate was changed from 254 to 254 ±
2 nm and 1 to 1 ± 0.1 ml/min. There was no considerable
change in the peak areas and retention times. Using 0.9
ml/min flow rate, the retention time for GFN and DSP
were found to be 2.69 and 5.34 min, respectively and
with 1.1 ml/min flow rate, retention times for GFN and
DSP were found to be 2.21 and 4.41 min, respectively

Voltage (mV)
Time (min)
Figure 2. Typical chromatogram for the standard solution of GFN and DSP.
Table 1. Linearity by regression analysis (n = 6).
Substance R 2 Slope Concentration range (µg/ml)
GFN 0.999 17.94 24-144
DSP 0.999 20.56 8-48
‘n’ is the number of determinations.
Table 2. Precision (% RSD).
Table 3. Recovery studies (n = 6).
Drug
GFN
DSP
Parameter GFN DSP
Intra-day precision 0.033 0.096
Inter-day precision 1.78 1.39
Analyst precision 0.08 0.19
Injection repeatability for tR 0 0.027
Injection repeatability for peak area 0.12 0.22
‘n’ is the number of determinations and RSD is relative standard deviation.
Concentration
(µg/ml)
Amount recovered
(µg/ml)
Sireesha and Prakash 1993
Recovery (%) RSD (%)
96 95.94 99.94 0.037
120 119.65 99.71 0.04
144 143.86 99.9 0.052
32 31.97 99.26 0.05
40 39.86 99.65 0.426
48 45.85 99.68 0.29
‘n’ is the number of determinations and RSD is relative standard deviation.

1994 Afr. J. Pharm. Pharmacol.
Voltage (mV)
Table 4. System suitability parameters (n = 6).
Parameter GFN DSP
Retention time (tR) 2.42 4.81
Asymmetry factor 1.7 1.2
Resolution - 13.2
Number of plates 3735 8926
‘n’ is the number of determinations.
Time (min)
Figure 3. Chromatogram for the sample solution of GFN and DSP.
Table 5. Assay of eye drops (n = 6).
Drug Label claim (mg/ml)
Amount found
(mg/ml)
Mean recovery (%) RSD (%)
GFN 3 3 99.7 ± 0.8 0.034
DSP 1 0.99 99.44 ± 0.4 0.032
‘n’ is the number of determinations and RSD is relative standard deviation.
without affecting the resolution of the drugs. When
detection wavelength was changed to 254 ± 2 nm, the
retention time for GFN and DSP were not changed from
the normal. System suitability parameters are shown in
Table 4.
Assay of the marketed formulation
According to ICH in the case of assay, demonstration of
specificity requires that the procedure is unaffected by
the presence of impurities or excipients. The assay value
of the marketed formulation was found to be within the
limits. The low RSD value indicated suitability of this
method for routine analysis of GFN and DSP in
pharmaceutical dosage forms. Chromatogram of the
sample shows that there was no interference from the
excipients present in the formulation (Figure 3); this
indicates the specificity of the method. The results are
shown in Table 5.

Conclusion
The method described in this paper for the simultaneous
estimation of GFN and DSP are found to be simple,
sensitive, accurate, precise, rapid, robust and
economical. The analytical conditions and the solvent
system developed provided good resolution within a short
analysis time. The RSD for all parameters was found to
be within the limits, which indicates the validity of method
and assay results obtained by this method are in fair
agreement. Thus, the developed method can be
proposed for routine analysis of GFN and DSP in
laboratories and for quality control purposes.
REFERENCES
Ali M.S, Ghori M, Saeed A (2002). Simultaneous Determination of
Ofloxacin, Tetrahydrozoline Hydrochloride, and Prednisolone Acetate
by High-Performance Liquid Chromatography., J. Chromatographic
Sci., 40(8): 429-433.
Balaji K, Raghunatha reddy GV, Madhusudan reddy K, Jayaramireddy
S (2008). Determination of prednisolone, dexamethasone and
hydrocortisone in pharmaceutical formulations and biological fluid
samples by voltammetric techniques using β- cyclodextrin modified
carbon paste electrode. Afr. J. Pharm. Pharmcol., 2(8): 157-166.
Chen Y, Zhou YP, Tan DH, Deng JJ, Xu LF (2006). Simultaneous
Determination of Ciprofloxacin hydrochloride and Dexamethasone
Acetate in Compound Ciprofloxacin Hydrochloride Ear Drops by
HPLC. J. Liaoning Univ., 4: 10.
Hyung WK, Donald JD (1995). Determination of Dexamethasone
sodium phosphate in the vitreous by high performance liquid
chromatography. Korean J. Opthalmol., 9: 79-83.
Indian Pharmacopoeia, Vol ІІ (2007). Published by The Indian
Pharmacopoeia commission, Ghaziabad, 1158: 1005-1007.
Iqbal MS, Shad MA, Ashraf MW, Bilal M, Saeed M (2006). Development
and validation of an HPLC method for the determination of
dexamethasone, dexamethasone sodium phosphate and
chloramphenicol in presence of each other in pharmaceutical
preparations. Chromatographia, 64: 219-222.
Sireesha and Prakash 1995
Joana S, Gilberto A, Ana F, Angelina P, Celeste L, Amilcar F (2011).
Development and validation of a fast isocratic liquid chromatography
method for the simultaneous determination of norfloxacin,
lomefloxacin and ciprofloxacin in human plasma. Biomed.
chromatogr., 25: 535-41.
Rele RV, Warkar CB (2010). Simultaneous Determination of
Ciprofloxacin Hydrochloride and Dexamethasone in Ophthalmic
Solution by Reversed Phase High Performance Liquid
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Syed MR, Nadia MSA, Mansour ME (2011). Evaluation of effects of
ciprofloxacin and gatifloxacin on neurotransmitter levels on cortex
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United States Pharmacoepia 23 (1995). The United States
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473.

African Journal of Pharmacy and Pharmacology Vol. 5(17), pp. 1996-2001, 8 November, 2011
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP11.190
ISSN 1996-0816 © 2011 Academic Journals
Full Length Research Paper
Anti-inflammatory and wound healing activity of
Fagonia schweinfurthii alcoholic extract herbal gel on
albino rats
Saleh I. Alqasoumi 1,2 , Hasan S. Yusufoglu 1 * and Aftab Alam 1
1 Department of Pharmacognosy, College of Pharmacy, Al Kharj University, Al Kharj, Kingdom of Saudi Arabia.
2 Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Kingdom of Saudi Arabia.
Accepted 21 September, 2011
Traditionally, a large number of plants are used for treatment of inflammation and wounds. In Asian and
African countries Fagonia schweinfurthii (Hadidi) (F. Zygophyllaceae) and the closely related herb, such
as Fagonia arabica are traditionally used for treatment of inflammation, open wounds, boils, skin
eruptions, allergies, etc. Hence, the present study was conducted to investigate the anti-inflammatory
and wound healing effects of 90% alcoholic extract of F. schweinfurthii formulated gel on carrageenan
induced rats paw edema and excision wound model, respectively. The effects were compared with the
anti-inflammatory diclofenac sodium ointment (Diclomax ® ) and the wound healing povidone-iodine
(Betadine ® ) drugs. The herbal gels and diclofenac sodium ointment were topically applied (0.5 g) to the
planter surface of the left hind paw and anti-inflammatory effect was observed within 3 h. The wound
healing effect was investigated by application of 0.5 g/wound of the F. schweinfurthii gel and Betadine ®
once daily for 19 days to the excision wound of albino rats and was observed at 4 days intervals. It was
observed that gel formulations have progressive anti-inflammatory effect and accelerate the wound
closer time. This study suggests that F. schweinfurthii plant extract gel formulation could be developed
as a therapeutic agent for anti-inflammatory and wound healing effects.
Key words: Fagonia schweinfurthii, herbal formulation, anti-inflammatory, wound healing.
INTRODUCTION
Fagonia schweinfurthii and its closely related species are
widely distributed in deserts and dry areas of India to
tropical Africa and Chile to South West U.S.A. They are
traditionally well known for the treatment of hemorrhoids,
inflammation, sores, leprosy, open wounds and fever in
the form of internal and external conventional formulation
(Miller et al., 1988). When the powder that is made up of
the whole plant of F. schweinfurthii is dusted on boils and
skin eruptions, it causes healing and when the whole
plant is boiled in water, its bath is useful for allergies and
other skin diseases and decoction is given orally as blood
purifier (Qureshi et al., 2010). The other species like
Fagonia bruguieri aqueous extract is claimed for anti-
*Corresponding author. E-mail: hasanagazar@hotmail.com.
Tel: +966-1-5886025, +966-535159935. Fax: +966-1-5886001.
allergy (Abdulaziz and Hussein, 2007). Fagonia cretica
methanol extract is claimed for good antimicrobial
potential (Anjum et al., 2007) and it exhibited strong free
radical scavenging properties against reactive oxygen
and nitrogen species (Rawal et al., 2004). The other
effects of Fagonia species include anti-inflammatory,
analgesic, antipyretic and thrombolytic activity (Prasad et
al., 2007; Satpute et al., 2009). Many chemical
constituent's, such as triterpenoids, saponins, flavonoid
glycosides, etc., have already been reported in different
Fagonia spp. (Shaker et al., 1999; Abdel-Khalik et al.,
2001; El-Wakil, 2007). Although, currently used antiinflammatory
and wound healings drugs are associated
with some severe side effects, herbal products are often
perceived as safe, because they are natural therefore,
the development of potent anti-inflammatory and wound
healer drugs with fewer side effects is necessary (Gesler,
1992). Inflammation is considered as a primary
physiologic defense mechanism that helps body to

protect itself against infection, burn, toxic chemicals,
allergens or other noxious stimuli. An uncontrolled and
persistent inflammation may act as an etiologic factor for
many of the chronic illnesses (Kumar et al., 2004).
Wound healing is the process of repairing injury of skin
and other soft tissue. This dynamic process is classically
divided into three overlapping phase “inflammation,
proliferation and remodeling” (Harding et al., 2002). Initial
stages of wound healing involve an acute inflammatory
phase followed by synthesis of collagen and other
extracellular matrix which are later remodeled to form
scar (Bhagavathula et al., 2009). Thus, the present study
aim to investigate wound healing and anti-inflammatory
activities to justify the traditional claims of this plant gel
form.
MATERIALS AND METHODS
Collection of plant and authentication
The plants of F. schweinfurthii was collected during December,
2009, from local area of Riyadh, Saudi Arabia and was kindly
authenticated by Dr. Mohammad Atiqur Rahman, taxonomist of the
Medicinal, Aromatic and Poisonous Plants Research Center
(MAPPRC), College of Pharmacy, King Saud University, Riyadh,
Saudi Arabia. A voucher specimen is deposited at the herbarium of
the College of Pharmacy, King Saud University.
Chemicals
Diclofenac sodium (1%) ointment (Diclomax ® ) and 10% povidoneiodine
(Betadine ® ) were purchased from a local pharmacy.
Triethanolamine and ethanol were obtained from Merck & Co. Inc
(USA). Carrageenan and carbapol-934 gel were supplied by Sigma
(USA).
Extraction of plant
The air dried powdered plant was extracted with 90% ethanol in a
soxhlet apparatus at 60°C. The extract was concentrated to syrupy
solution using rotary evaporator under reduced pressure at 40°C.
The thick solution was lyophilized using freeze drying system. The
yield (13. 5%) was used for the experimental studies.
Animals
Healthy Wister albino rats of either sex weighing ≈200 g, obtained
from the Experimental Animal Care Center, College of Pharmacy,
King Saud University, Riyadh were used. Rats were maintained
under controlled condition at temperature (22 ± 20°C), humidity
(55%) and light (12 h light/dark condition). The animals were
provided with Purina chow and drinking water ad libitum. The
experiments and procedure used in this study were approved by
the Ethical Committee of the College of Pharmacy, King Saud
University, Riyadh, KSA.
Acute toxicity study
The acute toxicity of F. schweinfurthii alcoholic extract was
determined in rats according to the previous method (Adedapo et
Alqasoumi et al. 1997
al., 2009) with slight modifications. Rats that fasted for 12 h were
randomly divided into four groups (n = 5). Graded doses of the
extract (300, 600, 1200 and 2400 mg/kg p.o.) were separately
administered to the rats. All the animals were then observed over a
period of 10 days for deaths and signs of acute toxicity.
Preparation of herbal gel
Herbal gels 10 and 20% were prepared separately according to the
method (Dey et al., 2009) with slight modifications. Carbapol-934
(1.8%) and sufficient amount of distilled water were mixed in a
separate beaker and soaked for 24 h at room temperature.
Triethanolamine was added drop-wise with constant stirring using
mechanical stirrer. After gel formulation, a weighed amount of (10
and 20 g) extract powder was incorporated in gelling agent
separately and mixed using glass rod. A similar procedure was
followed for control base gel without powdered extract.
Anti-inflammatory study
Carrageenan-induce rat paw edema model was used for antiinflammatory
study (Maswadeh et al., 2006). Twenty rats were
divided into four groups and fasted overnight with free access to
water before the experiment proceeds. Rats of the first, second and
third groups were treated with the control base, 10 and 20% gel
formulations. Rats of the forth group (standard) were treated with
marketed gel formulation (Diclomax®). Each formulation (0.5 g)
was applied to the planter surface of the left hind paw by gently
rubbing 50 times with the index finger. After 1 h, inflammation was
induced by subplanter injection of 0.1 ml of 1% carrageenan
solution in normal saline into the treated paw of all rats. The paw
volume of each rat was measured in milliliter using a
plethysmometer (Aptex, France) at 0 and 3 h post carrageenan
injections. The percentage anti-inflammatory activity was calculated
using the following equation:
Percentage anti-inflammatory activity = (V3h - V0h)/V0h × 100
where V3h is the paw volume after 3 h carrageenan injection and V0h
is the initial paw volume.
Wound healing study
The excision wound model was used to study the wound
contraction of F. schweinfurthii extract herbal formulations (Okoli et
al., 2009). At the beginning of the experiment, twenty rats were
anesthetized using diethyl ether and the dorsal skin of each rat was
shaved with an electric clipper and put in separate cage. After 12 h,
all animals were again anesthetized by diethyl ether and the shaved
areas were sterilized with 70% alcoholic solution and sketch wound
area (≈ 2.5 cm 2 ). A predetermined dorsal area was excised using
toothed forceps, scalpel and pointed scissors. A fresh surgical
blade was used for the perpendicular cut in each animal and the
tension of the skin was kept constant during the procedure. Animals
were divided into four groups (n = 5). Rats of the first, second and
third groups were treated with the control base, 10 and 20% gel
formulations. Rats of the forth group (standard) were treated with
marketed formulation (Betadine®). The base gel, extract gels and
standard drug (0.5 g, each) were applied topically on the wound
surface once a day for 19 days. The wound areas were traced on
graph paper (1 mm 2 ) immediately after the wound excision and
every 4 days until healing was accomplished. The reduction in the
wound size was calculated according to the following formula:
Wound contraction (%) = [(W0 - Wt)/W0] × 100

1998 Afr. J. Pharm. Pharmacol.
Table 1. Effect of Fagonia alcoholic extract gel formulations on Anti-inflammatory.
Treatment
Net increase in paw volume
(ml)
Reduction of edema
(%)
Control base 2.28 ± 0.06 -
10% gel 1.88 ± 0.08* 17.54
20% gel 1.10 ± 0.10*** 51.75
Diclomax ® 0.44 ± 0.06*** 80.70
Each value is the mean ± SEM, n = 5. Values differ significantly (*P < 0.05 and ***P<
0.001) from control base gel.
where W 0 is the wound diameter on day zero and W t is the wound
diameter on day t.
The time taken for 50% of wound closure (WC50) was calculated
by a plot of percentage wound closure against days.
Statistical analysis
The results were expressed as mean ± SEM. The data were
subjected to one-way ANOVA student t-test using graphPad Prism
5 software. P < 0.001 was considered as significant.
RESULTS AND DISCUSSION
Animal toxicity study
Oral administration of graded doses (300 to 2400 mg/kg
p.o.) of the alcoholic extract of F. schweinfurthii to rats,
did not produce any mortality and changes in behavior,
breathing, cutaneous effects, sensory nervous system
responses or gastrointestinal effects during the 10 days
observation period. The obtained results signify that the
use of the plant for treatment is safe. So the formulations
(10 and 20% gel) were selected for anti-inflammatory and
wound healing studies.
Anti-inflammatory study
The anti-inflammatory activity was expressed as "mean
increase in paw volume ± SEM" in terms of milliliter and
percentage inhibition in paw volume by different gel
formulation and standard drug. The 20% herbal gel
produced significant (P < 0.001) reduction of
Carrageenan-induced paw edema (1.10 ± 0.10 ml) as
compared to the control base gel group (2.28 ± 0.06 ml)
after 3 h from carrageenan injection. The reference drug
was found to be comparatively more potent as compared
to formulated gel (0.44 ± 0.06 ml). The percentage
inhibition of paw edema for 10 and 20% and Diclomax ®
was 17.54, 51.75 and 80.70%, respectively (Table 1).
The probable mechanism of inflammation action is biphasic,
the first phase is attributed to the release of
histamine, serotonin, 5-HT and kinins in the first hour;
while, the second accelerating phase of swelling is
related to the release of prostaglandin, bradykinins and
lysozymes-like substances in 2 to 3 h (Brooks and Day,
1991; Silva et al., 2005). Carrageenan-induced edema
involves the synthesis or release of pain and fever
mediators like prostaglandins (PGE), histamine,
bradykinins, leucotrienes and serotonin. This model has a
significant predictive test for anti-inflammatory agents
acting by the mediators of acute inflammation (Adedapo
et al., 2009). The results of this study indicated that F.
schweinfurthii extract gel formulation can be effective in
acute inflammatory disorders. The plant contains
polyphenolic compounds, saponins and flavonoid
glycosides that could be responsible for the antiinflammatory
activity either alone or may be due to
inhibition of inflammatory mediators in combination with
other constituents (Adamu et al., 2007).
Wound healing study
The percentage of wound contraction in the second, third
and fourth groups were 6.93 ± 0.11, 8.40 ± 0.20 and 9.06
± 0.11 on the 4 th day and 90.53 ± 0.10, 93.93 ± 0.09 and
95.53 ± 0.10 on the 19th day, respectively. The
percentage of wound contraction of the first group 6.13 ±
0.23 and 72.08 ± 0.16 on 4th and 19th days, respectively
was comparatively lower than the other three groups
(Table 2). The WC50 (time for 50% wound healing) of
control base, 10 and 20% herbal gel and standard drug
(Betadine®) was, 13.92, 11.06, 10.53 and 10.82%,
respectively (Table 3). The wound size decreases after
4th (A) and 19th (B) day of 10 and 20% gel and
Betadine® was clearly indicating the wound healing
effects (Figures 1 to 3). Wound healing is an elementary
response to tissue injury that it consequences in
restoration of tissue integrity, is mainly achieved by the
synthesis of the connective tissue matrix. It involves
regeneration of specialized cells by proliferation of
surviving cells characterized by the formation of
granulation tissue and wound contraction, which is largely
due to the action of myofibroblasts (Okoli et al., 2009). All
groups show the wound healing, including control base
gel, because of biological response regulating the body's
own cellular defense mechanisms which contributes in

Table 2. Effect of Fagonia alcoholic extract gel formulations on wound healing.
Alqasoumi et al. 1999
Treatment
4-days
Percentage wound contraction (mean ± SEM)
8-day 12 days 16 days 19 days
Control base 6.13 ± 0.23 17.96 ± 0.03 48.93 ± 0.11 55.33 ± 0.11 72.08 ± 0.16
10% gel 6.93 ± 0.11 32.93 ± 0.16 61.33 ± 0.23 80.40 ± 0.20 90.53 ± 0.10
20% gel 8.40 ± 0.20 36.60 ± 0.17 64.66 ± 0.11 83.46 ± 0.11 93.93 ± 0.09
Betadine ® 9.06 ± 0.11 24.46 ± 0.09 66.93 ± 0.11 85.86 ± 0.11 95.53 ± 0.10
Each value is the mean ± SEM, n = 5. Values of percentage wound reduction of each formulation differ when compared with
control base gel.
Table 3. Effect of Fagonia alcoholic extract gel
formulations on WC50.
Treatment WC50 (Days)
Control base 13.92
10% gel 11.06
20% gel 10.53
Betadine ® 10.82
Each value is the time taken for 50% wound closure (WC50).
Values of WC50 of each formulation differ when compared
with control base gel.
Figure 1. Excision wound healing of 10% Fagonia alcoholic
extract gel formulation treated rat on 4th (A) and 19th (B) days
observation.
Figure 2. Excision wound healing of 20% Fagonia lcoholic extract
gel formulation treated rat on 4th (A) and 19th (B) days
observation.

2000 Afr. J. Pharm. Pharmacol.
Figure 3. Excision wound healing effect of 10% povidone-iodine (Betadine ® ) treated
rat on 4th (A) and 19th (B) days observation.
wound healing and its repair (Malviya and Jain, 2009).
The formulated gel fasten the wound healing process
may be due to enhancing the cellular defense
mechanisms, proliferation, suppression of inflammation
and contraction of the collagen tissue and could be
delayed by reactive oxygen species or microbial infection
(Marwah et al., 2007). The polyphenolic compounds have
improved regeneration and organization of the new tissue
and hasten the wound healing process (Leite et al., 2002)
may be due to anti-inflammation, anti-oxidant and
antimicrobial activities. The plant contains polyphenolic
compounds including saponins, flavonoids, glycosides
(Kumar et al., 2004; Abdel-Khalik et al., 2001; Gesler,
1992) and these compounds showed the antioxidant and
anti-microbial (Anjum et al., 2007; Rawal et al., 2004) and
anti-inflammatory (present study). The result of the
present study revealed that the topical application of 10
and 20% extract gel on the experimentally excised wound
accelerate the wound healing process.
Conclusion
In conclusion, the plant is safe for use as no mortality
was recorded in the acute toxicity test. The two gel (10
and 20%) prepared with F. schweinfurthii reduced
significantly the formation of edema induced by
carrageenan and exhibited a good anti-inflammatory
effect comparable to those of Diclomax ® and exhibited a
good wound healing effect comparable to those of
Betadine®. The study has thus, provided some
rationalization for the folkloric use of the plant in several
communities for conditions, such as inflammation, boils,
skin eruptions and other skin diseases.
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African Journal of Pharmacy and Pharmacology Vol. 5(17), pp. 2002-2006, 8 November, 2011
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP11.229
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
The effects of maximal aerobic exercise on cortisol and
thyroid hormones in male field hockey players
Malik BEYLEROGLU
School of Physical Education and Sports, Sakarya University, Sakarya, Turkey. E-mail: malik-beyleroglu@hotmail.com.
Tel: + 90 264 2956646. Fax: + 90 264 2956642.
Accepted 20 July 2011
Some metabolic and endocrine functions might be influenced by exercise and it leads to changed
concentrations of hormone secretions in humans. In order to determine the changes of thyroid
hormones (THs) related to shuttle run exercise (SRE), 14 field hockey players and their thyroid
stimulating hormones (TSH) free T3 (fT3), free T4 (fT4) were included in this study, and cortisol levels
were measured three times; before the exercise, just after the exercise and one hour later after
exercise. The results of this study showed that there were no statistically significant differences among
three measurements in the serum levels of TSH and thyroid hormones. However, both fT3 and TSH
were significantly decreased in one hour later after exercise, whereas no change was observed in fT4.
Cortisol concentrations were slightly increased immediately after SRE. In conclusion, serum levels of
thyroid stimulating hormone and thyroid hormones were affected by maximal aerobic exercise.
Key words: Thyroid hormones (THs), thyroid stimulating hormone (TSH) free T3 (fT3), freeT4 (fT4), cortisol,
shuttle run exercise (SRE).
INTRODUCTION
Exercise affects the activity of many glands and the
production of their hormones. Thyroid hormone levels
influence the skeletal and cardiac muscle function,
pulmonary performance, metabolism, and the
neurophysiologic axes. Hypothalamus secretes thyroid
releasing hormone (TRH) for stimulation of anterior
pituitary gland to release thyroid releasing hormone
(TSH). TSH causes thyroid gland to secrete two
aminoacid based hormones: Three iodine atom
containing triiodothyronine (T3) and four iodine atom
containing thyroxine (T4). Thyroid hormones have many
important biological effects. It controls how quickly the
body burns energy, makes proteins, and how sensitive
the body should be to other hormones. These hormones
regulate the rate of metabolism and affect the growth and
rate of function of many other systems in the body
(Wartofsky, 1995; Bernet and Wartofsky, 2000; McMurray
and Hackney, 2000).
The secretion of thyroid hormones T3 and T4 are
controlled by negative feedback loops. When the level of
thyroid hormones (T3 and T4) in the blood is high, by
regulatory negative feedback loop TSH production is
reduced. Both T3 and T4 exist in unbounded (free) and
bounded forms (McMurray and Hackney, 2000).
Cortisol is a glucocorticoid and synthesized by adrenal
gland. Its primary functions are to increase protein
breakdown, inhibite glucose uptake and increase
lipolysis. The level of serum cortisol is effected by many
factor such as intensity, duration and timing of exercise,
type of exercise, age, altitude, environmental temperature
and psychology (Bernet and Wartofsky, 2000; Deligiannis
et al., 1993).
In spite of the fact that many studies have been
reported on the effect of exercise on neuroendocrine
secretions, there were disagreements among the results
of these studies. While the level of THS was constant in
some studies ((Bernet and Wartofsky, 2000; Deligiannis
et al., 1993), there was an increase in its level in the
other studies (Burtis et al., 2008; Deligiannis et al., 1993;
Ciloglu et al., 2005). Some studies (Bernet and
Wartofsky, 2000; Siddiqui et al., 1983) reported that free
T4 level was unchanged, whereas there were an
increase (Bernet and Wartofsky, 2000; Deligiannis et al.,
1993; Ciloglu et al., 2005; Licata et al., 1984) and a
decrease in its level after exercise (Bernet and
Wartofsky, 2000; Limanova et al., 1983; Hackney and
Dobridge, 2009). Free T3 levels following exercise were
found as increased (Bernet and Wartofsky, 2000;

Table 1. Mean values of subjects for age, body height and body weight (n = 14).
Variable Age (year) Body height (cm) Body weight (kg)
MSD 19.50±1.22 176.00±5.53 68.78±6.29
Beyleroglu 2003
Table 2. Levels of THS, fT3, fT4 and cortisol in association with pre- (Assay 1), just after (Assay 2) and one hour after exercise (Assay 3),
and P values.
Variable
Assay 1
Trial
Assay 2 Assay 3
Summary of two groups comparisons
p (1-2) p (2-3) p (1-3)
TSH (µIU/ml) 2.12±1.58 2.21±1.44 1.29±0.73 0.54** 0.002* 0.016*
fT3 (pg/ml) 3.86±0.55 4.28±1.25 3.42±0.61 0.52** 0.015* 0.018*
fT4 (ng/dl) 1.70±0.29 1.50±0.27 1.50±0.22 0.06** 0.92** 0.031*
Cortisol (µg/dl) 17.12±7.15 22.96±7.47 18.93±4.6 0.018* 0.11** 0.26**
*p0.05, non-significant.
Siddiqui et al., 1983) and decreased (Deligiannis et al.,
1993) or unaffected (Ciloglu et al., 2005; Siddiqui et al.,
1983; Licata et al., 1984).
Static or isometric exercise, usually of short duration
but of high intensity, uses previously stored energy
whereas more prolonged exercise must use energy
generated by the normal metabolic pathways. The
changes in concentrations of analyzes as a result of
exercise are largely due to shifts of fluid between the
intravascular and interstitial compartments, changes in
hormone concentrations stimulated by the change in
activity and loss of fluid due to sweating (Burtis et al.,
2008). Progressive shuttle run test is suitable for
endurance athletes and players of endurance sports
including football, rugby and field hockey and its objective
is to monitor the development of the athlete's maximum
oxygen uptake (Mackenzie, 2005). Here, the effects of
exhaustive exercise on cortisol and thyroid hormones
(THs) were investigated before, immediately and one
hour after exercise.
MATERIALS AND METHODS
The researcher used the quasi-experimental approach with onegroup
design and measurements were taken pre- post and one
hour after the exercise. Post sample was purposefully chosen from
14 elite male field hockey players that participated in this study. The
mean age of the participants was 19,50±1,22 years, mean height
was 176.00±5.53 cm, mean weight was 68.78±6.29 kg. The
average training experience of the participants was 7,21±1,36 years
(Table 1).
Blood sample collection
Blood samples were obtained following an overnight fasting state.
Samples were withdrawn three times (at rest, immediately after
exercise and 1 h post exercise) from antecubital vein into blood
tubes and separated from the cells by centrifugation at 3000 rpm for
10 min. Serum samples were stored at -70°C and then they were
analyzed.
Measurement of thyroid hormones
Samples were analyzed three times (Assays 1, 2 and 3) for thyroid
stimulating hormone (TSH), free triiodotironin (fT3), free thyroxine
(fT4) and cortisol by using commercial kit and analyzer (Immulite
2000, BioDPC, USA) with chemiluminescence method.
Exercise protocol
Progressive shuttle run test was conducted to ensure the maximal
exhaustion of the participants (Ciloglu et al., 2005).
Statistical analysis
Results were presented as mean±SD. SPSS 10.0 program was
used for statistical analysis. Comparison among multiple assays
was performed by non-parametric test Mann Whitney-U. A 2-tailed
p value p0.05). Mean serum fT4 of Assay 2 was lower
than Assay 1 (p>0.05). Mean serum TSH and fT3 of
Assay 3 were lower than both assays 2 and 1 (p0.05) and Assay 1 (p

2004 Afr. J. Pharm. Pharmacol.
5
4.5
4
3.5
3
2.5
2
1.5
1
assay 1 assay 2 assay 3
TSH (µIU/mL)
fT3 (pg/mL)
fT4 (ng/dL)
Figure 1. Graph of changes in THS, fT3, fT4 and cortisol levels.
26
24
22
20
18
16
14
Cortisol (µg/dl)
Assay 1 Assay 2 Assay 3
Figure 2. Graph of changes in cortisol levels before (Assay 1),
immediate (Assay 2) and one hour (Assay 3) after exercise.
Assay 1 (p>0,05) (Table 2 and Figure 1).
The hormonal response to exercise involves increased
sympathoadrenal activity, increased somatotropin,
corticotropin, β-endorphin, prolactin, vasopressin, and
possibly TSH secretion. The extent of these changes is
related to training, nutrition, and state of health; all of the
endocrine responses are reduced by exercise training
(Licata et al., 1984, Limanova et al., 1983).
A major function of thyroid hormones is their control of
the basal metabolic rate and calorigenesis through
increased oxygen consumption in tissue via the effects of
thyroid hormone on membrane transport and enhanced
mitochondrial metabolism (Burtis et al., 2008).
The effects of the exercise on circulating thyroid hormone
values remain controversial. The relationship between
exercise and thyroid hormone metabolism has been
studied by several groups of investigators prevıously.
In a study carried out by Fortunato et al. (2008), they
classified rats into five groups to elucidate the effects of a
session of acute exercise on the treadmill at 75% of
maximum oxygen consumption on thyroid function of
rats: Control (without exercise), and killed just after (0
min) or 30, 60, and 120 min after the end of the exercise
session. They reported that a significant increase in T3
occurred just after the exercise, with a gradual decrease
thereafter so that 120 min after the end of the exercise,
serum T3 was significantly lower than that of controls.
Total thyroxine T4 increased progressively reaching
values significantly higher than the control group at 120
min. T3/T4 ratio was significantly decreased 60 and 120
min after exercise, indicating impaired T4-to-T3
conversion. Brown adipose tissue (BAT) type 2
deiodinase activity (D2) was significantly lower at 30 min,
but pituitary D2 have remained unchanged. No change in
serum thyrotropin was detected, while serum corticoste-
rone was significantly higher 30 min after exercise. They
concluded that decreased liver D1 and BAT D2 might be
involved in the decreased T(4)-to-T(3) conversion
detected after an exercise session on the treadmill
(Hackney and Dobridge, 2009).
In an earlier study, Krotkiewski et al. (1984) measured
thyroid hormones before, during and after acute exercise
(60 min) or physical training (3 months) in obese women;
thyroid stimulating hormone concentration increased
during acute work and decreased immediately after. No
changes were seen during the two following days. T4
concentrations showed no changes. T3 decreased
slightly immediately after acute exercise, and after three
months of physical training (Fortunato et al., 2008).
Simsch et al. (2002) have investigated the influence of
different training intensities on Leptin and the
hypothalamic-thyroid-axis in highly trained rowers. They
measured TSH, fT3 and fT4 and reported that there was
no change of fT4 level and a significant reduction in TSH
and fT3 after resistance training. A significant increase of
TSH was found after endurance training (Simsch et al.,
2002).
In Huang et al. (2004) research, Twenty-six healthy
male military recruits aged 23 to 27 years with a mean of
25 years have been studied. All subjects had maintained
identical diet and physical activity for a week before the
test. Serum samples had been drawn before (baseline)
and immediately, 1, 4, 24, 24 and 48 h after maximal
exercise (on a treadmill with Bruce protocol). Specimens
had been analyzed to measure T3, T4, fT3, fT4 and TSH
in the same assays. No significant changes of serum
mean TH values before and after exercise have been
found except for TSH, which had increased significantly
immediately after exercise (1.72 vs. baseline 1.42 IU/L, p
< 0.01). They have reported that maximal treadmill
exercise had not greatly affected the determination of
concentrations of circulating THs (Huang et al., 2004).
It is known that measurements of changes in hormone
values after exercise may reflect only acute transcapillary
movements of water, which resolve shortly after exercise
ceases. The hemodynamics return to baseline within
minutes following termination of exercise. Exercise can
cause hemoconcentration (Beaumont, 1972).
In this study, circulating TSH and fT3 levels increased
immediately after exercise followed by a significant
decrease until the end of the study period. Our results
were consistent with other reports suggesting that

hemoconcentration could be a cause of changes in
circulating TSH and fT3 (Krotkiewski et al., 1984; Sowers
et al., 1977; Schmid et al., 1982).
Physical exercise has been reported to stimulate the
peripheral deiodination of T4 and an increased uptake of
T4 in the liver during exercise (Opstad et al., 1984).
Increased conversion of T4 to T3 by peripheral tissues
during training is improbable since there were no
significant changes in serum fT3 concentrations after
immediately exercise in the present study. Moreover,
both cortisol and catecholamine actions initiated by
exercise will also stimulate peripheral T4 deiodination
(Chopra et al., 1975; Nauman et al., 1980). Whether the
accelerated deiodination resulted in the increase in fT3
and minimal decrease in fT4 values observed
immediately after exercise (Assay 2) remains to be
clarified.
It has been reported that the type, intensity and
duration of the training regimes, as well as the training
background of the subjects, play a role in the changes
taking place in serum T4 and fT4 levels have suggested
that training may slightly impair thyroid function
(Pakarinen et al., 1988). The main findings of the present
study were the slight decrease in serum concentration of
fT4, slight increases in serum concentration of fT3 and
TSH immediately after exercise. One hour after exercise,
both fT3 and TSH have significantly decreased, fT4
remained unchanged. Since these changes were all
within the reported normal ranges for reference values,
they cannot have any clinical significance.
Although no statically significant differences have been
reported between pre- and post- exercise serum levels of
thyroid hormones in a study including 20 patients with
coronary artery disease (Siddiqui et al., 1983;), in our
study, in which 14 field hockey players were included,
while the concentrations of fT4 decreased, the
concentrations of TSH and fT3 immediately increased
after exercise.
The level of serum cortisol is effected by many factor
such as intensity, duration and timing of exercise, type of
exercise, age, altitude, environmental temperature and
psychology (Bernet and Wartofsky, 2000; McMurray and
Hackney, 2000). In this study, cortisol concentration
significantly increased immediately after exercise. High
serum levels of cortisol generated by the stress
procedure might also contribute to the post-stress
(exercise) depression of TSH levels (Sowers et al.,
1977).
Conclusion
There were no statically significant differences between
pre- and just after the exercise serum levels of TSH and
thyroid hormones. It has been appeared that the plasma
thyroid hormones and TSH are not highly affected
imediately after exercise. However one hour after the
Beyleroglu 2005
exercise, both fT3 and TSH significantly decreased. fT4
was also affected. Cortisol concentrations slightly
increased immediately after maximal aerobic exercise.
The previously studies is consistent in respect to plasma
cortisol level response to exercise. This can be
explained by the plasma level of cortisol effected by
several factors such as type, intensity, duration and
timing of exercise, age, gender, altitude, environmental
temperature, blood glucose level, focus of attention etc.
ACKNOWLEDGEMENTS
The author would like to thank Prof. Dr. Ahmet Aslan,
Prof. Dr. Mehmet Akif Ziyagil and Dr. Ahmet Celik for
their valuable support and comments.
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Ozmerdivenli R (2005). Exercise intensity and its effects on thyroid
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Deligiannis A, Karamouzis M, Kouidi E, Mougios V, Kallaras C (1993).
Plasma TSH, T3, T4 and cortisol responses to swimming at varying
water temperatures. Br. J. Med., 27(4): 247-250.
Fortunato RS, Ignacio DL, Padron AS, Peçanha R, Marassi MP,
Rosenthal D, Weneck-de-Castro JPS, Carvalho DP (2008). The
Effect of Acute Exercise Session on Thyroid Hormone Economy in
Rats. J. Endocrinol.,198(2): 347-53.
McMurray RG, Hackney AC (2000). Endocrine responses to exercise
and training In Garrett, W. E. and Kirkendall, D. T. (Eds.) Exercise
and Sport Science, Philedelphia: Lippincott Williams & Wilkins. pp,
135-162.
Hackney AC, Dobridge JD (2009). Thyroid hormones and the
interrelation of cortsiol and prolactin: influence of porolonged,
exhaustive exercise. Endokrynologia Polska, 60(4): 252–257.
Huang WS, Yu MD, Lee MS, Cheng CY, Yang SP, Chin HM, Wu SY
(2004). Effect of treadmill exercise on circulating thyroid hormone
measurements. Med. Princ. Pract., 13(1): 15-19.
Krotkiewski M, Sjostrom L, Sullivan L, Lundberg PA, Lindstedt G,
Wetterqvist H, Björntorp P (1984). The effect of acute and chronic
exercise in thyroid hormones in obesity. Acta Med. Scand., 216: 269-
275.
Licata GR, Scaglione S, Novo MA, Dichiara D, Vincenzo D (1984).
Behaviour of serum T3, rT3, TT4, FT4 and TSH levels afters after
exercise on a bicycle ergometer in healty euthyroid male young
subjects. Boll. Soc. Ital. Biol., 60(4): 753-759.
Limanova Z, Sonka J, Kratochvil O, Sonka K, Kanka J, Sprynarova S
(1983). Effects of exercise on serum cortisol and thyroid hormones.
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Eur. J. Clin. Invest., 10: 189-192.
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Pakarinen A, Alan M, Hakkinen K, Komi P (1988). Serum thyroid
hormones, thyrotropin and thyroxine binding globulin during
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Schmid P, Wolf W, Pilger E, Schwaberger E, Pessenhofer G, Pristautz
H, Leb G (1982). TSH, T3, rT3, and fT4 in maximal and submaximal
physical exercise. Eur. J. Appl. Physiol., 48: 31-39.
Siddiqui AR, Hinnefeld RB, Dillon T, Judson WE (1983). Immediate
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Sports Med., 17(3): 180-183.
Simsch C, Lormes W, Petersen KG, Baur S, Liu Y, Hackney AC,
Lehmann M, Steinacker J M (2002). Training intensity influences
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(6): 422-427.
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African Journal of Pharmacy and Pharmacology Vol. 5(17), pp. 2007-2012, 8 November, 2011
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP11.331
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Cationic liposomes as gene delivery system
Mohsen M. Mady
Department of Physics and Astronomy, College of Science, King Saud University, Riyadh-11451, Saudi Arabia.
E-mail: dr_mmady@yahoo.com or mmady@ksu.edu.sa. Tel: +966-1-4698600.
Accepted 27 July, 2011
Delivery of oligonucleotides (ONs) and genes to their intracellular targets is a prerequisite for their
successful use in medical therapy. Cationic liposomes are among the most commonly used and
promising delivery system for ONs and genes. Cationic-liposomes and their complexes with ON were
characterized according to complex size, zeta potential measurements, transmission electron
microscope (TEM) and confocal laser scanning microscope (CLSM). It is successfully demonstrated
that cationic liposomes, dispersed in 10% serum-containing growth medium, efficiently delivered ON to
HeLa cells. Indeed, intact ON was found in the cytoplasm and nucleus only when delivered by cationic
liposomes. The results suggest that cationic lipid-based delivery systems can be efficient for gene
delivery if their biophysical properties can be properly controlled.
Key words: Cationic liposomes, HeLa cells, gene delivery, characterization.
INTRODUCTION
Gene therapy is based on the introduction of specific
exogenous sequences of deoxyribonucleic acid (DNA)
into the target cells for production of the therapeutic gene
product (Crystal, 1995; Lasic and Templeton, 1996). The
prerequisite for successful gene therapy is efficient and
safe delivery of DNA into the cells. Because of the fast
progress of nucleic acid-based technologies in the
treatment of diseases, the call for appropriate delivery
vehicles becomes increasingly important. The ideal
vehicle should avoid immediate uptake by the
mononuclear phagocyte system and have prolonged
circulation in blood, thus increasing the probability of
reaching the desired targets. In addition, the vehicle
should be able to deliver its contents efficiently into the
cell cytoplasm, avoiding lysosomal degradation
(Shepushkin et al., 1997). The most widely used types of
vehicles for gene delivery are: viral (e.g., adenovirus,
retrovirus and adeno-associated virus) and non-viral (for
example, liposomes, polymer and peptides) (Lasic,
1999). Viral vectors are often highly efficient, but safety
Abbreviations: ONs, Oligonucleotides; TEM, transmission
electron microscope; CLSM, confocal laser scanning
microscope; DNA, deoxyribonucleic acid; DOTAP 1, 2 -
dioleoyl-3 trimethylammonium-propane; FCS, fetal calf serum;
PI, propidium iodide; DOPE, dioleoylphosphatidylethanolamine.
and immunogenicity are issues of potential concern, and
the limited transgene size often possesses a serious
obstacle (Mady et al., 2004). Nonviral vectors, on the
other hand, frequently face the problem of low
transduction efficiency.
Among the non-viral vectors, cationic liposomes are the
most widely used vectors. Although less efficient in
delivering the genes than the virus, they have many
important qualities such as being much less or nonimmunogenic,
have no known limitation in the size of the
DNA, can be custom-synthesized for targeting and easily
scalable for large-scale production. Moreover, the
liposome can deliver different kinds of DNA (super coiled
or linear) or ribonucleic acid (RNA) with or without
proteins, even to non-dividing cells and are usually
composed of biodegradable lipids. Also, covalent
attachment of target specific ligands on the liposome can
facilitate targeted delivery of genes (Mady et al., 2004).
These advantages have prompted researchers to explore
the applications of cationic liposomes in gene therapy
clinical trials (Li and Huang, 1997; Lasic and
Papahadjopoulos, 1998; Mady, 2007).
Cationic lipids were used for the first time, for gene
delivery, by Felgner et al (1987), and for ON delivery a
few years later by Bennett (1993). When cationic
liposomes are used, no encapsulation is needed because
the lipids bind electrostatically to negatively charged

2008 Afr. J. Pharm. Pharmacol.
nucleotides. Despite their widespread use as transfection
reagents, information about the interactions of cationic
lipids and polynucleotide is sparse (Mönkkönen and Urtti,
1998). Very little is understood about the events which
take place when cationic liposomes interact with mamma-
lian cells or the processes which result in the delivery of
nucleic acids. Three model of the interaction of cationic
lipid / polynucleotide complex with cells have been
proposed: (i) direct fusion with the plasma membrane
(Felgner et al., 1987; Lewis et al., 1996), (ii) endocytosis
and subsequent fusion or destabilization of endosome
membrane (Legendre and Szoka, 1992; Felgner et al.,
1994; Zhou and Huang, 1994; Zabner et al., 1995), and
(iii) translocation through pores across the plasma mem-
brane (Engberts and Hoekstra, 1995).
The physicochemical properties, such as particle sizes
and surface charges of the liposome-DNA and/or
oligonucleotides (ON) complexes may be important
factors to obtain a higher transfection efficiency of the
liposomal vectors. Although gene transfection of plasmid
and/or ON complexed with cationic liposomes is
investigated, little attention seems to be paid to
understanding their physico- chemical characteristics and
cellular uptake mechanisms. The intent in this study was
to characterize ON /liposome complexes in terms of ζ
potential and particle size and to see whether these
physicochemical properties have any influence on their
disposition characteristics and cellular uptake process. In
the presence of serum, we investigated that cationic
liposomes efficiently delivered ON to HeLa cells.
MATERIALS AND METHODS
1, 2 - dioleoyl-3-trimethylammonium-propane (DOTAP) was
purchased from Avanti Polar Lipids Inc. (Alabaster, Alabama, USA).
Fetal calf serum (FCS), L-Glutamin (200 nM solution), Penicillin
5000 units/streptomycin 5000 mg, and DMEM (Dulbecco's modified
Eagle's medium) was purchased from Bio Whittaker Europe,
Verviers, Belgium. HeLa cells were obtained from Children Hospital.
DNA- analogues of chimeric ON as the one used here will be
referred to as FIXDNA-DNA. A 5´FAM (Eurogentec EGT Group,
4102 Seraing- Belgium) labeled 68-mer of sequence (5´-TGT-CAA-
GCA-GAT-CGT-GGG-GGA-CCC-CTT-TTG-GGG-TCC-CCC-ACG-
ATC-TCC-TTG-ACA-GCG-CGT-TTT-CGC-GC-3´). Propidium
iodide (PI) was purchased from Molecular Probes (Eugene, Oreg.
USA). All other reagents were of analytical reagent grade.
Preparation of liposomes
Liposomes were formulated according to well-established methods
of extrusion (Olson et al., 1979). In short: the appropriate
phospholipid composition was mixed in organic solvent in a 50 ml
round flask. The organic solvent was evaporated to dryness by a
rotary evaporator. The resulting lipid suspension was extruded
through 100 nm polycarbonate membranes using mini- extruder
(Liposofast, Avestin Inc., Canada). Size measurement was done by
dynamic laser light scattering and the size was in the range of 100 nm.
Condensation of ON
DOTAP liposomes were added separately to 2.5 µg ON to achieve
the desired charge ratio (+/-). The fluorescence intensity of ON
(excitation at 492 nm and emission at 515 nm) was measured by
using Perkin Elmer Spectrofluorometer LS 50B (UK). The
hydrodynamic diameters of complex were measured by using
Zetasizer 3000 HS, Malvern Instruments, Germany.
Transmission electron microscopy
DOTAP/ON (8:1 +/-) complex was also characterized by using
negative stain electron microscope (EM 109, Zeiss, West
Germany). On a copper grid, the appropriate concentration from
each sample was added. Then add one drop of 20% uranyl acetate;
wait for 2 min at room temperature; remove the excess solution with
a filter paper; then the sample was examined under the
transmission electron microscope.
Zeta potential
In deionized water, we dispersed the pure ONs and their complexes
with DOTAP (1:8 -/+) and then measured the corresponding zetapotential
ζ (n=5) by using Zetasizer 3000 HS, Malvern Instruments,
Germany.
ON transfection experiment
To investigate the gene expression or transfer efficiency, HeLa cells
were grown on glass cover slips in six-well plate (10 5 -10 6 cells per
well) in DMEM medium supplemented with 10% FCS, 1% glutamine
and 1% penicillin-streptomycin solutions. The transfection system
(ON/DOTAP 1:8 -/+) complexes and cells were incubated for 6
hours at 37°C in 5% CO2. The cells were washed away by rinsing
three times with cold PBS. Cells nuclei were stained with PI stain.
Cells were fixed with formaldehyde. Turn the cover slip containing
the cells on a Moviol drop on a glass slide and examine with
confocal laser scanning microscope. We used True Confocal
Scanner, Leica DM R with A4, L5, N3 and Y5 filters, Leica, Wetzlar,
Germany and Leitz DM RXE upright microscope with a
Krypton/Argon laser (emission wavelengths of 488, 578, and 647
nm) was used. Images were converted to TIF-format with Scanware
5.1 Scion Corporation, Frederick, MD, USA. Cellular distribution of
the FAM-ON, complexed with DOTAP liposomes, was investigated
in HeLa cells following the kinetics of this process using confocal
laser scanning microscope (CLSM). In general, liposomes were in
the range of 100 nm in diameter. ON and liposomes were
appropriately diluted in 10 mM tris buffered saline (pH 7.8). The
final complex had a size of approximately 150 nm, which was
diluted with the appropriate cell cultured medium containing 10%
FCS and then added to HeLa cells.
RESULTS AND DISCUSSION
ON condensation
The condensing agents must not only efficiently
condense ON but also require the ability to be effectively
displaced from ON, allowing for subsequent
transcriptional and translational events to occur (Sorgi,

Relative Fluorescence fluorescence Intensity intensity
100
80
60
40
20
-2 0 2 4 6 8 10 12 14 16
DOTAP/FAM(+/-)
Figure 1. Condensation of FAM by DOTAP liposomes (Excitation
wavelength at 492 nm and emission wavelength at 515 nm).
Size(nm) Size (nm)
190
180
170
160
150
140
130
0 10 20 30 40 50
DOTAP/FAM(+/-)
Figure 2. Hydrodynamic diameters of DOTAP/FAM complexes
versus different complexes charge ratio (+/-); n = 3.
1998). In order to condense FAM-ON, increasing
amounts of DOTAP-liposomes were added to the FAM-
ON solution. As seen in Figure 1 the addition of DOTAP
to FAM solution resulted in a rapid decrease in
fluorescence intensity of FAM-ON. This loss in
fluorescence intensity can be attributed to the
condensation of ON. This is thought to occur due to
electrostatic interactions between cationic lipid and ON,
resulting in a charge neutralization of the complex and
the formation of a condensed structure. This condensed
structure, due to its diminished size, may be more readily
endocytosed by the cell, resulting in the increased levels
of transgene expression.
The complex is examined by several physical methods to
Mady 2009
gain insight into the nature of the interaction among the
components and the mechanism of the enhanced
transfection activity. We first looked at the effect of
cationic lipid on the complex size. Figure 2 shows that the
size of the complex was dependent on the charge ratio of
DOTAP/ON. As the DOTAP content increases, vesicle
size is reduced. Results indicate that, at a low charge
ratio (2.5:1 +/-), the size of the complex was 186.5 nm.
However, as the DOTAP content increases; complex size
is reduced to approx. 132 nm at a charge ratio (50:1 +/-).
The data indicates the condensation ability of ON by
DOTAP liposomes (Sternberg et al., 1994; Li et al., 1998;
Perrie and Gregoriadis, 2000).
There are several possibilities that may explain the
potentiation effect of the condensing agent on the
transfection activity of liposomes. First, on the basis of
the current endocytosis model, there is a size limitation
for particles to be taken up efficiently by cells (Machy and
Leserman, 1983). Direct size measurement of the
complexes showed that the condensing agent
significantly reduced the size of the complex formed over
a wide range of DOTAP/ON ratios.
Transmission electron microscopy
Negative stain electron microscopy is a useful method for
addressing questions concerning size distribution of
liposome, and although obtaining quantitative data is
laborious, negative staining is a reliable technique, which
is simple to perform and requires only limited specialized
equipment, which should be available in any electron
microscopy laboratory (Haschmeyer and Myers, 1972).
Morphological studies demonstrated that spherical
DOTAP/ON complexes are formed (Mönkkönen and Urtti
1998). Electron micrograph of DOTAP/ON (8:1 +/-)
complex was shown in Figure 3. The majority of the
particles in the pictures appeared spherical, small (≤ 100
nm in diameter), electron dense and some of them were
associated with low-density lipid membranes. These
complexes of small size should be more favorable to
enter the cells via an endocytosis pathway than the larger
ones.
Zeta potential measurements
Transfection complex formation is based on the
interaction of the positively charged lipid with the
negatively charged phosphate groups of the nucleic acid.
The information of the overall charge of transfection
complexes by zeta-potential measurements can be
speed up the development of better non-viral DNA
delivery vectors for gene therapy (Son et al., 2000; Mady
et al., 2004). The following physical chemical parameters
were used in the determination of zeta-potential: medium

2010 Afr. J. Pharm. Pharmacol.
Zeta-potential (mv)
Figure 3. Negative stain electron micrograph of DOTAP/ON
(8:1 +/-).
40
30
20
10
0
-10
-20
-30
FAM
DOTAP/FAM(5:1)
DOTAP/FAM (7.5:1)
Figure 4. Zeta potential measurements of different
formulations (n=5).
viscosity 0.89 cPoise, medium refractive index 1.333,
temperature 25°C and dielectric constant 79.
Results in Figure 4 show trends of increasing zetapotential
values with increasing DOTAP content. Values
of the zeta-potential of liposomes indirectly reflect vesicle
surface net charge and can therefore be used to evaluate
the extent of interaction of the liposomal surface cationic
charges with ON. On this basis, the zeta-potential of
DOTAP liposomes was investigated before and after
complexing with FAM-ON. Results show the negatively
zeta-potential of the naked FAM (-26.8 ± 4). After addition
of DOTAP to FAM in charge ratio of (5:1 +/-), the zetapotential
value becomes positive (15.2 ± 6.6 mV). Also, it
was more positive (33.5 ± 6) for DOTAP/FAM (7.5:1 +/-).
ON transfection
ONs are promising therapeutic agents against viral
infections and cancer. However, problems with their
inefficient delivery and inadequate stability have to be
solved before they can be used in therapy. Cellular
uptake of ON is highly inefficient, as their net negative
charge and high molecular weight prohibit efficient
transfer across cell membrane without the help of carrier
system (Bennett 1993; Ledley, 1995; Welz et al., 2000).
Confocal microscopy is a well-established technique for
the investigation of 3D structures in biological and
industrial materials (Mady et al., 2009). The basis of this
success is the optical sectioning capability of this type of
microscopy, which enables one to study 3D-structure of
intact specimens in their natural environment. CLSM has
the major advantages that it yields a very short depth of
focus, its transverse definition and the contrast of the
image are better than a standard microscope, the device
is very well-suited for optical cross-sectioning and with
the use of a laser beam, the intensity of illumination can
be very high. With CLSM one can slice incredibly clean,
thin optical sections out of thick fluorescent specimens;
view specimens in planes running parallel to the line of
sight; penetrate deep into light-scattering tissues; gain
impressive three-dimensional views at very high
resolution; and improve the precision of microphotometry.
Figure 5 shows that DOTAP-liposomes effectively
transport ON into the nucleus of HeLa cells, in the
presence of serum. DOTAP-liposomes improved the
cellular delivery of ON by protecting it in FCS, by
increasing the total uptake of ON in cells, and by
increasing the nuclear localization of ON in cells.
Complexation with cationic lipids facilitates the entry of
ON into cells. ON-cationic lipid complexes enter the cells
via endocytosis and they promote the nuclear entry of ON
(Bennet et al., 1992; Zelphati and Szoka, 1996a). After
their cellular delivery with cationic lipids, the ONs are
mostly seen in the nucleus, whereas the lipids are
entirely localized in the cytoplasm and, particulay, in
some perinuclear regions (Zelphati and Szoka, 1996a).
Zelphati and Szoka (1996b) showed that ONs were
released from the complexes as a result of lipid
intermixing in the endosomal wall.
The cationic lipid gene delivery system invariably
comprises three components: a cationic lipid, a neutral
co-lipid, which is most often dioleoylphosphatidylethano-
lamine (DOPE) and the plasmid DNA that encodes the
transgene of interest. Although the liposomal gene
delivery systems require a neutral co-lipid, usually DOPE
for optimal activity, the aggregation of phosphatidyle-
thanolamine containing liposomes by serum proteins has
been observed (Forbes et al., 1984). In the case of
cationic lipid based delivery systems, the charge density
of the lipid bilayer can significantly affect the colloidal
stability of the particles, with mole fractions of helper
lipids [for example, (DOPE)] of > 0.4 producing
considerable instability at elevated ionic strength. So, in
the present work, DOTAP liposomes are only used to
investigate how physicochemical methods can be used to

Figure 5. Confocal laser scanning microscopy of HeLa cells
incubated with DOTAP/FAM (8:1 +/-) complexes in DMEM
supplemented with 10% FCS, at 37°C and 5% CO2 after 6 h.
assess the functionality of the ON-lipid complexes in vivo.
Most mammalian cells require the addition of serum to
the culture medium for optimal growth and maintenance
of the cell lines in vitro. The presence of serum often
reduces the transfection efficiency of liposomal vectors
(Felgner et al., 1987; Felgner and Ringold, 1989; Gao
and Huang, 1995; Lee and Huang, 1997; Mady et al.,
2009). This may be due to the premature release of DNA
from the complexes or its degradation by the nucleases.
So, in the presence of serum, we investigated that
cationic liposomes efficiently delivered ON to HeLa cells.
Analytical methods are described that provide a
platform for systematically evaluating the effect of
formulation variables in the development of ON-lipid nonviral
gene therapy complexes. The methods encompass
those used to characterize the components, that is, ON
and liposomes, as well as those applied to the complexes
themselves. Several lines of evidence obtained from
studies on complex size; zeta-potential; electron
microscope and CLSM indicate that ON is entrapped
within the aqueous compartments, in between bilayers,
presumably bound to the cationic charges. The focus is
on physicochemical methods so that their parameters
can be assessed in relationship to the functionality of the
ON-lipid complexes in vivo (Perrie and Gregoriadis, 2000;
Hutchins, 2000).
More studies are needed to compare and investigate
the role of fusogenic helper lipids such as DOPE and
cholesterol or peptides to disrupt the endosome
membrane so as to facilitate the escape of DNA from
endosome. Also, covalent attachment of target specific
ligands on the liposome can facilitate targeted delivery of
genes (Mady et al., 2004). These advantages have
prompted researchers to explore the applications of
cationic liposomes in gene therapy clinical trials.
ACKNOWLEDGEMENT
Mady 2011
Author extends his appreciation to the Deanship of
Scientific Research at King Saud University for funding
the work through the research group project No RGP-
VPP-121.
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African Journal of Pharmacy and Pharmacology Vol. 5(17), pp. 2013-2017, 8 November, 2011
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP11.490
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Nitrous oxide versus pethidine with promethasine for
reducing labor pain
Batool Teimoori 1 , Nahid Sakhavar 1 , Masoome Mirteimoori 1 and Behzad Narouie 2 *
1 Department of Obstetrics and Gynecology, Health Promotion Research Center, Zahedan University of Medical
Sciences, Zahedan, Iran.
2 Clinical Research Development Center, Ali-ebne-Abitaleb hospital, Zahedan University of Medical Sciences, Zahedan,
Iran.
Accepted 17 October, 2011
Systemic opioids are widely used for the relief of labor pain. Self-administered nitrous oxide with
concentration of 50% is a new form of analgesia. The aim of this study was to compare the analgesic
efficacy and side effects of the patient controlled inhaled nitrous oxide (50% ‘‘Entonox’’) with systemic
intramuscular pethidine, in reducing pain during normal vaginal labor in Iranian population. In a
randomized controlled study, the analgesic efficacy of inhaled 50% nitrous oxide (Entonox) was
evaluated as compared to intra muscular pethidine for reducing labor pain among 100 women
undergoing normal vaginal delivery. Mean maternal age was 26.2 and 27.2 years in entonox and
pethidine groups, respectively. Duration of first and second stages was significantly shorter in patients
receiving nitrous oxide as analgesia as compared to pethidine group (P < 0.05). Pain severity according
to visual analog scale (VAS) score was significantly lower in patient that received nitrous oxide (P =
0.0001). We also showed significantly higher satisfaction of pain reduction in nitrous oxide group
during labor (P = 0.01). No significant difference was observed among the groups regarding neonatal
complications. Although, nitrous oxide is certainly not a potent analgesic, we found that it has more
beneficial effects than pethidine in parturient women which is yet to be cleared.
Key words: Entonox, labor pain, pethidine, nitrous oxide.
INTRODUCTION
Labor is one of the painful conditions that is considered to
be the most intense and stressful experiences (Melzack,
1984). In the last decades, changes have occurred in the
obstetric expectations and in their care. In developed
countries, the number of women requesting labor
analgesia is increasing, and in some communities, an
effective pain relief for childbirth is in great demand
(National Institute for Health and Clinical Excellence,
2008).
As it is cheap, simple to use and readily available,
systemic pethidine is widely used for relief of labor pain
(Hawkins and Beaty, 1999; Wilson et al., 1986; Morrison
et al., 1987). Use of parenteral opioids was found to be
between 39 and 56% in various hospital obstetrics units,
*Corresponding author. E-mail: b_narouie@yahoo.com.
Tel/Fax: +985413414103.
in the United States (Hawkins and Beaty, 1999; Fairlie et
al., 1999; Olofsson et al., 1996).
Systemic opioids lead to some adverse effects on both
mother and baby including dysphoria, sedation,
respiratory depression, nausea and vomiting and delayed
gastric emptying for the mother (Douglas and Levinson,
2001). As pethidine crosses the placenta, it may
accumulate in the fetal circulation (Gaylard et al., 1990)
causing early neonatal respiratory depression and
behavioral and feeding problems for even up to six weeks
after delivery (Belsey et al., 1981; Belfrage et al., 1981;
Nissen et al., 1997).
Self-administered 50% nitrous oxide “Entonox” is an
effective and safe form of analgesia, which has been
used by many emergency medical services for many
years. Nitrous oxide is an odorless, tasteless and inhaled
analgesic (Faddy and Garlick, 2005), and it was found to
be an effective analgesia for many women while also
being safe for the mothers and babies (Rooks, 2007).

2014 Afr. J. Pharm. Pharmacol.
Table 1. Descriptive data of entonox and pethidine subjects.
Variable Entonox (n = 50) Pethidine (n = 50) P-value
Age (year) 7.34 ± 26.2 6.02 ± 27.2 NS
Weight (kg) 0.38 ± 69 11.06 ± 73 NS
Gestation (week) 0.95 ± 38.44 0.92 ± 38.58 NS
Cervical dilation at Analgesic initiation (cm) 0.91 ± 4.46 0.89 ± 4.36 NS
Nowadays, nitrous oxide is widely used in many countries
for relieving labor pain (Rooks, 2007).
Unlike opioids, it does not depress respiration (Rosen,
2002). Nitrous oxide rapidly takes effect (Latto et al.,
1973) and is quickly reversible on discontinuation of
therapy (Latto et al., 1973; Einarsson et al., 1996).
Despite its wide and popular use in many countries,
nitrous oxide for the relief of labor pain is largely unknown
in Iran. In the present study, we aimed to compare the
analgesic efficacy and side effects of the patient
controlled, inhaled nitrous oxide (50% ‘‘Entonox’’) with
systemic intramuscular pethidine (the most popular drug
with opioid analgesic properties), in relieving pain during
normal vaginal labor in Iranian population.
MATERIALS AND METHODS
In this randomized clinical trial study, we evaluated the analgesic
efficacy of inhaled 50% nitrous oxide (Entonox) as compared to
intra muscular pethidine for relieving labor pain among 100 women
undergoing normal vaginal delivery in Ali-Ebne-Abitaleb hospital, in
Iran, from March 2007 to 2008. The study was reviewed and
approved by the ethics committee in Zahedan University of Medical
Sciences, and informed consents were obtained from all
participants. 100 pregnant women with gestational age ranging
from 38 to 42 weeks, and who were referred to in the early phase of
labor, were randomly enrolled in the study. Participants were
selected among non-complicated, term pregnancies with a normal
cephalic fetus, and were referred to in the active phase of labor with
cervical dilation less than 7 cm. Women who could not keep their
facial mask, have recent administration of local or systemic
analgesics and opioids, patients with altered mental status, vitamin
B12 deficiency receiving replacement therapy, any oxygenation
abnormalities, hemodynamically unstable patients and women
bearing any fetus abnormalities were excluded from the study.
Patients were randomly allocated in two groups. The number of
nulliparous women was comparable in two groups. Participants of
one group (Group A, n = 50) were medicated with entonox, where
women in the other group (Group B, n = 50) received pethidine for
relieving their labor pain. All women were trained for self
administration of entonox in group A and women in group B
received 1 mg/kg slowly intra-venuous injection of pethidine
combined with 25 mg promethazine.
Fifty percent nitrous oxide in 50% oxygen was premixed in a
single cylinder called by the trade name ‘‘Entonox’’ (Lieberman and
O’Donoghue, 2002). Entonox was self-administered by the laboring
woman using a face mask, when she determines that she needs it.
Patients were trained to administer face mask of entonox with the
initial of every uterine contraction and continue deep inspirations
while the contraction and pain exists.
Entonox administration can be started and stopped at any point
during labor, according to the needs and preferences of the woman.
It takes effect in about 50 s after the first breath, and the effect is
transient and gone when it is no longer needed (Rooks, 2007). The
flow of gas into the mask is initiated by the negative pressure of
inhalation, which opens a demand valve. This same demand valve
prevents the flow of gas when inhalation ceases, and the entonox
apparatus allows the exhaled gas to be scavenged, so it is not
released into the air, minimizing the exposure to others in the room.
Following different analgesic administrations, severity of labor
pain was evaluated according to the VAS score, numbering from 0
to 10 (0 = no pain and 10 = severe and non tolerable pain).
Parturients pain scored once before any analgesic administrations,
and they were requested to score their maximum pain following
each contraction. Total visual analog scale (VAS) score is the mean
of scores rated during labor.
Patients satisfaction of analgesia method was also evaluated by
verbal rating scale, scoring from 0 = not satisfied to 4 = complete
satisfaction.
All parturients were monitored for vital signs, arterial O2
saturation and fetal heart rate each 30 min during labor and
mothers were suggested to have left lateral position during labor for
prevention of supine hypotension.
Mothers’ somnolence and sedation was also evaluated by a
nurse in 10 min intervals according to Ramsy score, from 1 to 5. 1 =
completely awake, 2 = somnolence, 3 = irritable to sound, 4 =
irritable to touch and 5 = non responder.
Statistical analysis
Descriptive statistics were used to report demographic
characteristics with SPSS statistics package version 15. The Chisquare
test and Student t-test were used to compare the groups on
qualitative and quantitative variables, respectively.
RESULTS
A total of 100 pregnant women, including 50 primi-gravid women
were enrolled in the study. Mean maternal age was 26.2 and 27.2
in entonox and pethidine groups, respectively. Demographic data of
the participants is summarized in (Table 1). Patient’s characteristics
were comparable in two randomly allocated groups.
All participants underwent normal vaginal delivery and none of
them needed vacuum or forceps assistant. Duration of first and
second stages was significantly shorter in patients receiving nitrous
oxide as analgesia as compared to pethidine group (P < 0.05)
(Table 2).
VAS score before administration of any analgesic agent was
statistically equal between groups; however, it shows significant
difference at the end of both first and second stages of labor. Pain
severity according to VAS score was lower in patient that received
nitrous oxide. (P = 0.00)
There were no differences in blood pressure, heart rate and
respiratory rate before analgesia. Where, after the end of stage 1
and 2, nitrous oxide users had significantly lower heart rate and
respiratory rate. Blood pressure still remained equal in both groups

Table2. Comparison of labor outcomes in patients that received nitrous oxide and pethidine.
Labor outcomes Entonox Pethidine P-value
Duration
First stage (h) 3.12 ± 1.37 2.24 ± 1.07 0.001
Second stage (min) 3.44 ± 1.73 1.18 ± 1.00

2014 Afr. J. Pharm. Pharmacol.
entonox as analgesia. Thus, entonox led to women’s
higher satisfaction of their labor pain relief.
Our results were in line with previous studies (Douglas
and Levinson, 2001; Faddy and Garlick, 2005; Rooks,
2007; Latto et al., 1973; Bishop, 2007; Evans et al., 1995;
Jones et al., 1969; McAneny, 1963). However, in
contrast, one prospective non-randomized study, labor
pain was more severe in primi-gravid women that were
administered nitrous oxide as compared to those that
were given pethidine (Keskin et al., 2003).
A mixture of 50% nitrous oxide in oxygen “Entonox” is
available in a single cylinder as a patient controlled
inhaled analgesic (Faddy and Garlick, 2005). It has low
solubility in blood and is transported in solution without
binding to protein. Nitrous oxide rapidly takes effect (Latto
et al., 1973; Bishop, 2007), because it diffuses rapidly
through the alveolar arterial membrane and is excreted
unchanged, mainly through the lungs (Faddy and Garlick,
2005). As a result, it is quickly reversible on
discontinuation of therapy (Latto et al., 1973; Einarsson
et al., 1996; Bishop, 2007). It has shown that recovery
from sedative effects of nitrous oxide is faster when
compared with intravenous analgesia (Faddy and Garlick,
2005).
Rapid onset and quick reversibility, allows nitrous oxide
to be administered throughout the second stage of labor
without fear of effects on the newborn (Bishop, 2007).
Supervised by physicians, nurses or midwives, nitrous
oxide is widely used as a safe analgesic in many parts of
the world including Canada, Australia, Finland, United
Kingdom and New Zealand (43 to 49%) (STAKES, 2006;
NSW Department of Health, 2005; Biró et al., 2000).
The maximum effect of nitrous oxide appears at a
concentration of 70%, and it has been shown to relieve
labor pain in approximately two-thirds of women (Rooks,
2007).
The precise mechanism of action of nitrous oxide
analgesia remains uncertain. It may induce release of
endogenous opioid peptides in the periaqueductal gray
area of midbrain (Maze and Fuginaga, 2000).
Among various inhalation anesthetic agents studied for
labor analgesia, only nitrous oxide is used to any great
extent in modern obstetric practice. The reasons are
probably related to the ease of administration of nitrous
oxide, its lack of flammability, absence of pungent odor,
minimal toxicity, minimal depression of cardio vascular
system, lack of effect on uterine contractility and the fact
that it does not trigger malignant hyperthermia (Rooks,
2007). Entonox is administered either intermittently,
starting with the onset of pain with each contraction and
discontinuing as the contraction pain eases or abates, or
continuously, by inhaling both during and between
contractions (Rooks, 2007).
Intermittent administration of entonox, as used in this
study, is somehow problematic, because there is a lag of
approximately 50 s after the onset of administration
before the analgesic effect can be expected (Chan et al.,
1996). However, entonox is significantly beneficial if
administration initiates approximately 30 to 50 s before
each contraction (Waud and Waud, 1970).
Side effects induced by nitrous oxide are nausea and
vomiting reported in 5 to 36% (Rooks, 2007; Jones et al.,
1969; McAneny, 1963; Bergsjo and Lindbaek, 1971;
McGuinness and Rosen, 1984). Dizziness, dreams and
drowsiness reported in 0 to 24%, dry mouth from
breathing dry gas, buzzing in the ears and rarely,
numbness are also reported (Rooks, 2007; Bishop, 2007;
Jones et al., 1969; Bergsjo and Lindbaek, 1971;
McGuinness and Rosen, 1984).
The greater maternal risk of inhalation of nitrous oxide
is loss of consciousness. It is rare with 50% nitrous oxide.
The alveolar concentration for wakefulness for nitrous
oxide is between 50 and 70% in non-pregnant women
and probably lower in pregnant. So it is important that the
agent should be self-administered and not by anyone
else. It is also important that a mask is kept by parturient
and not fixed to the face. If it is not strapped, her hand
will fall away from her face, when she became too drowsy
rendering the device nonfunctional. Therefore, the nitrous
oxide concentration wills rapidly decline (Rooks, 2007).
Nitrous oxide rapidly transfer placenta, however, as
shown in our study, the fetus infants are clinically
unaffected. There have shown no significant differences
in Apgar scores or neonatal outcomes between babies
born to mothers who received nitrous oxide (Rooks,
2007; McAneny, 1963; Abboud et al., 1981; Stefani et al.,
1982).
When used intramuscularly, analgesic effect of
pethidine, one of the most frequently used opiate
agonists, starts within 10 to 20 min, and lasts for 2 to 4 h
(Lee et al., 1993).
As it is cheap, simple to use and readily available,
systemic pethidine is widely used for relief of labour pain.
Use of parental opioids was found to be between 39 and
56% in various hospital obstetrics units, in the United
States (Hawkins and Beaty, 1999). However, many
studies have suggested that intramuscular pethidine may
be ineffective at relieving labor pain (Wilson et al., 1986;
Morrison et al., 1987; Fairlie et al., 1990; Olofsson et al.,
1996) and it has been suggested that their use may even
be unethical and medically incorrect (Fairlie et al., 1999).
Systemic opioids lead to some adverse effects on both
mother and baby, including dysphoria, sedation,
respiratory depression, nausea and vomiting and delayed
gastric emptying for the mother (Douglas and Levinson,
2001). As pethidine crosses the placenta, it may
accumulate in the fetal circulation (Gaylard et al., 1990),
causing early neonatal respiratory depression and
behavioral and feeding problems for even up to six weeks
after delivery (Belsey et al., 1981; Belfrage et al., 1981;
Nissen et al., 1997).
Consistent with ours, an uncontrolled, observational
study on primi-gravid, showed that women judged nitrous
oxide to be more effective than opioids (Harrison et al.,

1987). The study suggests that, nitrous oxide is a useful
method for women who wish to cope with the earlier part
of labor "drug free”. Consistent with ours, this study
showed that labor was more rapid in the nitrous group;
however, it is unlikely that nitrous oxide causes more
rapid labor, and it is unlikely that opioids significantly slow
labor. It may be more effective for women whose labor is
shorter (Harrison et al., 1987).
Although, nitrous oxide is certainly not a potent
analgesic, it has more beneficial effects for many
parturient women as compared to pethidine. It is easy to
administer and safe for both mother and infant.
ACKNOWLEDGEMENTS
This study was supported by a dissertation grant from
School of Medicine, Zahedan University of Medical
Sciences, the authors would like to thank subjects who
willingly participated in the study and would like to
acknowledge the Clinical Research Development Center
of Ali-ebne-abitaleb Hospital, Zahedan University of
Medical Sciences for its help in preparing this manuscript.
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African Journal of Pharmacy and Pharmacology Vol. 5(17), pp. 2018-2026, 8 November, 2011
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP11.518
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Differential analysis of human kidney stone samples
using electrospray ionization mass spectrometry
Zhiquan Zhou 1 , Yongzhong Ouyang 2 , Xiangtai Zeng 3 , Tingting Zhang 1 , Bin Jia 2 , Xinglei Zhang 2 ,
Huanwen Chen 2 and Jianhua Ding 2 *
1 Institute of Information Engineering, Harbin Institute of Technology, Weihai, 264209, P. R. China.
2 Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation, Biology and Material Science, College of Chemistry,
East China Institute of Technology, Nanchang, 330013, P. R. China.
3 Department of Surgery, 2nd Hospital of Gannan Medical University, Xinfeng, 341600, P. R. China.
Accepted 21 October, 2011
Kidney stones may be caused by many factors, including ingestion of melamine for a relatively long time.
The diagnosis of melamine-induced kidney stones and the understanding of how the melamine is
involved in the formation of kidney stones are of practical importance. To establish a sensitive method
based on widely used electrospray ionization mass spectrometry (ESI-MS) for diagnosis of
melamine-induced kidney stones and to probe the differential formation of melamine-induced kidney
stones at molecular levels. Human kidney stones were collected in hospital from 6 groups of patients at
different ages. ESI-MS was employed as the main technique with the principal component analysis for
data processing. Using principal component analysis (PCA) of the ESI-MS fingerprints, a set of 21
melamine-induced kidney stone samples and 21 uric acid derived kidney stone samples were
successfully differentiated from the other groups, rendering ESI-MS method a potential platform for
differential analysis of the human kidney stones of various causes at molecular levels. The experimental
results also indicate that in addition to the melamine, the chemical compounds enwrapped in the
melamine-induced kidney stone samples are different from other kidney stone samples. These findings
suggest that ESI-MS is a useful tool for diagnosis of melamine-induced kidney stone samples and the
melamine-induced kidney stone could be formed by different mechanisms.
Key words: Toxicity of melamine, chemical profiling, uric acid, electrospray ionization-mass spectrometry
(ESI-MS), kidney stone, melamine, principal component analysis.
INTRODUCTION
Kidney stones, one of the most painful urologic disorders,
have beset humans for centuries. It is estimated that
about three percent of the world's population will suffer
from kidney stones in their lifetime (Johnson et al., 1979;
Coe et al., 1996). Annually, about 3 million people need
medical care due to kidney stone problems (Coe et al.,
1996; Hiatt et al., 1996). Kidney stones in human are
normally caused by many factors, such as the diet and
genetics (Anderson, 2002). For example, it is known that
*Corresponding author. E-mail: Dingjianh2004@126.com. Tel:
0086-791-3896370. Fax: 0086-791-3896370.
the ingestion of melamine illicitly used in food may cause
the formation of kidney stones in both humans (Lam et al.,
2009) and animals (Baynes et al., 2008). Although, the
formation mechanism for the melamine-induced kidney
stones remains unclear, unambiguous chemical profiling
of the melamine-induced human kidney stones is urgently
required for better medical care performance. Chinese
government covers all the medical expenditures for the
melamine-induced kidney stone diseases after the
melamine event, while patients suffering from uric
acid-induced kidney stones are not included (Chan and
Lai, 2009). Thus, accurate detection of melamine-induced
human kidney stones and the study of the formation
mechanism as well are of great significance to both clinics

Table 1. Clinical data of human kidney stone samples.
Zhou et al. 2019
Patient number Age Sex Quantity and period Stone location Imaging studies
Sample 1 26 month Male 30 - 50 g/day, 2 years Left ureter Nonopaque stone
Sample 2 36 month Female 30 - 50 g/day, 1.5 years Left kidney pelvis Nonopaque stone
Sample 3 7 years Male 30 - 50 g/day, 2.5 years Left kidney pelvis Radiopaque
Sample 4 17 month Male 30 - 50 g/day, 1.5 year Left kidney pelvis Radiopaque
Sample 5 48 years Female No drinking Right ureter Radiopaque
Sample 6 44 years Male No drinking Left kidney pelvis Radiopaque
diagnosis and economic interests.
X-ray inspection provides valuable information about
the size and location of the kidney stone, and thus, it is
the most common technique for clinical diagnosis of the
calcium oxalate stones (Herring, 1962; Pietrow and
Karellas, 2006). However, about 10% of radiolucent
kidney stones, such as melamine-induced stone, do not
contain enough calcium to be detected by standard X-ray
imaging methods. Currently, computed tomography (CT)
is considered as the gold standard diagnostic test for the
detection of kidney stones (Ferrandino et al., 2010;
Otnes,1983), and most of them are detectable by CT
except for very rare stones, which are composed of drug
residues in the urine (Pietrow and Karellas, 2006).
However, the CT scans impose a radiation exposure and
a high cost on patients, and thus, it is not the best choice
for clinical diagnosis. Ultrasound imaging is alternatively
useful for the detection of kidney stones as it gives details
about the presence of hydronephrosis, particularly, for
cases where X-ray/CT imaging is discouraged (Jia et al.,
2009; Sun et al., 2009). However, the results of ultrasound
imaging are highly dependent on the clinical observations
and statistics (Wen et al., 2009; Zhang et al., 2009).
Infrared spectroscopy and Raman spectroscopy detects
limited categories of urinary stones (Chiu et al., 2010;
Evan et al., 2005). The chemical elements in the stones,
such as calcium, phosphorus, oxygen, carbon, etc., can
be detected using scanning electron microscopy (SEM)
combined with Fourier transform infrared spectroscopy
(FTIR) (Marickar et al., 2009). However, the presence of
melamine in the stone samples can not be determined. So
far, there is no reliable method available for fast
differentiation of the melamine-induced kidney stone from
other types.
Recently, surface desorption atmospheric pressure
chemical ionization (SDAPCI) (Yang et al., 2009a, b, c;
Chen et al., 2007) has been successfully employed for
direct analysis of melamine tainted powdered milk
samples, with minimal sample pretreatment. This
provides the potential feasibility for detection of melamine
in kidney stone samples by SDAPCI-MS, which will be
described in other studies. Methods based on
electrospray ionization mass spectrometry (ESI-MS) have
been widely used for rapid differentiation of quality of
volatile liquid foods (De Souza et al., 2007; Mendonça et
al., 2008; Sanvido et al., 2010; Biasotoa et al., 2010;
Alves et al., 2010). In this study, an electrospray mass
spectrometric method has been applied for the chemical
profiling of kidney stone solution samples.
Melamine-induced kidney stones and uric acid derived
kidney stones have been differentiated by performing
principal component analysis (PCA) with the mass
spectral raw data, which were recorded under either
positive or negative ion detection mode. Since neither
time consuming steps for separation of the sample nor
expensive MS n instrument for molecular structure
identification is required for differentiation of the
melamine-induced kidney stone samples, the results
suggest that the ESI-MS-based method reported here can
be particularly useful for rapid differentiation of kidney
stones at the molecular level.
METHODOLOGY
Materials and sample preparation
The human kidney stone samples for 6 patients were provided by
the Hospital of Gannan Medical University. As shown in Table 1, the
patients for sample 1 to 4 are little children (not more than 7 years
old) who are possible victims of melamine tainted milk event (Xin
and Stone, 2008) that happened in China. There is no clear
evidence showing that the powder milks consumed by these
patients were contaminated with melamine. As recorded in the
clinical data sheet, patient 3, a 7 year old boy, had consumed
powdered milk for the longest time. The preliminary work performed
in a Canada lab using secondary ion mass spectrometry (Sodhi et
al., 2010) also shows that melamine indeed exists in the calculus
from the patient 3. Patients 5 and 6 are adults, who did not consume
any milk products. Therefore, the stone samples from patients 5 and
6 were used as the reference samples. The kidney stone samples (1
to 5 mg) were dissolved in 10 ml acetic acid/methanol solution (1:4,
v/v) and ultrasonicated for 20 min (10 W power) to assist dissolution.
The solution was then diluted 1000 times with methanol/water (1:1,
v/v) solvent for the ESI-MS analysis. Chemicals, such as methanol
(analytical reagent (AR) grade) and acetic acid (AR grade) were
bought from Chinese Chemical Reagent Co. Ltd. (Shanghai, China).
ESI-LTQ mass spectrometer
The experiments were carried out using a commercial available
linear ion trap mass spectrometer (LTQ-XL, Finnigan, San Jose, CA)

2020 Afr. J. Pharm. Pharmacol.
Figure 1. Mass spectrum recorded using positive ESI-MS from
different kidney stone solution samples. 1 to 6#: the stone from
patient 1 to 6, respectively.
installed with an ESI source. The ESI source and the LTQ mass
spectrometer were set to work under positive/negative ion detection
mode. The nebulizing gas (N2) pressure was 1 MPa, the ESI high
voltage was +4.5 kV/-3.5kV, and the temperature of the ion
introduction capillary was 350°C. Other parameters were set at
default values of the instrument and no further optimization of the
ESI-MS was performed.
Data acquisition and analysis
The full scan mass spectra were recorded for an average acquisition
time of 30 s. Collision induced dissociation (CID) was performed
with 20 to 37% collision energy (CE). The parent ions were isolated
with a mass window width of 1.5 Da, and the mass
spectrometry/mass spectrometry (MS/MS) spectra were collected
for a recording time of more than 30 s if necessary. Principal
component analysis (PCA) of the mass spectral fingerprint data was
performed in Matlab (version 7.0, Mathworks Inc., Natick, U.S.A.).
The mass spectral data were exported to Microsoft Excel and the
data were arranged using the mass to charge (m/z) values as
independent variables and using the relative abundance of the full
scan mass fingerprint (MS 1 ) as the dependent variables. The whole
mass spectra data were treated as a matrix X, in which the rows and
the columns corresponded to sample cases and m/z value variables,
respectively. All the mass spectral data expressed by the relative
abundance were directly used for the PCA. The ‘princomp’ function
included in the ‘Matlab Toolbox’ was used for the PCA processing.
When the PCA was completed, the scores of the first three principal
components (PCs) were exported to new spreadsheets, and then
Matlab was used to present the results of statistical analysis for
better visualization.
RESULTS AND DISCUSSION
ESI-MS analysis
Generally, different types of kidney stones vary in
chemical compositions, but most of the kidney stones are
composed of calcium oxalate, calcium phosphate and
magnesium ammonium phosphate (Moe, 2006; Millman
et al., 1982). Dissolved in acetic acid/methanol solution,
the corresponding metal salts, such as calcium acetic,
magnesium acetic, etc., were formed. ESI-MS provides
information-rich spectra and many species can be
simultaneously detected. Note that the solid kidney stones
must be dissolved into liquid form prior to ESI-MS
measurements. In order to avoid the contamination of the
ESI-MS instrument, the stone solutions were diluted 1000
times with methanol/water (1:1, v/v) solvent before they
were electrosprayed for further MS analysis.
The mass spectra of the six samples were recorded
under the positive ion detection mode. As shown in Figure
1, a set of peaks, such as m/z 127, 135, 177, 279, 301
and 377 appeared in all the six samples. It was known
that the peaks at m/z 279 and 301 were the protonated
dibutyl phthalate (DBP) (Hu et al., 2010) and [DBP+Na] + ,
respectively, due to the DBP used as plasticizers in the
instrument system. Although, the signal of m/z 127 was
recorded in all the six samples, the intensity of m/z 127
detected in the sample from patients 1, 5 and 6 was in
an order of magnitude less than those detected from
the others. Furthermore, in the CID experiments, the
precursor ions at m/z 127 detected from the sample 3
generated ions of m/z 43 (CH3N2 + ), 85 (C2H5N4 + ) and 110
(C3H4N5 + ) as the major fragments (Figure 2a) by the loss
of C2H4N4, CH2N2 and NH3, respectively. In the MS 3
experiment, a characteristic fragment of m/z 43 (CH3N2 + )
was successively produced from the fragmentation of m/z
85 (Figure 2b) or 110 (Figure 2c). These fragmentation
patterns match with those obtained with authentic
melamine samples and are in accordance with the
fragmentation pathways of protonated melamine
observed in previous studies (Zhu et al., 2009).
Consequently, the peak at m/z 127 detected from sample
3 was confirmed to the protonated melamine molecule
(M+H) + . However, these characteristic fragments could
not be produced from the other stones (samples 1, 2, 4, 5
and 6) (Figure 3), indicating that only sample 3 is the
melamine-induced kidney stone among these stones.
Upon negative ion detection mode, calcium and
magnesium acetate salts formed the corresponding
deprotonated ions, such as (CaAC2)AC - (m/z 217),

Figure 2. Mass spectra recorded by ESI-MS. (a) MS/MS spectrum of protonated melamine
(m/z 127) in sample 3; (b) MS/MS/MS spectrum of the ionic fragments (m/z 85) produced
from protonated melamine (m/z 127) in sample 3; (c) MS/MS/MS spectrum of the ionic
fragments (m/z 110) produced from protonated melamine (m/z 127) in sample 3; (d) MS/MS
spectrum of deprotonated uric acid (m/z 167) in sample 2.
Figure 3. MS/MS spectra of precursor ions (m/z 127) and MS/MS/MS spectra of ionic
fragments (m/z 85) produced from m/z 127 from different kidney stone solution samples. 1 to
6#: the stone from patient 1 to 6, respectively.
Zhou et al. 2021

2022 Afr. J. Pharm. Pharmacol.
Figure 4. Upon the negative ion detection mode, ESI mass
spectrum recorded from the kidney stone solution samples. 1 to
6#: the stone from patient 1 to 6, respectively.
(CaAC2)2AC - (m/z 375), (CaAC2)3AC - (m/z 533),
(MgAC2)AC - (m/z 201), (NaAC)AC - (m/z 141) and
(KAC)2AC - (m/z 255), as shown in the Figure 4. It is
noteworthy that significant abundant peaks at m/z 167 is
present only in sample 2, while other MS peaks for
sample 2 are much lower than those in other samples,
suggesting that sample 2 might have different chemical
contents. Upon CID, the precursor ions of m/z 167
generated the abundant fragments of m/z 124 (C4H2N3O2
by the loss of CHNO (Figure 2d). These characteristic
fragments were in good agreement with previous work of
uric acid (Dai et al., 2007). Furthermore, CID data also
matched with those recorded using authentic uric acid
compound under the same experimental conditions.
Therefore, the ions at m/z 167 in the samples were
assigned to be the deprotonated uric acid. In comparison
with other samples, abundance of m/z 167 in sample 2
suggests that the content of uric acid in kidney stone
samples 2 should be much higher than others.
Accordingly, the cause of the kidney stone for baby girl
(patient 2) could be diagnosed as uric acid.
In a related study, the same groups of samples were
investigated using time-of-flight secondary ion mass
- )
spectrometry (ToF-SIMS) (Sodhi et al., 2010). Polished
cross-sections of the samples were obtained for recording
SIMS mass spectra in both high spatial and high mass
resolution modes. The motivation was to distinguish
between different stone types using the distribution
patterns of the N-containing species obtained in the
ToF-SIMS experimental data. Although, some interesting
data were obtained, the SIMS technique was not able to
detect melamine molecules from the melamine-induced
stone sample. The results obtained using ESI-MS in this
work differentiated melamine-induced kidney stones from
the uric acid-based kidney stones, showing that ESI-MS
provides complementary information for the ToF-SIMS
results. The combination of these two techniques is
promising for the understanding of the formation
mechanism of the kidney stones. For the clinic diagnosis
purpose, however, ESI-MS is of obvious advantages,
including simple operation, low cost instrument and fast
analysis speed, especially when PCA is applied to
process the experimental data (demonstrated
subsequently).
Principal component analysis (PCA)
As mentioned earlier, the melamine and uric acid induced
human kidney stones can be differentiated under the
positive and/or negative mode of ESI-MS/MS experiments.
However, the sensitive CID measurements require an
instrument of advanced tandem MS capability. This
requirement demands expensive mass spectrometers. As
shown in the full scan mass spectra, more signals rather
than the melamine itself (m/z 127) were differentiable in
the mass spectral fingerprints. Once the differences are
clearly visualized, the melamine-induced kidney stones
can be reliably diagnosed without resorting to tandem MS
experiments, featured on site applications, using simple
and low cost portable mass spectrometers. Therefore,
PCA, a powerful tool for data compression and
information extraction (Jackson, 1980; Moore, 1981;
Tipping and Bishop, 1999) was employed to process the
ESI-MS data for differentiation of the samples.
As a result, a PCA score plot of six types of stone
samples are shown in Figure 5, among which a1 and a2
correspond to the data points of full ESI mass spectrum
recorded upon the positive ion detection mode. Note that,
there are 21 data points generated for the groups of all six
patients, using 21 pieces of the stone samples from the
same patient. As shown in Figure 5a1 and a2, a total of
126 data points were explained by the score graphs of
PC1-PC2 and PC2-PC3. 91.9% of the total variations
were represented and the percentages of variance exp-
lained by PC1, PC2 and PC3 were 54.73, 25.20 and 11.97%,
respectively. Except for samples 4 and 6 which can not be
distinguished from each other, others were all differentia-
ted from each other. The differentiation of samples 1, 2

Figure 5. PCA score results for differentiation of the human kidney stones. a1: score plot PC1-PC2 based on
positive ESI; a2: score plot PC2-PC3 based on positive ESI; b1: score plot PC1-PC2 under negative ESI ion
mode; b2: score plot PC2-PC3 under negative ESI ion mode. c1: 3D-score plot PC1-PC2 under positive ESI
c2: 3-D score plot under negative ESI.
(uric acid induced stones) and 3 (melamine-induced
stones) has been achieved. On the other hand, the
distribution of signs in each cluster from samples 4 to 6 is
restricted in a narrow scope. Thus, the PCs show strong
ability for differentiation of melamine and uric acid induced
stones from the other types of kidney stones. Although, a
few data points of sample 5 were mixed together with
samples 4 and 6 in the PC1 and PC2 plane (Figure 5a1),
all the data points of sample 5 were completely separated
from the other samples in PC3, as shown in Figure 5a2.
However, upon the negative ion detection mode, the six
different types of kidney stones were differentiated from
each other in the PC1 and PC2 plane (Figure 5b1), and
this could be clearly visualized in the corresponding 3-D
space (Figure 5c2). Therefore, negative ESI-MS is
proposed for differentiation of the kidney stone samples
without CID experiments, although the melamine induced
stones have been differentiated from the others under the
positive ion mode.
As shown in the clinical data (Table 1), the different
X-ray imaging properties (both the stone location and
transparency) of sample 1 from sample 5 might explain
the separation of samples 1 and 5 in the PCA score plots.
Zhou et al. 2023
In both Figure 5b1 and 5b2, 1.5-year history of powdered
milk intake was most likely to be the only factor for
differentiating sample 4 from sample 6 in the PCA score
plots, since the stones in patients 4 and 6 were both
located in the left kidney pelvis and were all radiopaque in
the X-ray imaging studied in the clinical examination.
Although, the stones in patient 5 were also radiopaque,
the location of the stone 5 was at the right ureter instead
of the left kidney pelvis. This could be the important factor
for differentiating sample 5 from either sample 4 or
sample 6. Similar to sample 5, the stone in sample 1 was
also located in the ureter (the left ureter), but it was
radiolucent in the X-ray imaging study, indicating that the
chemical composition of sample 1 should be different from
the opaque samples. In the PCA score plots, sample 2
was located remarkably far from the sample 3, because
sample 2 was uric acid induced kidney stones, which
should be chemically different from the melamine induced
kidney stones. Conclusively, the location and imaging
property of kidney stones are two of the most important
factors used to distinguish these kidney stones and
they play important roles in differentiating different types
of kidney stones after performing PCA.

2024 Afr. J. Pharm. Pharmacol.
Figure 6. PCA loading results for the PCs based on the negative
ESI.
Figure 6 presents the PCA loading plots of the 126 data
points upon negative ion detection. The major differential
peaks (that is, peaks of significant abundances in the PCA
loading plots, which correspond to the ions that
contributed most to differentiation of the samples) shown
in the PCA loading plots, could potentially be useful as
molecular markers for differentiating different kidney
stones. For example, the most predominant ions in the
loading of PC2, which corresponded to the deprotonated
uric acid (m/z 167), could serve as the most important
factor for differentiating the uric acid induced kidney stone
(patient 2) from the others. For this reason, typical
differential peaks at m/z 167, 255, 265, 279, 281, 282, 311,
337, 346, 353, 375 and 397 were selected as molecular
markers, because they were outstanding in the PCA
loading plots. Structural identification of all these
differential peaks is theoretically possible using tandem
mass spectrometry experiments, but it is beyond the
scope of this study. Also, Figure 7 gives the loading plots
of 126 data points under positive ion detection model. It is
clear that the peaks at m/z 135, 139, 149, 177, 191, 195,
198, 203, 207, 261, 285, 301, 377, 391 and 413
contributed most to differentiation of these samples, and
could be regarded as molecular markers for differentiating
different kidney stones. The absence of the ion at m/z 127
in the load plots suggests that the ion at m/z 127 is not the
important variable in the differentiation of these samples
in the full ESI scan mass spectra, although the
confirmation of melamine-contained sample requires the
MS/MS of the ion at 127 and MS 3 of the ionic fragments at
m/z 85 produced from the m/z 127. This is because, the
Figure 7. PCA loading results for the PCs based on the
positive ESI.
ion at m/z 127 can be observed in all the six samples in
the full ESI scan experiments, due to disturbance of
impurities.
In summary, the full mass spectra could be collected in
a few seconds using ESI mass spectrometry with minimal
sample pretreatment, indicating that ESI-MS affords high
analysis speed. Sufficient information used for
differentiation of different kidney stones could be provided,
even though the stone liquid solutions were diluted 1000
times before introducing into the ESI ionization,
demonstrating high sensitivity of the ESI-MS. Good
results for differentiation of melamine and uric acid
induced kidney stones from the others whenever the
positive or negative ion mode was used, to a certain
degree, and they showed the stability and reproducibility
of the ESI. Therefore, ESI-MS-based method proposed in
this study is indeed a practical technique for rapid
differentiation of kidney stones, with its high analysis
speed, high sensitivity and good reproducibility.
Conclusions
An ESI-MS-based method was applied for fast detection
of the melamine-induced and uric acid derived human
kidney stone samples with minimal sample pretreatment.
Using the tandem mass spectrometry, specific
compounds, such as melamine and uric acid have been
identified with high confidence. On the other hand, without
the need of tandem MS capability, the ESI-MS combined
with PCA is an analytical tool for differentiation of different
types of human kidney stones, and is especially useful for

the negative ESI-MS mass spectral fingerprints in the
analysis and detection of melamine-induced kidney
stones. Melamine, a basic compound, could not be
detected in the negative ESI mass spectra. However, it
has been experimentally demonstrated that many other
chemicals detected from the melamine-induced kidney
stone samples were different from those detected from
other types of kidney stone samples. These findings are
the sound bases at molecular level for differentiation of
the melamine-induced kidney stones from the others,
particularly in the negative ion detection mode, and they
provide useful hints for further study on the formation
mechanisms of melamine-induced kidney stones in the
pathological research.
ACKNOWLEDGEMENT
This work was jointly supported by the National
Instrumentation Program (No. 2011YQ170067) and the
Major Grant of Natural Science Foundation of Jiangxi
Province (No. 2010GZH002).
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African Journal of Pharmacy and Pharmacology Vol. 5(17), pp. 2027-2034, 8 November, 2011
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP11.522
ISSN 1996-0816 © 2011 Academic Journals
Full Length Research Paper
Radical scavenging and antioxidant activity of Hibiscus
rosasinensis extract
Rajesh Mandade 1 *, S. A. Sreenivas 2 , D. M. Sakarkar 3 and Avijit Choudhury 4
1 Pharmacology Department,S.N. Institute of Pharmacy, Pusad, India.
2 Guru Nanak Institute of Pharmacy,Ibrahimpattnam, Hyderabad (A.P.), India.
3 S.N. Institute of Pharmacy, Pusad, India.
4 Shree Dhanvantary Pharmaceutical Analysis and Research Centre, Kim, Surat, Gujarat, India.
Accepted 17 October, 2011
Free radicals induce numerous diseases by lipid peroxidation, protein peroxidation and DNA damage. It
has been reported that numerous plant extracts have antioxidant activities to scavenge free radicals. In
the present study, the antioxidant properties of crude extract of Hibiscus rosasinensis were examined,
using different in vitro analytical methodologies, such as total antioxidant activity determination by
ferric thiocyanate, hydrogen peroxide scavenging, 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH)
scavenging, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS + radical cation) radical cation
scavenging activity and superoxide anion radical scavenging by riboflavin-methionine-illuminate
system. Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and α-tocopherol were used
as the reference antioxidant radical scavenger compounds. The crude extract inhibited 94.58%
peroxidation of linoleic acid emulsion at 20 µg/ml concentration, while the standard antioxidants BHA,
BHT and α-tocopherol indicated an inhibition of 93.75, 96.66 and 83.33% at 60 µg/ml concentration,
respectively. The hydrogen peroxide radical, DPPH radical, ABTS + radical cation(s) and superoxide
anion radical scavenging activities of crude extract were also compared to BHA, BHT and α-tocopherol
as references antioxidant compounds. The present study shows that the crude extract is an effective
natural antioxidant component.
Key words: Hibiscus rosasinensis, antioxidant activity, free radical scavenging activity.
INTRODUCTION
Antioxidants help living organisms to deal with oxidative
stress, caused by free radical damage. Free radicals are
chemical species which contains one or more unpaired
electrons due to which they are highly unstable and
cause damage to other molecules by extracting electrons
from them in order to attain stability. Reactive oxygen
species (ROS) formed in vivo, such as superoxide anion,
hydroxyl radical and hydrogen peroxide, are highly reactive
and potentially damaging transient chemical species.
ROS are continuously produced in the human body, as
they are essential for energy supply, detoxification, chemi-
cal signaling and immune function. ROS are regulated
by endogenous superoxide dismutase, glutathione
*Corresponding author. E-mail: raj_mandade@rediffmail.com.
Tel: +917233249795 or +918888673088. Fax: 07233247308.
peroxidase and catalase, but due to over-production of
reactive species induced by exposure to external oxidant
substances or a failure in the defense mechanisms,
damage to cell structures, DNA, lipids and proteins
(Valko et al., 2006) occur which increases risk of more
than 30 different disease processes (Aruoma, 1998).
Hibiscus rosasinensis Linn (Malvaceae) is a glabrous
shrub widely cultivated in the tropics as an ornamental
plant and has several forms with varying colors of
flowers. In medicine, however the red flowered variety is
preferred (Adhirajan et al., 2003). The leaves and flowers
are observed to be promoters of hair growth and aid in
healing of ulcers (Jadhav et al., 2009). Aerial part of H.
rosasinensis has calcium channel blocking action (Gilani
et al., 2005). Recent reports have also shown antiammonemic
(Essa and Subramanian., 2007), antidiabetic
(Venkatesh et al., 2008), hypolipidemic (Kumar
et al., 2009), post-coital anti-fertility, cardio protective and

2028 Afr. J. Pharm. Pharmacol.
wound healing activities (Gauthaman et al., 2006, Nayak
et al., 2007).
The free radical neutralizing property of several plants
was reported by previous studies. The extracts from a
number of medicinal plants which are known to have
some biologically active principles are used in ayurvedic
preparations and these extracts are prepared in bulk for
commercial purpose. In the present study, the antioxidant
property of 80% aqueous-ethanol crude extract of H.
rosasinensis was examined by different in vitro analytical
methodologies, such as total antioxidant activity
determination by ferric thiocyanate, hydrogen peroxide
scavenging, 1,1-diphenyl-2-picryl-hydrazyl free radical
(DPPH) scavenging, 2,2′-azino-bis(3-ethylbenzthiazoline-
6-sulfonic acid) (ABTS +
radical cation) radical
scavenging activity and superoxide anion radical
scavenging by riboflavin-methionine-illuminate system.
MATERIAL AND METHODS
Plant
Aerial part of H. rosasinensis was collected from the botanical
garden of S. N. Institute of Pharmacy, Pusad, India. Identification
and authentication of the samples was done by using standard
botanical monographs. They were further confirmed at the
Department of Botany, R.S.T.M University Nagpur, India.
Preparation of crude extract
The plant material was cleaned off adulterants, shade dried and
was coarsely grounded. The powdered material (1 kg) was soaked
in 80% aqueous-ethanol for 3 days with occasional shaking. It was
filtered through a muslin cloth and then through a filter paper. This
procedure was repeated thrice and the combined filtrate was
evaporated on a rotary evaporator under reduced pressure (-760
mmHg) to a thick, semi-solid mass of dark brown color, that is, the
crude extract with a yield of approximately 10% (Gilani et al., 2005).
Phytochemical screening
The preliminary phytochemical studies (Adhirajan et al., 2003) were
conducted for the aforementioned crude extracts of H. rosasinensis
to find out the presence of sterols, carbohydrates and glycosides,
tannins and flavonoid using standard analytical procedures (Gupta
et al., 2009) (Table 1).
Estimation of total phenolic compounds
Total phenol
The total phenolic content in the crude extract of H. rosasinensis
was determined spectrophotometrically with Folin-Ciocalteu reagent
using the modified method of Wolfe et al. (2003). An aliquot of the
crude extract (0.5 ml) was mixed with 2.5 ml of 10% Folin-Ciocalteu
reagent and 2 ml of Na2CO3 (75% w/v). The resulting mixture was
vortexed for 15 s and incubated at 40°C for 30 min for colour
development. The absorbance of the samples was measured at
765 nm using spectrophotometer at UV/visible light. Total phenolic
content was expressed as mg/g tannic acid equivalent from the
calibration curve using the equation:
Y = 0.1216x, R2 = 0.936512
Where x was the absorbance and Y was the tannic acid equivalent
(mg/g).
The experiment was conducted in triplicate and the results are
reported as mean ± SD values.
Total flavonoid
The method of Ordon et al. (2006) was used to estimate the total
flavonoid contents of the crude extract solution based on the
formation of a complex flavonoid-aluminium. A volume of 0.5 ml of
2% AlCl3 ethanol solution was added to 0.5 ml of crude extract
solution. After 1 h of incubation at the room temperature, the
absorbance was measured at 420 nm using UV-VIS
spectrophotometer. All determinations were done in triplicate and
values were calculated from calibration curve obtained from
quercetin using the equation:
Y = 0.0255x, R2 = 0.9812
where x was the absorbance and Y the quercetin equivalent (mg/g).
Total flavonols
Total flavonol content was determined by adopting the procedure
described by Kumaran and Karunakaran (2007). The reacting
mixture consisted of 2.0 ml of the sample, 2.0 ml of AlCl3 prepared
in ethanol and 3.0 ml of (50 g/L) sodium acetate solution. The
absorption at 440 nm was read after 2.5 h at 20°C. Total flavonoid
content was calculated as quercetin (mg/g) equivalent from the
calibration curve using the equation:
Y= 0.0255x, R2 = 0.9812
where x was the absorbance and Y the quercetin equivalent (mg/g).
Total proanthocyandins
Total proanthocyandins was determined based on Sun et al. (1998)
procedure. 3 ml of vanillin-methanol (4% v/v) and 1.5 ml of
hydrochloric acid was added to 0.5 ml of 1 mg/ml crude extract
solution and then vortexed. The absorbance of resulting mixture
was measured at 500 nm after 15 min at room temperature. Total
proanthocyandin content was expressed as catechin equivalents
(mg/g) using the following equation from the calibration curve:
Y = 0.5825x, R2 = 0.9277
where x was the absorbance and Y the catechin equivalent (mg/g).
Total antioxidant activity determination by ferric thiocyanate
method (FTC)
The total antioxidant activity of the crude extract and standard
antioxidants (BHA, BHT and α-tocopherol) was determined
according to the ferric thiocyanate method (Mitsuda et al., 1996) as
described by Gulcin (2006b). For stock solutions, 10 mg of the
extract was dissolved in 10 ml distillate water. Then, the solution
which contains 20 µg/ml concentration of the extract solution in
2.5 ml of sodium phosphate buffer (0.04 M, pH 7.0) was added to
2.5 ml of linoleic acid emulsion in sodium phosphate buffer (0.04 M,
pH 7.0). Therefore, 5 ml of the linoleic acid emulsion was prepared
by mixing and homogenising 15.5 µl of linoleic acid, 17.5 mg/g of

tween-20 as emulsifier and 5 ml phosphate buffer (pH 7.0).
On the other hand, 5 ml of control was composed of 2.5 ml of
linoleic acid emulsion and 2.5 ml sodium phosphate buffer (0.04 M,
pH 7.0). The mixed solution (5 ml) was incubated at 37°C in
polyethylene flask. The peroxide level was determined by reading
the absorbance at 500 nm in a spectrophotometer after reaction
with FeCl2 (3.5%) and thiocyanate (30%) at intervals during
incubation. During the linoleic acid peroxidation, peroxides are formed
and that leads to the oxidation of Fe 2+ –Fe 3+ . The latter ions form a
complex with ammonium thiocyanate and this complex has a
maximum absorbance at 500 nm. This step was repeated every
5 h. The percentage inhibition values were calculated at this point
(30 h). High absorbance indicates high linoleic acid emulsion
peroxidation (Table 2). The solutions without the extract were used
as blank samples. Total antioxidant activity determination was
performed in triplicate. The inhibition percentage of lipid peroxidation in
linoleic acid emulsion was calculated by following equation:
Inhibition of lipid peroxidation (%)=100-(As/Ac×100)
AC is the absorbance of control reaction which contains only linoleic
acid emulsion and sodium phosphate buffer and AS is the
absorbance in the presence of sample crude extract or standard
compounds (Gulcin, 2006a, b).
Hydrogen peroxide scavenging activity
The hydrogen peroxide scavenging assay was carried out following
the procedure of Ruch et al. (1989). For this aim, a solution of H2O2
(43 mM) was prepared in phosphate buffer (0.1 M, pH 7.4). Crude
extract at 20 µg/ml concentration in 3.4 ml phosphate buffer was
added to 0.6 ml of H2O2 solution (0.6 ml, 43 mM). The absorbance
value of the reaction mixture was recorded at 230 nm. Blank
solution contains sodium phosphate buffer without H2O2. The
concentration of hydrogen peroxide (mM) in the assay medium was
determined using a standard curve (r 2 : 0.9895):
Absorbance = 0.038 × [H2O2] + 0.4397
The percentage of H2O2 scavenging of crude extract and standard
compounds were calculated using the following equation:
H2O2 scavenging effect (%) = (1 – As/Ac) × 100
where AC is the absorbance of the control and AS is the absorbance
in the presence of the sample extract or standards (Gulcin, 2006b;
Elmastas et al., 2005).
DPPH free radical scavenging activity
The DPPH free radical scavenging activity of crude extract was
determined according to the method described by Gulcin (2006b)
with slight modifications. In its radical form, DPPH absorbs at
517 nm, but upon reduction by an antioxidant or a radical species
its absorption decreases and thereby the bleaching rate of a stable
free radical, DPPH in the presence of the sample is monitored at a
characteristic wavelength.
Briefly, 0.1 mM solution of DPPH was prepared in ethanol and
0.5 ml of this solution was added 1.5 ml of the extract solution in
ethanol at different concentrations (20 to 60 µg/ml). These solutions
were vortexes thoroughly and incubated in dark, half hour later, the
absorbance was measured at 517 nm against blank samples.
Mandade et al. 2029
Lower absorbance of the reaction mixture indicates higher DPPH
free radical scavenging activity. A standard curve was prepared
using different concentrations of DPPH. The DPPH concentration
scavenging capacity was expressed as mM in the reaction medium
and calculated from the calibration curve determined by linear
regression (r 2 : 0.9845):
Absorbance = 9.692 × [DPPH] + 0.215
The capability to scavenge the DPPH radical was calculated using
the following equation:
DPPH scavenging effect (%) = (1- As/Ac) × 100
where AC is the absorbance of the control which contains DPPH
solution and AS is the absorbance in the presence of the extracts
(Gulcin et al., 2004c; Elmastas et al., 2006).
ABTS + radical cation(s) decolorization assay
The spectrophotometric analysis of ABTS + radical cation(s)
scavenging activity was determined according to Re et al. (1999)
method. This method is based on the ability of antioxidants to
quench the long-lived ABTS radical cation, a blue/green
chromophore with characteristic absorption at 734 nm, in
comparison to that of BHA, BHT and α-tocopherol. The ABTS + was
produced by reacting 2 mM ABTS + radical cation(s) in H2O with
2.45 mM potassium persulfate (K2S2O8), stored in the dark at room
temperature for 4 h. Before usage, the ABTS + solution was diluted
to get an absorbance of 0.750 ± 0.025 at 734 nm with sodium
phosphate buffer (0.1 M, pH 7.4). Then, 1 ml of ABTS + solution was
added 3 ml of the extract solution in ethanol at different
concentrations (20 to 60 µg/ml).
After 30 min, percentage inhibition at 734 nm was calculated for
each concentration, relative to a blank absorbance. Solvent blanks
were run in each assay. The extent of decolorization is calculated
as percentage reduction of absorbance. For preparation of a
standard curve, different concentrations of ABTS + radical cation(s)
were used. The ABTS + radical cation(s) concentration (mM) in the
reaction medium was calculated from the following calibration
curve, determined by linear regression (r 2 : 0.9841):
Absorbance = 4.6788 × [ABTS + ] + 0.199
The scavenging capability of ABTS + radical was calculated using
the following equation:
ABTS scavenging effect (%) = (1- As/Ac) × 100
where AC is the initial concentration of the ABTS + radical cation(s)
and AS is absorbance of the remaining concentration of ABTS +
radical cation(s) in the presence of the extract.
Superoxide anion radical scavenging activity
Superoxide radicals were generated by the method of Beauchamp
and Fridovich (1971) described by Zhishen et al. (1999) with slight
modification. Superoxide radicals are generated in riboflavin,
methionine, illuminate and assayed by the reduction of nitroblue
tetrazolium (NBT) to form blue formazan (NBT 2+ ). All solutions were
prepared in 0.05 M phosphate buffer (pH 7.8). The photo-induced
reactions were performed using fluorescent lamps (20 W). The
concentration of the extract in the reaction mixture was 20 µg/ml.

2030 Afr. J. Pharm. Pharmacol.
Absorbance (500 ηm)
Table 1. Preliminary phytochemical screening.
Chemical constituent Crude extract
Alkaloids -
Sterols +
Carbohydrates and glycosides +
Fixed oil and fats -
Tannins and phenolic +
Proteins +
Triterpenoids +
Flavonoids +
3
2
1
0
0 20 40 60 80
Incubation Time (h)
Control
α-Tocopherol
BHA
20 µg/ml
40 µg/ml
60 µg/ml
Figure 1. Total ferric reducing power (FRAP) of different
concentrations (20 to 60 µg/ml) of extract of H. rosasinensis and
reference antioxidants; BHA, BHT and α-tocopherol. Total ferric
reducing power determined according to the ferric ions (Fe 3+ )ferrous
ions (Fe 2+ ) transformation. The reducing power was
estimated based on the absorbance reading at 700 nm with a
spectrophotometer. Values are expressed as mean ± standard
deviation of three replicate determinations.
The total volume of the reactant mixture was 3 ml and the
concentrations of the riboflavin, methionine and NBT was 1.33 × 10 -
5 , 4.46 × 10 -5 and 8.15 × 10 -8 M, respectively. The reactant was
illuminated at 25°C for 40 min. The photochemically reduced
riboflavin generated O2 . This reduced NBT to form blue formazan.
The unilluminated reaction mixture was used as a blank. The
absorbance was measured at 560 nm. The extract was added to
the reaction mixture, in which O2 was scavenged, thereby inhibiting
the NBT reduction. Decreased absorbance of the reaction mixture
indicates increased superoxide anion scavenging activity. The
inhibition percentage of superoxide anion generation was
calculated by using the following formula:
O2 .- scavenging effect (%) = (1- As/Ac) × 100
where AC is the absorbance of the l-ascorbic acid and AS is the
absorbance of the extract or standards (Gulcin et al., 2003, 2004b).
RESULTS AND DISCUSSION
A wide variety of in vitro methods have been set up to
assess radical scavenging ability and antioxidant activity.
Antioxidant capacity is widely used as a parameter for
medicinal bioactive components. Different artificial free
radical species, such as ABTS + radical cation, DPPH
radical, as well as biological radicals or H2O2 scavenging
activity have been used.
Polyphenol are the major plant compounds with high
level of antioxidant activity. This activity could be due to
their ability to adsorb, neutralize and to quench free
radicals (Duh et al., 1999). Their ability as free radical
scavenger could also be attributed to their redox
properties, presence of conjugated ring structures and
carboxylic group which have been reported to inhibit lipid
peroxidation (Rice-Evans et al., 1995).
In the present study, it was found that the crude extract
of H. rosasinensis contains high level of phenol content
that might account for the strong activity observed
against ABTS + radical cation(s) and H2O2 radicals. This
scavenging activity may be due to the presence of
hydroxyl groups attached to the aromatic ring structures
and thus, help to quench the radicals (Vinson et al.,
1998). On the other hand, the activity depicted in DPPH
and superoxide anion may be as a result of the content of
flavonoid which has been reported to possess high
antioxidant activity.
Total antioxidant activity determination in linoleic
acid emulsion system by ferric thiocyanate method
Lipid peroxidation contains a series of free radicalmediated
chain reaction processes and is also
associated with several types of biological damage. The
total antioxidant activity of the crude extract, BHA, BHT
and α-tocopherol in the linoleic acid system was
determined by the ferric thiocyanate method. This
method measures the amount of peroxide produced
during the initial stages of oxidation, which is the primary
product of lipid oxidation.
Crude extract showed effective antioxidant activity in
this system. The effect of different concentration (20 to
60 µg/ml) of the extract on lipid peroxidation of linoleic
acid emulsion is as shown in Figure 1.
At these concentrations of the extract caused 94.58,
95.00 and 95.88% lipid peroxidation inhibition of linoleic
acid emulsion. Their activities are greater than 60 µg/ml
concentration of BHA (93.75%), α-tocopherol (83.33%),
but close to BHT (96.66%). Consequently, these results
clearly indicate that the extract has an effective and
powerful antioxidant activity by ferric thiocyanate method.
Hydrogen peroxide scavenging activity
Hydrogen peroxide has strong oxidizing properties. It can

Table 2. Polyphenol contents of crude extract of H. rosasinensis.
Antioxidant compound Total antioxidant compound
Phenol contents 49.44 mg/g tannic acid equivalent
Proanthocyandins 9.15 mg/g catechin equivalent
Flavonols 5.5 mg/g quercetin equivalent
Flavonoids 4.8 mg/g quercetin equivalent
Table 3. Ability of crude extract to scavenge hydrogen peroxide when compared with BHA,
BHT and α-tocopherol.
H2O2 scavenging activity (%) Superoxide scavenging activity (%)
BHA 38.2 ± 2.8 76.4 ± 5.3
BHT 36.3 ± 3.2 72.2 ± 6.4
α-Tocopherol 41.2 ± 2.7 24.1 ± 3.2
Extract 51.9 ± 4.5 68.2 ± 3.4
be formed in vivo by many oxidizing enzymes, such as
superoxide dismutase and can cross cellular membranes
and may slowly oxidize a number of intracellular
compounds. The ability of crude extract to scavenge
hydrogen peroxide when compared with BHA, BHT and
α-tocopherol is as shown in Table 3.
Hydrogen peroxide scavenging activity of the extract at
the used concentration of 20 µg/ml was found to be
51.9 ± 4.5%. On the other hand, BHA, BHT and αtocopherol
exhibited 38.2 ± 2.8, 36.3 ± 3.2 and
41.2 ± 2.7% hydrogen peroxide scavenging activity at the
same concentration, respectively. These results showed
that the extract had an effective hydrogen peroxide
scavenging activity. At the aforementioned concentration,
the hydrogen peroxide scavenging effect of the extract
and three standard compounds decreased in the order of
the extract > α-tocopherol > BHA > BHT. Hydrogen
peroxide itself is not very reactive; however, it can
sometimes be toxic to cell, because it may give rise to
hydroxyl radical in the cells.
Radical scavenging activity
Radical scavenging activities are very important due to
the deleterious role of free radicals in foods and in
biological systems. Diverse methods are currently used
to assess the antioxidant activity of plant phenolic
compounds. Chemical assays are based on the ability to
scavenge synthetic free radicals, using a variety of
radical-generating systems and methods for detection of
the oxidation end-point. ABTS + radical cation(s) or DPPH
radical scavenging methods are common
spectrophotometric procedures for determining the
antioxidant capacities of components.
These chromogens (the violet DPPH radical and the
blue green ABTS + radical cation(s) are easy to use,
Mandade et al. 2031
have a high sensitivity and allow for rapid analysis of the
antioxidant activity of a large number of samples. These
assays have been applied to determine the antioxidant
activity of pure components (Awika et al., 2003, Yu et al.,
2002; van den Berg et al., 2000b). DPPH has been
widely used to evaluate the free radical scavenging
effectiveness of various antioxidant substances (Ozcelik
et al., 2003). DPPH is usually used as a reagent to
evaluate free radical scavenging activity of antioxidants.
DPPH is a stable free radical and accepts an electron
or hydrogen radical to become a stable diamagnetic
molecule (Soares et al., 1997). In the DPPH assay, the
antioxidants are able to reduce the stable radical DPPH
to non-radical form, DPPH-H. The purple colored
alcoholic solution of DPPH radicals changes color to
yellow in the presence of a hydrogen-donating
antioxidant which could be measured at 517 nm.
Figure 2 illustrates a significant decrease (P < 0.01) in
the concentration of DPPH radical due to the scavenging
ability of the extract and standards. BHA, BHT and αtocopherol
were used as references. The scavenging
effect of the extract and standards on the DPPH radical
decreased in the order of BHA > Extract > BHT > αtocopherol
which were 86.63, 84.01, 71.54 and 59.55%,
at the concentration of 60 µg/m, respectively. DPPH free
radical scavenging activity of the extract also increased
with an increasing concentration (r 2 : 8246).
Generation of the ABTS + radical cation(s) forms the
basis of one of the spectrophotometric methods that have
been applied to the measurement of the total antioxidant
activity of pure substances solutions, aqueous mixtures
and beverages (Miller, 1996). A more appropriate format
for the assay is a decolorization technique, in that the
radical is generated directly in a stable form prior to
reaction with putative antioxidants. The improved
technique for the generation of ABTS + described here
involves the direct production of the blue/green ABTS +

2032 Afr. J. Pharm. Pharmacol.
Absorbance (517 ηηm)
3
2
1
0
0 20 40 60 80
Concentration µg/ml (µg/ml)
Extract
BHT
BHA
α-Tocopherol
Figure 2. DPPH free radical scavenging activity of different concentrations (20 60
60 µg/ml) of extract of H. rosasinensis and reference antioxidants; BHA, BHT and
α-tocopherol.
Absorbance (734 ηηm)
0.8
0.6
0.4
0.2
0.0
0 20 40 60 80
Concentration µg/ml (µg/ml)
Extract
BHA
BHT
α-Tocopherol
Figure 3. ABTS free radical scavenging activity of different concentrations (20 to
60 µg/ml) of extract of H. rosasinensis and reference antioxidants; BHA, BHT and
α-tocopherol.
chromophore through the reaction between ABTS +
radical cation(s) and potassium persulfate.
All the tested compounds exhibited affectual radical
cation scavenging activity. As seen in Figure 3, the
extract had effective ABTS + radical scavenging activity in
a concentration-dependent manner (20 to 60 µg/ml).
There is a significant decrease (P < 0.01) in all the
concentration of ABTS + due to the scavenging capacity
of all the extract concentrations. Also, the scavenging
effect of the extract and standards on the ABTS + radical
cation(s) decreased in the order: BHA > BHT > Extract
> α-tocopherol, which were 97.33, 97.06, 89.2 and
73.33% at the concentration of 60 µg/ml, respectively.
Superoxide anion are a precursor to active free radicals
and plays an important role in the formation of other
ROS, such as hydrogen peroxide, hydroxyl radical and
singlet oxygen, which induce oxidative damage in lipids,
proteins and DNA (Pietta, 2000). Superoxide radical is
normally formed first, and its effects can be magnified,
because it produces other kinds of free radicals and
oxidizing agents. Superoxide anions have the potential of
reacting with biological macromolecules and have been
implicated in several pathophysiological processes due to
its transformation into more reactive species, such as
hydroxyl radical that initiate lipid peroxidation. Also,
superoxide has been observed to directly initiate lipid
peroxidation (Wickens, 2001). In addition, it has been
reported that antioxidant properties of some flavonoids
are effective mainly via scavenging of superoxide anion
radical. Superoxide anion derived from dissolved oxygen

iboflavin/methionine/illuminate system and reduces NBT
in this system. In this method, superoxide anion reduces
the yellow dye (NBT 2+ ) to produce the blue formazan
which is measured spectrophotometrically at 560 nm.
Antioxidants are able to inhibit the blue NBT formation
(Parejo et al., 2002) and the decrease of absorbance at
560 nm indicates the consumption of superoxide anion in
the reaction mixture. Table 3 shows the inhibition
percentage of superoxide radical generation by 20 µg/ml
concentration of crude extract and antioxidant standards.
The inhibition of superoxide radical generation results of
extract and standards were found to be similar
statistically. As shown in Table 3, the percentage
inhibition of superoxide anion radical generation by
20 µg/ml concentration of the extract was found as
68.2 ± 3.4 %.
On the other hand, at the same concentration, BHA,
BHT and α-tocopherol exhibited 76.4 ± 5.3, 72.2 ± 6.4
and 24.1 ± 3.2% superoxide anion radical scavenging
activity, respectively. According to these results, crude
extract had similar superoxide anion radical scavenging
activity to BHA and BHT; however, it had higher
superoxide anion radical scavenging activity than αtocopherol.
Conclusion
According to data obtained from the present study, 80%
aqueous-ethanol crude extract of H. rosasinensis extract
was found to be an effective antioxidant in different in
vitro assays, including total antioxidant activity
determination by ferric thiocyanate, DPPH radical,
ABTS + radical cation(s) radical, superoxide anion radical
scavenging and hydrogen peroxide scavenging when it is
compared to standard antioxidant compounds, such as
BHA, BHT and α-tocopherol.
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African Journal of Pharmacy and Pharmacology Vol. 5(17), pp. 2035-2041, 8 November, 2011
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP11.511
ISSN 1996-0816 © 2011 Academic Journals
Full Length Research Paper
Therapeutic monitoring of isoniazid, rifampicin,
ethambutol and pyrazinamide serum levels in the
treatment of active pulmonary tuberculosis and
determinants of their serum concentrations
Servet Kayhan 1,2 * and Alper Akgüneş 1,3
1 Chest Diseases and Thoracic Surgery Hospital, Samsun, Turkey.
2 Department of Pulmonary Disease And Tuberculosis, Ministry of Health, Chest Diseases and Thoracic Surgery
Hospital, Samsun, Turkey.
3 Department of Microbiology, Ministry of Health, Chest Diseases and Thoracic Surgery Hospital, Samsun, Turkey.
Accepted 12 October, 2011
Inadequate serum levels of antimycobacterial drugs have been associated with treatment failure,
relapse and acquired drug resistance as well as high concentrations of these drugs may cause
intolerance and toxic effects. We objected in this study to determine serum concentrations of antituberculosis
drugs and to know the determinants of their concentrations. Venous blood samples was
obtained 2 and 6 h after drug ingestion, and serum levels of drugs were analysed using high
performance liquid chromatography. Among 49 enrolled active pulmonary tuberculosis patients, the
prevalances of a low concentration of isoniazid, rifampicin, ethambutol and pyrazinamide were 28.6,
75.5, 18.4 and 20.4%, respectively. 2 h ısoniazid (INH) concentration was found to be associated with
sex (p = 0.005), correlated with body mass index (r = -0.390) and associated with drug dose (mg/kg) (p =
0.000). By Independent samples t-test analysis, low 2 h rifampicin concentration was found to be
associated with sex (p = 0.000) and smoking cigarette (p = 0.004). In conclusion, the results of this
study have shown that, low 2 h serum INH and rifampicin (RIF) concentration are common and It may be
necessary to optimise drug doses by therapeutic drug monitoring.
Key words: Therapeutic drug monitoring, tuberculosis, isoniazid, rifampicin, ethambutol, pyrazinamide.
INTRODUCTION
The World Health Organisation estimated the global
burden of tuberculosis(TB) in 2010 as around 14 million
prevalance and 2.38 million deaths from this curable
infectious disease (WHO, 2010). Despite directly
observed therapy (DOT) in TB control programs,
treatment failure, relapse, acquired drug resistance,
increasing in number of multidrug resistant cases and
drug toxicities remain ongoing complications in some TB
patients. Serum concentrations of anti-tuberculosis drugs
have been associated with many factors such as
malabsorption, smoking status, comorbidity of diabetes
mellitus and HIV, alcohol consumption,
hypoalbuminaemia, calculated creatinine clearance by
*Corresponding author. E-mail: servet-kayhan@hotmail.com.tr.
Cockcroft-Gault equation for EMB (Cockroft and Gault,
1976), liver and kidney dysfunctions, low dose per
kilogram of body weight, changes in drug formulation,
age and gender (Um et al., 2007; Mcllleron et al., 2006;
Narita et al., 2001). Low serum concentrations of antituberculosis
drugs has been reported as a reason for
treatment failure, relapse and acquired drug resistance in
previous studies (Heysell et al., 2010; Mehta et al., 2001).
Low serum levels can be a consequence of
malabsorption, inaccurate dosing, altered metabolism, or
drug interactions, but in most instances low serum levels
can be readily corrected with dose adjustment. TDM is
currently recommended in TB treatment guidelines as
optional (Peloquin, 2002; Blumberg et al., 2003).
Although certain patients infected with HIV and thus
prone to malabsorption, are at higher risk for low drug
levels, studies of TDM that included patients responding

2036 Afr. J. Pharm. Pharmacol.
well to anti-TB medications found lower than expected
drug levels of isoniazid and rifampin in many patients with
adequate clinical response (Mcllleron et al., 2006; Narita
et al., 2001; Holland et al., 2009; Holdiness, 1984). In the
present study, we measured serum concentrations of
anti-tuberculosis drugs (INH, RIF, EMB and PZA) using
HPLC method at 2 and 6 h after drug ingestion. The aims
of the present study were to determine the prevalance of
low serum concentrations of antituberculosis drugs and to
identify the determinants of the serum concentrations of
these drugs in TB patients.
MATERIALS AND METHODS
Study subjects and patients
The present study was performed at a tuberculosis referral center,
Samsun (Turkey) Chest Diseases and Thoracic Surgery Hospital
and all the patients were informed about the study and they gave
their consent to this investigation. 49 adult patients enrolled in the
study between January 2011 to June 2011. All the patients were
bacteriologically confirmed as active pulmonary tuberculosis. Study
subjects had to have received daily standart anti-tuberculosis
drugs: Isoniazid (INH) (daily oral 300 mg), rifampicin (RIF) (daily
oral 600 mg), EMB (weight adjusted daily oral 1000 or 1500 mg)
and PZA (weight adjusted daily oral 1500 or 2000 mg) for at least 7
days before serum drug level measurements. The same trade
marks of drugs were used in all study subjects and the antituberculosis
drugs were single drug products rather than fixed-dose
combinations. Patients with any of the following were excluded from
the study: HIV co-infection, diabetes mellitus, a history of diarrhea,
hepatic, gastrointestinal or renal disease, regular alcohol
consumption and any medication that could affect drug serum
concentrations (e.g. antacids and theophylline) .
Sample preparation
The specific drugs were administered to patients under fasting
conditions and directly observed therapy by the nurses. Venous
blood samples were obtained in an EDTA tube, 2 h after drug
ingestion to estimate peak serum levels of the four drugs and
second samples were taken after 6 h to rule out the possibility of
delayed absorption. After collection, blood samples were
centrifuged at 4000 rpm for a period of 15 min immediately. Plasma
was frozen after centrifugation and stored at -20°C until analysis
and transferred to the reference laboratory on dry ice according to
published recommendations (Holdiness, 1984).
High performance liquid chromatography (HPLC)
Samples were deproteinized by trichloroacetic acid for INH and by
acetonitrile for EMB, RIF and PZA. Solutions were injected to HPLC
system with an LC-20AT pump and 20AC-HT autosampler
(Shimadzu, Japan); results were analysed by using reversed phase
technique, SPD-20A UVdetector and a VP-ODS(150 × 3.9 mm)
Nova-pak C18 4 um (Waters) column (Moussa et al., 2002). The
gradient elution created using 10 mmol potasium hidrojen
phosphate (pH 6.24). Mobile phase A was 10 mmol potasium
hydrogen phosphate and mobile phase B was acetonitrile for INH,
RIF and PZA analysis. Samples were eluted at a flow rate of 0.8
ml/min. EMB mobile phase consisted methanol and deionized water
(70/30) and flow rate was 1,0 ml/min. The limit of quantitation
values in this system for INH, RIF, EMB and PZA were 2.0, 1.5, 1.4
and 1.6 µg/ml, respectively.
Reference serum levels of anti-tuberculosis drugs
INH, RIF, EMB and PZA reach peak serum concentrations
approximately 2 h after ingestion and may delay in malabsorption.
Using published reference ranges, low 2 h drug levels were defined
as follows: INH < 3 µg/ml (300 mg/day); RIF < 8 µg/ml (600
mg/day); EMB < 2 µg/ml (weight adjusted daily dose; 25 mg/kg) and
PZA < 20 µg/ml (weight adjusted daily oral dose; 25 mg/kg). The
patients were treated with oral and standart dose of drugs.
Therapeutic serum levels for INH, RIF, EMB and PZA were 3 to 6, 8
to 24, 2 to 6 and 20 to 50 µg/ml, respectively (Heysell et al., 2010;
Peloquin, 2002).
Statistical analysis of determinants
Statistical analysis was performed using SPSS 15.0 for Windows
(SPSS Inc., Chicago, IL, USA). Variabbles are represented as
mean ± SD (standart deviation) when normally distributed and as
medians (range) in other cases. The potential determinants were
age, sex, smoking status, serum albumin, heamoglobin and drug
dose per kilogram of body weight for all drugs, calculated creatinine
clearance (CCr) for EMB, as it requires renal elimination.
Independent samples T-test was used to compare drug plasma
levels with sex and smoking behaviour variables. The other
variables were statistically analysed with Pearson correlation.
Statistical significance was accepted at P

Kayhan and Akgüneş 2037
Table 1. Comparison of median serum concentration at 2 h after medication administration as estimate of peak serum concentration
levels and expected range, therapeutic drug monitoring.
Medication Median serum concentration (µg/ml) Expected serum concentration range (µg/ml)
Isoniazid (daily 300 mg) 3.83 ± 2.09 3-6
Rifampicin (daily 600 mg) 6.13 ± 4.27 8-24
Ethambutol (weight adjusted) 3.68 ± 2.41 2-6
Pyrazinamide (weight adjusted) 32.20 ± 16.96 20-50
Table 2. Study variables and their correlation with drug serum concentrations (µg/ml).
Variable
Means± SD or
n
Test used
Age (years) 41.51±16.90 PC
Analysis INH
result
p = 0.160
r = -0.204
Analysis RIF
result
p = 0.193
r = -0.189
Analysis
EMB result
p = 0.726
r = 0.051
Analysis
PZA result
p = 0.106
r = -0.234
Sex (males/females) 31/18 IST p = 0.005 p = 0.000 p = 0.570 p = 0.002
Body mass index (kg/m 2 ) 22.50±4.34 PC
p = 0.006
r = 0.390
p = 0.935
r = -0.012
p = 0.317
r = 0.146
p = 0.139
r = -0.214
Smoking status smoker/ nonsmoker IST p = 0.532 p = 0.004 p = 0.551 p = 0.391
'Drug dose(mg/kg)
Isoniazid 4.49±1.14
Rifampicin 9.92±2.21
Ethambutol 24.16±5.15
Pyrazinamide 28.98±8.65
Serum Albumin (g/dl) 3.87±0.54 PC
Heamoglobin 12.60±1.51 PC
PC
p = 0.000
r = 0.487
p = 0.303
r = -0.150
p = 0.303
r = -0.150
p = 0.039
r = 0.295
p = 0.734
r = -0.012
p = 0.083
r = -0250
Creatinine clearence* 118.73±37.08 PC - -
p = 0.827
r = -0.155
p = 0.889
r = 0.020
p = 0.581
r = 0.081
p = 0.816
r = 0.034
p = 0.005
r = 0.395
p = 0.550
r = -0.087
p = 0.005
r = -0.203
*By Cockcroft-Gault equation: in men= (140-age) × (weight in kg)/ (72 × serum creatinine) and in women = 0.85 × (140-age) × (weight in kg)/(72 × serum
creatinine) used for ethambutol; PC = Pearson correlation; IST = Independent samples T-test.
significantly associated with drug dose (mg/kg) (p =
0.000) and there was a correlation between these two
variables (r = 0.487). 2 h INH concentration was not
associated with age, serum albumin, smoking status and
heamoglobin levels (Table 2).
Serum RIF concentration
The mean serum concentration of RIF was 6.13 ± 4.27
µg/ml and the mean RIF dose was 9.92 ± 2.21 mg/kg
(Tables 1 and 2; Figure 3). The prevalance of a low 2 h
RIF concentration was 75.5% (37/49) in this study (Figure
1) . RIF concentrations did not reach to therapeutic levels
at 6 h in these patients. 2 h RIF concentration showed
difference according to sex (p = 0.000). The mean RIF
concentration of males (4.23 µg/ml) was lower than
females (9.41 µg/ml). By Independent samples T-test
analysis, 2 h RIF concentration was found to be
associated with cigarette smoking (p = 0.004). The mean
serum RIF level of smokers (4.50 µg/ml) was lower than
non-smokers (7.50 µg/ml). 2 h RIF serum concentration
was not associated with the other variables (age, body
mass index, serum albumin, drug dose (mg/kg) and
-

2038 Afr. J. Pharm. Pharmacol.
PYRAZINAMIDE
ETHAMBUTOL
RIFAMPICIN
ISONIAZID
20.4%
18.4%
28.6%
75.5%
61.2%
65.3%
55.1%
18.4%
16.3%
24.5%
16.3%
0 100
Figure 1. Results of serum concentration 2 h after oral drug administration levels
of antituberculosis medications . Frequencies(%) are reported for low, within
target (therapeutic), and high levels corresponding to levels below, within, or
above the expected range for each medication.
Figure 2. Isoniazid(INH) serum levels of each patient (low

heamoglobin) (Table 2).
Serum EMB concentration
Figure 4. Ethambutol (EMB) serum levels of each patient < 2 µg/ml ; 18.4% (9/49) , n = 49.
The mean serum concentration of EMB was 3.68 ± 2.41
µg/ml and the mean EMB dose was 24.16 ±5.15 mg/kg
(Tables 1 and 2). The prevalance of a low 2 h EMB
concentration was 18.4% (9/49) (Figures 1 and 4). A
delayed absortion was observed in seven of nine low 2 h
EMB concentrations and they reached to therapeutic
serum levels at 6 h measurements. 2 h EMB
concentration was not found to be associated with any of
variables including calculated creatinine clearence in the
present study (Table 2).
Serum PZA concentration
The mean serum concentration of PZA was 32.20 ±
16.96 µg/ml and the mean PZA dose was 28.98 ± 8.65
mg/kg (Tables 1 and 2; Figure 5). The prevalance of a
low 2 h PZA concentration was 20.4% (10/49) (Figure
1). There was a difference between 2 h PZA
concentration and sex variable (p = 0.002). The mean
concentration of males (26.57 µg/ml) was lower than
females (41.88 µg/ml). Drug dose (mg/kg) was found to
be statistically significant in serum PZA concentration.
There was a correlation between 2 h serum PZA
concentration and drug dose (p = 0.005 and r = 0.395)
(Table 2).
Treatment outcomes
Complete outcomes were available for all patients. Fourty
nine patients succesfully completed treatment. No patient
Kayhan and Akgüneş 2039
had died during therapy and follow up. There was not any
treatment failure among the study cases. Median time to
completion of therapy among all 49 patients was 7.3
months (inter quartille range [IQR] 6 to 9 months). We did
not achieve a correlation between low antimycobacterial
drug levels and the time to completion of therapy. There
were no reports of relapse of infection over a median of 5
months (IQR 3 to 8 months) from the conclusion of
treatment. No patient had documented acquisition of
medication resistance in follow up TB cultures while on
treatment.
The 2 h value after ingestion of tuberculosis drugs is
the peak serum concentration of drugs in normal
patterns. Should the 2 and 6 h values be roughly the
same, perhaps somewhat below the expected ranges, or
should the 6 h levels be higher than the 2 h, delayed
absorption is most likely. In these situations, it is possible
that the actual peak occurred between the two intervals.
It is recommended to take the drugs under fasting
positions, especially for isoniazid and rifampicin.
Malabsorption is most likely if both 2 and 6 h serum drug
levels below the expected ranges, and consideration
should be given higher doses (Peloquin, 2002, Peloquin
et al., 1993). Second blood samples were taken after 6 h
to rule out the possibility of delayed absorption secondary
to poor gastric emptying in this study. We observed some
delayed absorptions and therapeutic drug levels after 6 h
in EMB (14.28%, 7/49), PZA (4.08%, 2/49), INH (2.04%,
1/49) and RIF (0%) respectively. The frequency of
delayed absorption has been rare in other cohorts for
which 2 and 6 h measurements were performed (Holland
et al., 2009).
According to previous studies, the prevalences of low
concentrations of anti-tuberculosis drugs showed wide
variations in TB patients, that is, 2 to 48% for INH, 5 to
78% for RIF and 2 to 41% for EMB (Mcllleron et al., 2006;
Mehta et al., 2001; Peloquin et al., 1996; Holland et al.,
2009; Chang et al., 2008; Fahimi et al., 2011). According

2040 Afr. J. Pharm. Pharmacol.
Figure 5. Pyrazinamide(PZA) serum levels of each patients < 20 µg/ml 20.4% (10/49) n = 49.
to previous studies, serum concentrations of INH are
related to many factors, that is, age, sex, a prior history of
TB, serum haemoglobin levels, laxative use, HIV
infection, fixed-drug combinations formulation, fasting
and weight-adjusted dose (Heysell et al., 2010; Mehta et
al., 2001; Peloquin, 2002; Kimerling et al., 1998). In our
study prevalance of low concentration of INH was 28.6%.
Sex, body mass index and dose of drug (mg/kg) were
effective factors in serum INH levels in this study.
Previous studies revealed that weight-adjusted dose
and a higher serum albumin level were associated with a
higher RIF concentration (Heysell et al., 2010; Mehta et
al., 2001). Patients with diabetes were at significantly
increased risk of having a low rifampin level. Diabetes
was significantly associated with slow response in a
study population, and, among persons with a slow
response with diabetes, 2 h levels of rifampsin were
significantly more likely to be below than the expected
ranges. Hyperglycemia can decrease gastric hydrochloric
acid secretion, which results in a higher gastric pH and
reduced rifampin absorption (Heysell et al., 2010). In this
study, patients enrolled to study were normoglycemic but
serum RIF levels were low in 75.5% of patients. Sex (to
be male) and smoking cigarette were found to be
associated variables in low RIF serum concentrations in
our study. Diabetic patients are at greater risk for incident
TB and are more likely to have poor TB treatment
outcomes, which may partially be explained by
inadequate pharmacotherapy (Jeon and Murray, 2008).
Dose-titration studies of rifampisin confirm a continuously
increasing response of early bactericidal activity by
measurement of sputum colony counts with
corresponding increase in rifampicin dose (Sirgel et al.,
2005; Diacon et al., 2007). As a result a second drug
dose adjustment has to be done to prevent from slow
response to therapy especially in patients with low RIF
serum levels.
A TDM study from South Korea reported low 2 h EMB
concentration ratio as 22.4% and an association between
EMB concentration and calculated creatinine clearance
(Mcllleron et al., 2006). We observed delayed EMB
absortion in seven of nine patients with low 2 h EMB
concentrations. 2 h EMB concentration was not found to
be associated with any of variables including calculated
creatinine clearence in our study. We have found the
prevalance of a low 2 h PZA concentration as 20.4%. The
mean concentration of males was lower than females.
Another result of the study is a correlation between 2 h
serum PZA concentration and drug dose. These
variables have accordance with previous studies (Um et
al., 2007; Mcllleron et al., 2006; Tappero et al., 2005).
Although an existence of high prevalance of low
antimycobacterial drug concentrations, treatment
outcomes included succesfull therapy results in the
present study. Early diagnosis, performing a directly
observing therapy in hospital, absence of extensive
disease and drug resistance may play role in this
condition. This study may support that all four antituberculosis
drugs should be dosed as mg/kg, smoking is
a negative factor in therapeutic RIF serum
concentrations. This study does not have an analysis
about TDM and slow response to therapy with clinical
outcomes in long periods of TB treatment. Another
limitation of present study is not having results of second
TDM after dose adjustment in low serum drug
concentrations. Thus further studies are required to

determine the nature of these conditions.
Conclusıon
A low serum concentration of at least one
antituberculosis drug was found in about all of the
patients included in this study. Several risk factors of drug
concentration variation were also identified. It is clear
that, therapeutic drug monitoring is necessary to optimise
drug doses in tuberculosis treatment.
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UPCOMING CONFERENCES
American Association of Pharmaceutical Scientists Annual meeting and
Exposition (AAPS), Chicago, USA, 14 Oct 2012
International Conference on Pharmacy and Pharmacology, Bangkok,
Thailand, 26 Dec 2012

Conferences and Advert
October 2012
American Association of Pharmaceutical Scientists Annual meeting and Exposition
(AAPS), Chicago, USA, 14 Oct 2012
December 2012
International Conference on Pharmacy and Pharmacology, Bangkok, Thailand, 26
Dec 2012

African Journal of
Pharmacy and
Pharmacology
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