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African Journal of<br />
Pharmacy and<br />
Pharmacology<br />
Volume 5 Number 17 8 November, 2011<br />
ISSN 1996-081
ABOUT AJPP<br />
The African Journal of Pharmacy and Pharmacology (AJPP) is published weekly (one volume per year) by<br />
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Sharmilah Pamela Seetulsingh- Goorah<br />
Associate Professor,<br />
Department of Health Sciences<br />
Faculty of Science,<br />
University of Mauritius,<br />
Reduit,<br />
Mauritius<br />
Himanshu Gupta<br />
University of Colorado- Anschutz Medical Campus,<br />
Department of Pharmaceutical Sciences, School of<br />
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USA<br />
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Molecular Cardiovascular Research Program<br />
College of Medicine<br />
Arizona Health Sciences Center<br />
University of Arizona<br />
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University of São Paulo.<br />
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School of life science, Xinjiang University,<br />
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Peking University First Hospital<br />
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Guangdong Cardiovascular Institute, Guangdong General<br />
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Faculty of Engineering and Applied Science,<br />
Memorial University of Newfoundland,<br />
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Medical Intensive Care Unit<br />
University hospital Ibn Sina, Univesity Mohamed V<br />
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Dr. Dong Hui<br />
Department of Gynaecology and Obstetrics, the 1st<br />
hospital, NanFang University,<br />
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Prof. Ma Hui<br />
School of Medicine, Lanzhou University,<br />
China.<br />
Prof. Gu HuiJun<br />
School of Medicine, Taizhou university,<br />
China.<br />
Dr. Chan Kim Wei<br />
Research Officer<br />
Laboratory of Molecular Biomedicine,<br />
Institute of Bioscience, Universiti Putra,<br />
Malaysia.<br />
Dr. Fen Cun<br />
Professor, Department of Pharmacology, Xinjiang<br />
University,<br />
China.<br />
Dr. Sirajunnisa Razack<br />
Department of Chemical Engineering, Annamalai<br />
University,<br />
Annamalai Nagar, Tamilnadu,<br />
India.<br />
Prof. Ehab S. EL Desoky<br />
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Assiut University, Assiut,<br />
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Dr. Yakisich, J. Sebastian<br />
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R54<br />
Karolinska University Hospital, Huddinge<br />
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Environmental Pathology LEEPA<br />
University of Chile Medical School,<br />
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Dr. Sirajunnisa Razack<br />
Department of Chemical Engineering,<br />
Annamalai University, Annamalai Nagar, Tamilnadu,<br />
India.<br />
Dr. Yasar Tatar<br />
Marmara Unıversıty,<br />
Turkey.<br />
Dr Nafisa Hassan Ali<br />
Assistant Professor, Dow institude of medical technology<br />
Dow University of Health Sciences,Chand bbi Road, Karachi,<br />
Pakistan.<br />
Dr. Krishnan Namboori P. K.<br />
Computational Chemistry Group, Computational<br />
Engineering and Networking,<br />
Amrita Vishwa Vidyapeetham, Amritanagar, Coimbatore-<br />
641 112<br />
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Prof. Osman Ghani<br />
University of Sargodha,<br />
Pakistan.<br />
Dr. Liu Xiaoji<br />
School of Medicine, Shihezi University,<br />
China.
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African Journal of Pharmacy and Pharmacology<br />
International Journal of Medicine and Medical Sciences<br />
Table of Contents: Volume 5 Number 17 8 November, 2011<br />
ences<br />
Research Articles<br />
ARTICLES<br />
A review on Hyssopus officinalis L.: Composition and biological activities 1959<br />
Fatemeh Fathiazad and Sanaz Hamedeyazdan<br />
Identification of phenolic compounds and assessment of in<br />
vitroantioxidants activity of 30% ethanolic extracts derived from two<br />
Phyllanthus species indigenous to Malaysia 1967<br />
Elrashid Saleh Mahdi, Azmin Mohd Noor, Mohamed Hameem Sakeena,<br />
Ghassan Z. Abdullah, Muthanna Abdulkarim and Munavvar Abdul Sattar<br />
Cultural importance and antibacterial activity of Ziziphus mucronata<br />
(Willd.) in the Umlazi community in Durban 1979<br />
R. M. Coopoosamy, K. K. Naidoo and N. J. Ndlazi<br />
Altitude-related changes in activities of carbon metabolism enzymes<br />
and secondary plant products-menthoforon an active pharmaceutical<br />
constituents yield in pippermint (Mentha piperita L. Var. Kukarail) 1983<br />
A. Misra and N. K. Srivastava<br />
Simultaneous determination of gatifloxacin and dexamethasone sodium<br />
phosphate in bulk and pharmaceutical formulations by HPLC 1990<br />
K. R. Sireesha and K. Prakash<br />
Anti-inflammatory and wound healing activity of Fagonia schweinfurthii<br />
alcoholic extract herbal gel on albino rats 1996<br />
Saleh I. Alqasoumi, Hasan S. Yusufoglu and Aftab Alam
Table of Contents: Volume 5 Number 17 8 November, 2011<br />
ences<br />
ARTICLES<br />
The effects of maximal aerobic exercise on cortisol and thyroid hormones<br />
in male field hockey players 2002<br />
Malik BEYLEROGLU<br />
Cationic liposomes as gene delivery system 2007<br />
Mohsen M. Mady<br />
Nitrous oxide versus pethidine with promethasine for reducing labor<br />
pain 2013<br />
Batool Teimoori, Nahid Sakhavar, Masoome Mirteimoori and<br />
Behzad Narouie<br />
Differential analysis of human kidney stone samples using electrospray<br />
ionization mass spectrometry 2018<br />
Zhiquan Zhou, Yongzhong Ouyang, Xiangtai Zeng, Tingting Zhang, Bin Jia,<br />
Xinglei Zhang, Huanwen Chen and Jianhua Ding<br />
Radical scavenging and antioxidant activity of Hibiscus rosasinensis<br />
extract 2027<br />
Rajesh Mandade, S. A. Sreenivas, D. M. Sakarkar and Avijit Choudhury<br />
Therapeutic monitoring of isoniazid, rifampicin, ethambutol and<br />
pyrazinamide serum levels in the treatment of active pulmonary<br />
tuberculosis and determinants of their serum concentrations 1713<br />
Servet Kayhan and Alper Akgüneş
African Journal of Pharmacy and Pharmacology Vol. 5(17), pp. 1959-1966, 8 November, 2011<br />
Available online at http://www.academicjournals.org/AJPP<br />
DOI: 10.5897/AJPP11.527<br />
ISSN 1996-0816 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Review<br />
A review on Hyssopus officinalis L.: Composition and<br />
biological activities<br />
Fatemeh Fathiazad* and Sanaz Hamedeyazdan<br />
Department of Pharmacognosy, Faculty of Pharmacy, Tabriz University of Medical Sciences, Iran.<br />
Accepted 5 October, 2011<br />
Hyssopus officinalis L. (Hyssop) is one of the most popular herbal preparations, mainly distributed in<br />
the East Mediterranean to central Asia. The plant has been used traditionally for medicinal purposes;<br />
generally, these therapeutic uses and health benefits of hyssop are largely based on folklore rather<br />
than on scientific substantiation, making it a good candidate to gather documentations, including the<br />
phytochemical content, in vitro experiments, animal models and human studies available in the recent<br />
scientific studies. A literature review on the chemical and biological aspects of the plant indicates that<br />
the main constituents of H. officinalis include several polyphenolic compounds, primarily the<br />
flavonoids apigenin, quercetin, diosmin, luteolin and their glucosides followed by other phenolic<br />
compounds chlorogenic, protocatechuic, ferulic, syringic, p-hydroxybenzoic and caffeic acids. Reports<br />
on the essential oils extracted from aerial parts of H. officinalis revealed several principal components,<br />
including terpenoids pinocamphone, isopinocamphone and β-pinene. Hyssop has moderate antioxidant<br />
and antimicrobial activity against Gram positive and negative bacteria activities together with antifungal<br />
and insecticidal antiviral properties in vitro. Animal model studies indicate myorelaxant, antiplatelet and<br />
α-glucosidase inhibitory activities for this plant. However, human studies, adverse reactions and<br />
clinical trials examining the reported properties of hyssop are absent and needs more attention to<br />
determine whether biological differences in findings of the studies reflect the different isolation<br />
procedures, different types of plant material used, collection time, locations or different chemotypes.<br />
Key words: Hyssopus officinalis L., phenolic compounds, essential oil, extract.<br />
INTRODUCTION<br />
One of the most frequently consumed herbal remedies<br />
available today is the hyssop preparations prepared from<br />
Hyssopus officinalis (L) which is gaining increased<br />
importance as a minty flavor, condiment and spices in<br />
food industries as well (Dragland et al., 2003; Jung et al.,<br />
2004; Lugasi et al., 2006). Not surprisingly, like many<br />
other herbal preparations used in traditional medicinal<br />
cultures, the therapeutic uses and health benefits of<br />
hyssop are largely based on folklore rather than on<br />
scientific substantiation. Regardless of the wide range of<br />
literatures suggesting health benefits of herbal remedies<br />
associated with hyssop, evidence-based information<br />
regarding the effects of hyssop is quite limited.<br />
Developing an efficient herbal remedy is reliant to a<br />
*Corresponding author. E-mail: fathiazad@tbzmed.ac.ir. Tel:<br />
+98 411 6692405. Fax: +98 411 3344798.<br />
better understanding of the relationship between<br />
chemical constituents and biological properties of the<br />
natural product. In view of these aspects, natural<br />
products, particularly higher plant species, continue to be<br />
important sources of medicine and supplementary health<br />
products which represent a challenge to science due to<br />
their various properties, including chemical diversity,<br />
synergism to biological activity and variable<br />
compositions. Herein, we tried to gather detailed<br />
documentations of the available scientific papers related<br />
to the bioactivity and potential health benefits of hyssop<br />
ensuring a high quality herbal medicine to meet the ever<br />
more demands of the public.<br />
NOMENCLATURE<br />
The genus Hyssopus comprises aromatic perennial herbs<br />
or subshrubs that are mainly cultivated, but can also be
1960 Afr. J. Pharm. Pharmacol.<br />
found in the wild. The inflorescence is 20 to 25 cm long,<br />
false spikelike, composed of 4 to 10 flowered<br />
pseudoverticils in the terminal. The root of H. officinalis is<br />
a strongly branching, multi-headed tap root. The stems<br />
are 0.5 to 0.7 m in height, erect or decumbent dividing<br />
into many woody stems. The leaves are opposite, shiny<br />
dark-green, entire-edged and lanceolate or oblong,<br />
obtuse to acuminate that are 2 to 4 cm long and 0.5 to 1<br />
cm wide. Hyssopus L. comprises of about 10 to 12<br />
species distributed mainly in the East Mediterranean to<br />
central Asia. H. officinalis L. (Family: Lamiaceae alt.<br />
Labiatae) has a long history of medicinal use as<br />
carminative, tonic, antiseptic, expectorant and cough<br />
reliever. Despite having a slightly bitter taste, H. officinalis<br />
is often used as a minty flavor and condiment in food<br />
industries. The merit of the traditional use of H. officinalis<br />
has been supported by some prior studies from the<br />
genus Hyssopus, providing several biologically active<br />
constituents especially main compounds from essential<br />
oils. Although, a great body of papers refers to the<br />
composition of H. officinalis oil, far too little attention has<br />
been paid to the chemical constituent structures present<br />
in the plant. Herein, we offer documentations including<br />
the phytochemical content, in vitro experiments, animal<br />
models and human studies available in the recent<br />
scientific literatures.<br />
PHYTOCHEMICAL CONSTITUENTS<br />
The phytochemical study of the aerial parts of H.<br />
officinalis cultivated in Xinjiang, China, revealed isolation<br />
of two new flavonoid glycosides and nine other known<br />
flavonoids from the ethanolic extract of the plant. The<br />
new compounds were identified as; quercetin 7-O-b-Dapiofuranosyl-(1→2)-b-dxylopyranoside<br />
(1) and quercetin<br />
7-O-b-D-apiofuranosyl-(1→2)-b-D-xylopyranoside 30-Ob-D-glucopyranoside<br />
(2), together with nine known<br />
flavonoids apigenin (3), apigenin 7-O-b-Dglucopyranoside<br />
(4), apigenin 7-O-b-Dglucuronopyranoside<br />
methyl ester (5), luteolin (6),<br />
apigenin 7-O-b-D-glucuronide (7), apigenin 7-O-b-Dglucuronopyranoside<br />
butyl ester (8), luteolin 7-O-b-Dglucopyranosid<br />
(9), diosmin (10) and acacetin 7-O-a-Lrhamnopyranosyl-(1→6)-b-D-glucopyranoside<br />
(11). The<br />
free radical scavenging activity of the compounds 1 to 11<br />
was also determined using 2,2-diphenyl-1-picrylhydrazyl<br />
(DPPH). The isolated compounds were found to possess<br />
noble radical scavenging activity. Out of the isolated<br />
compounds, 1, 2, 6 and 9 with IC50 values in the range<br />
of 2.81 to 10.41 mmol/L exhibited stronger scavenging<br />
activity on DPPH assay than butylated hydroxytoluene<br />
and L-ascorbic acid as standards (Wang and Yang,<br />
2010).<br />
Mario et al. (1997) revealed the presence of the most<br />
widespread class of secondary metabolites, flavonoids, in<br />
H. officinalis L. using high-performance liquid<br />
chromatography and magnetic-resonance imaging (NMR)<br />
spectroscopy. The major flavone, diosmin, was present in<br />
the plant with 51 and 40.5% in sepals and leaves,<br />
respectively that were identified as the total content of<br />
diosmin in whole plant. Nonetheless, there were changes<br />
in diosmin levels during the development of hyssop<br />
leaves, stems and roots. The other identified compound<br />
in the plant was considered to be isoferulyl D-glucose<br />
ester (Marin et al., 1998). Previously, Hilal et al. (1979)<br />
reported isolation of seven glycosides of flavanone type<br />
from H. officinalis where the aglycon of the glycosides<br />
were determined as 5,4'-dihydroxy-7,3'-dimethoxy<br />
flavanone.<br />
The content of free phenolic acids (PhAs) for ten<br />
popular medicinal plants used in Polish phytotherapy<br />
including H. officinalis belonging to the family Lamiaceae<br />
were determined by a rapid, selective and accurate<br />
extraction method combining solid-phase extraction and<br />
high-performance liquid chromatography. Considering the<br />
findings of the study, methanolic extract of the H.<br />
officinalis was shown to be rich in phenolic compounds,<br />
especially high in chlorogenic, protocatechuic, ferulic,<br />
syringic, p-hydroxybenzoic and caffeic acids followed by<br />
vanillic, p-coumaric, rosmarinic and gentisic acids<br />
(Murakami et al., 1998; Varga et al., 1998a; Kochan et<br />
al., 1999; Zgorka and Głowniak, 2001). Elsewhere, the<br />
presence of caffeic acid and its derivatives in the roots of<br />
H. officinalis L. cultivated in Romania with a content of<br />
1.69% was reported. Additionally, rosmarinic acid, ferulic<br />
acid and phenylpropanic compounds were also identified<br />
in the plant by chromatography and spectrophotometric<br />
analyses (Benedec et al., 2002). Later, Proestos et al.<br />
(2005) employed reversed phase high-performance liquid<br />
chromatography with UV detection for the identification<br />
and quantification of the phenolic compounds for some<br />
plant extracts including H. officinalis. The most abundant<br />
phenolic acids in H. officinalis were considered to be<br />
ferulic acid (13.2 mg/100 g of dry sample) and caffeic<br />
acid (6.5 mg/100 g of dry sample). Moreover, syringic,<br />
gentisic and p-hydroxybenzoic acids along with two<br />
flavonoids (+)-catechin and apigenin were also detected<br />
in the genus H. officinalis (Proestos et al., 2005).<br />
ESSENTIAL OIL<br />
Garg et al. (1999) reported on the characteristics of the<br />
oil of H. officinalis L. ssp. officinalis cultivated in the North<br />
Indian plains as an annual crop. The GC and GC-MS<br />
analysis of the colourless essential oil led to the<br />
identification of 21 compounds representing 95.6% of the<br />
oil, comprising seven monoterpene hydrocarbons<br />
(32.3%), five oxygenated monoterpenes (60.5%), one<br />
phenol (0.2%) and six sesquiterpene hydrocarbons<br />
(0.35%). The major constituents of the camphorous<br />
predominant monoterpenes of the oil were<br />
pinocamphone (49.1%) >β-pinene (18.4%)
isopinocamphone (9.7%) (Shah et al., 1986; Garg et al.,<br />
1999). Myrtenol methyl ether, myrtenic acid, methyl<br />
myrtenate, pinic acid, cis-pinic acid, (+)-2hydroxyisopinocamphone,<br />
pinonic acid and cis-pinonic<br />
acid were identified for the first time in H. officinalis oil by<br />
Joulain (Joulain, 1976; Joulain and Ragault, 1976). The<br />
analysis of the composition of two essential oils from H.<br />
officinalis L. ssp. officinalis grown in two different<br />
localities near Urbino (Marche, Italy) revealed major<br />
essential oil components as pinocamphone (34 and<br />
18.5%), isopinocamphone (3.2 and 29%) and β-pinene<br />
(10.5 and 10.8%). However, they showed detectable<br />
differences in the ratio of<br />
pinocamphone/isopinocamphone and in the percentage<br />
of linalool (0.2 and 7.9%) and camphor (0.3 and 5.3%).<br />
All the same, the essential oils exhibited antifungal<br />
activity against 13 strains of phytopathogenic fungi; the<br />
essential oil of the plants grown at 1000 m above sea<br />
level was superior (Daniele et al., 2004). Another study<br />
performed with the H. officinalis from U.P. Himalaya<br />
explained the presence of isopinocamphone 38.1%,<br />
pinocarvone 20.3%, 1-8-cineole 12.2% and β-pinene<br />
10.2% as the main compounds and the total 47 chemical<br />
constituents represented 98.56% of the total oil (Shah,<br />
1991). Salma et al. (2002) identified H. officinalis as a<br />
new source of essential oil in Egypt that was<br />
characterized by high content of β-pinene (19.60%),<br />
pinocamphone (19.20%) and camphor (16.3%). The<br />
highest yield of oil production was determined at the<br />
flowering stage of growth, in July (Salma et al., 2002).<br />
Bulgarian and Italian essential oils of H. officinalis L. were<br />
analyzed and the main difference between these two<br />
kinds of hyssop oils was in the higher quantity of<br />
terpenoids in Bulgarian oil. Isopinocamphone and its<br />
biogenetic precursor β-pinene, camphor, 1,8-cineole,<br />
cubenene and germacrene B were detected in the<br />
Bulgarian oil, whereas in the case of Italian hyssop oil, βpinene<br />
was the minor component and phenyl propanoids,<br />
safrole and benzyl benzoates, were the predominant<br />
constituents of the oil (Manitto et al., 2004). Garcia-<br />
Vallejo et al. (1995) examined the volatile oil of H.<br />
officinalis grown in Spain by gas chromatography (GC)<br />
and gas chromatography/mass spectrometry (GC/MS)<br />
and reported a high content of 1,8-cineole (52.89%) and<br />
β-pinene (16.82%) as the main components of the oil. In<br />
another study, Özer et al. (2005) analyzed the essential<br />
oil of Hyssopus officinalis L. subsp. angustifolius (Bieb.)<br />
Arcangeli wild-growing in the Eastern Anatolian region of<br />
Turkey. The essential oil of this plant demonstrated the<br />
presence of many monoterpenes that were identified by<br />
gas chromatography; about thirty-four components were<br />
characterized, representing 91.0% of the total<br />
components detected. The main components were<br />
identified as pinocarvone (36.3%), pinocamphone<br />
(19.6%), β-pinene (10.6%), 1,8-cineole (7.2%) and<br />
isopinocamphone (5.3%) (Hold and Sirisoma, 2002; Ozer<br />
et al., 2005). Salvatore et al. (1997) performed detailed<br />
Fathiazad and Hamedeyazdan 1961<br />
examination of the essential oil of H. officinalis L. var.<br />
decumbens from the High-Provence Alps in Banon,<br />
France. Linalool (49.6%), 1,8-cineole (13.3%), limonene<br />
(5.4%), β-caryophyllene (2.8%), β-pinene (3.0%) and αpinene<br />
(2.4%) were identified as the major components<br />
of the essential oil, while iso-pinocamphone and<br />
pinocamphone were present at a lower content level<br />
suggesting the existence of different chemotypes in that<br />
province (Salvatore et al., 1997). Analysis of the essential<br />
oils of H. officinalis L. var. decumbens (HOD) from<br />
France (Banon) and H. officinalis (HO) from Italy by GC<br />
and GC/MS exhibited notable differences in the amounts<br />
of components. The bicyclic monoterpene ketones,<br />
pinocamphone and isopinocamphone, were present in<br />
HO, but their percentages were very low in HOD, where<br />
instead linalool (49.6%), 1,8-cineole (13.3%) and<br />
limonene (5.4%) were predominant (Salvatore et al.,<br />
1998). Chemical analysis for three essential oils of<br />
endemic H. officinalis cultivated in Yugoslavia: f. albus<br />
Alef., f. cyaneus Alef. and f. ruber Mill. showed that<br />
components mainly composed of cis- and transpinocamphone<br />
and pinocarvone, together with lesser<br />
amounts of germacrene D, bicyclogermacrene, elemol<br />
and spathulenol (Chalchat et al., 2001). Furthermore, the<br />
presence of aliphatic fatty acids, such as palmitic acid<br />
15.60%, stearic acid 10.73%, linolenic acid 63.98%,<br />
arachidic acid 2.64% and eicosadienoic acid 0.68% in the<br />
Romanian hyssop oil was determined (Benedec et al.,<br />
2002). In our previous study for the essential oil from<br />
Iran, the main constituents were myrtenyl acetate<br />
(74.08%), camphor (6.76%), germacrene (3.39%),<br />
spathulenol (2.14%), caryophyllen oxide (2.13%) and βcaryophyllene<br />
(2.10%) with lesser amounts of cis-sabinol<br />
(1.75%), β- bourbonene (1.47%) and bornyl acetate<br />
(1.42%) (Fathiazad et al., 2011).<br />
Kerrola et al. (1994) investigated the volatile<br />
compounds of the four phenotypes of H. officinalis L.<br />
differentiated by the color of the corolla, by Soxhlet<br />
extraction and Supercritical Fluid Extraction (SFE). The<br />
main components of all extracts were identified as<br />
pinocamphone, isopinocamphone, and pinocarvone.<br />
However, differences in the quantity of the constituents<br />
were worth mentioning; the lower amount of<br />
monoterpene hydrocarbons and a higher amount of<br />
oxygenated hydrocarbons were obtained in the SFE<br />
(Kerrola et al., 1994). Detailed examination of the SFE of<br />
the hyssop oil was undertaken by Kazazi et al. (2007) at<br />
various pressures, temperatures, extraction (dynamic and<br />
static), times and modifier (methanol) concentrations.<br />
Considering the impacts of different factors during the<br />
extraction, it was shown that the composition of the<br />
extracted oils was significantly influenced by the<br />
operating conditions. Major components of the extracts<br />
under different SFE conditions were sabinene (4.2 to<br />
17.1%, w/w), iso-pinocamphene (0.9 to 16.5%) and<br />
pinocamphene (0.7 to 13.6%). Consequently, SFE<br />
offered more choices with parameters for the extraction
1962 Afr. J. Pharm. Pharmacol.<br />
of different components of the hyssop oil (Kazazi et al.,<br />
2007). Kazazi and Rezaei (2007) in another study<br />
evaluated effects of various parameters on the selective<br />
extraction of compounds from hyssop using SFE and<br />
hydrodistillation. Sabinene, pinocamphene and isopinocamphene<br />
were the major compounds applying SFE<br />
with different operational conditions. The optimized<br />
conditions of SFE for the highest extraction selectivity of<br />
pinocamphene and iso-pinocamphene were achieved at<br />
100 atmosphere, 45°C temperature, with 4.5 µl (0.14%,<br />
w/w) methanol, dynamic extraction time of 20 min and<br />
static extraction time of 25 min. Nonetheless, the results<br />
of the study suggested that the hyssop collected from<br />
Iran could be a special chemotype with a high sabinene<br />
concentration (11.04%) (Kazazi and Rezaei, 2009). More<br />
recently, Langa et al. (2009) studied the effects of<br />
pressure, temperature and flow rate of CO2, as well as<br />
the particle size of the vegetable material, on the yield<br />
and composition of the SFE of essential oil from H.<br />
officinalis in comparison with HD extraction. The major<br />
compounds for both techniques were 1,8-cineol<br />
(eucalyptol) (60 to 75%) followed by terpinen-4-ol (4 to<br />
10%), pinocarvone (2 to 6%) and β-pinene (1 to 6%). In<br />
spite of the major similar compounds with comparable oil<br />
yields for both SFE and HD methods, heavier compounds<br />
were detected for the oil obtained from SFE technique<br />
(Toth et al., 1989; Langa et al., 2009).<br />
On the whole, the essential oil content may vary<br />
considerably within a single species from one growth<br />
season to another, affected by extraction method,<br />
climatic parameters and agrotechnical factors<br />
(Benhammou et al., 2008; Ghalem and Mohamed, 2009;<br />
Xu et al., 2011). Additionally, many plants have various<br />
chemotypes that differ in their both quantitative and<br />
qualitative diversity in the composition of essential oils<br />
obtained (Varga et al., 1998b). Further studies are<br />
mandatory to determine the origin of the differences<br />
observed during examinations.<br />
IN VITRO STUDIES<br />
Antimicrobial and antioxidant activities<br />
Mazzanti et al. (1998) published a paper in which they<br />
reported that essential oil of both H. officinalis L. and H.<br />
officinalis L. var decumbens possessed strong<br />
antimicrobial activity in vitro. The findings of the study<br />
showed that all yeasts including seven strains of Candida<br />
albicans, Candida krusei and Candida tropicalis were<br />
strongly inhibited by both species. In liquid medium the<br />
minimal inhibitory concentration (MIC) of H. officinalis L.<br />
was 41.2% v/v for bacteria and between 0.6 and 1.2% v/v<br />
for yeasts, while the MIC of var. decumbens was<br />
between 0.15 and 0.6% v/v for the Gram positive<br />
bacteria, 0.3 and 1.2% v/v for the Gram negative bacteria<br />
and 0.15 and 0.3% v/v for the yeasts. Regarding the<br />
contribution of pure components to the antimicrobial<br />
activity of the oils, pinocamphone and isopinocamphone<br />
were present in H. officinalis L. (4.4 and 43.3%,<br />
respectively), and instead linalol (51.7%), 1,8-cineole<br />
(12.3%) and limonene (5.1%) were predominant in var.<br />
decumbens representing the special microbiological<br />
properties of the essential oils. On the whole, the effect of<br />
var. decumbens was generally bactericidal. Linalol and<br />
1,8-cineole, may contribute to the greater antimicrobial<br />
activity of var. decumbens compared to H. officinalis L.,<br />
while limonene may be responsible for the antimycotic<br />
action observed in both oils (Mazzanti et al., 1998).<br />
Marino et al. (2001) evaluated three groups of essential<br />
oils including hyssop oil for their inhibitory effects against<br />
nine strains of Gram negative bacteria and six strains of<br />
Gram positive bacteria. On the contrary to the previously<br />
published paper by Mazzanti et al. (1998) the findings of<br />
the study exhibited that the hyssop oil in general was less<br />
inhibitory against different strains of bacteria, suggesting<br />
variation in the composition of the essential oils according<br />
to the environmental conditions and plant chemotypes<br />
(Marino et al., 2001).<br />
Recently, Kizil et al. (2010) evaluated antimicrobial and<br />
antioxidant activities of the essential oil of H. officinalis<br />
(L.) collected from wild in the Southeast Anatolian,<br />
Turkey. Isopinocamphone (57.27%), (-)-β-pinene<br />
(7.23%), (-)-terpinen-4-ol (7.13%), pinocarvone (6.49%),<br />
carvacrol (3.02%), p-cymene (2.81%) and myrtenal<br />
(2.32%) were determined as the major components of the<br />
hydrodistilled essential oil by GC-MS analysis. The<br />
essential oil with 5 and 10 µl concentrations was carried<br />
out for anti-microbial disc diffusion tests. The results of<br />
the study were indicative of the oils strong antimicrobial<br />
activities against Staphylococcus pyogenes,<br />
Staphylococcus aureus, C. albicans and Escherichia coli,<br />
but not against Pseudomonas aeruginosa. The<br />
antioxidant activity of H. officinalis essential oil was lower<br />
as compared to butylated hydroxytoluene and ascorbic<br />
acid. Generally, hyssop essential oil showed relatively<br />
low antioxidant activity and good antimicrobial activity<br />
against some test organisms (Kizil et al., 2010). In<br />
addition to the all cited papers indicating the importance<br />
of this genus for its antibacterial activity, publication of a<br />
patented product identified as KR 2005073080 of 2005-<br />
07-13 is an extra confirmation of the fact. The invention<br />
comprises of an anti-acne composition which exhibits<br />
excellent anti-bacterial activity to propionbacterium acnes<br />
as causative bacteria of acne, while no adverse reaction<br />
to human body by comprising essential oil extracted from<br />
plants including H. officinalis as effective ingredient.<br />
Besides, another patent product has also been reported<br />
as JP 2004262861 of 2004-09-24 with the aim of<br />
cosmetics skin-conditioning and antiwrinkle topical<br />
formulations containing 0.01% H. officinalis and 50%<br />
ethanolic extract (Handa, 2004; In Hong et al., 2005).<br />
The essential oil of H. officinalis L. subsp. angustifolius<br />
and methanolic extract of the plant were examined for
their in vitro antimicrobial and antioxidant activities.<br />
Although, the methanol extract in the DPPH assay<br />
provided 50% inhibition at a concentration of 117.0 µg/ml<br />
and 40% inhibition at the concentration of 2 g/L in linoleic<br />
acid test system, it showed no effective activities in the<br />
antimicrobial assays, whereas, the essential oil exhibited<br />
activity against eight bacteria, ten fungi and yeast, C.<br />
albicans, with MIC values ranging from 15.625 to 250<br />
µl/ml; no distinctive anti oxidant properties were achieved<br />
for the essential oil (Ozer et al., 2006).<br />
Ebrahimzadeh et al. (2010) employed six different in<br />
vitro methods for evaluating antioxidant and free radical<br />
scavenging activities of methanolic extract of the aerial<br />
parts of H. officinalis L. var. angustifolius along with three<br />
other plants. They showed that it showed potent to<br />
modrate antioxidant activities in reducing powers and<br />
DPPH radical-scavenging as well as Fe2+ chelating<br />
ability assays, respectively. Although, in the case of nitric<br />
oxide and hydrogen peroxide scavenging and ferric<br />
thiocyanate methods, the results for the anti oxidant<br />
activities of H. officinalis L. extracts were very low and<br />
weak (Ebrahimzadeh et al., 2010). SFE extraction of<br />
antioxidant fractions from certain Lamiaceae herbs with<br />
their antioxidant capacity was evaluated (Babovic et al.,<br />
2010). Antioxidant activity of the obtained extracts,<br />
including H. officinalis were determined by measuring<br />
their ability to scavenge stable DPPH free radical and<br />
reactive hydroxyl radical during the Fenton reaction<br />
trapped by 5,5-dimethyl-1-pyroline-N-oxide, using<br />
electron spin resonance spectroscopy. According to the<br />
results of the study, hyssop extract showed much weaker<br />
antioxidant activity as compared to the rosemary, sage,<br />
and thyme extracts in different methods of antioxidant<br />
evaluations (Dragland et al., 2003; Fernandez-Lopez et<br />
al., 2003; Babovic et al., 2010). Ludmila and Viera (2005)<br />
assessed the antiradical activity and the reduction power<br />
of H. officinalis extracts in another study. All the extracts<br />
showed high activities by both evaluation criteria.<br />
Besides, among the phenolic acids, gallic acid was found<br />
to be the most active component in scavenging free<br />
radicals and caffeic acid had the highest reducing power.<br />
In a study conducted by Glamoålija et al. (2005),<br />
essential oil of the H. officinalis L. was evaluated for its<br />
antifungal activity against Mycogone perniciosa (Mang),<br />
one of the major pathogenic diseases of the cultivated<br />
mushroom Agaricus bisporus (Lange) Imbach in Serbia.<br />
The findings of the study revealed its positional antifungal<br />
activity with minimal inhibitory quantity of 5 µl/ml and a<br />
minimal fungicidal quantity of 15 to 20 µl/ml. These kinds<br />
of studies have been placed in the focus of medical and<br />
aromatic plants investigations for their antifungal<br />
properties since relative biological control systems are<br />
not much used in mushroom cultivation (Ghfir et al.,<br />
1994; Ghfir et al., 1997; Glamoålija et al., 2005; Raila et<br />
al., 2009). Twelve essential oils from Mediterranean<br />
aromatic plants were tested at different doses against<br />
four fungi: Botrytis cinerea, Penicillium italicum,<br />
Fathiazad and Hamedeyazdan 1963<br />
Phytophthora citrophthora, and Rhizopus stolonifer. The<br />
findings of the study revealed weak to moderate fungicide<br />
activities in the case of hyssop oil; however, these<br />
essential oils together with hyssop oil could be<br />
considered as natural preservatives for food products<br />
(Camele et al., 2010). Motiejunaite and Kalediene (2003)<br />
carried out an antifungal screening for essential oils of<br />
some Lamiacae plants using agar-diffusion method. In<br />
most cases including H. officinalis L. a complete inhibition<br />
of Aspergillus niger growth was observed at 0.5 to 1.5%<br />
v/v concentrations.<br />
Pavela (2004) investigated insecticidal activities of<br />
eight medicinal plants including H. officinalis in third instar<br />
larvae of Egyptian cottonworm (Spodoptera littoralis).<br />
Methanolic extract of H. officinalis at the concentration of<br />
10 % (w/v) significantly affected the growth indexes which<br />
showed a certain degree of larval toxicity with 1.78 LC50<br />
and a range of 1.66 and 1.82 confidence interval of 95%<br />
(Pavela, 2004).<br />
Anti viral activity<br />
Crude extracts of the dried leaves of H. officinalis were<br />
also tested for its effectiveness on inhibition of human<br />
immunodeficiency virus (HIV) replication. Not only a safe<br />
non-toxic activity was determined for the uninfected Molt-<br />
3 cells, but also, a strong anti-HIV activity was revealed<br />
as measured by inhibition of syncytia formation, HIV<br />
reverse transcriptase (RT) and p17 and p24 antigen<br />
expression. In the experiment, either extracts from direct<br />
extraction, after removal of tannins or from the residue<br />
after dialysis of the crude extract, also showed good<br />
antiviral activity. Eventually, Kreis et al. (1990) concluded<br />
that the hyssop extracts contained caffeic acid,<br />
unidentified tannins, and possibly a third class of<br />
unidentified higher molecular weight compounds which<br />
exhibited strong anti-HIV activity, and might be useful in<br />
the treatment of patients with AIDS (Kreis et al., 1990).<br />
In another study conducted by Gollapudi et al. (1995), an<br />
isolated polysaccharide (MAR-10) from the aqueous<br />
extract of H. officinalis was examined for its activity<br />
against HIV-1 (SF strain) in HUT78 T cell line and<br />
primary cultures of peripheral blood mononuclear cells.<br />
They demonstrated that the MAR-10 inhibited HIV-1<br />
replication in a concentration-dependent manner with no<br />
substantial direct toxicity or effect on lymphocyte<br />
functions or CD4+ and CD8+ T cell counts (Gollapudi et<br />
al., 1995).<br />
Other activities<br />
Methanolic and hexane extracts of twelve plants including<br />
H. officinalis that are used in traditional European<br />
medicine to treat different central nervous system<br />
disorders were tested for the symptomatic treatment of
1964 Afr. J. Pharm. Pharmacol.<br />
Alzheimer’s disease using Ellman’s colorimetric method.<br />
Since the therapy of early and moderate stages of<br />
Alzheimer’s disease is mainly based on the choline<br />
esterase inhibitors; effects of the plant extracts on<br />
acetylcholinesterase (AChE) and butyrylcholinesterase<br />
(BuChE) inhibitors were investigated (Wszelaki et al.,<br />
2010). Ultimately, H. officinalis revealed no significant<br />
inhibitory activity, as the methanolic and hexane extracts<br />
showed 5.2 ± 8.2 and 29.6 ± 2.3 AChE inhibition (%) and<br />
11.5 ± 0.5 and 23.2 ± 2.0 BuChE inhibitions (%) at the<br />
concentrations of 100 mg/ml -1 , respectively (Wszelaki et<br />
al., 2010).<br />
Animal model studies<br />
Matsuura et al. (2004) evaluated aqueous methanol<br />
extracts of dried H. officinalis leaves for their αglucosidase<br />
inhibitory activity. The active principles<br />
against α-glucosidase, prepared from rat small intestine<br />
acetone powders, were isolated and the amount of<br />
glucose derived from sucrose in the reaction mixture was<br />
measured. The extract showed inhibitory activity which<br />
led to the further isolation and identification of the<br />
responsible compounds for the α-glucosidase inhibitory<br />
activity. The structures of the two isolated compounds<br />
were determined to be (7S,8S)-syringoylglycerol-9-O-(60-<br />
O-cinnamoyl)-β-d-glucopyranoside (1) and (7S,8S)syringoylglycerol<br />
9-O-β-d-glucopyranoside (2) by analysis<br />
of physical and spectroscopic data together with chemical<br />
syntheses that exhibited 53 and 54% inhibitory activity at<br />
the concentration of 3 × 10 -3 M for the compounds 1 and<br />
2, respectively (Matsuura et al., 2004). In another study,<br />
Miyazaki et al. (2003) evaluated the α-glucosidase<br />
inhibitory effects of the hyssop extracts on<br />
hyperglycemia, intestinal carbohydrate absorption by<br />
examining the inhibitory effects on intestinal carbohydrate<br />
absorption in rat everted gut sac and carbohydrateloaded<br />
hyperglycemia in mice. According to the results, in<br />
the presence of 0.5 and 1.0 mg/ml hyssop extracts, the<br />
carbohydrate-loaded excessive increase in blood glucose<br />
was inhibited within 120 min, suggesting that hyssop<br />
might be a useful supplemental food for inhibiting of<br />
postprandial hyperglycemia (Miyazaki et al., 2003).<br />
Confirming this idea, they had provided a patented<br />
product identified as JP 2004256467 of 2003-50400 from<br />
the derivatives of this plant as α-glucosidase inhibitors<br />
(Miyazaki et al., 2004). Elsewhere, the inhibitory activities<br />
of some plants used in Lebanon traditional medicine<br />
containing H. officinalis extracts against angiotensin<br />
converting enzyme (ACE) and digestive enzymes related<br />
to diabetes were investigated (Loizzo et al., 2008). They<br />
demonstrated that the H. officinalis chloroform extract<br />
was active only on the α-glucosidase enzyme, with IC50<br />
values ranging from 127.3 to 908.4 µg/ml. At the same<br />
time the n-hexane extract of the H. officinalis exhibited a<br />
strong inhibitory potency against ACE (IC50 values of<br />
52.0 µg/ml) (Loizzo et al., 2008). Churl et al. (2005)<br />
carried out an experiment to study the simulative or<br />
sedative effects of some inhaling essential oils by using a<br />
forced swimming test with mice. Inhalation of the hyssop<br />
oil (P < 0.01) increased the immobile state in mice that<br />
were artificially over agitated by an intra parental injection<br />
of caffeine (a psycho-stimulant). Accordingly, the authors<br />
stated that the inhaling fragrant hyssop oil possessed<br />
sedative effects (Churl et al., 2005).<br />
Essential oils extracted from different plants including<br />
H. officinalis were tested for their antiplatelet activity and<br />
inhibition of clot retraction in guinea pig and rat plasma.<br />
As Tognolini et al. (2006) mentioned in their study,<br />
phenylpropanoid moiety is a favorable chemical feature<br />
for the inhibition of platelet aggregation and lack of this<br />
moiety in the hyssop oil is responsible for the oil to be<br />
inactive.<br />
The myorelaxant effect of the hyssop essential oil on<br />
isolated preparations of guinea-pig and rabbit intestinal<br />
musculature was determined (Lu et al., 2002).<br />
Isopinocamphone, the major component of the essential<br />
oil, was considered to be responsible of the relaxing<br />
effect. Accordingly, essential oil and isopinocamphone<br />
inhibited the acetylcholine- and BaCl2-induced<br />
contractions in guinea-pig ileum in a concentrationdependent<br />
manner (IC50 42.4 and 61.9 µg/ml to<br />
acetylcholine; 48.3 and 70.4 µg/ml to BaCl2), whereas<br />
limonene or β-pinene left tissue contraction was<br />
unchanged. Nonetheless, synergic actions among the<br />
other several components of the essential oil could not be<br />
excluded. They believed that the myorelaxant activity<br />
induced by the hyssop oil could originate from its<br />
interaction with the plasma membrane and subsequent<br />
alteration of the ionic channels. Considering the inactivity<br />
of the β-pinene and limonene, it had been suggested that<br />
the interaction not only depends on the lipophilicity of the<br />
essential oil and its components, but also on the chemical<br />
structure of the components of the essential oil (Lu et al.,<br />
2002). Mazzanti et al. (1998) reported the spasmolytic<br />
activity of the essential oil from H. officinalis L. var.<br />
decumbens. The essential oil and its major pure<br />
components, linalool, 1,8-cineole and limonene inhibited<br />
the acetylcholine- and BaCl2-induced contractions on<br />
isolated guinea-pig ileum with IC50 values of 37, 60, 10<br />
and 51 µg/ml, correspondingly. Generally, it has been<br />
suggested that the hyssop oil would provide us with<br />
valuable feature of myorelaxant activity in antispasmodic<br />
remedies (Mazzanti et al., 1998).<br />
Cytotoxicity of the essential oils of H. officinalis L. (HO)<br />
and H. officinalis var. decumbens (HOD) was evaluated<br />
using the brine shrimp (Artemia salina Leach) test. The<br />
percent of nauphii dead within 24 h was reported to<br />
determine the cytotoxic activity of the essential oils.<br />
Accordingly, HOD with LC50 of 156.03 µg/ml lower than<br />
HO 191.06 mg/ml revealed stronger cytotoxic activity<br />
probably in support of linalool rich in HOD (Renzini et al.,<br />
1999).
CONCLUSION<br />
Concisely, these issues furnish the background for the<br />
experiments on the associated basic studies for the H.<br />
officinalis (L.) which is an important source of bioactive<br />
substances of medicinal interest. Nevertheless, several<br />
experimental studies are required to confirm the<br />
therapeutic potential of this plant and determine whether<br />
biological differences reflect the different isolation<br />
procedures, different types of plant material used,<br />
collection time, locations or different chemotypes.<br />
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African Journal of Pharmacy and Pharmacology Vol. 5(17), pp. 1967-1978, 8 November, 2011<br />
Available online at http://www.academicjournals.org/AJPP<br />
DOI: 10.5897/AJPP11.034<br />
ISSN 1996-0816 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Identification of phenolic compounds and<br />
assessment of in vitro antioxidants activity of 30%<br />
ethanolic extracts derived from two Phyllanthus<br />
species indigenous to Malaysia<br />
Elrashid Saleh Mahdi 1 *, Azmin Mohd Noor 1 , Mohamed Hameem Sakeena 1 , Ghassan Z.<br />
Abdullah 1 , Muthanna Abdulkarim 1 and Munavvar Abdul Sattar 2<br />
1 Department of Pharmaceutical Technology, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden<br />
11800 Pulau Pinang, Malaysia.<br />
2 Department of Physiology, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden 11800 Pulau Pinang,<br />
Malaysia.<br />
Accepted 21 July, 2011<br />
Phenolic compounds were identified in 30% ethanolic extracts derived from Phyllanthus niruri (P. niruri)<br />
and Phyllanthus urinaria (P. urinaria) using high performance liquid chromatography (HPLC) assay. In<br />
vitro antioxidants activity of the extracts was studied based on total phenolic contents (TPC) using<br />
Folin-Ciocalteu reagent and their scavenging activity towards radical 2,2-diphenyl-1-picrylhydrazyl<br />
(DPPH). The HPLC results show that gallic acid (GA), corilagin (Cor) and ellagic acid (EA) were the<br />
major components of the extracts and their quantifications in P. niruri were 11.867 � 0.130, 89.579 �<br />
0.602 and 37.309 � 0.033 mg/g of extract respectively and in P. urinaria were 8.710 � 0.091, 56.382 �<br />
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<br />
277.98 � 1.04 mg of gallic acid equivalent per gram of extract respectively and their scavenging based<br />
on IC50 was 32.64 and 25.00 �g mass of the extract compared to the IC50 of references standards GA<br />
(3.28 �g) and EA (2.99 �g). The results revealed the extracts as a potential source of natural<br />
antioxidants that can be utilized in cosmetics as skin antiaging, sun-blocking and whitening agents.<br />
Key words: Corilagin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), ellagic acid, Folin-Ciocalteu, gallic acid.<br />
INTRODUCTION<br />
Reactive oxygen species (ROS) and free radicals (FR)<br />
are small molecules naturally generated as by-product of<br />
cellular metabolism. Exposure to environmental hazards<br />
such as radiations, chemicals and gases increases their<br />
production in the body to toxicity levels (Govindarajan et<br />
al., 2005). Endogenous antioxidants in the body, such as<br />
glutathione and α-tocopherol can maintain and counteract<br />
the produced FR and ROS when they are within the<br />
physiological limit (Halliwell, 1996). Improper balance<br />
between the oxidants (FR and ROS) and antioxidants in<br />
*Corresponding author. E-mail: elrashidm@yahoo.com.<br />
favour of the oxidants, is potentially leading to oxidative<br />
stress state (Sies, 1997). Oxidative stress is a fragmental<br />
state of DNA and cellular membrane damage, thus can<br />
ultimately lead to cells and tissues death due to proteins<br />
denaturation and lipids peroxidation (Ratnam et al.,<br />
2006). Consequently, make way for various human<br />
generative diseases like myocardial infarction, heart<br />
failure, hypertension, atherosclerosis, Parkinson‟s<br />
disease, Alzheimer‟s disease, muscular dystrophy,<br />
multiple sclerosis, diabetes, rheumatoid arthritis, chronic<br />
inflammatory diseases, sickle cell anaemia, acute renal<br />
failure, cancers and premature aging (Ferrari et al., 2004;<br />
Halliwell, 1987; Lefer and Granger, 2000; Nath and<br />
Norby, 2000; Pham-Huy et al., 2008; Praticò and Delanty,
1968 Afr. J. Pharm. Pharmacol.<br />
2000; Ratnam et al., 2006). Antioxidants are compounds<br />
capable of scavenging FR/ROS by terminating oxidative<br />
reaction chain in the biological tissues and hence can<br />
prevent cellular damage and oxidative stress associated<br />
with free radical induced generative diseases (Ratnam et<br />
al., 2006). Recognized dietary antioxidants such as<br />
ascorbic acid (vitamin C) and �-tocopherols (Vitamin E)<br />
can pick up and neutralize FR and ROS, prevent and<br />
reversed age related disorder and diseases (Halliwell,<br />
1996). Plants secondary metabolites such as flavonoids<br />
and polyphenols compounds exhibited important<br />
commercial and biological role due to their antioxidants<br />
activity (Agati et al., 2007; Bendini et al., 2007; Di<br />
Mambro and Fonseca, 2005; Rice-Evans et al., 1997).<br />
They are good electron donors and having potentials<br />
redox pattern that can scavenge FR and ROS and<br />
prevent their harmful effects (Pietta et al., 1998). They<br />
are relatively stable due to resonance, delocalization and<br />
formation of side conjugated system with the hydroxyl<br />
group attached to the aromatic ring (Srinivasan et al.,<br />
2007). Therefore, they have been the major research<br />
issues for the last two decades (Gourine et al., 2010).<br />
Consequently, their commercial application as food<br />
supplements, food preservatives in nutraceuticals and<br />
skin anti-aging, sun-blocking and whitening agents in<br />
cosmeceuticals is highly increased (Peschel et al., 2006).<br />
Phyllanthus urinaria and Phyllanthus niruri belong to<br />
widely distributed genus “Phyllanthus”, family<br />
Phyllanthaceae (Samuel et al., 2005). The genus is found<br />
all over the world in the tropical and subtropical countries.<br />
More than 750 species of genus Phylanthus have been<br />
described (Calixto et al., 1998; Wehtje et al., 1992). The<br />
genus “Phyllanthus” has been traditionally used internally<br />
to treat a broad spectrum of diseases such as diarrhoea,<br />
hepatitis, diabetes, abdominal pain, and kidney disorder<br />
(Chularojmontri et al., 2005; Mellinger et al., 2005). It is<br />
also used topically as a poultice to treat skin ulcers,<br />
sores, itching and wounds healing. The phytochemical<br />
compounds of many of Phyllanthus species such as<br />
tannins, ellagitannins flavonoids have been isolated and<br />
characterized (Ahmeda, 2005; Chang et al., 2003; Fang<br />
et al., 2008; Liu et al., 1999; Murugaiyah and Chan,<br />
2007). The potential pharmacological effects of the many<br />
of these isolated compounds have been assessed<br />
(Ambali et al., 2010; Calixto et al., 1998; Krithika et al.,<br />
2009). Several studies have shown the antioxidants<br />
activity of various Phyllanthus species using different<br />
solvents and methods of extractions (Chularojmontri et<br />
al., 2005; Fang et al., 2008; Harish and Shivanandappa,<br />
2006). Various phenolic compounds with antioxidants<br />
effect have been identified in P. niruri and P. urinaria<br />
(Harish and Shivanandappa, 2006; Markom et al., 2007;<br />
Murugaiyah and Chan, 2007). P. urinaria and P. niruri,<br />
being investigated in this study are found in Malaysia.<br />
The two species are closely-related in appearance and<br />
traditionally uses and locally known as “Dukung anak”<br />
which means carry baby; because the plants carry the<br />
fruits on their backs and underneath the feathered-like<br />
leaves (Ahmeda, 2005; Markom et al., 2007; Ong and<br />
Norzalina, 1999). Our aim of this study was to identify<br />
and quantify the major components of the extracts<br />
chromatographically using high performance liquid<br />
chromatography (HPLC) and to assess an in vitro<br />
antioxidants activity in terms of total phenolic content and<br />
scavenging activity toward the radical 2, 2-Diphenyl-1-<br />
Picrylhydrazyl (DPPH) and to compare it to the<br />
scavenging activity of gallic and ellagic acids as control<br />
positive reference standards.<br />
MATERIALS AND METHODS<br />
Reagents<br />
Folin-Denis‟ reagent, sodium carbonate 99%, 2,2-Diphenyl-1picrylhydrazyl<br />
(DPPH 95%), gallic acid (GA, 99%) and ellagic acid<br />
(EA, 95%) were purchased from Sigma–Aldrich (St. Louis, MO.,<br />
USA). Methanol was purchased from J. T. Baker (Philipsburg,<br />
USA), formic acid 98 to 100% assay from Merck (Darmstadt,<br />
Germany). Ethanol (99.7%) was purchased from Brightchem Sdn<br />
Bhd (Malaysia), sodium hydroxide from R & M Marketing (Essex,<br />
UK) and corilagin in-house prepared working standard was<br />
obtained as a gift from Nova Laboratories Sdn Bhd (Malaysia).<br />
Extracts<br />
The extracts of P. urinaria and P. niruri were prepared by Nova<br />
Laboratories Sdn. Bhd. (Malaysia) by dissolving 50 g of dried<br />
powdered materials from the aerial part of each plant (P. niruri and<br />
P. urinaria) in 500 ml of 30% ethanol at 60°C water bath for 1 h.<br />
The 30% ethanol liquid extract was filtered using Whatman No. 1<br />
filter paper. The residue was re-extracted with another 500 ml of<br />
30% ethanol. The two filtrates were combined and dried using<br />
rotatory evaporator at 60°C.<br />
Identification of major phenolic compounds<br />
GA external reference standard was prepared by dissolving 10 mg<br />
into 100 ml of distilled water. 10 mg EA of external reference<br />
standard was transferred into 100 ml volumetric flask and dissolved<br />
into 10 ml of sodium hydroxide 0.1 M and the volume was<br />
completed to 100 ml with water. Serial dilutions from the two<br />
references standards solutions of GA and EA were prepared in the<br />
range of concentration from 0.5.0 to 16 µg/ml. Corilagin (Cor)<br />
working standard was prepared in concentration of 250 µg /ml<br />
methanol. The solutions were ultra sonicated at ambient<br />
temperature for 10 min and filtered through nylon membrane filters<br />
47 mm 0.45 µm (Whatman, UK) before HPLC analysis was<br />
performed. The calibration curves were plotted with six<br />
concentrations each of the standard solution of GA and EA versus<br />
the areas under the peaks. The GA and EA standard curves<br />
equations were used to quantify in the extracts. Since corilagin is<br />
commercially unavailable and the small quantity available was<br />
enough only for identification purposes and triplicate runs was used<br />
to quantify corilagin in the extracts.<br />
Samples solutions<br />
10 mg of each extracts were weighed and transferred into 10 ml<br />
volumetric flasks. Each sample of P. niruri and P. urinaria was
Table 1. Liquid chromatography (LC) time programme of binary gradient pumps modules.<br />
Time (min)<br />
Module action<br />
Pump A (% concentration) Pump B (% concentration)<br />
0.00 0 100<br />
2.00 5 95<br />
5.00 30 70<br />
8.00 34 66<br />
11.00 45 55<br />
14.00 45 55<br />
17.00 0 100<br />
20.00 0 100<br />
dissolved in formic acid 0.2%, vortexed for 20 s and then ultra<br />
sonicated at ambient temperature for 10 min at room temperature.<br />
The resulting samples solutions were filtered through nylon<br />
membrane filter 47 mm 0.45 µm diameter (Whatman, UK) before<br />
HPLC analysis were performed.<br />
Detection and quantification limits (DL and QL)<br />
The DL and QL were estimated based on the standard deviation of<br />
the response and slope (Guideline, 2005). The standard deviation<br />
(�) of the responses of the lowest concentration in calibration curve<br />
of six runs (n = 6) and the slope of the calibration curves of GA and<br />
EA (S) were used to calculate DL and QL using Equations 1 and 2,<br />
respectively (Guideline, 2005). The sensitivity of the method was<br />
evaluated by the relative standard deviation (RSD %) of mean area<br />
under the peaks of the reference standards. The selectivity of the<br />
method and suitability of system were evaluated by comparing the<br />
retention time of the standards peaks to the samples peaks. The<br />
means of retention time of the GA and EA in the samples solutions<br />
were compared to external reference standards GA and EA mean<br />
retention time and were analyzed for null hypothesis using student‟s<br />
paired t-test.<br />
Chromatographic condition<br />
The HPLC analysis was performed using LC 20 AD Class LCsolution<br />
software, connected to SPD-20A UV/VIS detector, binary<br />
pump and temperature controlled column oven, (Shimadzu, Japan).<br />
Thermo Hypersil Gold TM (250 × 4.6 mm i.d., 5 µm) reversed phase<br />
column was used for all separations. The column oven temperature<br />
was set at 40°C and the external reference standards solutions and<br />
extracts were eluted with a binary gradient mode at a UV<br />
wavelength of 270 nm and 20 �l volume of injection (Rangkadilok et<br />
al., 2005). Methanol (solvent A) and formic acid 0.2% (solvent B)<br />
were used as mobile phase at flow rate of 1 ml/min. The liquid<br />
chromatography (LC) time programme of modules of the binary<br />
gradient pumps was set as in Table 1 and the retention time of the<br />
peaks was specified by band of 0.2 min.<br />
(1)<br />
(2)<br />
Determination of total phenolic content (TPC)<br />
Mahdi et al. 1969<br />
The total phenolic content (TPC) of the extracts was studied<br />
spectrophotometrically by folin–ciocalteu reagent assay with slight<br />
modification (Bajpai et al., 2009). GA standard curve was plotted in<br />
the concentrations range of 50, 100, 150, 200, 250 and 500 mg/L in<br />
ethanol:water (10/90) solution and their corresponding<br />
absorbencies were measured at 765 nm visible wavelength. 10 mg<br />
of each extracts material were transferred in 25 ml volumetric flask<br />
and dissolved into ethanol:water (30:70). 100 µl from the samples<br />
solutions were placed in screw-capped test tubes, 500 µl of the<br />
Folin–Ciocalteu reagent and 1500 µl of distilled water (1/15 dilution)<br />
were added to the samples solutions respectively. The test tubes<br />
were properly shaken before incubated at room temperature for 1<br />
min. After 1 min, 1000 µl of 20% sodium carbonate (Na2CO3)<br />
aqueous solution was added. The final mixture was vortexed for 10<br />
s and then incubated for 2 h at room temperature. After 2 h, the<br />
absorbance was measured at 765 nm UV wavelength using Hitachi<br />
U-2000 spectrophotometer, Perkin Elmer Lambda, USA. The<br />
procedure was carried out in triplicate manner (n = 3) and the TPC<br />
the extracts was calculated using Equation 3. The results were<br />
expressed as milligram gallic acid equivalent per gram dry extract<br />
weight (mg GAE/g DW).<br />
The experimental TPC were compared to the predicted TPC based<br />
on the quantified composes of the extracts. GA contains three<br />
hydroxyl group attached to aromatic ring as was used; the<br />
reference standard is equivalent to unity. The number of GA<br />
equivalent to each composes in the extracts was obtained by<br />
dividing the number of hydroxyl group in the composes by the<br />
number of hydroxyl group of GA (GAE). The predicted TPC were<br />
evaluated by summation of the total quantitative amount of each<br />
composes equivalent to GA multiplied by its quantified amount in<br />
the extract as shown by expression 4. In which, PTPC was the<br />
predicted TPC, COC was the content of composes in the extract<br />
and GAE was the GA equivalent of the composes.<br />
Determination of radical scavenging activity<br />
The powdered extracts and EA reference standard were dissolved<br />
in 99.7% ethanol in the concentration range of 0.1 to 2.0 mg/ml.<br />
(3)<br />
(4)
1970 Afr. J. Pharm. Pharmacol.<br />
0.0 2.5<br />
5.0 5.0 7.5 10.0 12.5 15.0 17.5 min<br />
12.5 15.0<br />
min<br />
Figure 1. P. niruri chromatogram showing the peaks of Compound 1 gallic acid at a retention time 8.522 min, Compound 2, corilagin at<br />
12.196 min, Compound 3 expected to be geraniin at 12.406 min and compound 5, ellagic acid at 16.963 min.<br />
While GA reference standard was dissolved in distilled water in the<br />
range of 0.2 to 2.0 mg/ml. The extracts and the reference standards<br />
solutions of GA and EA were allowed to react with 2,2-Diphenyl-1picrylhydrazyl<br />
(DPPH) (Fang et al., 2008; Krithika et al., 2009). 0.3<br />
ml of 0.394 mg/ml DPPH and 2.4 ml of 99.7% ethanol were mixed<br />
in screw-capped test tubes. 0.1 ml of the extracts and the reference<br />
standards were added to a separate reaction mixture in the test<br />
tubes and allowed to stand for 30 min in the dark. The scavenging<br />
activity of the mixtures was measured at 517 nm visible wavelength.<br />
The experiment was carried in triplicates (n = 3) for each<br />
concentration of the reference standard and the extracts and the<br />
percentages of scavenging activity of the extracts were calculated<br />
using Equation 5, where Sc. A is the scavenging activity, Ao is the<br />
absorbance of the blank mixture (the absorbance of reaction<br />
mixture without extract or the reference standards), Am was the<br />
absorbance of the reaction mixture with the extract or the reference<br />
standards. The results of scavenging activity of the extracts and the<br />
reference standards were plotted against their dose of the extracts<br />
and the standards in the mixtures (µg). The dose that inhibits 50%<br />
of DPPH radical activity (IC50) were calculated from the equation of<br />
the scavenging activity plotted curves. The scavenging activities of<br />
the extracts were compared with GA and EA scavenging activity as<br />
positive control standards. The relative scavenging activity of the<br />
extracts to the reference standards GA and EA scavenging activity<br />
were analyzed for null hypothesis using student‟s t-test.<br />
Statistical analysis<br />
Results were expressed as means�standard deviation (SD) of<br />
triplicates measurements. Student‟s t-test was used to analyse the<br />
(5)<br />
data and p-value
Mahdi et al. 1971<br />
Figure 2. P. urinaria chromatogram shows the peaks of Compound 1, gallic acid at a retention time 8.436 min, Compound 2<br />
corilagin at 12.144 min, Compound 3 expected to be geraniin at 12.353 min and Compound 5 ellagic acid at 16.868 min.<br />
0.0<br />
0.0<br />
2.5 5.0 7.5 10.0 12.5 15.0 17.5 min<br />
2.5 5.0 7.5 10.0 12.5 15.0 17.5 min<br />
Figure 3. The chromatogram of external references standards mixture of gallic acid Compound 1, corilagin Compound 2 and ellagic A<br />
Compound 5 peaks at 8.477, 12.183 and 16.961 min, respectively.<br />
min, respectively in P. urinaria chromatogram (Figure 2).<br />
The chromatograms of GA, Cor and EA reference<br />
standards mixture shows peaks of retention‟s time at<br />
8.477, 12.183 and 16.961 min, respectively (Figure 3).<br />
Figure 4 shows the chemical structure of the phenolic<br />
compounds identified in the extracts and geraniin which<br />
is structurally closed to corilagin, its peak could be much<br />
closed to the corilagin peak such as Compound 3 in the<br />
chromatograms (Figures 1 and 2). The expectation was<br />
compared to previously identification of geraniin closed to<br />
corilagin in previous identification of phenolic compounds<br />
described previously (Thitilertdecha et al., 2010). It was<br />
cleared that these compounds are large molecules, have<br />
high molecular and their solubility are varied from water<br />
soluble compound such as GA to lower water soluble EA.<br />
It was also cleared from the chromatogram (Figure 1) and
1972 Afr. J. Pharm. Pharmacol.<br />
HO<br />
HO<br />
O<br />
HO<br />
OH<br />
O<br />
O<br />
HO<br />
OH<br />
Compound 1 Gallic Acid<br />
Figure 4. Chemical structures of the major compounds identified in the extracts.<br />
that corilagin was the major compound in the extracts<br />
followed by Compound 3 which was expected to be<br />
geraniin, then EA and lastly GA. The important of<br />
polyphenols compounds is that they have amphiphilic<br />
properties which facilitate their antioxidants mechanism in<br />
both water and lipid phase (Sies and Stahl, 2004).<br />
Therefore, their application in cosmeceuticals will have<br />
important role in scavenging the ROS and FR results<br />
from excessive exposures to solar UV radiations. As it<br />
was known that the antioxidants properties of the<br />
polyphenolic compounds is because of the presence of<br />
hydroxyl group attached to aromatic ring as electron<br />
donating group (Ng et al., 2000). The more electron<br />
donating group available in the antioxidants compound,<br />
the more potent and stronger scavenging activity<br />
(Srinivasan et al., 2007). Based on this, geraniin which<br />
possess eleven hydroxyl groups attached to aromatic will<br />
be more potent compared to corilagin which has nine<br />
OH<br />
OH HO<br />
O<br />
O<br />
OH<br />
OH<br />
O<br />
O<br />
O<br />
O<br />
O<br />
O<br />
OH<br />
OH<br />
OH<br />
O<br />
O<br />
O<br />
OH<br />
OH<br />
OH<br />
OH<br />
OH<br />
OH<br />
Compound 2 Corilagin<br />
Compound 3 expected to be Geraniin Compound 5 ellagic acid<br />
HO<br />
HO<br />
O<br />
OH<br />
HO<br />
O<br />
O O<br />
O<br />
O<br />
O<br />
OH<br />
O<br />
OH<br />
O<br />
OH<br />
O O<br />
hydroxyl groups and consequently, corilagin is a strong<br />
antioxidant compared to EA and GA which has four and<br />
three hydroxyl groups, respectively Figure 4.<br />
Furthermore, carboxylic acid group in GA can provide<br />
additional attack sites for free radicals and thus prevent<br />
them from attacking cell membrane and denaturation of<br />
protein (Srinivasan et al., 2007). Similarly, EA which<br />
contains two lactones‟ groups have contribution to its<br />
antioxidant activity.(Barch et al., 1996). Hence, the<br />
variation in content of these active ingredients in the<br />
extracts will affect the antioxidant activity. The<br />
quantification of major compounds in extracts was shown<br />
in Table 2. These compounds were also found in other<br />
plant extracts with content almost similar the P. niruri and<br />
P. urinaria and their uses as antioxidants material were<br />
explored (Rangkadilok et al., 2007). These polyphenolic<br />
compounds have been shown as antioxidants able to<br />
scavenge the free radicals reactive oxygen species (Fang<br />
OH<br />
OH<br />
OH<br />
OH<br />
OH<br />
OH
Table 2. Quantitative results of major active ingredients in the extracts (n = 3).<br />
Dry weight of the extract (mg/g) P. niruri P. urinaria<br />
Gallic acid (GA) 11.867�0.130 8.709�0.091<br />
Corilagin (Cor) 89.579�0.602 56.382� 0.364<br />
Ellagic acid (EA) 37.309�0.033 27.880�0.263<br />
Mahdi et al. 1973<br />
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<br />
extracts.<br />
Retention time<br />
(min)<br />
GA standard (n<br />
= 6)<br />
EA standard (n<br />
= 6)<br />
P. niruri (n = 3) P. urinaria (n = 3)<br />
GA EA GA EA<br />
Mean (min) 8.398 16.897 8.526 16.964 8.406 16.861<br />
SD 0.013 0.042 0.033 0.031 0.012 0.010<br />
RSD (%) 0.154 0.249 0.387 0.183 0.143 0.059<br />
P-value - - 0.530 0.127 0.420 0.324<br />
et al., 2008; Lin et al., 2008). They also can absorb the<br />
UV radiations and act as sun-blocking and whitening<br />
agents. It can penetrate the skin, pick up and neutralized<br />
the excessively generated ROS and FR due to the<br />
exposure to UV radiation and act as skin antiaging. To<br />
utilized the extracts as skin cosmetics, they were<br />
subjected to further treatment such as identification of<br />
TPC and scavenging activity towards stable radical<br />
DPPH.<br />
Detection and quantification limits (DL and QL)<br />
The HPLC analysis results in the present study show the<br />
linearity of the method by references standards at six<br />
levels of concentrations in the range of 0.5 to 16 �g/ml by<br />
the equations of the calibration curves of reference<br />
standards GA and EA. The calibration curve of reference<br />
standard GA equation was y = 4978.79 + 60900.7 × x, R 2<br />
= 0.9994. While the EA equation was y = 54933.8 +<br />
36348.1 × x with regression factor R 2 = 0.9981. The DL of<br />
the two external reference standards GA and EA were<br />
0.021 and 0.076 �g/ml, respectively. While the QL of the<br />
two external reference standards GA and EA were O.070<br />
and 0.252 �g/ml, respectively. The lower values of DL<br />
and QL reflect the sensitivity of the method. Furthermore,<br />
the sensitivity of the method was confirmed by the result<br />
of the relative standard deviation (RSD%) of the mean<br />
area under peak of both GA and EA reference standards<br />
1.34 and 1.63%, respectively. It was also cleared that the<br />
DL and QL of GA is lower than EA. Hence the method<br />
was more sensitive to GA compared to EA.<br />
Selectivity of the HPLC method<br />
The selectivity of the HPLC method was evaluated by<br />
comparing the retentions time of the external references<br />
standards GA and EA with retentions time of GA and EA<br />
active ingredients in the extracts Table 3. The small value<br />
of the relative standard deviation (RSD %) show the<br />
selectivity of the method. The SD of the means of the<br />
retention time is less than the retention time band set in<br />
the instrument which was 0.2 min. In addition, the<br />
statistical result also shows that the paired t-test tested<br />
satisfies the null hypothesis for two-sided with p-value ><br />
0.05 and 95% confidence interval of the difference<br />
between the external reference standards retention time<br />
and the sample solution (Table 3). The proposed HPLC<br />
method was efficient, simple, rapid, cost effect and safe<br />
for the column on long uses since small amount of formic<br />
acid was utilized. Since the proposed HPLC method<br />
identified and quantified high content of phenolic<br />
compounds in the extracts, the extracts were subjected<br />
for further study to evaluate their antioxidants activities<br />
with respect to TPC and scavenging activity towards<br />
DPPH.<br />
Total phenolic content<br />
The quantification of the total phenolic content of P.<br />
urinaria and P. niruri were expressed as means ± SD and<br />
was found to be 277.98 � 1.04 and 262.10 � 1.04 mg of<br />
GAE/g dry weight of plant extract, respectively. The<br />
present study also revealed that P. urinaria and P. niruri<br />
extracts possessed high TPC compared to previous<br />
studies of extract from P. niruri (Harish and<br />
Shivanandappa, 2006; Kumaran and Joel, 2007). The<br />
TPC of the P. niruri was lower compared to the predict<br />
TPC based on the quantity of the major active ingredients<br />
in the extract; while the TPC of P. urinaria was higher<br />
compared to the predicted TPC which based on the
1974 Afr. J. Pharm. Pharmacol.<br />
Table 4. Predicted TPC of the extracts based on the polyphenolic contents<br />
compared to the experimental TPC.<br />
Compose P. niruri (GAE/g DW) P. urinaria (GAE/g DW)<br />
GA 11.87�0.13 8.71�0.09<br />
Corilagin 268.74�1.81 169.15�1.09<br />
EA 48.50�0.04 36.24�0.34<br />
Predicted TPC 329.11�1.98 214.10�1.53<br />
Experimental TPC 262.10� 1.04 277.98�1.04<br />
GA has 3 OH groups is equivalent to 1 GAE/g DW, corilagin has 9 OH groups. Therefore<br />
= 3GAE/g DW equivalent and EG has 4 OH groups = 1.33 GAE/g DW.<br />
number of hydroxyl groups in each of the phenolic<br />
compounds to the number of hydroxyl groups in GA and<br />
its content in the extracts (Table 4). The variations in the<br />
predicted and experimental TPC of P. urinaria could be<br />
due to presence of much major and high content of<br />
unidentified compounds. Such compounds were 6, 7 and<br />
8 which were found only in P. urinaria and Compound 4<br />
which was expected to be geraniin with highest peak in<br />
P. urinaria chromatogram (Figure 2) compared to P. niruri<br />
chromatogram (Figure 1). Therefore, its high content in P.<br />
urinaria certainly will increase its TPC. This also could be<br />
reason of P. urinaria has high phenolic content despite it<br />
contain less amount of GA, Cor and EA compared to P.<br />
niruri. In case of P. niruri, the predicted and experimental<br />
TPC results (Table 4) were closely relevant since only<br />
Compound 4 was not quantified (Figure 1). The predicted<br />
TPC gives quick estimation to the TPC of the extracts<br />
despite that it required more study and broad application<br />
before been normalize as a measure.<br />
Scavenging activity<br />
The scavenging activity of P. niruri towards the stable<br />
radical DPPH was calculated as IC50 from the plotted<br />
graph equation, Y = -1684.82/X + 101.618, with<br />
correlation coefficient factor R 2 = 0.9938 (Figure 5). The<br />
IC50 is the dose in mass of the antioxidants materials<br />
(extract or reference standard) necessary to inhibit the<br />
initial DPPH radical activity by 50%. The smaller value of<br />
IC50 means high scavenging activity and potent<br />
antioxidants compound. The IC50 of P. niruri was 32.64 �g<br />
mass of the extract and its inhibition capacity to DPPH<br />
was 91.57% at dose of 200 �g. The IC50 of P. urinaria<br />
was 25.00 �g mass of the extract and was inhibited<br />
93.81% of DPPH scavenging activity at a dose of 200 �g<br />
based on the curve equation Y = -1288.4/X + 101.528<br />
with regression coefficient R 2 = 0.9982 (Figure 6) . The<br />
results of scavenging activities of P. niruri and P. urinaria<br />
were compared to the scavenging activity of the control<br />
positive reference standard GA and EA. The IC50 of GA<br />
and EA were 3.28 μg and 2.99 �g mass of the reference<br />
standard, respectively. The maximum scavenging<br />
capacity of the positive control external references<br />
standards GA and EA against DPPH was 95.95% and<br />
94.01% at a dose of 200 μg and 100 �g respectively<br />
(Figures 7 and 8). The scavenging activity curves of the<br />
extracts and the reference standards were non-linear with<br />
increasing mass of the extract or the reference standards<br />
in the form Y = A/X + B. These curves show the<br />
maximum scavenging activity of the extracts and the<br />
control reference standards and behave as biological<br />
curves. The inhibition capacity was the maximum<br />
percentage of scavenging activity of the extract/reference<br />
standard towards DPPH. The maximum inhibition<br />
capacity never reaches 100% due to the plateau feature<br />
of the scavenging activity profile of both the extracts and<br />
the control positive standards which is the characteristic<br />
of biological activity. The profiles were typical biological<br />
activity curve and closely resemble enzyme kinetic curve.<br />
The results of scavenging activity of the extracts and the<br />
references standards reported in Table 5 show that the<br />
scavenging activity of both extracts were much lower<br />
compared to the control reference standards compare to<br />
the TPC of the extracts. According to the TPC of the<br />
extracts, they must have lower IC50 than actually<br />
obtained. This might be because the high TPC phenolic<br />
content of the extracts was due to high molecular weight<br />
and bulky molecule corilagin and geraniin compared to<br />
the planar GA and EA used as reference standards. The<br />
bulky molecules, corilagin and geraniin might have some<br />
steric hindrance to donate the phenolic hydrogen of the<br />
hydroxyl group to DPPH compared to the relatively small<br />
molecules GA and EA (Figure 4). Hence corilagin and<br />
geraniin might be slower in scavenger DPPH. While in<br />
case of GA and EA which were small planar compounds<br />
geometry enable them to participate easily in the reaction<br />
with DPPH. Therefore, the scavenging activity of the<br />
extracts was mainly due to GA and EA in the extracts.<br />
This can be predicted also from the relative scavenging<br />
activity of the extracts to the reference standards. The<br />
relative scavenging activity (RScA) was the ratio of the<br />
scavenging activities of the extracts to the scavenging<br />
activity of the control positive standard. From the relative<br />
scavenging activity (RScA) of the extract to GA and EA.
Table 5. Antioxidants activity based on IC50 (�g) and the capacity of inhibition<br />
to DPPH (%) of the extracts compared to the reference standards.<br />
Extract/standard IC50<br />
Capacity of DPPH inhibition<br />
(%)<br />
P. niruri 32.64 91.57<br />
P. urinaria 25.00 93.81<br />
Gallic acid 3.28 95.95<br />
Ellagic acid 2.99 94.01<br />
p-value - 0.00<br />
Table 6. Relative scavenging activity (RScA) of extracts to GA and<br />
EA scavenging activity.<br />
RScA P. niruri P. urinaria<br />
GA 9.96 7.63<br />
EA 10.90 8.35<br />
Mean�SD 10.43�0.67 7.99�0.51<br />
P-value of RScA 0.029 0.029<br />
Figure 5. P. niruri scavenging activity profile against 2, 2-diphenyl-1-picrylhydrazyl radical<br />
(DPPH), the curve equation Y = -1684.82/X + 101.618, correlation coefficient R 2 = 0.9938 and<br />
IC50 = 32.64 �g.<br />
standards (Table 6), the RScA of P. urinaria was 7.630 GA<br />
and contained 8.709 �g GA /mg dry weight of the extract.<br />
While in P. niruri, the RScA was 9.960 and the content of<br />
GA was 11.867�g /mg dry weight of P. niruri. The<br />
percentage ratio of the RScA of GA to its contents in P.<br />
niruri and P. urinaria extracts were approximately 83.9<br />
and 87.6%, respectively. This result shows that the<br />
scavenging activity of the extracts was mainly due to GA.<br />
Similarly, the contribution of EA in the scavenging activity<br />
Phyllanthus niruri extract (in �g)<br />
Mahdi et al. 1975<br />
of the P. niruri and P. urinaria extracts was 22.5 and<br />
23.1%, respectively. Therefore, the scavenging activity of<br />
both extracts towards DPPH was due to the GA and EA<br />
in the extracts. Hence DPPH was not suitable indicator<br />
for scavenging activity of the extracts contains such bulky<br />
molecules like corilagin and geraniin. The inhibition<br />
capacity and the relative scavenging activity were<br />
significantly different and the null hypothesis was rejected<br />
for both p-value
1976 Afr. J. Pharm. Pharmacol.<br />
Phyllanthus urinaria extract (in �g)<br />
Figure 6. P. urinaria scavenging activity profile against 2, 2-diphenyl-1-picrylhydrazyl radical<br />
(DPPH), the curve equation Y = -1288.4/X + 101.528, correlation coefficient R 2 = 0.9982 and IC50<br />
= 25.00 �g.<br />
Gallic acid extract (in �g)<br />
Figure 7. Gallic acid scavenging activity profile against 2, 2, diphenyl-1-picrylhydrazyl radical<br />
(DPPH), the curve equation Y = -138.615/X + 92.3058, correlation coefficient R 2 = 0.9395 and<br />
IC50 = 3.28 �g.<br />
Ellegic acid extract (in �g)<br />
Figure 8. Ellagic acid scavenging activity profile against 2, 2-diphenyl-1-picrylhydrazyl radical<br />
(DPPH), the curve equation Y = -126.56/X + 92.27, correlation coefficient R 2 = 0.9394 and<br />
IC50 = 2.99 �g.
The results of scavenging activity (Table 5) consequently<br />
show that P. urinaria was more potent compared to P.<br />
niruri based on IC50 value and capacity of scavenging<br />
activity. The result was consistent with the TPC finding<br />
which showed P. urinaria with high TPC compared to P.<br />
niruri. Therefore P. urinaria was strong inhibitor to DPPH<br />
activity compared to P. niruri as well as it has high total<br />
phenolic content (TPC). This result was also consistent<br />
with the previous finding which is, the higher the TPC, the<br />
stronger scavenging to DPPH (Tawaha et al., 2007;<br />
Zheng and Wang, 2001). Similarly, it was also noticed<br />
that EA IC50 was lower compared to GA IC50, hence it<br />
was more potent than GA. EA also retuned high<br />
scavenging capacity towards DPPH since it inhibited<br />
94.01% of DPPH activity at a dose of 100 �g compare to<br />
GA which inhibited 95.95% of DPPH activity but at a dose<br />
of 200 �g (Figure 7 and 8). This might be because of<br />
differences in the number of hydroxyl group attached to<br />
the aromatic ring in GA and EA chemical structures and<br />
hence more availability of donating the phenolic hydrogen<br />
of hydroxyl group as scavenger in the reaction (Figure 4).<br />
The results of scavenging activity of GA and EA from this<br />
study and the scavenging activity of P. niruri and P.<br />
urinaria were comparable to the previous study of plants<br />
material of similar content of our extracts (Rangkadilok et<br />
al., 2007). The scavenging capacity of the extracts from<br />
this study was higher at higher dose as compared to<br />
previous (Kumaran and Joel, 2007).<br />
Conclusions<br />
The proposed HPLC method successfully identified and<br />
quantified the phenolic compounds of the extracts. The<br />
method was quite simple, rapid, sensitive, selective, cost<br />
effective procedure, friendly and safe to the column and<br />
instrument on long uses. The HPLC analysis, TPC and<br />
the scavenging activities results show that the extracts<br />
contain high phenolic materials comparable to the<br />
references standards gallic acid and ellagic acid. DPPH<br />
was not a good indicator of scavenging activity of the<br />
extract because it mainly composes bulky molecules<br />
such as corilagin and geraniin. The result of antioxidants<br />
evaluation is revealed to the extracts as a potential<br />
natural source of antioxidants. The high antioxidants<br />
properties of the extracts might make them versatile in<br />
various fields of nutraceuticals, pharmaceuticals and<br />
cosmeceuticals applications. Specifically, our interest in<br />
this time is formulation of these extracts as skin<br />
antiaging, sun-blocking and whitening agents.<br />
ACKNOWLEDGEMENTS<br />
The authors thank Biotechnology Directorate Malaysia for<br />
allocation of the research grant, Universiti Sains Malaysia<br />
(USM) for financial support for Elrashid and Nova<br />
Mahdi et al. 1977<br />
Laboratories Sdn.Bhd. (Malaysia) for supplying the<br />
extracts materials and corilagin working standard.<br />
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African Journal of Pharmacy and Pharmacology Vol. 5(17), pp. 1979-1982, 8 November, 2011<br />
Available online at http://www.academicjournals.org/AJPP<br />
DOI: 10.5897/AJPP11.045<br />
ISSN 1996-0816 ©2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Cultural importance and antibacterial activity of<br />
Ziziphus mucronata (Willd.) in the Umlazi community in<br />
Durban<br />
R. M. Coopoosamy 1 *, K. K. Naidoo 1 and N. J. Ndlazi 2<br />
1 Department of Nature Conservation, Mangosuthu University of Technology, P. O. Box 12363, Jacobs, 4026, Durban,<br />
KwaZulu-Natal, South Africa.<br />
2 Faculty of Natural Science, Mangosuthu University of Technology, P. O. Box 12363, Jacobs, 4026, Durban, KwaZulu-<br />
Natal, South Africa.<br />
Accepted 21 October, 2011<br />
The Zulu community of Umlazi, Durban makes extensive use of Ziziphus mucronata (Buffalo thorn) for<br />
predominantly cultural purposes. There are many superstitions and beliefs associated with the tree. A<br />
validation of its antimicrobial properties from leaf, bark and root extracts were attempted against three<br />
Gram positive and three Gram negative bacteria. In addition, an antifungal assay was attempted, using<br />
ethanol, aqueous and boiled aqueous extracts on six fungal species. Acetone and ethyl acetate<br />
samples showed greater inhibition of mostly Gram positive bacteria. Extracts from the leaves showed<br />
the greatest inhibition, while root extracts showed the least inhibition. Ethanol extracts were found to<br />
possess greater antifungal activity than aqueous and boiled aqueous extracts. Extracts were most<br />
effective against Aspergillus flavus and Aspergillus glaucus with little effect on Candida albicans and<br />
Candida tropicalis. It was evident that the bark and leaves of Z. mucronata possess greater<br />
antimicrobial properties than the root and can be used as a substitute for other extensively harvested<br />
species demonstrating similar properties.<br />
Key words: Ziziphus mucronata, antimicrobial, cultural importance.<br />
INTRODUCTION<br />
In recent years, the interest of traditional medicine from<br />
different cultures has increased significantly in<br />
industrialized countries, with prescription drugs worldwide<br />
originating from tropical flora (Nelson-Harrison et al.,<br />
2002). Traditional medicine plays an important role in<br />
many areas of South Africa, where communities do not<br />
have access to proper healthcare facilities. In Southern<br />
Africa, it is estimated that 70% of the black population<br />
consults traditional healers for health hazard problems<br />
and utilize traditional medicines, most of which is derived<br />
from plant species indigenous to the region (Jager et al.,<br />
1995). One of such community, living in Umlazi, Durban<br />
makes extensive use of buffalo thorn for predominantly<br />
cultural purposes.<br />
*Corresponding author. E-mail: rogercoopoosamy@gmail.com.<br />
Tel: +27 82 200 3342. Fax: +27 31 907 7665.<br />
Africans have many beliefs and superstitions attached to<br />
this tree. Zulus use the buffalo thorn in connection with<br />
burial rites. It was once customary that when a Zulu chief<br />
died, the tree was planted on his grave as a reminder or<br />
symbol of where the chief lies, hence, the name<br />
umLahlankosi (that which buries the chief, Pers. Comm:<br />
Fakazi). A twig from the tree was and is still used to<br />
attract and carry the spirit of the deceased from the place<br />
of death to the new resting place. When a stock owner<br />
died and was buried, according to custom, within the<br />
cattle or goat kraal, some branches were placed on the<br />
grave so that the animals nibbled on leaves and twigs,<br />
and so understood that their master had died (Palmer<br />
and Pitman, 1972). In other parts, Africans drag a branch<br />
round the village to protect it from evil spirits, as it is<br />
believed to keep evil spirits away (Pers. Comm: Fakazi).<br />
In Botswana as well as most parts of South Africa, the<br />
residents believed the buffalo thorn to be immune against<br />
lightning; anyone standing under one in a storm would be
1980 Afr. J. Pharm. Pharmacol.<br />
safe. It is also believed that if it is felled in summer, a<br />
drought, hail or lightning will certainly follow (Pers.<br />
Comm: Fakazi). A decoction of the glutinous roots is<br />
commonly administered as a sedative for all sorts of<br />
pains as well as dysentery (Nadembega et al., 2011). A<br />
concoction of the bark and the leaves is used for<br />
respiratory ailments and other septic swellings of the<br />
skin. Pastes of the root and leaves can be applied to treat<br />
boils, swollen glands, wounds and sores (Hutchings et<br />
al., 1996). Steam baths from the bark are used to purify<br />
and improve complexion (Palmer and Pitman, 1972). A<br />
study using root extracts from buffalo thorn showed<br />
activity against dermatophytes causing skin diseases<br />
(Adamu et al., 2006). Furthermore, aqueous and<br />
methanol extracts of the stem bark showed antifungal<br />
activity against Candida albicans (Gundidza, 1986). In<br />
East Africa, roots are used for treating snake bites<br />
(Hutchings et al., 1996). It has been reported that rural<br />
communities in Burkina Faso use a decoction of the root<br />
to prevent obesity and stave off hunger pangs<br />
(Nadembega et al., 2011).<br />
Programmes for the screening of plant remedies are<br />
important for validating the traditional use of herbal<br />
remedies and for providing leads in the discovery of new<br />
active principles. It is evident that buffalo thorn is used<br />
sparingly by traditional healers in Umlazi, possibly<br />
because of its scarcity in the area as well as the fact that<br />
there is an abundance of other species exhibiting high<br />
medicinal value. Although, some work has shown<br />
antifungal properties of the plant, not much focus has<br />
been placed on the antibacterial properties. Validation of<br />
the antibacterial and antifungal properties of buffalo thorn<br />
may thus ease existing pressure on other intensively<br />
harvested medicinal plant species and intensify efforts to<br />
re-introduce this species in previously occupied habitats<br />
where it has become almost extinct due to<br />
overharvesting.<br />
METHODOLOGY<br />
Plants species were located with the assistance of Mr Fakazi and<br />
samples were collected from Umlazi, Durban, South Africa.<br />
Antibacterial assay<br />
One kilogram of dried material (leaf, root and bark) was crushed<br />
and placed in a 2 L conical flask containing one of the three<br />
mediums, that is, water, ethyl acetate and acetone, for extraction<br />
based on varying polarity of solvents. The media were left for 72 h<br />
in an orbital shaker at 20 shakes per minute. After 72 h, the extracts<br />
were filtered. The extracts were then used for further tests.<br />
The plant extract was then tested for antibacterial properties<br />
against three strains of Gram positive (Bacillus subtilis, Micrococcus<br />
kristinae and Staphylococcus aureus) and three strains of Gram<br />
negative bacteria (Escherichia coli, Proteus vulgaris and<br />
Enterobacter aerogenes) for antibacterial activity using the cupplate<br />
method. Each organism was prepared by diluting in 24 h old<br />
broth cultures with sterile nutrient broth. The cultures were then<br />
diluted 100 fold to give approximately 10 6 bacteria ml -1 . Cultures<br />
were incubated for 72 h at 60°C. Each treatment was done in<br />
triplicate and the mean values were calculated.<br />
Antifungal assay<br />
Buffalo thorn leaf, bark and root samples (approximately 1 kg of<br />
each) were cut into small pieces and crushed in a homogenizer.<br />
The plant materials were soaked in ethanol (95% v/v) and in<br />
distilled water in 2 L conical flasks for 3 weeks. The extracts (water<br />
and ethanol) obtained were evaporated at reduced pressure (45°C)<br />
to a residue. Extracts for testing ethanol and aqueous extract were<br />
prepared in three different concentrations. The stock solutions were<br />
prepared by dissolving 100 mg of dry extract in 1 ml of ethanol and<br />
water separately in order to obtain a concentration of 100 mg/ml<br />
dilutions (1:10, 1:100 and 1:500). These stock solutions were then<br />
used in phosphate buffer at pH 6.0 to evaluate the antifungal<br />
activity (Champion et al., 1992). The solutions were then tested for<br />
antifungal activity using the following fungal cultures: Aspergillus<br />
flavus, Aspergillus glaucus, Candida albicans, Candida tropicalis,<br />
Trichophyton mentagrophytes, and Trichophyton rubrum. Plates<br />
containing potato dextrose agar served as controls. All tests were<br />
done in triplicate and the mean values were calculated.<br />
RESULTS AND DISCUSSION<br />
Water did not serve as a good extraction medium with<br />
only bark extract samples inhibiting B. subtilus and M.<br />
kristinae (Table 1). Acetone and ethyl acetate samples<br />
showed greater inhibition of mostly Gram positive<br />
bacteria (Tables 2 and 3). Acetone extracts from leaves<br />
showed the greatest inhibition while extracts from roots<br />
showed the least inhibition (Table 2). All Gram positive<br />
bacteria and one Gram negative bacteria, that is, E. coli<br />
were inhibited by acetone extracts obtained from the<br />
leaves. Staphylococcus aureus was the only Gram<br />
positive bacteria not inhibited by acetone extracts from<br />
bark (Table 2).<br />
All Gram positive bacteria were inhibited by ethyl<br />
acetate extracts from leaves and bark while extracts from<br />
roots inhibited only S. aureus (Table 3). In contrast, none<br />
of the Gram negative bacteria were inhibited by ethyl<br />
acetate extracts of the different plant parts (Table 3). The<br />
results of the present study are similar to findings<br />
obtained by other researchers who showed that<br />
antibacterial activity was more prevalent in Gram positive<br />
strains (Coopoosamy and Magwa, 2007; Grierson and<br />
Afolayan, 1999).<br />
The mixed extracts of buffalo thorn showed similar<br />
activities to that of the leaves (Tables 1, 2 and 3).<br />
However, the mixed extract was less effective when<br />
compared with the leaf extract. This can be explained by<br />
the fact that these samples contained extracts from<br />
leaves, bark and roots; extracts from bark and root being<br />
less effective.<br />
The antifungal activities (Table 4) of the ethanol<br />
extracts were found to be more effective than aqueous<br />
and boiled aqueous extracts. Extracts were most<br />
effective against A. flavus and A. glaucus with little effect<br />
on C. albicans and C. tropicalis (Table 4). No effects
Table 1. Minimal inhibitory concentration (MIC) of Z. mucronata in water extract.<br />
Bacteria<br />
Gra<br />
m<br />
+/-<br />
Leave<br />
Stem bark<br />
Plant part<br />
Roots<br />
Coopoosamy et al. 1981<br />
Mixture of leaves,<br />
bark and roots<br />
B. subtilis + Na Na Na 4.0 5.0 Na Na Na Na 6.0 4.0 Na<br />
M. kristinae + Na Na Na 4.0 4.0 Na Na Na Na Na Na Na<br />
S. aureus + Na Na Na Na Na Na Na Na Na Na 6.0 Na<br />
E. coli - Na Na Na Na Na Na Na Na Na Na Na Na<br />
P. vulgaris - Na Na Na Na Na Na Na Na Na Na Na Na<br />
E. aerogenes - Na Na Na Na Na Na Na Na Na Na Na Na<br />
Na = No activity; all activities were done in triplicate.<br />
Table 2. Minimal inhibitory concentration (MIC) of Z. mucronata in acetone extract.<br />
Bacteria<br />
Gra<br />
m +/-<br />
Leaves<br />
Stem bark<br />
Plant part<br />
Roots<br />
Mixture of leaves,<br />
bark and roots<br />
B. subtilis + 3.0 4.0 3.0 2.0 3.0 Na 5.0 Na Na 3.0 4.0 Na<br />
M. kristinae + 4.0 3.0 5.0 3.0 3.0 4.0 Na Na Na 4.0 Na Na<br />
S. aureus + 2.0 4.0 4.0 Na Na Na Na Na Na 4.0 5.0 Na<br />
E. coli - 3.0 3.0 3.0 Na Na Na Na Na Na Na Na Na<br />
P. vulgaris - Na Na Na Na Na Na Na Na Na Na Na Na<br />
E. aerogenes - Na Na Na Na Na Na Na Na Na Na Na Na<br />
Na= No activity; all activities were done in triplicate.<br />
Table 3. Minimal inhibitory concentration (MIC) of Z. mucronata in ethyl-acetate extract.<br />
Bacteria<br />
Gram<br />
+/-<br />
Leaves<br />
Stem bark<br />
Plant part<br />
Roots<br />
Mixture of leaves,<br />
bark and roots<br />
B. subtilis + 4.0 3.0 3.0 2.0 3.0 Na 5.0 Na Na 4.0 4.0 2.0<br />
M. kristinae + 5.0 4.0 5.0 4.0 4.0 5.0 Na Na Na 4.0 Na 3.0<br />
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<br />
E. coli - Na Na Na Na Na Na Na Na Na Na Na Na<br />
P. vulgaris - Na Na Na Na Na Na Na Na Na Na Na Na<br />
E. aerogenes - Na Na Na Na Na Na Na Na Na Na Na Na<br />
Na = No activity; all activities were done in triplicate.<br />
were recorded for T. mentagrophtes and T. rubrum<br />
(Table 4).<br />
This contradicts a study by Adamu et al. (2006) where<br />
methanol extracts of root samples showed antifungal<br />
activity against T. rubrum, T. mentagrophytes, Aspergillus<br />
fumigatus and Microsporum canis. Unfortunately, those<br />
researchers did not test leaf nor bark samples based on<br />
their results on modifications of existing methods.<br />
Growth inhibition (zone of inhibition) was recorded as<br />
very high (++++), high (+++), medium (++) and low<br />
(+),which indicated zones of inhibition between 41 to 50,<br />
31 to 40, 21 to 30 and 11 to 20 mm, respectively.<br />
These zones of inhibition were determined from the<br />
central point to the point where growth inhibition has<br />
occurred and measured, respectively. The ethanol extract<br />
and boiled aqueous extract of the leaves were noted to<br />
have more antimicrobial effects as compared to the<br />
aqueous extracts.<br />
The high zones of inhibition noted in the ethanol<br />
extracts (using a 1:10 concentration) suggest further<br />
explanation of the possibility of using this plant against<br />
certain ailments caused by the aforementioned fungal
1982 Afr. J. Pharm. Pharmacol.<br />
organisms.<br />
Conclusion<br />
Table 4. Effect of ethanol, aqueous extracts and boiled aqueous extracts obtained from Z. mucronata on different fungal<br />
species (Tests were done in triplicate).<br />
Fungal species<br />
Ethanol extract<br />
1:10 1:100 1:500<br />
Aqueous extract<br />
1:10 1:100 1:500<br />
Boiled aqueous extract<br />
1:10 1:100 1:500<br />
A. flavus ++ ++ ++ ++ - - +++ + -<br />
A. glaucus +++ + - ++ + - +++ - -<br />
C. albicans + - - - - - ++ - -<br />
C. tropicalis + - - - - - - - -<br />
T. mentagrophytes - - - - - - - - -<br />
T. rubrum - - - - - - - - -<br />
- = Negative antifungal activity; + = Positive antifungal activity (low inhibition); ++ = Positive antifungal activity (medium<br />
inhibition); +++ = Positive antifungal activity (high inhibition); ++++ = Positive antifungal activity (very high inhibition). Plates<br />
containing potato dextrose agar served as controls. Controls did not show any inhibition of any of the test fungal species.<br />
It was evident that Ziziphus mucronata does possess<br />
antimicrobial properties and can be used as a substitute<br />
for other extensively harvested species demonstrating<br />
similar properties. Although, the roots of the plant did not<br />
yield positive results, the study showed that leaves and<br />
bark possesses greater antimicrobial properties.<br />
Therefore, this study was important as it may help to<br />
sustain remnants of the existing population in Umlazi.<br />
However, further investigations are needed, including<br />
purification and identification of the active compounds<br />
present in the leaves and bark.<br />
ACKNOWLEDGEMENTS<br />
The researchers are greatly appreciative for the help from<br />
Mr Bheki Fakazi for the site identification and traditional<br />
information pertaining to Ziziphus mucronata in Umlazi,<br />
Durban. Further acknowledgement goes to the Research<br />
Directorate of Mangosuthu University of Technology for<br />
providing fund towards this investigation.<br />
REFERENCES<br />
Adamu HM, Abayeh OJ, Ibok NU, Kafu SE (2006). Antifungal activity of<br />
extracts of some Cassia, Datarium and Ziziphus species against<br />
dermatophytes. Nat. Prod. Rad., 5: 357-360.<br />
Champion RH, Burton J L, Ebling FJG (1992). Textbook of Dermatology<br />
(5 th edition). London, Blackwell., 3: 1130-1175.<br />
Coopoosamy RM, Magwa ML (2007). Traditional use, antibacterial<br />
activity and antifungal activity of crude extract of Aloe excelsa. Afr. J.<br />
Biotech., 6: 2406-2410.<br />
Grierson DS, Afolayan AJ (1999). An Ethnobotanical study of plants<br />
used in treatment of wounds in the Eastern Cape, South Africa. J.<br />
Ethnopharmacol., 67: 327-332.<br />
Gundidza M (1986). Screening of extracts from Zimbabwean higher<br />
plants. Part 2: Antifungal properties. Fitoterapia, 57: 11-113.<br />
Hutchings A, Scott AH, Lewis G, Cunningham AB (1996). Zulu<br />
Medicinal plants: An inventory. University of Natal Press. pp. 53-54.<br />
Jager AK, Hutchings A, Van Staden J (1995). Screening of Zulu<br />
medicinal plants for prostaglandin-synthesis inhibitors. J.<br />
Ethnopharmacol., 52: 95-100.<br />
Nadembega P, Boussim JI, Nikiema JB, Poli F, Antognoni F (2011).<br />
Medicinal plants in Baskoure, Kourittenga Province, Burkina Faso: An<br />
ethnobotanical study. J. Ethnopharmacol., 133: 378-395.<br />
Nelson-Harrison ST, King SR, Limbach C, Jackson C, Galiwango A,<br />
Kato SK, Kanyerezi BR (2002). Ethnobotanical research into the 21 st<br />
century. In: Iwu MM, Woottron JC (Eds.), Ethnomedicine and Drug<br />
Discovery. Elsevier, Amsterdam, pp. 283-307<br />
Palmer E, Pitman N (1972). Trees of Southern Africa covering all known<br />
indigenous species in the Republic of South Africa, South-West<br />
Africa, Botswana, Lesotho and Swaziland. Volume 1. A.A. Balkema,<br />
Cape Town.<br />
Pers. Comm. Mr Fakazi BT (Principal): Kwa Mathanda High School,<br />
Durban.
African Journal of Pharmacy and Pharmacology Vol. 5(17), pp. 1983-1989, 8 November, 2011<br />
Available online at http://www.academicjournals.org/AJPP<br />
DOI: 10.5897/AJPP11.118<br />
ISSN 1996-0816 ©2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Altitude-related changes in activities of carbon<br />
metabolism enzymes and secondary plant productsmenthoforon<br />
an active pharmaceutical constituents<br />
yield in pippermint (Mentha piperita L. Var. Kukarail)<br />
A. Misra* and N. K. Srivastava<br />
Central Institute of Medicinal and Aromatic Plants, P. O. CIMAP, Lucknow-226015, India.<br />
Accepted 15 September, 2011<br />
Activities of some enzymes related to carbon metabolism were studied in different ecotypes of<br />
pippermint (Mentha piperita L. Var. Kukarail), growing at 950 and 1,250 m above mean sea level.<br />
Activities of peroxidase and superoxide dismutase (SOD) and geranyl pyro phosphate (GPP) are<br />
significantly higher in higher altitude cultivated cultivars. The GPP which converts to geranyl geranyl<br />
pyro phosphate (GGPP) comprises of two subunits, the smaller one and the large subunits.<br />
Electrophoretic bands of PCR showed the gene specific primers. In higher altitude, the smaller and<br />
large subunits of the key enzyme are present where as in the plain cultivated plants, the larger subunits<br />
are absent and the only smaller subunits are more conspicuously synthesized. The plain cultivated<br />
plants at 0.05 ppm Zn showed the higher carbon dioxide exchange rate (0.65 µg(CO2)m- 2 s -1 ) and sacride<br />
formation 0.470 µg(CH2O)m -2 s -1 ). The higher altitude pippermints plants at 0.1 Zn mg/L grown plants,<br />
showed higher total amounts of monoterpne oil(s) (0.67%) and the increased was 26% over control, with<br />
higher menthol, menthyl actate and of medicinal uses menthofuron contents in comparison to plain<br />
cultivations.xide-dismutase (SOD) and peroxidise iso-enzymes with altitude were studied, where as<br />
activities of SOD did not show a significant difference with change in altitude. RFPD of the two altitude<br />
grown cultivars showed the different lines of conspicuous lane bandings. The key enzymes are from<br />
geranyl pyro phosphate (GPP).<br />
Key words: Peroxidase, superoxide dismutase, peroxidise iso-enzymes, gernul geranul pyruphosphate,<br />
phosphoenolpyruvate carboxylase; ribulose-1,5-bisphosphate, carboxylase/oxygenase.<br />
INTRODUCTION<br />
Peppermint (Mentha piperita L) was mostly affected by<br />
the leaf blight disease of Alternaria alternata. Therefore, a<br />
protocol has been established by tissue culture<br />
techniques, to get an improved and resistant variety of<br />
Mentha piperita L. var. Kukarail. Further, an adaptation to<br />
environment refers to the capacity of organisms or cells<br />
to alter their phenotype in response to changes. Plants<br />
display enormous plasticity to survive under changing<br />
environmental variables with altitude (Clements et al.,<br />
1950; Billings et al., 1961; Tranquillini, 1964). This<br />
plasticity in variety is according to altitude cropping<br />
response to environmental variables, This may involve<br />
*Corresponding author. E-mail: amisracimap@yahoo.co.in.<br />
the changes at anatomical, morphological, physiological<br />
and biochemical levels to enable plants to combat ‘harsh’<br />
climatic conditions at high altitude and maintain a<br />
reasonably efficient carbon harvesting system to<br />
compensate for the relatively short growing period<br />
(Tranquillini 1964, Larcher, 1995; Purohit, 2003).<br />
Plasticity in each of these responses could be of special<br />
significance under specific environment. Morphological<br />
plasticity is related to high competition in productive<br />
environments, whereas species acclimate through<br />
physiological plasticity in unproductive environments<br />
(Cordell et al., 1998). Mountain plants evolved in<br />
response to their particular altitude environment differ in<br />
physiological response to the respective ecotypes from<br />
lowland areas (Billings et al., 1961; Hiesey et al., 1971;
1984 Afr. J. Pharm. Pharmacol.<br />
Mächler et al., 1977; Körner and Diemer, 1987; Cordell et<br />
al., 1998; Hovenden and Schimanski, 2000; Hovenden<br />
and Schoor, 2003; Kumar et al., 2005, 2006; Vats and<br />
Kumar, 2006). However, there is little information on<br />
altitude related changes in enzymatic activities related to<br />
carbon metabolism except for enzymes such as<br />
peroxidase , superoxide-dismutase(SOD) and peroxidise<br />
iso-enzymes and geranyl geranyl pyro phosphate<br />
(GGPP), geranyl geranyl pyro phosphate (GGPP),<br />
comprises of two subunits, the smaller one and the large<br />
subunits. Further apart from the monoterpenes<br />
biosynthesis, the primary carbon metabolism, that is, the<br />
ribulose-1, 5-bisphosphate carboxylase/oxygenase<br />
(RuBPCO) plays an important role in carbon<br />
sequestration and carbon capturing for carbon balancing.<br />
The higher RuBPCO activity was reported in high altitude<br />
ecotypes of Selinum vaginatum (Pandey et al., 1984).<br />
Lower activation state of RuBPCO but higher total<br />
carboxylase activity in barley, pea, and wheat at high<br />
elevation suggested responsiveness of the enzyme to<br />
low partial pressures of CO2 at high altitude (Kumar et al.,<br />
2004). While low temperature could stimulate RuBPCO<br />
activity in C4 plant Atriplex (Osmond et al., 1982), neither<br />
high altitude nor chilling could enhance RuBPCO capacity<br />
in C4 plants Bouteloua gracilis and Muhlenbergia<br />
montanum (Pittermann and Sage, 2000, 2001).<br />
Increased phosphoenolpyruvate carboxylase (PEPC)<br />
activity was reported in C3 species Glycine soja with<br />
increase in elevation from about 500 to 3 650 m, though<br />
the implication of this fact was not discussed (Pandey<br />
and Purohit, 1980). Earlier, we reported that crop plants<br />
like barley and wheat when grown at high altitude<br />
significantly increased carboxylase and oxygenase<br />
activities of RuBPCO and activities of PEPC, aspartate<br />
aminotransferase (AspAT), and glutamine synthetase<br />
(GS) as compared to those grown at low altitude (Kumar<br />
et al., 2006). The objective of the present study was to<br />
find the response of some carbon capturing enzymes for<br />
secondary plant products production in altitudinal<br />
ecotypes of wild species, using Rumex nepalensis as the<br />
target plant that has its natural distribution spread along a<br />
wide altitudinal gradient in Himalaya (Bahar, 2002).<br />
Keeping in view the facts that a study has been made to<br />
raise the disease free and efficient genotype of M.<br />
piperita L. Var. Kukarail at high altitude at Purera and in<br />
plains to see the carbon sequestration.<br />
MATERIALS AND METHODS<br />
Plant<br />
Plant tips (5 to 6 inches) with of efficient and disease free M.<br />
piperita L. Var. Kukarail genotype were obtained from the the tissue<br />
culture techniques as described previously (Saxena et al., 2008) in<br />
CIMAP, Lucknow, India. Uniform cuttings were initially planted in<br />
10000 cm 3 earthen pots filled with purified silica sand (Agarwala<br />
and Sharma, 1961), for the development of roots. After 15 days,<br />
rooted cuttings were transferred to 2500 cm 3 pots. The salts used<br />
in nutrient solution culture were purified for Zn (Hewitt, 1952).<br />
Hoagland and Arnon’s (1952) nutrient solution was used in the<br />
experiment except Fe which was supplied as Fe-EDTA. Three pots<br />
each of Zn treatments ranging from 0.0 to 1.0 g Zn ml -1 were<br />
maintained in controlled glass house condition at ambient<br />
temperature (30±5°C) and irradiance (between 800 and 1000 �mol<br />
m -2 s -1 ). The nutrient solution in each treatment was added at<br />
alternate days. With onset of deficiency and toxicity (after 20 days)<br />
growth observation and detailed physiological and biochemical data<br />
with growth attributes were performed.<br />
CO2 exchange rate (PN)<br />
CO2 exchange rate was measured using a computerized portable<br />
photosynthesis system (Srivastava and Misra, 1991) (Model LiCOR<br />
6000, LiCOR, USA).<br />
Chlorophyll (Chl) content<br />
A known mass of leaf tissue (3 rd leaf) was extracted with 80%<br />
acetone and observance was recorded on spectrophotometer (Pye<br />
Unicham PU8610, USA) for determination of Chl a and b according<br />
to Arnon (1949)., and carotenoids were calculated as described by<br />
Deming-Adams(1992).<br />
Growth attributes and Zn analysis<br />
Leaf fresh and shoot dry mass and area (area meter LICOR Li-<br />
3000) were recorded. For tissue elemental analysis 1.g. dried leaf<br />
samples were digested with I N HCl at 60°C for 24 h. Aliquot<br />
samples of the clear digest were diluted with water (10 cm 3 ) and<br />
analyzed for Zn by atomic absorption spectrophotometer (Pye<br />
Unicam SP 2800) (Misra and Sharma, 1991).<br />
For antioxidant reactive peroxidase enzyme assays of peroxidise,<br />
Iso-enzymes of peroxidase, SOD and GGPP enzymes, frozen leaf<br />
samples were ground with a mortar and pestle in extraction buffer<br />
containing 5 ml 0.1 M phosphate buffer (pH 6.8), as described<br />
previously (Shanon et al., 1960) and peroxidise iso-enzymes by<br />
polyacrylamide gel electrophoresis (PAGE). Leaf samples of<br />
Mentha piperita were collected for enzymatic studies from three<br />
different altitudinal locations, including Purera (1,250 m,<br />
32°17'41"N, 7°10'76"E), and of plains ( 950 m, 32°20'47''N,<br />
77°13'17"E). Fully developed young leaves were harvested<br />
between 09:00 and 10:00 h on a clear sunny day and stored in<br />
liquid nitrogen for further use. All the assays were performed in the<br />
Institute’s laboratory at Lucknow. The range for photosynthetic<br />
photon flux density varied from 1500 to 1700 and 2200 to 2500<br />
μmol m -2 s -1 for Purera, and Plains, respectively. Mean monthly day<br />
temperatures during the month of data recording at these localities<br />
were 9.2±2.2 and 22.2±3.2°C, respectively. Further, using 2 g of<br />
fresh chopped leaves at 3rd position, were homogenized with 5 ml<br />
of 0.1 M phosphate buffer (pH 6.8). Each treatment was replicated<br />
3 times and assayed on SDS page electrophoresis. Superoxide<br />
dismutase (SOD) activity was assayed by the method of Henary<br />
et.al. (1976). Geranyl pyrophosphate synthtase (GPP) assayed was<br />
as described previously (Misra and Sharma, 1991). RFPD through<br />
PCR and cDNA analysis were performed as described by Saxena<br />
et al. (2008).<br />
Estimation of essential monoterpene oil(s)<br />
Geranium oil estimation was done by steam distillation of 100 g<br />
freshly plucked leaves in a clevenger’s apparatus (Clevenger,
Figure 1. Disease free plants produced through plant tissue<br />
culture technique in the culture tubes.<br />
(a)<br />
(b)<br />
Figure 2. The plants with full growth in the controlled conditions at<br />
glass hous. (a) At hills cultivation; (b) At plains cultivation.<br />
1928). The oil constituents mainly geraniol, citronellol and other<br />
associated oil contents were determined by gas liquid<br />
chromatography (Perkin –Elemer model 3920 B). The stainless<br />
steel column was packed with 10% carbowax (20 mesh) on<br />
Misra and Srivastava 1985<br />
Figure 3. Effect of peroxidise activity in peppermint cultivated at<br />
hiher altitude- Purera and plains: Series#1 to 4 at hills, and<br />
series#5 to 8 are at plains cultivation.<br />
chromosorb WNAW. Injector and detector temperature was<br />
maintained at 200°C. The flow of H2 was 0.47 cm S -1 data<br />
processing for area % was done on a Hewllet Packard integrator<br />
model HP-33%.<br />
Statistical analysis<br />
The results were statistically analyzed for the least significant<br />
differences (LSD) using the layout of a complete randomized<br />
design (CRD). Further, the results were analyzed for the correlation<br />
coefficient to determine the relationship among the characters<br />
studied, using the relationship Y= a+b x.<br />
RESULTS AND DISCUSSION<br />
Disease free somaclonal variants of Mentha piperita Var.<br />
Kukarail were obtained from tissue culture techniques invitro<br />
from agar medium at controlled condition (Figure 1),<br />
and further planted in plains and at higher altitudes of<br />
CIMAP Resource Center at Purera.<br />
Peroxidase, isoenzymes of peroxidises, increased with<br />
increase in altitude and was higher by 60%, at Purera,<br />
than in plants grown at Plains (Figure 1). Similar trend<br />
was shown by and an antioxidant enzyme - SoD and<br />
activities of GPP (Figure 2). Activity also showed similar<br />
trend with almost double activity c-DNA at Purera as<br />
compared to plains (Figure 3). Three out of four probable<br />
enzymes of antioxidants activities in monoterpene<br />
synthesis during primary plant products and secondary<br />
plant products- the monoterpenes metabolism, including<br />
Peroxidise, iso-enzymes of peroxidises (Figure 5) and<br />
SOD exhibited lower activities at plains, compared to<br />
Purera (Figure 2). The fourth probable enzyme of GPP of<br />
involved in Carbon metabolism monoterpene metabolism,<br />
showed higher activity by 43% at Purera, compared to<br />
plains (Figure 3).<br />
GPP activity, which is associated with the capacity of
1986 Afr. J. Pharm. Pharmacol.<br />
Figure 4. RNA isolated from leaves and shoeing the bands in<br />
electrophoresis.<br />
Figure 5. Native polyacrylamide gel electrophoresis (PAGE): Zn treatment 0.00 to 1.0 Ug/ml. Showed the<br />
peroxidase isoenzyme band profiles in Peppermints cultivation at plains (#1 to 4) and at higher altitudes<br />
of Purera (#5 to 7b and unnumbered the last band).<br />
carbon fixation, may increase with altitude (Chabot et al.,<br />
1972). Temperature drops consistently with altitude and<br />
may impart a major impetus to shape leaf’s<br />
photosynthetic response at high elevation. Response of<br />
GPP to temperature is largely explained by the function<br />
of its activating enzyme, GPP activase which has a low<br />
temperature optimum (Robinson and Portis, 1989; Crafts-<br />
Brandner et al., 1997). RuBPCO activase is instrumental<br />
in maintaining high GPP activity at low temperatures<br />
(Pearcy, 1977). GPP was transformed into GGPP<br />
through GPP synthase enzyme. GPP synthase is<br />
composed of two subunits, one larger subunits and<br />
another of smaller subunits. Here in our studies the plains<br />
cultivated peppermint is composed of only smaller<br />
subunits which showed the lesser activity of GPP of plain<br />
crops (Figure 4). The same trend was also obtained in
Figure 6. Electrophoretic bands after PCR with gene specific primer.<br />
cDNA analysis with RFPD lesser bands then the<br />
cultivations of higher altitudes of Purera (Figure 6). Again<br />
the total photosynthesis, saccarides formation, and total<br />
oil production was 2 folds much higher than the plain<br />
cultivations (Table 1). The same production of<br />
menthofuron was obtained in the higher cultivation of<br />
peppermints. Further, CD values indicated significant<br />
differences at p
1988 Afr. J. Pharm. Pharmacol.<br />
Table 1. Effect of M. pipeperita cultivation on growth parameters.<br />
Harvesting<br />
Growth attribute<br />
Plane side Hill side LSD LSD<br />
1 st Harvest 2 nd Harvest 3 rd Harvest 4 th Harvest 5 th Harvest 6 th Harvest At 5% At 1%<br />
Plant height (cm) 57.0 58.0 61.0* 63.4** 64.1** 59.0 2.5 4.1<br />
No. of branches 9 10* 13** 10* 10* 8 1.1 3.2<br />
Fresh mass (g plant -1 ) 218.8 238.6* 224.8 282.5** 215.5** 196.2 11.1 16.3<br />
Dry mass (g plant -1 ) 14.11 16.33* 16.81* 19.36** 18.46** 15.85 2.10 3.30<br />
Leaf area (cm 2 ) 8.2 12.1* 25.2** 40.3** 37.2** 11.2 3.5 6.2<br />
Chl a (g kg -1 (FM)) 0.68 0.79* 0.94** 1.48** 1.01** 0.82* 0.11 0.15<br />
Chl b (g kg -1 (FM)) 0.50 0.56 0.61* 0.79** 0.40 0.29 0.08 0.12<br />
Chl a/b 1.36 1.41 1.54 1.87 2.53 2.83 - -<br />
PN (μg(CO2) m -2 s -1 ) 0.15 0.19* 0.75** 0.82** 0.71** 0.42** 0.03 0.06<br />
Saccharides (μg (CH2O)<br />
m -2 s -1 )<br />
0.102 0.129 0.510 0.558 0.483 0.286 - -<br />
Oil % 0.35 0.36 0.47* 0.56** 0.46 0.47 0.02 0.04<br />
Menthone % of total oil 21 27** 27** 25** 38** 37** 0.01<br />
Menthol % of total oil 0.59 59 67** 67** 69** 69** 0.01<br />
Menthofuron % of total oil 5 10** 9.** 19** 18** 17** 0.04<br />
Fe (mg kg -1 ) 98 112 142** 537** 419** 312** 21<br />
Mn (mg kg -1 ) 26 37** 41** 98** 62** 53** 9<br />
Zn (mg kg -1 ) 12 19* 34** 64** 41** 36** 7<br />
Cu (mg kg -1 ) 7 9 11** 12** 7 5 3<br />
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.<br />
possible source. Further, these enzymatic alterations<br />
could provide adaptive advantage to plant in order to<br />
conserve carbon at high elevation. The high total oil<br />
contents, menthofuron at higher altitude cropping of<br />
peppermints at Purera leads to value addition. This<br />
higher menthofurn enrich plants at higher altitude than<br />
the plains, leads to control of dangerous smoking and as<br />
a good reliever to smoker if added into the candies.<br />
Further, it is highly available in candies of states, which<br />
have menthofuron as an additive in candies.<br />
REFERENCES<br />
Arnon DI (1949). Copper enzymes in isolated chloroplasts.<br />
Polyphe¬noloxidase in Beta vulgaris. Plant Physiol., 24: 1-15.<br />
Bahar N (2002). Studies on morphological biomass and root characters<br />
of Rumex nepalensis Spreng., in temperate regions of Himalayas.<br />
Indian Forester, 128: 707-708.<br />
Billings WD, Clebsch EEC, Mooney HA (1961). Effects of low<br />
concentrations of carbon dioxide on photosynthesis rates of two<br />
races of Oxyria. Science, 133: 1834.<br />
Chabot BF, Chabot JF, Billings WD (1972). Ribulose-1,5-diphosphate<br />
carboxylase activity in arctic and alpine populations of Oxyria digyna.<br />
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African Journal of Pharmacy and Pharmacology Vol. 5(17), pp. 1990-1995, 8 November, 2011<br />
Available online at http://www.academicjournals.org/AJPP<br />
DOI: 10.5897/AJPP11.172<br />
ISSN 1996-0816 ©2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Simultaneous determination of gatifloxacin and<br />
dexamethasone sodium phosphate in bulk and<br />
pharmaceutical formulations by HPLC<br />
K. R. Sireesha 1 * and K. Prakash 2<br />
1 S. N. Vanitha Pharmacy Mahavidyalaya, Exhibition grounds, Hyderabad (AP), India.<br />
2 Nirmala College of pharmacy, Mangalagiri, Guntur (AP), India.<br />
Accepted 20 September, 2011<br />
A liquid chromatography method was developed and validated for the simultaneous determination of<br />
gatifloxacin and dexamethasone sodium phosphate in bulk and pharmaceutical formulations. Optimum<br />
separation was achieved in less than 5 min using a C18 column (250 × 4.6 mm i.d, 5 µ particle size) by<br />
isocratic elution. UV detection was carried out at 254 nm. Developed method was economical in terms<br />
of the time taken and amount of solvent consumed for each analysis. It was also validated with respect<br />
to linearity, limit of detection, limit of quantification, precision, accuracy, specificity, robustness and<br />
system suitability. The limits of detection for gatifloxacin and dexamethasone sodium phosphate were<br />
0.397 and 0.11 µg/ml, respectively. Limits of quantification were found to be 1.203 and 0.342 µg/ml for<br />
gatifloxacin and dexamethasone sodium phosphate, respectively. The developed method was<br />
successfully applied to the simultaneous determination of gatifloxacin and dexamethasone sodium<br />
phosphate in bulk and pharmaceutical formulations.<br />
Key words: Gatifloxacin, dexamethasone sodium phosphate, high performance liquid chromatography (HPLC),<br />
isocratic elution.<br />
INTRODUCTION<br />
Dexamethasone sodium phosphate (DSP) is a highly<br />
selective glucocorticoid which is widely used in ocular<br />
inflammatory diseases. It’s chemical name is 9- fluoro-<br />
11b, 17, 21-trihydroxy-16α- methylpregna-1,4- diene-<br />
3,20-dione 21-(dihydrogen phosphate) disodium salt<br />
(Indian Pharmacoepia, 2007; Balaji et al., 2008).<br />
Gatifloxacin (GFN) is a fourth generation fluoroquinolone<br />
antibiotic used in bacterial infections. It is chemically 1cyclopropyl-6-fluoro-<br />
1,4- dihydro-8-methoxy-7-(3methylpiperazin-1-yl)-<br />
4-oxo-3-quinoline carboxylic acid<br />
(USP, 1995; Sayed et al., 2011). Dexamethasone in<br />
combination with Gatifloxacin is used in several antiinfective<br />
eye preparations to treat acute and sub acute<br />
conjunctivitis caused by susceptible strains of the<br />
following aerobic gram positive and negative bacteria<br />
such as Staphyloccocus aureus, Staphyloccocus<br />
*Corresponding author. E-mail: rupak1@rediffmail.com. Tel:<br />
08686654459.<br />
epidermidis, Streptococcus pneumonia and Haemophilus<br />
influenza.<br />
In the literature, methods were described for the<br />
individual estimation of fluoroquinolones and<br />
dexamethasone in aqueous samples and biological fluids<br />
by liquid chromatography (Chen et al., 2006; Hyung and<br />
Donald, 1995) liquid chromatography-fluorescence<br />
detection (Joana et al., 2011). A few methods were also<br />
given for the simultaneous determination of<br />
Dexamethasone with other drugs such as<br />
Chloremphenicol (Iqbal et al., 2006), ciprofloxacin (Rele<br />
et al., 2010) and ofloxacin (Ali et al., 2002).But simulta-<br />
neous determination of these drugs in pharmaceutical<br />
formulations has not been reported in the literature. So<br />
an attempt was made to develop a HPLC method for the<br />
estimation of these drugs available as eye drops.<br />
The purpose of the present study was to develop a<br />
simple, sensitive, precise and accurate HPLC method for<br />
simultaneous determination of GFN and DSP in bulk and<br />
pharmaceutical formulations. The developed method has<br />
been validated (USP, 1995; ICH Q2B 2003) by evaluation
of the system suitability, specificity, linearity, limit of<br />
detection and quantitation, precision, accuracy and<br />
recovery. The validated method was applied to the<br />
commercially available pharmaceutical formulations<br />
containing both the drugs.<br />
MATERIALS AND METHODS<br />
DSP and GFN were obtained as gift samples from Ajanta Pharma<br />
Ltd, Mumbai. HPLC grade acetonitrile was purchased from SD<br />
Fine-chemicals, India. Triple distilled water was used during the<br />
study. The pharmaceutical formulations containing 3 mg/ml of GFN<br />
and 1 mg/ml DSP (ZIGAT-D eye drops, FDC proxima, India.) were<br />
purchased from local market.<br />
Instrumentation<br />
A high performance liquid chromatograph (Shimadzu-10 AT VP)<br />
equipped with two pumps (Model-10AT VP) and Shimadzu UV-<br />
Visible detector (SPD-10AT VP), ultrasonic bath (Spincotech Pvt.<br />
Ltd, India).<br />
Chromatographic conditions<br />
For chromatographic analysis, a Hypersil C18, (250 × 4.6 mm i.d, 5<br />
µ particle size) was used. Separation was carried out by isocratic<br />
elution. The mobile phase consisting of a mixture of mixed<br />
phosphate buffer (pH 6.8) and acetonitrile (ACN) in the ratio of<br />
60:40 was filtered under vacuum from 0.45 membrane filter and<br />
degassed in ultrasonic bath for 30 min before passing through the<br />
instrument. The injection volume was 20 µl and the flow rate was 1<br />
ml/min. UV detection was carried out at 254 nm.<br />
Preparation of standard solution<br />
Stock standard solutions of GFN and DSP were prepared in the<br />
mobile phase at a concentration of 1200 and 400 µg/ml. The<br />
working standard solutions were prepared by serial dilution of stock<br />
solutions with the mobile phase.<br />
Sample preparation<br />
Sample solutions of GFN and DSP were prepared at a<br />
concentration of 1200 and 400 µg/ml by diluting 10 ml of the<br />
ophthalmic solution to 25 ml with the mobile phase. From this, 1 ml<br />
was taken and diluted to 10 ml to get a concentration of 120 and 40<br />
µg/ml of GFN and DSP.<br />
Validation<br />
The developed analytical method was validated as per International<br />
Conference on Harmonization (ICH) and United States<br />
Pharmacopeia (USP) guidelines for the parameters like linearity,<br />
limit of detection (LOD), limit of quantification (LOQ), precision,<br />
specificity, accuracy, robustness and system suitability.<br />
Linearity<br />
Six working standard solutions of each analyte in the concentration<br />
range of 24 to 144 µg/ml for GFN and 8 to 48 µg/ml for DSP was<br />
prepared in triplicate and injected. Calibration curves were<br />
constructed by plotting concentration versus mean peak area.<br />
Limits of detection and quantification<br />
Sireesha and Prakash 1991<br />
Limits of detection (LOD) and quantification (LOQ) were calculated<br />
based on the standard deviation of the response and slope of the<br />
calibration curve. LOD and LOQ are calculated from the formulae<br />
3.3 and 10 σ/s, respectively, where σ is the standard deviation of yintercepts<br />
of the regression line and s is the slope of the calibration<br />
curve.<br />
Precision<br />
Method precision<br />
The precision of the method was evaluated in terms of intermediate<br />
precision, that is, intra-day and inter-day precision and by different<br />
analysts. For intra-day precision, three different concentrations of<br />
GFN and DSP in the linearity range were prepared in triplicate and<br />
were analyzed during the same day. For inter-day precision the<br />
same concentrations were analyzed on three consecutive days.<br />
The relative standard deviations (RSD) values for GFN and DSP<br />
showed that the precision of the method was satisfactory.<br />
System precision<br />
System precision was analyzed by injection repeatability. This was<br />
examined by analyzing six injections of the mixture containing GFN<br />
and DSP at 120 and 40 µg/ml, respectively. The RSD were<br />
calculated from the peak areas and retention times of GFN and<br />
DSP.<br />
Accuracy<br />
Accuracy of the method was determined by recovery studies. These<br />
studies were carried out by addition of known amounts of GFN and<br />
DSP to a sample solution of known concentration and comparing<br />
calculated and measured concentrations. A sample solution<br />
containing GFN and DSP (1.2 and 0.4 mg/ml, respectively) was<br />
prepared by diluting 10 of the ophthalmic solution to 25 ml in<br />
volumetric flask and make-up of the solution with the mobile phase,<br />
samples (0.4 ml) of the filtered solution were transferred to 10 ml<br />
volumetric flasks containing 0.4, 0.6 and 0.8 ml of GFN and DSP<br />
standard solution, dilutions were made and analyzed. The<br />
percentage recovery and the percentage RSD was calculated and<br />
found to be within the limits.<br />
Specificity<br />
Specificity of an analytical method may be defined as the ability of<br />
the method to measure accurately and specifically the analyte in<br />
presence of additional components, such as matrix, impurities,<br />
degradation products and other related substances.<br />
Robustness<br />
Robustness of the method is a measure of capacity of the method<br />
to remain unaffected by small but deliberate variations in method<br />
parameters and provides an indication of the variability during<br />
normal usage. Robustness of the method was evaluated by varying<br />
method parameters, such as detection wavelength and flow rate.<br />
Detection wavelength was changed from 254 nm to 254 ± 2 nm and<br />
flow rate was changed from 1 ml/min to 1 ± 0.1 ml/min. Effect of<br />
these changed parameters was studied by injecting the sample into<br />
the system.
1992 Afr. J. Pharm. Pharmacol.<br />
System suitability<br />
Voltage (mV)<br />
50<br />
Figure 1. Chromatogram for blank.<br />
System suitability was established in order to determine the adequate<br />
resolution and reproducibility of the proposed method.<br />
Suitability parameters including retention factor, resolution,<br />
asymmetry factor and plate number were investigated.<br />
Assay of the marketed formulation<br />
The developed method was applied to the simultaneous<br />
determination of GFN and DSP in pharmaceutical formulations.<br />
Sample was analyzed by performing six independent<br />
determinations and each series was injected in triplicate.<br />
RESULTS AND DISCUSSION<br />
Mobile phase optimization<br />
Chromatographic parameters were optimized to develop<br />
a HPLC method for simultaneous determination of GFN<br />
and DSP with short analysis time (< 5 min) and<br />
acceptable resolution (RS > 2). Various compositions of<br />
mobile phases like methanol: buffer and ACN: buffer in<br />
different ratios were tried.<br />
But with mixed phosphate buffer (pH 6.8) and ACN in<br />
the ratio of 60:40 at a flow rate of 1 ml/min symmetrical<br />
peaks with good resolution were obtained.<br />
Chromatogram for the mobile phase (blank<br />
chromatogram) is shown in (Figure 1) and shows no<br />
interference with the drug peaks. The optimum<br />
wavelength for detection was set at 254 nm at which<br />
Time (min)<br />
better detector response for both drugs was obtained.<br />
The retention times were 2.42 and 4.81 min for GFN and<br />
DSP, respectively (Figure 2).<br />
Validation<br />
Calibration graphs were constructed by plotting the peak<br />
area versus their corresponding concentrations. Good<br />
linearity was obtained in the range of 24 to 144 µg/ml and<br />
8 to 48 µg/ml for GFN and DSP. The results are shown in<br />
Table 1. LOD and LOQ were calculated from the slope<br />
and standard deviation y-intercepts of the regression line<br />
of the calibration curve. For GFN it was found to be 0.397<br />
and 1.203 µg/ml and for DSP 0.11 and 0.342 µg/ml,<br />
respectively. The precision of the method and instrument<br />
precision was evaluated and relative standard deviation<br />
(RSD) values were calculated. The RSD values for GFN<br />
and DSP showed that the precision of the method was<br />
satisfactory. The results are shown in Table 2. The<br />
accuracy of the method was determined by recovery<br />
studies. The recoveries were close to 100% for GFN and<br />
DSP; the results are as shown in Table 3. Developed<br />
method was found to be robust when the detection<br />
wavelength and flow rate was changed from 254 to 254 ±<br />
2 nm and 1 to 1 ± 0.1 ml/min. There was no considerable<br />
change in the peak areas and retention times. Using 0.9<br />
ml/min flow rate, the retention time for GFN and DSP<br />
were found to be 2.69 and 5.34 min, respectively and<br />
with 1.1 ml/min flow rate, retention times for GFN and<br />
DSP were found to be 2.21 and 4.41 min, respectively
Voltage (mV)<br />
Time (min)<br />
Figure 2. Typical chromatogram for the standard solution of GFN and DSP.<br />
Table 1. Linearity by regression analysis (n = 6).<br />
Substance R 2 Slope Concentration range (µg/ml)<br />
GFN 0.999 17.94 24-144<br />
DSP 0.999 20.56 8-48<br />
‘n’ is the number of determinations.<br />
Table 2. Precision (% RSD).<br />
Table 3. Recovery studies (n = 6).<br />
Drug<br />
GFN<br />
DSP<br />
Parameter GFN DSP<br />
Intra-day precision 0.033 0.096<br />
Inter-day precision 1.78 1.39<br />
Analyst precision 0.08 0.19<br />
Injection repeatability for tR 0 0.027<br />
Injection repeatability for peak area 0.12 0.22<br />
‘n’ is the number of determinations and RSD is relative standard deviation.<br />
Concentration<br />
(µg/ml)<br />
Amount recovered<br />
(µg/ml)<br />
Sireesha and Prakash 1993<br />
Recovery (%) RSD (%)<br />
96 95.94 99.94 0.037<br />
120 119.65 99.71 0.04<br />
144 143.86 99.9 0.052<br />
32 31.97 99.26 0.05<br />
40 39.86 99.65 0.426<br />
48 45.85 99.68 0.29<br />
‘n’ is the number of determinations and RSD is relative standard deviation.
1994 Afr. J. Pharm. Pharmacol.<br />
Voltage (mV)<br />
Table 4. System suitability parameters (n = 6).<br />
Parameter GFN DSP<br />
Retention time (tR) 2.42 4.81<br />
Asymmetry factor 1.7 1.2<br />
Resolution - 13.2<br />
Number of plates 3735 8926<br />
‘n’ is the number of determinations.<br />
Time (min)<br />
Figure 3. Chromatogram for the sample solution of GFN and DSP.<br />
Table 5. Assay of eye drops (n = 6).<br />
Drug Label claim (mg/ml)<br />
Amount found<br />
(mg/ml)<br />
Mean recovery (%) RSD (%)<br />
GFN 3 3 99.7 ± 0.8 0.034<br />
DSP 1 0.99 99.44 ± 0.4 0.032<br />
‘n’ is the number of determinations and RSD is relative standard deviation.<br />
without affecting the resolution of the drugs. When<br />
detection wavelength was changed to 254 ± 2 nm, the<br />
retention time for GFN and DSP were not changed from<br />
the normal. System suitability parameters are shown in<br />
Table 4.<br />
Assay of the marketed formulation<br />
According to ICH in the case of assay, demonstration of<br />
specificity requires that the procedure is unaffected by<br />
the presence of impurities or excipients. The assay value<br />
of the marketed formulation was found to be within the<br />
limits. The low RSD value indicated suitability of this<br />
method for routine analysis of GFN and DSP in<br />
pharmaceutical dosage forms. Chromatogram of the<br />
sample shows that there was no interference from the<br />
excipients present in the formulation (Figure 3); this<br />
indicates the specificity of the method. The results are<br />
shown in Table 5.
Conclusion<br />
The method described in this paper for the simultaneous<br />
estimation of GFN and DSP are found to be simple,<br />
sensitive, accurate, precise, rapid, robust and<br />
economical. The analytical conditions and the solvent<br />
system developed provided good resolution within a short<br />
analysis time. The RSD for all parameters was found to<br />
be within the limits, which indicates the validity of method<br />
and assay results obtained by this method are in fair<br />
agreement. Thus, the developed method can be<br />
proposed for routine analysis of GFN and DSP in<br />
laboratories and for quality control purposes.<br />
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African Journal of Pharmacy and Pharmacology Vol. 5(17), pp. 1996-2001, 8 November, 2011<br />
Available online at http://www.academicjournals.org/AJPP<br />
DOI: 10.5897/AJPP11.190<br />
ISSN 1996-0816 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Anti-inflammatory and wound healing activity of<br />
Fagonia schweinfurthii alcoholic extract herbal gel on<br />
albino rats<br />
Saleh I. Alqasoumi 1,2 , Hasan S. Yusufoglu 1 * and Aftab Alam 1<br />
1 Department of Pharmacognosy, College of Pharmacy, Al Kharj University, Al Kharj, Kingdom of Saudi Arabia.<br />
2 Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Kingdom of Saudi Arabia.<br />
Accepted 21 September, 2011<br />
Traditionally, a large number of plants are used for treatment of inflammation and wounds. In Asian and<br />
African countries Fagonia schweinfurthii (Hadidi) (F. Zygophyllaceae) and the closely related herb, such<br />
as Fagonia arabica are traditionally used for treatment of inflammation, open wounds, boils, skin<br />
eruptions, allergies, etc. Hence, the present study was conducted to investigate the anti-inflammatory<br />
and wound healing effects of 90% alcoholic extract of F. schweinfurthii formulated gel on carrageenan<br />
induced rats paw edema and excision wound model, respectively. The effects were compared with the<br />
anti-inflammatory diclofenac sodium ointment (Diclomax ® ) and the wound healing povidone-iodine<br />
(Betadine ® ) drugs. The herbal gels and diclofenac sodium ointment were topically applied (0.5 g) to the<br />
planter surface of the left hind paw and anti-inflammatory effect was observed within 3 h. The wound<br />
healing effect was investigated by application of 0.5 g/wound of the F. schweinfurthii gel and Betadine ®<br />
once daily for 19 days to the excision wound of albino rats and was observed at 4 days intervals. It was<br />
observed that gel formulations have progressive anti-inflammatory effect and accelerate the wound<br />
closer time. This study suggests that F. schweinfurthii plant extract gel formulation could be developed<br />
as a therapeutic agent for anti-inflammatory and wound healing effects.<br />
Key words: Fagonia schweinfurthii, herbal formulation, anti-inflammatory, wound healing.<br />
INTRODUCTION<br />
Fagonia schweinfurthii and its closely related species are<br />
widely distributed in deserts and dry areas of India to<br />
tropical Africa and Chile to South West U.S.A. They are<br />
traditionally well known for the treatment of hemorrhoids,<br />
inflammation, sores, leprosy, open wounds and fever in<br />
the form of internal and external conventional formulation<br />
(Miller et al., 1988). When the powder that is made up of<br />
the whole plant of F. schweinfurthii is dusted on boils and<br />
skin eruptions, it causes healing and when the whole<br />
plant is boiled in water, its bath is useful for allergies and<br />
other skin diseases and decoction is given orally as blood<br />
purifier (Qureshi et al., 2010). The other species like<br />
Fagonia bruguieri aqueous extract is claimed for anti-<br />
*Corresponding author. E-mail: hasanagazar@hotmail.com.<br />
Tel: +966-1-5886025, +966-535159935. Fax: +966-1-5886001.<br />
allergy (Abdulaziz and Hussein, 2007). Fagonia cretica<br />
methanol extract is claimed for good antimicrobial<br />
potential (Anjum et al., 2007) and it exhibited strong free<br />
radical scavenging properties against reactive oxygen<br />
and nitrogen species (Rawal et al., 2004). The other<br />
effects of Fagonia species include anti-inflammatory,<br />
analgesic, antipyretic and thrombolytic activity (Prasad et<br />
al., 2007; Satpute et al., 2009). Many chemical<br />
constituent's, such as triterpenoids, saponins, flavonoid<br />
glycosides, etc., have already been reported in different<br />
Fagonia spp. (Shaker et al., 1999; Abdel-Khalik et al.,<br />
2001; El-Wakil, 2007). Although, currently used antiinflammatory<br />
and wound healings drugs are associated<br />
with some severe side effects, herbal products are often<br />
perceived as safe, because they are natural therefore,<br />
the development of potent anti-inflammatory and wound<br />
healer drugs with fewer side effects is necessary (Gesler,<br />
1992). Inflammation is considered as a primary<br />
physiologic defense mechanism that helps body to
protect itself against infection, burn, toxic chemicals,<br />
allergens or other noxious stimuli. An uncontrolled and<br />
persistent inflammation may act as an etiologic factor for<br />
many of the chronic illnesses (Kumar et al., 2004).<br />
Wound healing is the process of repairing injury of skin<br />
and other soft tissue. This dynamic process is classically<br />
divided into three overlapping phase “inflammation,<br />
proliferation and remodeling” (Harding et al., 2002). Initial<br />
stages of wound healing involve an acute inflammatory<br />
phase followed by synthesis of collagen and other<br />
extracellular matrix which are later remodeled to form<br />
scar (Bhagavathula et al., 2009). Thus, the present study<br />
aim to investigate wound healing and anti-inflammatory<br />
activities to justify the traditional claims of this plant gel<br />
form.<br />
MATERIALS AND METHODS<br />
Collection of plant and authentication<br />
The plants of F. schweinfurthii was collected during December,<br />
2009, from local area of Riyadh, Saudi Arabia and was kindly<br />
authenticated by Dr. Mohammad Atiqur Rahman, taxonomist of the<br />
Medicinal, Aromatic and Poisonous Plants Research Center<br />
(MAPPRC), College of Pharmacy, King Saud University, Riyadh,<br />
Saudi Arabia. A voucher specimen is deposited at the herbarium of<br />
the College of Pharmacy, King Saud University.<br />
Chemicals<br />
Diclofenac sodium (1%) ointment (Diclomax ® ) and 10% povidoneiodine<br />
(Betadine ® ) were purchased from a local pharmacy.<br />
Triethanolamine and ethanol were obtained from Merck & Co. Inc<br />
(USA). Carrageenan and carbapol-934 gel were supplied by Sigma<br />
(USA).<br />
Extraction of plant<br />
The air dried powdered plant was extracted with 90% ethanol in a<br />
soxhlet apparatus at 60°C. The extract was concentrated to syrupy<br />
solution using rotary evaporator under reduced pressure at 40°C.<br />
The thick solution was lyophilized using freeze drying system. The<br />
yield (13. 5%) was used for the experimental studies.<br />
Animals<br />
Healthy Wister albino rats of either sex weighing ≈200 g, obtained<br />
from the Experimental Animal Care Center, College of Pharmacy,<br />
King Saud University, Riyadh were used. Rats were maintained<br />
under controlled condition at temperature (22 ± 20°C), humidity<br />
(55%) and light (12 h light/dark condition). The animals were<br />
provided with Purina chow and drinking water ad libitum. The<br />
experiments and procedure used in this study were approved by<br />
the Ethical Committee of the College of Pharmacy, King Saud<br />
University, Riyadh, KSA.<br />
Acute toxicity study<br />
The acute toxicity of F. schweinfurthii alcoholic extract was<br />
determined in rats according to the previous method (Adedapo et<br />
Alqasoumi et al. 1997<br />
al., 2009) with slight modifications. Rats that fasted for 12 h were<br />
randomly divided into four groups (n = 5). Graded doses of the<br />
extract (300, 600, 1200 and 2400 mg/kg p.o.) were separately<br />
administered to the rats. All the animals were then observed over a<br />
period of 10 days for deaths and signs of acute toxicity.<br />
Preparation of herbal gel<br />
Herbal gels 10 and 20% were prepared separately according to the<br />
method (Dey et al., 2009) with slight modifications. Carbapol-934<br />
(1.8%) and sufficient amount of distilled water were mixed in a<br />
separate beaker and soaked for 24 h at room temperature.<br />
Triethanolamine was added drop-wise with constant stirring using<br />
mechanical stirrer. After gel formulation, a weighed amount of (10<br />
and 20 g) extract powder was incorporated in gelling agent<br />
separately and mixed using glass rod. A similar procedure was<br />
followed for control base gel without powdered extract.<br />
Anti-inflammatory study<br />
Carrageenan-induce rat paw edema model was used for antiinflammatory<br />
study (Maswadeh et al., 2006). Twenty rats were<br />
divided into four groups and fasted overnight with free access to<br />
water before the experiment proceeds. Rats of the first, second and<br />
third groups were treated with the control base, 10 and 20% gel<br />
formulations. Rats of the forth group (standard) were treated with<br />
marketed gel formulation (Diclomax®). Each formulation (0.5 g)<br />
was applied to the planter surface of the left hind paw by gently<br />
rubbing 50 times with the index finger. After 1 h, inflammation was<br />
induced by subplanter injection of 0.1 ml of 1% carrageenan<br />
solution in normal saline into the treated paw of all rats. The paw<br />
volume of each rat was measured in milliliter using a<br />
plethysmometer (Aptex, France) at 0 and 3 h post carrageenan<br />
injections. The percentage anti-inflammatory activity was calculated<br />
using the following equation:<br />
Percentage anti-inflammatory activity = (V3h - V0h)/V0h × 100<br />
where V3h is the paw volume after 3 h carrageenan injection and V0h<br />
is the initial paw volume.<br />
Wound healing study<br />
The excision wound model was used to study the wound<br />
contraction of F. schweinfurthii extract herbal formulations (Okoli et<br />
al., 2009). At the beginning of the experiment, twenty rats were<br />
anesthetized using diethyl ether and the dorsal skin of each rat was<br />
shaved with an electric clipper and put in separate cage. After 12 h,<br />
all animals were again anesthetized by diethyl ether and the shaved<br />
areas were sterilized with 70% alcoholic solution and sketch wound<br />
area (≈ 2.5 cm 2 ). A predetermined dorsal area was excised using<br />
toothed forceps, scalpel and pointed scissors. A fresh surgical<br />
blade was used for the perpendicular cut in each animal and the<br />
tension of the skin was kept constant during the procedure. Animals<br />
were divided into four groups (n = 5). Rats of the first, second and<br />
third groups were treated with the control base, 10 and 20% gel<br />
formulations. Rats of the forth group (standard) were treated with<br />
marketed formulation (Betadine®). The base gel, extract gels and<br />
standard drug (0.5 g, each) were applied topically on the wound<br />
surface once a day for 19 days. The wound areas were traced on<br />
graph paper (1 mm 2 ) immediately after the wound excision and<br />
every 4 days until healing was accomplished. The reduction in the<br />
wound size was calculated according to the following formula:<br />
Wound contraction (%) = [(W0 - Wt)/W0] × 100
1998 Afr. J. Pharm. Pharmacol.<br />
Table 1. Effect of Fagonia alcoholic extract gel formulations on Anti-inflammatory.<br />
Treatment<br />
Net increase in paw volume<br />
(ml)<br />
Reduction of edema<br />
(%)<br />
Control base 2.28 ± 0.06 -<br />
10% gel 1.88 ± 0.08* 17.54<br />
20% gel 1.10 ± 0.10*** 51.75<br />
Diclomax ® 0.44 ± 0.06*** 80.70<br />
Each value is the mean ± SEM, n = 5. Values differ significantly (*P < 0.05 and ***P<<br />
0.001) from control base gel.<br />
where W 0 is the wound diameter on day zero and W t is the wound<br />
diameter on day t.<br />
The time taken for 50% of wound closure (WC50) was calculated<br />
by a plot of percentage wound closure against days.<br />
Statistical analysis<br />
The results were expressed as mean ± SEM. The data were<br />
subjected to one-way ANOVA student t-test using graphPad Prism<br />
5 software. P < 0.001 was considered as significant.<br />
RESULTS AND DISCUSSION<br />
Animal toxicity study<br />
Oral administration of graded doses (300 to 2400 mg/kg<br />
p.o.) of the alcoholic extract of F. schweinfurthii to rats,<br />
did not produce any mortality and changes in behavior,<br />
breathing, cutaneous effects, sensory nervous system<br />
responses or gastrointestinal effects during the 10 days<br />
observation period. The obtained results signify that the<br />
use of the plant for treatment is safe. So the formulations<br />
(10 and 20% gel) were selected for anti-inflammatory and<br />
wound healing studies.<br />
Anti-inflammatory study<br />
The anti-inflammatory activity was expressed as "mean<br />
increase in paw volume ± SEM" in terms of milliliter and<br />
percentage inhibition in paw volume by different gel<br />
formulation and standard drug. The 20% herbal gel<br />
produced significant (P < 0.001) reduction of<br />
Carrageenan-induced paw edema (1.10 ± 0.10 ml) as<br />
compared to the control base gel group (2.28 ± 0.06 ml)<br />
after 3 h from carrageenan injection. The reference drug<br />
was found to be comparatively more potent as compared<br />
to formulated gel (0.44 ± 0.06 ml). The percentage<br />
inhibition of paw edema for 10 and 20% and Diclomax ®<br />
was 17.54, 51.75 and 80.70%, respectively (Table 1).<br />
The probable mechanism of inflammation action is biphasic,<br />
the first phase is attributed to the release of<br />
histamine, serotonin, 5-HT and kinins in the first hour;<br />
while, the second accelerating phase of swelling is<br />
related to the release of prostaglandin, bradykinins and<br />
lysozymes-like substances in 2 to 3 h (Brooks and Day,<br />
1991; Silva et al., 2005). Carrageenan-induced edema<br />
involves the synthesis or release of pain and fever<br />
mediators like prostaglandins (PGE), histamine,<br />
bradykinins, leucotrienes and serotonin. This model has a<br />
significant predictive test for anti-inflammatory agents<br />
acting by the mediators of acute inflammation (Adedapo<br />
et al., 2009). The results of this study indicated that F.<br />
schweinfurthii extract gel formulation can be effective in<br />
acute inflammatory disorders. The plant contains<br />
polyphenolic compounds, saponins and flavonoid<br />
glycosides that could be responsible for the antiinflammatory<br />
activity either alone or may be due to<br />
inhibition of inflammatory mediators in combination with<br />
other constituents (Adamu et al., 2007).<br />
Wound healing study<br />
The percentage of wound contraction in the second, third<br />
and fourth groups were 6.93 ± 0.11, 8.40 ± 0.20 and 9.06<br />
± 0.11 on the 4 th day and 90.53 ± 0.10, 93.93 ± 0.09 and<br />
95.53 ± 0.10 on the 19th day, respectively. The<br />
percentage of wound contraction of the first group 6.13 ±<br />
0.23 and 72.08 ± 0.16 on 4th and 19th days, respectively<br />
was comparatively lower than the other three groups<br />
(Table 2). The WC50 (time for 50% wound healing) of<br />
control base, 10 and 20% herbal gel and standard drug<br />
(Betadine®) was, 13.92, 11.06, 10.53 and 10.82%,<br />
respectively (Table 3). The wound size decreases after<br />
4th (A) and 19th (B) day of 10 and 20% gel and<br />
Betadine® was clearly indicating the wound healing<br />
effects (Figures 1 to 3). Wound healing is an elementary<br />
response to tissue injury that it consequences in<br />
restoration of tissue integrity, is mainly achieved by the<br />
synthesis of the connective tissue matrix. It involves<br />
regeneration of specialized cells by proliferation of<br />
surviving cells characterized by the formation of<br />
granulation tissue and wound contraction, which is largely<br />
due to the action of myofibroblasts (Okoli et al., 2009). All<br />
groups show the wound healing, including control base<br />
gel, because of biological response regulating the body's<br />
own cellular defense mechanisms which contributes in
Table 2. Effect of Fagonia alcoholic extract gel formulations on wound healing.<br />
Alqasoumi et al. 1999<br />
Treatment<br />
4-days<br />
Percentage wound contraction (mean ± SEM)<br />
8-day 12 days 16 days 19 days<br />
Control base 6.13 ± 0.23 17.96 ± 0.03 48.93 ± 0.11 55.33 ± 0.11 72.08 ± 0.16<br />
10% gel 6.93 ± 0.11 32.93 ± 0.16 61.33 ± 0.23 80.40 ± 0.20 90.53 ± 0.10<br />
20% gel 8.40 ± 0.20 36.60 ± 0.17 64.66 ± 0.11 83.46 ± 0.11 93.93 ± 0.09<br />
Betadine ® 9.06 ± 0.11 24.46 ± 0.09 66.93 ± 0.11 85.86 ± 0.11 95.53 ± 0.10<br />
Each value is the mean ± SEM, n = 5. Values of percentage wound reduction of each formulation differ when compared with<br />
control base gel.<br />
Table 3. Effect of Fagonia alcoholic extract gel<br />
formulations on WC50.<br />
Treatment WC50 (Days)<br />
Control base 13.92<br />
10% gel 11.06<br />
20% gel 10.53<br />
Betadine ® 10.82<br />
Each value is the time taken for 50% wound closure (WC50).<br />
Values of WC50 of each formulation differ when compared<br />
with control base gel.<br />
Figure 1. Excision wound healing of 10% Fagonia alcoholic<br />
extract gel formulation treated rat on 4th (A) and 19th (B) days<br />
observation.<br />
Figure 2. Excision wound healing of 20% Fagonia lcoholic extract<br />
gel formulation treated rat on 4th (A) and 19th (B) days<br />
observation.
2000 Afr. J. Pharm. Pharmacol.<br />
Figure 3. Excision wound healing effect of 10% povidone-iodine (Betadine ® ) treated<br />
rat on 4th (A) and 19th (B) days observation.<br />
wound healing and its repair (Malviya and Jain, 2009).<br />
The formulated gel fasten the wound healing process<br />
may be due to enhancing the cellular defense<br />
mechanisms, proliferation, suppression of inflammation<br />
and contraction of the collagen tissue and could be<br />
delayed by reactive oxygen species or microbial infection<br />
(Marwah et al., 2007). The polyphenolic compounds have<br />
improved regeneration and organization of the new tissue<br />
and hasten the wound healing process (Leite et al., 2002)<br />
may be due to anti-inflammation, anti-oxidant and<br />
antimicrobial activities. The plant contains polyphenolic<br />
compounds including saponins, flavonoids, glycosides<br />
(Kumar et al., 2004; Abdel-Khalik et al., 2001; Gesler,<br />
1992) and these compounds showed the antioxidant and<br />
anti-microbial (Anjum et al., 2007; Rawal et al., 2004) and<br />
anti-inflammatory (present study). The result of the<br />
present study revealed that the topical application of 10<br />
and 20% extract gel on the experimentally excised wound<br />
accelerate the wound healing process.<br />
Conclusion<br />
In conclusion, the plant is safe for use as no mortality<br />
was recorded in the acute toxicity test. The two gel (10<br />
and 20%) prepared with F. schweinfurthii reduced<br />
significantly the formation of edema induced by<br />
carrageenan and exhibited a good anti-inflammatory<br />
effect comparable to those of Diclomax ® and exhibited a<br />
good wound healing effect comparable to those of<br />
Betadine®. The study has thus, provided some<br />
rationalization for the folkloric use of the plant in several<br />
communities for conditions, such as inflammation, boils,<br />
skin eruptions and other skin diseases.<br />
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Abdel-Khalik SM, Miyase T, Melek FR, el-Ashaal HA (2001). Further<br />
saponins from Fagonia cretica. Die. Pharmazie., 56(3): 247-250.<br />
Abdulaziz AM, Hussein EK (2007). Fagonia bruguieri freeze-dried<br />
extract as anti-allergic treatment. Int. Appl., No. PCT/IB2005/003712 .<br />
Adamu A, Abdurahman EM, Ibrahim H, Abubakar MS, Magaji MG, Yaro<br />
AH (2007). Effect of aqueous methanolic stem bark extract of Maerua<br />
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Adedapo AA, Sofidiya MO, Afolayan AJ (2009). Anti-inflammatory and<br />
analgesic activities of the aqueous extracts of Margaritaria discoidea<br />
(Euphorbiaceae) stem bark in experimental animal models. Rev. Biol.<br />
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Anjum MI, Ahmed E, Jabbar A, Malik A, Ashraf M, Moazzam M, Rasool<br />
MA (2007). Antimicrobial constituents from Fagonia cretica. J. Chem.<br />
Soc. Pak., 29(6): 634-639.<br />
Bhagavathula N, Warner RL, DaSilva M, McClintock SD, Barron A,<br />
Aslam MN, Johnson KJ, Varani J (2009). A combination of curcumin<br />
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Brooks PM, Day RO (1991). Non steroidal anti-inflammatory Drugs<br />
difference and similarities. N. Engl. J. Med., 324(24): 1716- 1725.<br />
Dey S, Mazumdar B, Patel JR (2009). Enhanced percutaneous<br />
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J. Pharm. Sci. Drug. Res., 1(1): 13-18.<br />
El-Wakil EA (2007). Phytochemical and molluscicidal investigations of<br />
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Gesler WM (1992). Therapeutic landscapes: medical issues in light of<br />
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Harding KG, Morris HL, Patel GK (2002). Clinical Review: Science,<br />
Medicine and the future healing chronic wounds. B.M.J. 324: 160-<br />
163.<br />
Kumar V, Abbas AK, Fausto N (2004). Robbins and Cotran (Ed),<br />
Pathologic basis of disease, 7th edition, Elsevier Saunders,<br />
Philadelphia, Pennsylvania, pp. 47‐ 86.<br />
Leite SN, Palhano G, Almeida S, Biavatti MW (2002). Wound healing<br />
activity and systemic effects of Vernonia scorpioides extract in guinea<br />
pig. Fitoterapia, 73(6): 496-500.<br />
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Maswadeh HM, Semreen MH, Naddaf AR (2006). Anti-inflammatory<br />
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Okoli CO, Eziken AC, Akah PA, Udegbunam SO, Okoye TC, Mbanu<br />
TP, Ugwu E (2009). Studies on Wound Healing and Antiulcer<br />
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African Journal of Pharmacy and Pharmacology Vol. 5(17), pp. 2002-2006, 8 November, 2011<br />
Available online at http://www.academicjournals.org/AJPP<br />
DOI: 10.5897/AJPP11.229<br />
ISSN 1996-0816 ©2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
The effects of maximal aerobic exercise on cortisol and<br />
thyroid hormones in male field hockey players<br />
Malik BEYLEROGLU<br />
School of Physical Education and Sports, Sakarya University, Sakarya, Turkey. E-mail: malik-beyleroglu@hotmail.com.<br />
Tel: + 90 264 2956646. Fax: + 90 264 2956642.<br />
Accepted 20 July 2011<br />
Some metabolic and endocrine functions might be influenced by exercise and it leads to changed<br />
concentrations of hormone secretions in humans. In order to determine the changes of thyroid<br />
hormones (THs) related to shuttle run exercise (SRE), 14 field hockey players and their thyroid<br />
stimulating hormones (TSH) free T3 (fT3), free T4 (fT4) were included in this study, and cortisol levels<br />
were measured three times; before the exercise, just after the exercise and one hour later after<br />
exercise. The results of this study showed that there were no statistically significant differences among<br />
three measurements in the serum levels of TSH and thyroid hormones. However, both fT3 and TSH<br />
were significantly decreased in one hour later after exercise, whereas no change was observed in fT4.<br />
Cortisol concentrations were slightly increased immediately after SRE. In conclusion, serum levels of<br />
thyroid stimulating hormone and thyroid hormones were affected by maximal aerobic exercise.<br />
Key words: Thyroid hormones (THs), thyroid stimulating hormone (TSH) free T3 (fT3), freeT4 (fT4), cortisol,<br />
shuttle run exercise (SRE).<br />
INTRODUCTION<br />
Exercise affects the activity of many glands and the<br />
production of their hormones. Thyroid hormone levels<br />
influence the skeletal and cardiac muscle function,<br />
pulmonary performance, metabolism, and the<br />
neurophysiologic axes. Hypothalamus secretes thyroid<br />
releasing hormone (TRH) for stimulation of anterior<br />
pituitary gland to release thyroid releasing hormone<br />
(TSH). TSH causes thyroid gland to secrete two<br />
aminoacid based hormones: Three iodine atom<br />
containing triiodothyronine (T3) and four iodine atom<br />
containing thyroxine (T4). Thyroid hormones have many<br />
important biological effects. It controls how quickly the<br />
body burns energy, makes proteins, and how sensitive<br />
the body should be to other hormones. These hormones<br />
regulate the rate of metabolism and affect the growth and<br />
rate of function of many other systems in the body<br />
(Wartofsky, 1995; Bernet and Wartofsky, 2000; McMurray<br />
and Hackney, 2000).<br />
The secretion of thyroid hormones T3 and T4 are<br />
controlled by negative feedback loops. When the level of<br />
thyroid hormones (T3 and T4) in the blood is high, by<br />
regulatory negative feedback loop TSH production is<br />
reduced. Both T3 and T4 exist in unbounded (free) and<br />
bounded forms (McMurray and Hackney, 2000).<br />
Cortisol is a glucocorticoid and synthesized by adrenal<br />
gland. Its primary functions are to increase protein<br />
breakdown, inhibite glucose uptake and increase<br />
lipolysis. The level of serum cortisol is effected by many<br />
factor such as intensity, duration and timing of exercise,<br />
type of exercise, age, altitude, environmental temperature<br />
and psychology (Bernet and Wartofsky, 2000; Deligiannis<br />
et al., 1993).<br />
In spite of the fact that many studies have been<br />
reported on the effect of exercise on neuroendocrine<br />
secretions, there were disagreements among the results<br />
of these studies. While the level of THS was constant in<br />
some studies ((Bernet and Wartofsky, 2000; Deligiannis<br />
et al., 1993), there was an increase in its level in the<br />
other studies (Burtis et al., 2008; Deligiannis et al., 1993;<br />
Ciloglu et al., 2005). Some studies (Bernet and<br />
Wartofsky, 2000; Siddiqui et al., 1983) reported that free<br />
T4 level was unchanged, whereas there were an<br />
increase (Bernet and Wartofsky, 2000; Deligiannis et al.,<br />
1993; Ciloglu et al., 2005; Licata et al., 1984) and a<br />
decrease in its level after exercise (Bernet and<br />
Wartofsky, 2000; Limanova et al., 1983; Hackney and<br />
Dobridge, 2009). Free T3 levels following exercise were<br />
found as increased (Bernet and Wartofsky, 2000;
Table 1. Mean values of subjects for age, body height and body weight (n = 14).<br />
Variable Age (year) Body height (cm) Body weight (kg)<br />
MSD 19.50±1.22 176.00±5.53 68.78±6.29<br />
Beyleroglu 2003<br />
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),<br />
and P values.<br />
Variable<br />
Assay 1<br />
Trial<br />
Assay 2 Assay 3<br />
Summary of two groups comparisons<br />
p (1-2) p (2-3) p (1-3)<br />
TSH (µIU/ml) 2.12±1.58 2.21±1.44 1.29±0.73 0.54** 0.002* 0.016*<br />
fT3 (pg/ml) 3.86±0.55 4.28±1.25 3.42±0.61 0.52** 0.015* 0.018*<br />
fT4 (ng/dl) 1.70±0.29 1.50±0.27 1.50±0.22 0.06** 0.92** 0.031*<br />
Cortisol (µg/dl) 17.12±7.15 22.96±7.47 18.93±4.6 0.018* 0.11** 0.26**<br />
*p0.05, non-significant.<br />
Siddiqui et al., 1983) and decreased (Deligiannis et al.,<br />
1993) or unaffected (Ciloglu et al., 2005; Siddiqui et al.,<br />
1983; Licata et al., 1984).<br />
Static or isometric exercise, usually of short duration<br />
but of high intensity, uses previously stored energy<br />
whereas more prolonged exercise must use energy<br />
generated by the normal metabolic pathways. The<br />
changes in concentrations of analyzes as a result of<br />
exercise are largely due to shifts of fluid between the<br />
intravascular and interstitial compartments, changes in<br />
hormone concentrations stimulated by the change in<br />
activity and loss of fluid due to sweating (Burtis et al.,<br />
2008). Progressive shuttle run test is suitable for<br />
endurance athletes and players of endurance sports<br />
including football, rugby and field hockey and its objective<br />
is to monitor the development of the athlete's maximum<br />
oxygen uptake (Mackenzie, 2005). Here, the effects of<br />
exhaustive exercise on cortisol and thyroid hormones<br />
(THs) were investigated before, immediately and one<br />
hour after exercise.<br />
MATERIALS AND METHODS<br />
The researcher used the quasi-experimental approach with onegroup<br />
design and measurements were taken pre- post and one<br />
hour after the exercise. Post sample was purposefully chosen from<br />
14 elite male field hockey players that participated in this study. The<br />
mean age of the participants was 19,50±1,22 years, mean height<br />
was 176.00±5.53 cm, mean weight was 68.78±6.29 kg. The<br />
average training experience of the participants was 7,21±1,36 years<br />
(Table 1).<br />
Blood sample collection<br />
Blood samples were obtained following an overnight fasting state.<br />
Samples were withdrawn three times (at rest, immediately after<br />
exercise and 1 h post exercise) from antecubital vein into blood<br />
tubes and separated from the cells by centrifugation at 3000 rpm for<br />
10 min. Serum samples were stored at -70°C and then they were<br />
analyzed.<br />
Measurement of thyroid hormones<br />
Samples were analyzed three times (Assays 1, 2 and 3) for thyroid<br />
stimulating hormone (TSH), free triiodotironin (fT3), free thyroxine<br />
(fT4) and cortisol by using commercial kit and analyzer (Immulite<br />
2000, BioDPC, USA) with chemiluminescence method.<br />
Exercise protocol<br />
Progressive shuttle run test was conducted to ensure the maximal<br />
exhaustion of the participants (Ciloglu et al., 2005).<br />
Statistical analysis<br />
Results were presented as mean±SD. SPSS 10.0 program was<br />
used for statistical analysis. Comparison among multiple assays<br />
was performed by non-parametric test Mann Whitney-U. A 2-tailed<br />
p value p0.05). Mean serum fT4 of Assay 2 was lower<br />
than Assay 1 (p>0.05). Mean serum TSH and fT3 of<br />
Assay 3 were lower than both assays 2 and 1 (p0.05) and Assay 1 (p
2004 Afr. J. Pharm. Pharmacol.<br />
5<br />
4.5<br />
4<br />
3.5<br />
3<br />
2.5<br />
2<br />
1.5<br />
1<br />
assay 1 assay 2 assay 3<br />
TSH (µIU/mL)<br />
fT3 (pg/mL)<br />
fT4 (ng/dL)<br />
Figure 1. Graph of changes in THS, fT3, fT4 and cortisol levels.<br />
26<br />
24<br />
22<br />
20<br />
18<br />
16<br />
14<br />
Cortisol (µg/dl)<br />
Assay 1 Assay 2 Assay 3<br />
Figure 2. Graph of changes in cortisol levels before (Assay 1),<br />
immediate (Assay 2) and one hour (Assay 3) after exercise.<br />
Assay 1 (p>0,05) (Table 2 and Figure 1).<br />
The hormonal response to exercise involves increased<br />
sympathoadrenal activity, increased somatotropin,<br />
corticotropin, β-endorphin, prolactin, vasopressin, and<br />
possibly TSH secretion. The extent of these changes is<br />
related to training, nutrition, and state of health; all of the<br />
endocrine responses are reduced by exercise training<br />
(Licata et al., 1984, Limanova et al., 1983).<br />
A major function of thyroid hormones is their control of<br />
the basal metabolic rate and calorigenesis through<br />
increased oxygen consumption in tissue via the effects of<br />
thyroid hormone on membrane transport and enhanced<br />
mitochondrial metabolism (Burtis et al., 2008).<br />
The effects of the exercise on circulating thyroid hormone<br />
values remain controversial. The relationship between<br />
exercise and thyroid hormone metabolism has been<br />
studied by several groups of investigators prevıously.<br />
In a study carried out by Fortunato et al. (2008), they<br />
classified rats into five groups to elucidate the effects of a<br />
session of acute exercise on the treadmill at 75% of<br />
maximum oxygen consumption on thyroid function of<br />
rats: Control (without exercise), and killed just after (0<br />
min) or 30, 60, and 120 min after the end of the exercise<br />
session. They reported that a significant increase in T3<br />
occurred just after the exercise, with a gradual decrease<br />
thereafter so that 120 min after the end of the exercise,<br />
serum T3 was significantly lower than that of controls.<br />
Total thyroxine T4 increased progressively reaching<br />
values significantly higher than the control group at 120<br />
min. T3/T4 ratio was significantly decreased 60 and 120<br />
min after exercise, indicating impaired T4-to-T3<br />
conversion. Brown adipose tissue (BAT) type 2<br />
deiodinase activity (D2) was significantly lower at 30 min,<br />
but pituitary D2 have remained unchanged. No change in<br />
serum thyrotropin was detected, while serum corticoste-<br />
rone was significantly higher 30 min after exercise. They<br />
concluded that decreased liver D1 and BAT D2 might be<br />
involved in the decreased T(4)-to-T(3) conversion<br />
detected after an exercise session on the treadmill<br />
(Hackney and Dobridge, 2009).<br />
In an earlier study, Krotkiewski et al. (1984) measured<br />
thyroid hormones before, during and after acute exercise<br />
(60 min) or physical training (3 months) in obese women;<br />
thyroid stimulating hormone concentration increased<br />
during acute work and decreased immediately after. No<br />
changes were seen during the two following days. T4<br />
concentrations showed no changes. T3 decreased<br />
slightly immediately after acute exercise, and after three<br />
months of physical training (Fortunato et al., 2008).<br />
Simsch et al. (2002) have investigated the influence of<br />
different training intensities on Leptin and the<br />
hypothalamic-thyroid-axis in highly trained rowers. They<br />
measured TSH, fT3 and fT4 and reported that there was<br />
no change of fT4 level and a significant reduction in TSH<br />
and fT3 after resistance training. A significant increase of<br />
TSH was found after endurance training (Simsch et al.,<br />
2002).<br />
In Huang et al. (2004) research, Twenty-six healthy<br />
male military recruits aged 23 to 27 years with a mean of<br />
25 years have been studied. All subjects had maintained<br />
identical diet and physical activity for a week before the<br />
test. Serum samples had been drawn before (baseline)<br />
and immediately, 1, 4, 24, 24 and 48 h after maximal<br />
exercise (on a treadmill with Bruce protocol). Specimens<br />
had been analyzed to measure T3, T4, fT3, fT4 and TSH<br />
in the same assays. No significant changes of serum<br />
mean TH values before and after exercise have been<br />
found except for TSH, which had increased significantly<br />
immediately after exercise (1.72 vs. baseline 1.42 IU/L, p<br />
< 0.01). They have reported that maximal treadmill<br />
exercise had not greatly affected the determination of<br />
concentrations of circulating THs (Huang et al., 2004).<br />
It is known that measurements of changes in hormone<br />
values after exercise may reflect only acute transcapillary<br />
movements of water, which resolve shortly after exercise<br />
ceases. The hemodynamics return to baseline within<br />
minutes following termination of exercise. Exercise can<br />
cause hemoconcentration (Beaumont, 1972).<br />
In this study, circulating TSH and fT3 levels increased<br />
immediately after exercise followed by a significant<br />
decrease until the end of the study period. Our results<br />
were consistent with other reports suggesting that
hemoconcentration could be a cause of changes in<br />
circulating TSH and fT3 (Krotkiewski et al., 1984; Sowers<br />
et al., 1977; Schmid et al., 1982).<br />
Physical exercise has been reported to stimulate the<br />
peripheral deiodination of T4 and an increased uptake of<br />
T4 in the liver during exercise (Opstad et al., 1984).<br />
Increased conversion of T4 to T3 by peripheral tissues<br />
during training is improbable since there were no<br />
significant changes in serum fT3 concentrations after<br />
immediately exercise in the present study. Moreover,<br />
both cortisol and catecholamine actions initiated by<br />
exercise will also stimulate peripheral T4 deiodination<br />
(Chopra et al., 1975; Nauman et al., 1980). Whether the<br />
accelerated deiodination resulted in the increase in fT3<br />
and minimal decrease in fT4 values observed<br />
immediately after exercise (Assay 2) remains to be<br />
clarified.<br />
It has been reported that the type, intensity and<br />
duration of the training regimes, as well as the training<br />
background of the subjects, play a role in the changes<br />
taking place in serum T4 and fT4 levels have suggested<br />
that training may slightly impair thyroid function<br />
(Pakarinen et al., 1988). The main findings of the present<br />
study were the slight decrease in serum concentration of<br />
fT4, slight increases in serum concentration of fT3 and<br />
TSH immediately after exercise. One hour after exercise,<br />
both fT3 and TSH have significantly decreased, fT4<br />
remained unchanged. Since these changes were all<br />
within the reported normal ranges for reference values,<br />
they cannot have any clinical significance.<br />
Although no statically significant differences have been<br />
reported between pre- and post- exercise serum levels of<br />
thyroid hormones in a study including 20 patients with<br />
coronary artery disease (Siddiqui et al., 1983;), in our<br />
study, in which 14 field hockey players were included,<br />
while the concentrations of fT4 decreased, the<br />
concentrations of TSH and fT3 immediately increased<br />
after exercise.<br />
The level of serum cortisol is effected by many factor<br />
such as intensity, duration and timing of exercise, type of<br />
exercise, age, altitude, environmental temperature and<br />
psychology (Bernet and Wartofsky, 2000; McMurray and<br />
Hackney, 2000). In this study, cortisol concentration<br />
significantly increased immediately after exercise. High<br />
serum levels of cortisol generated by the stress<br />
procedure might also contribute to the post-stress<br />
(exercise) depression of TSH levels (Sowers et al.,<br />
1977).<br />
Conclusion<br />
There were no statically significant differences between<br />
pre- and just after the exercise serum levels of TSH and<br />
thyroid hormones. It has been appeared that the plasma<br />
thyroid hormones and TSH are not highly affected<br />
imediately after exercise. However one hour after the<br />
Beyleroglu 2005<br />
exercise, both fT3 and TSH significantly decreased. fT4<br />
was also affected. Cortisol concentrations slightly<br />
increased immediately after maximal aerobic exercise.<br />
The previously studies is consistent in respect to plasma<br />
cortisol level response to exercise. This can be<br />
explained by the plasma level of cortisol effected by<br />
several factors such as type, intensity, duration and<br />
timing of exercise, age, gender, altitude, environmental<br />
temperature, blood glucose level, focus of attention etc.<br />
ACKNOWLEDGEMENTS<br />
The author would like to thank Prof. Dr. Ahmet Aslan,<br />
Prof. Dr. Mehmet Akif Ziyagil and Dr. Ahmet Celik for<br />
their valuable support and comments.<br />
REFERENCES<br />
Beaumont W (1972). Evaluation of hemoconcentration from hematocrit<br />
measurements. J. Appl. Physiol., 32: 712–713.<br />
Burtis CA, Ashwood ER, Bruns DE (2008). Tietz Fundamentals of<br />
Clinical Chemistry, 6th ed. St. Louis: Saunders, Elsevier.<br />
Chopra IJ, Williums DE, Orgiazzi J, Solomon DH (1975). Opposite<br />
effects of dexamethasone on serum reverse T3 and T3. J. Clin.<br />
Endocrinol. Metab., 41: 911-920.<br />
Ciloglu F, Peker I, Pehlivan A, Karacabey K, Ilhan N, Saygin O,<br />
Ozmerdivenli R (2005). Exercise intensity and its effects on thyroid<br />
hormones. Neuroendorcrinology Lett., 26: 830-834.<br />
Deligiannis A, Karamouzis M, Kouidi E, Mougios V, Kallaras C (1993).<br />
Plasma TSH, T3, T4 and cortisol responses to swimming at varying<br />
water temperatures. Br. J. Med., 27(4): 247-250.<br />
Fortunato RS, Ignacio DL, Padron AS, Peçanha R, Marassi MP,<br />
Rosenthal D, Weneck-de-Castro JPS, Carvalho DP (2008). The<br />
Effect of Acute Exercise Session on Thyroid Hormone Economy in<br />
Rats. J. Endocrinol.,198(2): 347-53.<br />
McMurray RG, Hackney AC (2000). Endocrine responses to exercise<br />
and training In Garrett, W. E. and Kirkendall, D. T. (Eds.) Exercise<br />
and Sport Science, Philedelphia: Lippincott Williams & Wilkins. pp,<br />
135-162.<br />
Hackney AC, Dobridge JD (2009). Thyroid hormones and the<br />
interrelation of cortsiol and prolactin: influence of porolonged,<br />
exhaustive exercise. Endokrynologia Polska, 60(4): 252–257.<br />
Huang WS, Yu MD, Lee MS, Cheng CY, Yang SP, Chin HM, Wu SY<br />
(2004). Effect of treadmill exercise on circulating thyroid hormone<br />
measurements. Med. Princ. Pract., 13(1): 15-19.<br />
Krotkiewski M, Sjostrom L, Sullivan L, Lundberg PA, Lindstedt G,<br />
Wetterqvist H, Björntorp P (1984). The effect of acute and chronic<br />
exercise in thyroid hormones in obesity. Acta Med. Scand., 216: 269-<br />
275.<br />
Licata GR, Scaglione S, Novo MA, Dichiara D, Vincenzo D (1984).<br />
Behaviour of serum T3, rT3, TT4, FT4 and TSH levels afters after<br />
exercise on a bicycle ergometer in healty euthyroid male young<br />
subjects. Boll. Soc. Ital. Biol., 60(4): 753-759.<br />
Limanova Z, Sonka J, Kratochvil O, Sonka K, Kanka J, Sprynarova S<br />
(1983). Effects of exercise on serum cortisol and thyroid hormones.<br />
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Mackenzie B (2005). 101 performance evaulation tests. Electric Word<br />
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Nauman A, Kaminski T, Herbaczynska-Cedro K (1980). In vivo and in<br />
vitro effects of adrenaline on conversion of thyroxine to<br />
triiodothyronine and to reverse-triiodothyronine in dog liver and heart.<br />
Eur. J. Clin. Invest., 10: 189-192.<br />
Opstad PK, Falch D, Okedalen O, Fonnum F, Wergeland R (1984). The<br />
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Pakarinen A, Alan M, Hakkinen K, Komi P (1988). Serum thyroid<br />
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Schmid P, Wolf W, Pilger E, Schwaberger E, Pessenhofer G, Pristautz<br />
H, Leb G (1982). TSH, T3, rT3, and fT4 in maximal and submaximal<br />
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Siddiqui AR, Hinnefeld RB, Dillon T, Judson WE (1983). Immediate<br />
effects of heavy exercise on the circulating thyroid hormones. Brit. J.<br />
Sports Med., 17(3): 180-183.<br />
Simsch C, Lormes W, Petersen KG, Baur S, Liu Y, Hackney AC,<br />
Lehmann M, Steinacker J M (2002). Training intensity influences<br />
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The effect of stressful diagnostic studies and surgery on anterior<br />
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African Journal of Pharmacy and Pharmacology Vol. 5(17), pp. 2007-2012, 8 November, 2011<br />
Available online at http://www.academicjournals.org/AJPP<br />
DOI: 10.5897/AJPP11.331<br />
ISSN 1996-0816 ©2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Cationic liposomes as gene delivery system<br />
Mohsen M. Mady<br />
Department of Physics and Astronomy, College of Science, King Saud University, Riyadh-11451, Saudi Arabia.<br />
E-mail: dr_mmady@yahoo.com or mmady@ksu.edu.sa. Tel: +966-1-4698600.<br />
Accepted 27 July, 2011<br />
Delivery of oligonucleotides (ONs) and genes to their intracellular targets is a prerequisite for their<br />
successful use in medical therapy. Cationic liposomes are among the most commonly used and<br />
promising delivery system for ONs and genes. Cationic-liposomes and their complexes with ON were<br />
characterized according to complex size, zeta potential measurements, transmission electron<br />
microscope (TEM) and confocal laser scanning microscope (CLSM). It is successfully demonstrated<br />
that cationic liposomes, dispersed in 10% serum-containing growth medium, efficiently delivered ON to<br />
HeLa cells. Indeed, intact ON was found in the cytoplasm and nucleus only when delivered by cationic<br />
liposomes. The results suggest that cationic lipid-based delivery systems can be efficient for gene<br />
delivery if their biophysical properties can be properly controlled.<br />
Key words: Cationic liposomes, HeLa cells, gene delivery, characterization.<br />
INTRODUCTION<br />
Gene therapy is based on the introduction of specific<br />
exogenous sequences of deoxyribonucleic acid (DNA)<br />
into the target cells for production of the therapeutic gene<br />
product (Crystal, 1995; Lasic and Templeton, 1996). The<br />
prerequisite for successful gene therapy is efficient and<br />
safe delivery of DNA into the cells. Because of the fast<br />
progress of nucleic acid-based technologies in the<br />
treatment of diseases, the call for appropriate delivery<br />
vehicles becomes increasingly important. The ideal<br />
vehicle should avoid immediate uptake by the<br />
mononuclear phagocyte system and have prolonged<br />
circulation in blood, thus increasing the probability of<br />
reaching the desired targets. In addition, the vehicle<br />
should be able to deliver its contents efficiently into the<br />
cell cytoplasm, avoiding lysosomal degradation<br />
(Shepushkin et al., 1997). The most widely used types of<br />
vehicles for gene delivery are: viral (e.g., adenovirus,<br />
retrovirus and adeno-associated virus) and non-viral (for<br />
example, liposomes, polymer and peptides) (Lasic,<br />
1999). Viral vectors are often highly efficient, but safety<br />
Abbreviations: ONs, Oligonucleotides; TEM, transmission<br />
electron microscope; CLSM, confocal laser scanning<br />
microscope; DNA, deoxyribonucleic acid; DOTAP 1, 2 -<br />
dioleoyl-3 trimethylammonium-propane; FCS, fetal calf serum;<br />
PI, propidium iodide; DOPE, dioleoylphosphatidylethanolamine.<br />
and immunogenicity are issues of potential concern, and<br />
the limited transgene size often possesses a serious<br />
obstacle (Mady et al., 2004). Nonviral vectors, on the<br />
other hand, frequently face the problem of low<br />
transduction efficiency.<br />
Among the non-viral vectors, cationic liposomes are the<br />
most widely used vectors. Although less efficient in<br />
delivering the genes than the virus, they have many<br />
important qualities such as being much less or nonimmunogenic,<br />
have no known limitation in the size of the<br />
DNA, can be custom-synthesized for targeting and easily<br />
scalable for large-scale production. Moreover, the<br />
liposome can deliver different kinds of DNA (super coiled<br />
or linear) or ribonucleic acid (RNA) with or without<br />
proteins, even to non-dividing cells and are usually<br />
composed of biodegradable lipids. Also, covalent<br />
attachment of target specific ligands on the liposome can<br />
facilitate targeted delivery of genes (Mady et al., 2004).<br />
These advantages have prompted researchers to explore<br />
the applications of cationic liposomes in gene therapy<br />
clinical trials (Li and Huang, 1997; Lasic and<br />
Papahadjopoulos, 1998; Mady, 2007).<br />
Cationic lipids were used for the first time, for gene<br />
delivery, by Felgner et al (1987), and for ON delivery a<br />
few years later by Bennett (1993). When cationic<br />
liposomes are used, no encapsulation is needed because<br />
the lipids bind electrostatically to negatively charged
2008 Afr. J. Pharm. Pharmacol.<br />
nucleotides. Despite their widespread use as transfection<br />
reagents, information about the interactions of cationic<br />
lipids and polynucleotide is sparse (Mönkkönen and Urtti,<br />
1998). Very little is understood about the events which<br />
take place when cationic liposomes interact with mamma-<br />
lian cells or the processes which result in the delivery of<br />
nucleic acids. Three model of the interaction of cationic<br />
lipid / polynucleotide complex with cells have been<br />
proposed: (i) direct fusion with the plasma membrane<br />
(Felgner et al., 1987; Lewis et al., 1996), (ii) endocytosis<br />
and subsequent fusion or destabilization of endosome<br />
membrane (Legendre and Szoka, 1992; Felgner et al.,<br />
1994; Zhou and Huang, 1994; Zabner et al., 1995), and<br />
(iii) translocation through pores across the plasma mem-<br />
brane (Engberts and Hoekstra, 1995).<br />
The physicochemical properties, such as particle sizes<br />
and surface charges of the liposome-DNA and/or<br />
oligonucleotides (ON) complexes may be important<br />
factors to obtain a higher transfection efficiency of the<br />
liposomal vectors. Although gene transfection of plasmid<br />
and/or ON complexed with cationic liposomes is<br />
investigated, little attention seems to be paid to<br />
understanding their physico- chemical characteristics and<br />
cellular uptake mechanisms. The intent in this study was<br />
to characterize ON /liposome complexes in terms of ζ<br />
potential and particle size and to see whether these<br />
physicochemical properties have any influence on their<br />
disposition characteristics and cellular uptake process. In<br />
the presence of serum, we investigated that cationic<br />
liposomes efficiently delivered ON to HeLa cells.<br />
MATERIALS AND METHODS<br />
1, 2 - dioleoyl-3-trimethylammonium-propane (DOTAP) was<br />
purchased from Avanti Polar Lipids Inc. (Alabaster, Alabama, USA).<br />
Fetal calf serum (FCS), L-Glutamin (200 nM solution), Penicillin<br />
5000 units/streptomycin 5000 mg, and DMEM (Dulbecco's modified<br />
Eagle's medium) was purchased from Bio Whittaker Europe,<br />
Verviers, Belgium. HeLa cells were obtained from Children Hospital.<br />
DNA- analogues of chimeric ON as the one used here will be<br />
referred to as FIXDNA-DNA. A 5´FAM (Eurogentec EGT Group,<br />
4102 Seraing- Belgium) labeled 68-mer of sequence (5´-TGT-CAA-<br />
GCA-GAT-CGT-GGG-GGA-CCC-CTT-TTG-GGG-TCC-CCC-ACG-<br />
ATC-TCC-TTG-ACA-GCG-CGT-TTT-CGC-GC-3´). Propidium<br />
iodide (PI) was purchased from Molecular Probes (Eugene, Oreg.<br />
USA). All other reagents were of analytical reagent grade.<br />
Preparation of liposomes<br />
Liposomes were formulated according to well-established methods<br />
of extrusion (Olson et al., 1979). In short: the appropriate<br />
phospholipid composition was mixed in organic solvent in a 50 ml<br />
round flask. The organic solvent was evaporated to dryness by a<br />
rotary evaporator. The resulting lipid suspension was extruded<br />
through 100 nm polycarbonate membranes using mini- extruder<br />
(Liposofast, Avestin Inc., Canada). Size measurement was done by<br />
dynamic laser light scattering and the size was in the range of 100 nm.<br />
Condensation of ON<br />
DOTAP liposomes were added separately to 2.5 µg ON to achieve<br />
the desired charge ratio (+/-). The fluorescence intensity of ON<br />
(excitation at 492 nm and emission at 515 nm) was measured by<br />
using Perkin Elmer Spectrofluorometer LS 50B (UK). The<br />
hydrodynamic diameters of complex were measured by using<br />
Zetasizer 3000 HS, Malvern Instruments, Germany.<br />
Transmission electron microscopy<br />
DOTAP/ON (8:1 +/-) complex was also characterized by using<br />
negative stain electron microscope (EM 109, Zeiss, West<br />
Germany). On a copper grid, the appropriate concentration from<br />
each sample was added. Then add one drop of 20% uranyl acetate;<br />
wait for 2 min at room temperature; remove the excess solution with<br />
a filter paper; then the sample was examined under the<br />
transmission electron microscope.<br />
Zeta potential<br />
In deionized water, we dispersed the pure ONs and their complexes<br />
with DOTAP (1:8 -/+) and then measured the corresponding zetapotential<br />
ζ (n=5) by using Zetasizer 3000 HS, Malvern Instruments,<br />
Germany.<br />
ON transfection experiment<br />
To investigate the gene expression or transfer efficiency, HeLa cells<br />
were grown on glass cover slips in six-well plate (10 5 -10 6 cells per<br />
well) in DMEM medium supplemented with 10% FCS, 1% glutamine<br />
and 1% penicillin-streptomycin solutions. The transfection system<br />
(ON/DOTAP 1:8 -/+) complexes and cells were incubated for 6<br />
hours at 37°C in 5% CO2. The cells were washed away by rinsing<br />
three times with cold PBS. Cells nuclei were stained with PI stain.<br />
Cells were fixed with formaldehyde. Turn the cover slip containing<br />
the cells on a Moviol drop on a glass slide and examine with<br />
confocal laser scanning microscope. We used True Confocal<br />
Scanner, Leica DM R with A4, L5, N3 and Y5 filters, Leica, Wetzlar,<br />
Germany and Leitz DM RXE upright microscope with a<br />
Krypton/Argon laser (emission wavelengths of 488, 578, and 647<br />
nm) was used. Images were converted to TIF-format with Scanware<br />
5.1 Scion Corporation, Frederick, MD, USA. Cellular distribution of<br />
the FAM-ON, complexed with DOTAP liposomes, was investigated<br />
in HeLa cells following the kinetics of this process using confocal<br />
laser scanning microscope (CLSM). In general, liposomes were in<br />
the range of 100 nm in diameter. ON and liposomes were<br />
appropriately diluted in 10 mM tris buffered saline (pH 7.8). The<br />
final complex had a size of approximately 150 nm, which was<br />
diluted with the appropriate cell cultured medium containing 10%<br />
FCS and then added to HeLa cells.<br />
RESULTS AND DISCUSSION<br />
ON condensation<br />
The condensing agents must not only efficiently<br />
condense ON but also require the ability to be effectively<br />
displaced from ON, allowing for subsequent<br />
transcriptional and translational events to occur (Sorgi,
Relative Fluorescence fluorescence Intensity intensity<br />
100<br />
80<br />
60<br />
40<br />
20<br />
-2 0 2 4 6 8 10 12 14 16<br />
DOTAP/FAM(+/-)<br />
Figure 1. Condensation of FAM by DOTAP liposomes (Excitation<br />
wavelength at 492 nm and emission wavelength at 515 nm).<br />
Size(nm) Size (nm)<br />
190<br />
180<br />
170<br />
160<br />
150<br />
140<br />
130<br />
0 10 20 30 40 50<br />
DOTAP/FAM(+/-)<br />
Figure 2. Hydrodynamic diameters of DOTAP/FAM complexes<br />
versus different complexes charge ratio (+/-); n = 3.<br />
1998). In order to condense FAM-ON, increasing<br />
amounts of DOTAP-liposomes were added to the FAM-<br />
ON solution. As seen in Figure 1 the addition of DOTAP<br />
to FAM solution resulted in a rapid decrease in<br />
fluorescence intensity of FAM-ON. This loss in<br />
fluorescence intensity can be attributed to the<br />
condensation of ON. This is thought to occur due to<br />
electrostatic interactions between cationic lipid and ON,<br />
resulting in a charge neutralization of the complex and<br />
the formation of a condensed structure. This condensed<br />
structure, due to its diminished size, may be more readily<br />
endocytosed by the cell, resulting in the increased levels<br />
of transgene expression.<br />
The complex is examined by several physical methods to<br />
Mady 2009<br />
gain insight into the nature of the interaction among the<br />
components and the mechanism of the enhanced<br />
transfection activity. We first looked at the effect of<br />
cationic lipid on the complex size. Figure 2 shows that the<br />
size of the complex was dependent on the charge ratio of<br />
DOTAP/ON. As the DOTAP content increases, vesicle<br />
size is reduced. Results indicate that, at a low charge<br />
ratio (2.5:1 +/-), the size of the complex was 186.5 nm.<br />
However, as the DOTAP content increases; complex size<br />
is reduced to approx. 132 nm at a charge ratio (50:1 +/-).<br />
The data indicates the condensation ability of ON by<br />
DOTAP liposomes (Sternberg et al., 1994; Li et al., 1998;<br />
Perrie and Gregoriadis, 2000).<br />
There are several possibilities that may explain the<br />
potentiation effect of the condensing agent on the<br />
transfection activity of liposomes. First, on the basis of<br />
the current endocytosis model, there is a size limitation<br />
for particles to be taken up efficiently by cells (Machy and<br />
Leserman, 1983). Direct size measurement of the<br />
complexes showed that the condensing agent<br />
significantly reduced the size of the complex formed over<br />
a wide range of DOTAP/ON ratios.<br />
Transmission electron microscopy<br />
Negative stain electron microscopy is a useful method for<br />
addressing questions concerning size distribution of<br />
liposome, and although obtaining quantitative data is<br />
laborious, negative staining is a reliable technique, which<br />
is simple to perform and requires only limited specialized<br />
equipment, which should be available in any electron<br />
microscopy laboratory (Haschmeyer and Myers, 1972).<br />
Morphological studies demonstrated that spherical<br />
DOTAP/ON complexes are formed (Mönkkönen and Urtti<br />
1998). Electron micrograph of DOTAP/ON (8:1 +/-)<br />
complex was shown in Figure 3. The majority of the<br />
particles in the pictures appeared spherical, small (≤ 100<br />
nm in diameter), electron dense and some of them were<br />
associated with low-density lipid membranes. These<br />
complexes of small size should be more favorable to<br />
enter the cells via an endocytosis pathway than the larger<br />
ones.<br />
Zeta potential measurements<br />
Transfection complex formation is based on the<br />
interaction of the positively charged lipid with the<br />
negatively charged phosphate groups of the nucleic acid.<br />
The information of the overall charge of transfection<br />
complexes by zeta-potential measurements can be<br />
speed up the development of better non-viral DNA<br />
delivery vectors for gene therapy (Son et al., 2000; Mady<br />
et al., 2004). The following physical chemical parameters<br />
were used in the determination of zeta-potential: medium
2010 Afr. J. Pharm. Pharmacol.<br />
Zeta-potential (mv)<br />
Figure 3. Negative stain electron micrograph of DOTAP/ON<br />
(8:1 +/-).<br />
40<br />
30<br />
20<br />
10<br />
0<br />
-10<br />
-20<br />
-30<br />
FAM<br />
DOTAP/FAM(5:1)<br />
DOTAP/FAM (7.5:1)<br />
Figure 4. Zeta potential measurements of different<br />
formulations (n=5).<br />
viscosity 0.89 cPoise, medium refractive index 1.333,<br />
temperature 25°C and dielectric constant 79.<br />
Results in Figure 4 show trends of increasing zetapotential<br />
values with increasing DOTAP content. Values<br />
of the zeta-potential of liposomes indirectly reflect vesicle<br />
surface net charge and can therefore be used to evaluate<br />
the extent of interaction of the liposomal surface cationic<br />
charges with ON. On this basis, the zeta-potential of<br />
DOTAP liposomes was investigated before and after<br />
complexing with FAM-ON. Results show the negatively<br />
zeta-potential of the naked FAM (-26.8 ± 4). After addition<br />
of DOTAP to FAM in charge ratio of (5:1 +/-), the zetapotential<br />
value becomes positive (15.2 ± 6.6 mV). Also, it<br />
was more positive (33.5 ± 6) for DOTAP/FAM (7.5:1 +/-).<br />
ON transfection<br />
ONs are promising therapeutic agents against viral<br />
infections and cancer. However, problems with their<br />
inefficient delivery and inadequate stability have to be<br />
solved before they can be used in therapy. Cellular<br />
uptake of ON is highly inefficient, as their net negative<br />
charge and high molecular weight prohibit efficient<br />
transfer across cell membrane without the help of carrier<br />
system (Bennett 1993; Ledley, 1995; Welz et al., 2000).<br />
Confocal microscopy is a well-established technique for<br />
the investigation of 3D structures in biological and<br />
industrial materials (Mady et al., 2009). The basis of this<br />
success is the optical sectioning capability of this type of<br />
microscopy, which enables one to study 3D-structure of<br />
intact specimens in their natural environment. CLSM has<br />
the major advantages that it yields a very short depth of<br />
focus, its transverse definition and the contrast of the<br />
image are better than a standard microscope, the device<br />
is very well-suited for optical cross-sectioning and with<br />
the use of a laser beam, the intensity of illumination can<br />
be very high. With CLSM one can slice incredibly clean,<br />
thin optical sections out of thick fluorescent specimens;<br />
view specimens in planes running parallel to the line of<br />
sight; penetrate deep into light-scattering tissues; gain<br />
impressive three-dimensional views at very high<br />
resolution; and improve the precision of microphotometry.<br />
Figure 5 shows that DOTAP-liposomes effectively<br />
transport ON into the nucleus of HeLa cells, in the<br />
presence of serum. DOTAP-liposomes improved the<br />
cellular delivery of ON by protecting it in FCS, by<br />
increasing the total uptake of ON in cells, and by<br />
increasing the nuclear localization of ON in cells.<br />
Complexation with cationic lipids facilitates the entry of<br />
ON into cells. ON-cationic lipid complexes enter the cells<br />
via endocytosis and they promote the nuclear entry of ON<br />
(Bennet et al., 1992; Zelphati and Szoka, 1996a). After<br />
their cellular delivery with cationic lipids, the ONs are<br />
mostly seen in the nucleus, whereas the lipids are<br />
entirely localized in the cytoplasm and, particulay, in<br />
some perinuclear regions (Zelphati and Szoka, 1996a).<br />
Zelphati and Szoka (1996b) showed that ONs were<br />
released from the complexes as a result of lipid<br />
intermixing in the endosomal wall.<br />
The cationic lipid gene delivery system invariably<br />
comprises three components: a cationic lipid, a neutral<br />
co-lipid, which is most often dioleoylphosphatidylethano-<br />
lamine (DOPE) and the plasmid DNA that encodes the<br />
transgene of interest. Although the liposomal gene<br />
delivery systems require a neutral co-lipid, usually DOPE<br />
for optimal activity, the aggregation of phosphatidyle-<br />
thanolamine containing liposomes by serum proteins has<br />
been observed (Forbes et al., 1984). In the case of<br />
cationic lipid based delivery systems, the charge density<br />
of the lipid bilayer can significantly affect the colloidal<br />
stability of the particles, with mole fractions of helper<br />
lipids [for example, (DOPE)] of > 0.4 producing<br />
considerable instability at elevated ionic strength. So, in<br />
the present work, DOTAP liposomes are only used to<br />
investigate how physicochemical methods can be used to
Figure 5. Confocal laser scanning microscopy of HeLa cells<br />
incubated with DOTAP/FAM (8:1 +/-) complexes in DMEM<br />
supplemented with 10% FCS, at 37°C and 5% CO2 after 6 h.<br />
assess the functionality of the ON-lipid complexes in vivo.<br />
Most mammalian cells require the addition of serum to<br />
the culture medium for optimal growth and maintenance<br />
of the cell lines in vitro. The presence of serum often<br />
reduces the transfection efficiency of liposomal vectors<br />
(Felgner et al., 1987; Felgner and Ringold, 1989; Gao<br />
and Huang, 1995; Lee and Huang, 1997; Mady et al.,<br />
2009). This may be due to the premature release of DNA<br />
from the complexes or its degradation by the nucleases.<br />
So, in the presence of serum, we investigated that<br />
cationic liposomes efficiently delivered ON to HeLa cells.<br />
Analytical methods are described that provide a<br />
platform for systematically evaluating the effect of<br />
formulation variables in the development of ON-lipid nonviral<br />
gene therapy complexes. The methods encompass<br />
those used to characterize the components, that is, ON<br />
and liposomes, as well as those applied to the complexes<br />
themselves. Several lines of evidence obtained from<br />
studies on complex size; zeta-potential; electron<br />
microscope and CLSM indicate that ON is entrapped<br />
within the aqueous compartments, in between bilayers,<br />
presumably bound to the cationic charges. The focus is<br />
on physicochemical methods so that their parameters<br />
can be assessed in relationship to the functionality of the<br />
ON-lipid complexes in vivo (Perrie and Gregoriadis, 2000;<br />
Hutchins, 2000).<br />
More studies are needed to compare and investigate<br />
the role of fusogenic helper lipids such as DOPE and<br />
cholesterol or peptides to disrupt the endosome<br />
membrane so as to facilitate the escape of DNA from<br />
endosome. Also, covalent attachment of target specific<br />
ligands on the liposome can facilitate targeted delivery of<br />
genes (Mady et al., 2004). These advantages have<br />
prompted researchers to explore the applications of<br />
cationic liposomes in gene therapy clinical trials.<br />
ACKNOWLEDGEMENT<br />
Mady 2011<br />
Author extends his appreciation to the Deanship of<br />
Scientific Research at King Saud University for funding<br />
the work through the research group project No RGP-<br />
VPP-121.<br />
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African Journal of Pharmacy and Pharmacology Vol. 5(17), pp. 2013-2017, 8 November, 2011<br />
Available online at http://www.academicjournals.org/AJPP<br />
DOI: 10.5897/AJPP11.490<br />
ISSN 1996-0816 ©2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Nitrous oxide versus pethidine with promethasine for<br />
reducing labor pain<br />
Batool Teimoori 1 , Nahid Sakhavar 1 , Masoome Mirteimoori 1 and Behzad Narouie 2 *<br />
1 Department of Obstetrics and Gynecology, Health Promotion Research Center, Zahedan University of Medical<br />
Sciences, Zahedan, Iran.<br />
2 Clinical Research Development Center, Ali-ebne-Abitaleb hospital, Zahedan University of Medical Sciences, Zahedan,<br />
Iran.<br />
Accepted 17 October, 2011<br />
Systemic opioids are widely used for the relief of labor pain. Self-administered nitrous oxide with<br />
concentration of 50% is a new form of analgesia. The aim of this study was to compare the analgesic<br />
efficacy and side effects of the patient controlled inhaled nitrous oxide (50% ‘‘Entonox’’) with systemic<br />
intramuscular pethidine, in reducing pain during normal vaginal labor in Iranian population. In a<br />
randomized controlled study, the analgesic efficacy of inhaled 50% nitrous oxide (Entonox) was<br />
evaluated as compared to intra muscular pethidine for reducing labor pain among 100 women<br />
undergoing normal vaginal delivery. Mean maternal age was 26.2 and 27.2 years in entonox and<br />
pethidine groups, respectively. Duration of first and second stages was significantly shorter in patients<br />
receiving nitrous oxide as analgesia as compared to pethidine group (P < 0.05). Pain severity according<br />
to visual analog scale (VAS) score was significantly lower in patient that received nitrous oxide (P =<br />
0.0001). We also showed significantly higher satisfaction of pain reduction in nitrous oxide group<br />
during labor (P = 0.01). No significant difference was observed among the groups regarding neonatal<br />
complications. Although, nitrous oxide is certainly not a potent analgesic, we found that it has more<br />
beneficial effects than pethidine in parturient women which is yet to be cleared.<br />
Key words: Entonox, labor pain, pethidine, nitrous oxide.<br />
INTRODUCTION<br />
Labor is one of the painful conditions that is considered to<br />
be the most intense and stressful experiences (Melzack,<br />
1984). In the last decades, changes have occurred in the<br />
obstetric expectations and in their care. In developed<br />
countries, the number of women requesting labor<br />
analgesia is increasing, and in some communities, an<br />
effective pain relief for childbirth is in great demand<br />
(National Institute for Health and Clinical Excellence,<br />
2008).<br />
As it is cheap, simple to use and readily available,<br />
systemic pethidine is widely used for relief of labor pain<br />
(Hawkins and Beaty, 1999; Wilson et al., 1986; Morrison<br />
et al., 1987). Use of parenteral opioids was found to be<br />
between 39 and 56% in various hospital obstetrics units,<br />
*Corresponding author. E-mail: b_narouie@yahoo.com.<br />
Tel/Fax: +985413414103.<br />
in the United States (Hawkins and Beaty, 1999; Fairlie et<br />
al., 1999; Olofsson et al., 1996).<br />
Systemic opioids lead to some adverse effects on both<br />
mother and baby including dysphoria, sedation,<br />
respiratory depression, nausea and vomiting and delayed<br />
gastric emptying for the mother (Douglas and Levinson,<br />
2001). As pethidine crosses the placenta, it may<br />
accumulate in the fetal circulation (Gaylard et al., 1990)<br />
causing early neonatal respiratory depression and<br />
behavioral and feeding problems for even up to six weeks<br />
after delivery (Belsey et al., 1981; Belfrage et al., 1981;<br />
Nissen et al., 1997).<br />
Self-administered 50% nitrous oxide “Entonox” is an<br />
effective and safe form of analgesia, which has been<br />
used by many emergency medical services for many<br />
years. Nitrous oxide is an odorless, tasteless and inhaled<br />
analgesic (Faddy and Garlick, 2005), and it was found to<br />
be an effective analgesia for many women while also<br />
being safe for the mothers and babies (Rooks, 2007).
2014 Afr. J. Pharm. Pharmacol.<br />
Table 1. Descriptive data of entonox and pethidine subjects.<br />
Variable Entonox (n = 50) Pethidine (n = 50) P-value<br />
Age (year) 7.34 ± 26.2 6.02 ± 27.2 NS<br />
Weight (kg) 0.38 ± 69 11.06 ± 73 NS<br />
Gestation (week) 0.95 ± 38.44 0.92 ± 38.58 NS<br />
Cervical dilation at Analgesic initiation (cm) 0.91 ± 4.46 0.89 ± 4.36 NS<br />
Nowadays, nitrous oxide is widely used in many countries<br />
for relieving labor pain (Rooks, 2007).<br />
Unlike opioids, it does not depress respiration (Rosen,<br />
2002). Nitrous oxide rapidly takes effect (Latto et al.,<br />
1973) and is quickly reversible on discontinuation of<br />
therapy (Latto et al., 1973; Einarsson et al., 1996).<br />
Despite its wide and popular use in many countries,<br />
nitrous oxide for the relief of labor pain is largely unknown<br />
in Iran. In the present study, we aimed to compare the<br />
analgesic efficacy and side effects of the patient<br />
controlled, inhaled nitrous oxide (50% ‘‘Entonox’’) with<br />
systemic intramuscular pethidine (the most popular drug<br />
with opioid analgesic properties), in relieving pain during<br />
normal vaginal labor in Iranian population.<br />
MATERIALS AND METHODS<br />
In this randomized clinical trial study, we evaluated the analgesic<br />
efficacy of inhaled 50% nitrous oxide (Entonox) as compared to<br />
intra muscular pethidine for relieving labor pain among 100 women<br />
undergoing normal vaginal delivery in Ali-Ebne-Abitaleb hospital, in<br />
Iran, from March 2007 to 2008. The study was reviewed and<br />
approved by the ethics committee in Zahedan University of Medical<br />
Sciences, and informed consents were obtained from all<br />
participants. 100 pregnant women with gestational age ranging<br />
from 38 to 42 weeks, and who were referred to in the early phase of<br />
labor, were randomly enrolled in the study. Participants were<br />
selected among non-complicated, term pregnancies with a normal<br />
cephalic fetus, and were referred to in the active phase of labor with<br />
cervical dilation less than 7 cm. Women who could not keep their<br />
facial mask, have recent administration of local or systemic<br />
analgesics and opioids, patients with altered mental status, vitamin<br />
B12 deficiency receiving replacement therapy, any oxygenation<br />
abnormalities, hemodynamically unstable patients and women<br />
bearing any fetus abnormalities were excluded from the study.<br />
Patients were randomly allocated in two groups. The number of<br />
nulliparous women was comparable in two groups. Participants of<br />
one group (Group A, n = 50) were medicated with entonox, where<br />
women in the other group (Group B, n = 50) received pethidine for<br />
relieving their labor pain. All women were trained for self<br />
administration of entonox in group A and women in group B<br />
received 1 mg/kg slowly intra-venuous injection of pethidine<br />
combined with 25 mg promethazine.<br />
Fifty percent nitrous oxide in 50% oxygen was premixed in a<br />
single cylinder called by the trade name ‘‘Entonox’’ (Lieberman and<br />
O’Donoghue, 2002). Entonox was self-administered by the laboring<br />
woman using a face mask, when she determines that she needs it.<br />
Patients were trained to administer face mask of entonox with the<br />
initial of every uterine contraction and continue deep inspirations<br />
while the contraction and pain exists.<br />
Entonox administration can be started and stopped at any point<br />
during labor, according to the needs and preferences of the woman.<br />
It takes effect in about 50 s after the first breath, and the effect is<br />
transient and gone when it is no longer needed (Rooks, 2007). The<br />
flow of gas into the mask is initiated by the negative pressure of<br />
inhalation, which opens a demand valve. This same demand valve<br />
prevents the flow of gas when inhalation ceases, and the entonox<br />
apparatus allows the exhaled gas to be scavenged, so it is not<br />
released into the air, minimizing the exposure to others in the room.<br />
Following different analgesic administrations, severity of labor<br />
pain was evaluated according to the VAS score, numbering from 0<br />
to 10 (0 = no pain and 10 = severe and non tolerable pain).<br />
Parturients pain scored once before any analgesic administrations,<br />
and they were requested to score their maximum pain following<br />
each contraction. Total visual analog scale (VAS) score is the mean<br />
of scores rated during labor.<br />
Patients satisfaction of analgesia method was also evaluated by<br />
verbal rating scale, scoring from 0 = not satisfied to 4 = complete<br />
satisfaction.<br />
All parturients were monitored for vital signs, arterial O2<br />
saturation and fetal heart rate each 30 min during labor and<br />
mothers were suggested to have left lateral position during labor for<br />
prevention of supine hypotension.<br />
Mothers’ somnolence and sedation was also evaluated by a<br />
nurse in 10 min intervals according to Ramsy score, from 1 to 5. 1 =<br />
completely awake, 2 = somnolence, 3 = irritable to sound, 4 =<br />
irritable to touch and 5 = non responder.<br />
Statistical analysis<br />
Descriptive statistics were used to report demographic<br />
characteristics with SPSS statistics package version 15. The Chisquare<br />
test and Student t-test were used to compare the groups on<br />
qualitative and quantitative variables, respectively.<br />
RESULTS<br />
A total of 100 pregnant women, including 50 primi-gravid women<br />
were enrolled in the study. Mean maternal age was 26.2 and 27.2<br />
in entonox and pethidine groups, respectively. Demographic data of<br />
the participants is summarized in (Table 1). Patient’s characteristics<br />
were comparable in two randomly allocated groups.<br />
All participants underwent normal vaginal delivery and none of<br />
them needed vacuum or forceps assistant. Duration of first and<br />
second stages was significantly shorter in patients receiving nitrous<br />
oxide as analgesia as compared to pethidine group (P < 0.05)<br />
(Table 2).<br />
VAS score before administration of any analgesic agent was<br />
statistically equal between groups; however, it shows significant<br />
difference at the end of both first and second stages of labor. Pain<br />
severity according to VAS score was lower in patient that received<br />
nitrous oxide. (P = 0.00)<br />
There were no differences in blood pressure, heart rate and<br />
respiratory rate before analgesia. Where, after the end of stage 1<br />
and 2, nitrous oxide users had significantly lower heart rate and<br />
respiratory rate. Blood pressure still remained equal in both groups
Table2. Comparison of labor outcomes in patients that received nitrous oxide and pethidine.<br />
Labor outcomes Entonox Pethidine P-value<br />
Duration<br />
First stage (h) 3.12 ± 1.37 2.24 ± 1.07 0.001<br />
Second stage (min) 3.44 ± 1.73 1.18 ± 1.00
2014 Afr. J. Pharm. Pharmacol.<br />
entonox as analgesia. Thus, entonox led to women’s<br />
higher satisfaction of their labor pain relief.<br />
Our results were in line with previous studies (Douglas<br />
and Levinson, 2001; Faddy and Garlick, 2005; Rooks,<br />
2007; Latto et al., 1973; Bishop, 2007; Evans et al., 1995;<br />
Jones et al., 1969; McAneny, 1963). However, in<br />
contrast, one prospective non-randomized study, labor<br />
pain was more severe in primi-gravid women that were<br />
administered nitrous oxide as compared to those that<br />
were given pethidine (Keskin et al., 2003).<br />
A mixture of 50% nitrous oxide in oxygen “Entonox” is<br />
available in a single cylinder as a patient controlled<br />
inhaled analgesic (Faddy and Garlick, 2005). It has low<br />
solubility in blood and is transported in solution without<br />
binding to protein. Nitrous oxide rapidly takes effect (Latto<br />
et al., 1973; Bishop, 2007), because it diffuses rapidly<br />
through the alveolar arterial membrane and is excreted<br />
unchanged, mainly through the lungs (Faddy and Garlick,<br />
2005). As a result, it is quickly reversible on<br />
discontinuation of therapy (Latto et al., 1973; Einarsson<br />
et al., 1996; Bishop, 2007). It has shown that recovery<br />
from sedative effects of nitrous oxide is faster when<br />
compared with intravenous analgesia (Faddy and Garlick,<br />
2005).<br />
Rapid onset and quick reversibility, allows nitrous oxide<br />
to be administered throughout the second stage of labor<br />
without fear of effects on the newborn (Bishop, 2007).<br />
Supervised by physicians, nurses or midwives, nitrous<br />
oxide is widely used as a safe analgesic in many parts of<br />
the world including Canada, Australia, Finland, United<br />
Kingdom and New Zealand (43 to 49%) (STAKES, 2006;<br />
NSW Department of Health, 2005; Biró et al., 2000).<br />
The maximum effect of nitrous oxide appears at a<br />
concentration of 70%, and it has been shown to relieve<br />
labor pain in approximately two-thirds of women (Rooks,<br />
2007).<br />
The precise mechanism of action of nitrous oxide<br />
analgesia remains uncertain. It may induce release of<br />
endogenous opioid peptides in the periaqueductal gray<br />
area of midbrain (Maze and Fuginaga, 2000).<br />
Among various inhalation anesthetic agents studied for<br />
labor analgesia, only nitrous oxide is used to any great<br />
extent in modern obstetric practice. The reasons are<br />
probably related to the ease of administration of nitrous<br />
oxide, its lack of flammability, absence of pungent odor,<br />
minimal toxicity, minimal depression of cardio vascular<br />
system, lack of effect on uterine contractility and the fact<br />
that it does not trigger malignant hyperthermia (Rooks,<br />
2007). Entonox is administered either intermittently,<br />
starting with the onset of pain with each contraction and<br />
discontinuing as the contraction pain eases or abates, or<br />
continuously, by inhaling both during and between<br />
contractions (Rooks, 2007).<br />
Intermittent administration of entonox, as used in this<br />
study, is somehow problematic, because there is a lag of<br />
approximately 50 s after the onset of administration<br />
before the analgesic effect can be expected (Chan et al.,<br />
1996). However, entonox is significantly beneficial if<br />
administration initiates approximately 30 to 50 s before<br />
each contraction (Waud and Waud, 1970).<br />
Side effects induced by nitrous oxide are nausea and<br />
vomiting reported in 5 to 36% (Rooks, 2007; Jones et al.,<br />
1969; McAneny, 1963; Bergsjo and Lindbaek, 1971;<br />
McGuinness and Rosen, 1984). Dizziness, dreams and<br />
drowsiness reported in 0 to 24%, dry mouth from<br />
breathing dry gas, buzzing in the ears and rarely,<br />
numbness are also reported (Rooks, 2007; Bishop, 2007;<br />
Jones et al., 1969; Bergsjo and Lindbaek, 1971;<br />
McGuinness and Rosen, 1984).<br />
The greater maternal risk of inhalation of nitrous oxide<br />
is loss of consciousness. It is rare with 50% nitrous oxide.<br />
The alveolar concentration for wakefulness for nitrous<br />
oxide is between 50 and 70% in non-pregnant women<br />
and probably lower in pregnant. So it is important that the<br />
agent should be self-administered and not by anyone<br />
else. It is also important that a mask is kept by parturient<br />
and not fixed to the face. If it is not strapped, her hand<br />
will fall away from her face, when she became too drowsy<br />
rendering the device nonfunctional. Therefore, the nitrous<br />
oxide concentration wills rapidly decline (Rooks, 2007).<br />
Nitrous oxide rapidly transfer placenta, however, as<br />
shown in our study, the fetus infants are clinically<br />
unaffected. There have shown no significant differences<br />
in Apgar scores or neonatal outcomes between babies<br />
born to mothers who received nitrous oxide (Rooks,<br />
2007; McAneny, 1963; Abboud et al., 1981; Stefani et al.,<br />
1982).<br />
When used intramuscularly, analgesic effect of<br />
pethidine, one of the most frequently used opiate<br />
agonists, starts within 10 to 20 min, and lasts for 2 to 4 h<br />
(Lee et al., 1993).<br />
As it is cheap, simple to use and readily available,<br />
systemic pethidine is widely used for relief of labour pain.<br />
Use of parental opioids was found to be between 39 and<br />
56% in various hospital obstetrics units, in the United<br />
States (Hawkins and Beaty, 1999). However, many<br />
studies have suggested that intramuscular pethidine may<br />
be ineffective at relieving labor pain (Wilson et al., 1986;<br />
Morrison et al., 1987; Fairlie et al., 1990; Olofsson et al.,<br />
1996) and it has been suggested that their use may even<br />
be unethical and medically incorrect (Fairlie et al., 1999).<br />
Systemic opioids lead to some adverse effects on both<br />
mother and baby, including dysphoria, sedation,<br />
respiratory depression, nausea and vomiting and delayed<br />
gastric emptying for the mother (Douglas and Levinson,<br />
2001). As pethidine crosses the placenta, it may<br />
accumulate in the fetal circulation (Gaylard et al., 1990),<br />
causing early neonatal respiratory depression and<br />
behavioral and feeding problems for even up to six weeks<br />
after delivery (Belsey et al., 1981; Belfrage et al., 1981;<br />
Nissen et al., 1997).<br />
Consistent with ours, an uncontrolled, observational<br />
study on primi-gravid, showed that women judged nitrous<br />
oxide to be more effective than opioids (Harrison et al.,
1987). The study suggests that, nitrous oxide is a useful<br />
method for women who wish to cope with the earlier part<br />
of labor "drug free”. Consistent with ours, this study<br />
showed that labor was more rapid in the nitrous group;<br />
however, it is unlikely that nitrous oxide causes more<br />
rapid labor, and it is unlikely that opioids significantly slow<br />
labor. It may be more effective for women whose labor is<br />
shorter (Harrison et al., 1987).<br />
Although, nitrous oxide is certainly not a potent<br />
analgesic, it has more beneficial effects for many<br />
parturient women as compared to pethidine. It is easy to<br />
administer and safe for both mother and infant.<br />
ACKNOWLEDGEMENTS<br />
This study was supported by a dissertation grant from<br />
School of Medicine, Zahedan University of Medical<br />
Sciences, the authors would like to thank subjects who<br />
willingly participated in the study and would like to<br />
acknowledge the Clinical Research Development Center<br />
of Ali-ebne-abitaleb Hospital, Zahedan University of<br />
Medical Sciences for its help in preparing this manuscript.<br />
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African Journal of Pharmacy and Pharmacology Vol. 5(17), pp. 2018-2026, 8 November, 2011<br />
Available online at http://www.academicjournals.org/AJPP<br />
DOI: 10.5897/AJPP11.518<br />
ISSN 1996-0816 ©2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Differential analysis of human kidney stone samples<br />
using electrospray ionization mass spectrometry<br />
Zhiquan Zhou 1 , Yongzhong Ouyang 2 , Xiangtai Zeng 3 , Tingting Zhang 1 , Bin Jia 2 , Xinglei Zhang 2 ,<br />
Huanwen Chen 2 and Jianhua Ding 2 *<br />
1 Institute of Information Engineering, Harbin Institute of Technology, Weihai, 264209, P. R. China.<br />
2 Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation, Biology and Material Science, College of Chemistry,<br />
East China Institute of Technology, Nanchang, 330013, P. R. China.<br />
3 Department of Surgery, 2nd Hospital of Gannan Medical University, Xinfeng, 341600, P. R. China.<br />
Accepted 21 October, 2011<br />
Kidney stones may be caused by many factors, including ingestion of melamine for a relatively long time.<br />
The diagnosis of melamine-induced kidney stones and the understanding of how the melamine is<br />
involved in the formation of kidney stones are of practical importance. To establish a sensitive method<br />
based on widely used electrospray ionization mass spectrometry (ESI-MS) for diagnosis of<br />
melamine-induced kidney stones and to probe the differential formation of melamine-induced kidney<br />
stones at molecular levels. Human kidney stones were collected in hospital from 6 groups of patients at<br />
different ages. ESI-MS was employed as the main technique with the principal component analysis for<br />
data processing. Using principal component analysis (PCA) of the ESI-MS fingerprints, a set of 21<br />
melamine-induced kidney stone samples and 21 uric acid derived kidney stone samples were<br />
successfully differentiated from the other groups, rendering ESI-MS method a potential platform for<br />
differential analysis of the human kidney stones of various causes at molecular levels. The experimental<br />
results also indicate that in addition to the melamine, the chemical compounds enwrapped in the<br />
melamine-induced kidney stone samples are different from other kidney stone samples. These findings<br />
suggest that ESI-MS is a useful tool for diagnosis of melamine-induced kidney stone samples and the<br />
melamine-induced kidney stone could be formed by different mechanisms.<br />
Key words: Toxicity of melamine, chemical profiling, uric acid, electrospray ionization-mass spectrometry<br />
(ESI-MS), kidney stone, melamine, principal component analysis.<br />
INTRODUCTION<br />
Kidney stones, one of the most painful urologic disorders,<br />
have beset humans for centuries. It is estimated that<br />
about three percent of the world's population will suffer<br />
from kidney stones in their lifetime (Johnson et al., 1979;<br />
Coe et al., 1996). Annually, about 3 million people need<br />
medical care due to kidney stone problems (Coe et al.,<br />
1996; Hiatt et al., 1996). Kidney stones in human are<br />
normally caused by many factors, such as the diet and<br />
genetics (Anderson, 2002). For example, it is known that<br />
*Corresponding author. E-mail: Dingjianh2004@126.com. Tel:<br />
0086-791-3896370. Fax: 0086-791-3896370.<br />
the ingestion of melamine illicitly used in food may cause<br />
the formation of kidney stones in both humans (Lam et al.,<br />
2009) and animals (Baynes et al., 2008). Although, the<br />
formation mechanism for the melamine-induced kidney<br />
stones remains unclear, unambiguous chemical profiling<br />
of the melamine-induced human kidney stones is urgently<br />
required for better medical care performance. Chinese<br />
government covers all the medical expenditures for the<br />
melamine-induced kidney stone diseases after the<br />
melamine event, while patients suffering from uric<br />
acid-induced kidney stones are not included (Chan and<br />
Lai, 2009). Thus, accurate detection of melamine-induced<br />
human kidney stones and the study of the formation<br />
mechanism as well are of great significance to both clinics
Table 1. Clinical data of human kidney stone samples.<br />
Zhou et al. 2019<br />
Patient number Age Sex Quantity and period Stone location Imaging studies<br />
Sample 1 26 month Male 30 - 50 g/day, 2 years Left ureter Nonopaque stone<br />
Sample 2 36 month Female 30 - 50 g/day, 1.5 years Left kidney pelvis Nonopaque stone<br />
Sample 3 7 years Male 30 - 50 g/day, 2.5 years Left kidney pelvis Radiopaque<br />
Sample 4 17 month Male 30 - 50 g/day, 1.5 year Left kidney pelvis Radiopaque<br />
Sample 5 48 years Female No drinking Right ureter Radiopaque<br />
Sample 6 44 years Male No drinking Left kidney pelvis Radiopaque<br />
diagnosis and economic interests.<br />
X-ray inspection provides valuable information about<br />
the size and location of the kidney stone, and thus, it is<br />
the most common technique for clinical diagnosis of the<br />
calcium oxalate stones (Herring, 1962; Pietrow and<br />
Karellas, 2006). However, about 10% of radiolucent<br />
kidney stones, such as melamine-induced stone, do not<br />
contain enough calcium to be detected by standard X-ray<br />
imaging methods. Currently, computed tomography (CT)<br />
is considered as the gold standard diagnostic test for the<br />
detection of kidney stones (Ferrandino et al., 2010;<br />
Otnes,1983), and most of them are detectable by CT<br />
except for very rare stones, which are composed of drug<br />
residues in the urine (Pietrow and Karellas, 2006).<br />
However, the CT scans impose a radiation exposure and<br />
a high cost on patients, and thus, it is not the best choice<br />
for clinical diagnosis. Ultrasound imaging is alternatively<br />
useful for the detection of kidney stones as it gives details<br />
about the presence of hydronephrosis, particularly, for<br />
cases where X-ray/CT imaging is discouraged (Jia et al.,<br />
2009; Sun et al., 2009). However, the results of ultrasound<br />
imaging are highly dependent on the clinical observations<br />
and statistics (Wen et al., 2009; Zhang et al., 2009).<br />
Infrared spectroscopy and Raman spectroscopy detects<br />
limited categories of urinary stones (Chiu et al., 2010;<br />
Evan et al., 2005). The chemical elements in the stones,<br />
such as calcium, phosphorus, oxygen, carbon, etc., can<br />
be detected using scanning electron microscopy (SEM)<br />
combined with Fourier transform infrared spectroscopy<br />
(FTIR) (Marickar et al., 2009). However, the presence of<br />
melamine in the stone samples can not be determined. So<br />
far, there is no reliable method available for fast<br />
differentiation of the melamine-induced kidney stone from<br />
other types.<br />
Recently, surface desorption atmospheric pressure<br />
chemical ionization (SDAPCI) (Yang et al., 2009a, b, c;<br />
Chen et al., 2007) has been successfully employed for<br />
direct analysis of melamine tainted powdered milk<br />
samples, with minimal sample pretreatment. This<br />
provides the potential feasibility for detection of melamine<br />
in kidney stone samples by SDAPCI-MS, which will be<br />
described in other studies. Methods based on<br />
electrospray ionization mass spectrometry (ESI-MS) have<br />
been widely used for rapid differentiation of quality of<br />
volatile liquid foods (De Souza et al., 2007; Mendonça et<br />
al., 2008; Sanvido et al., 2010; Biasotoa et al., 2010;<br />
Alves et al., 2010). In this study, an electrospray mass<br />
spectrometric method has been applied for the chemical<br />
profiling of kidney stone solution samples.<br />
Melamine-induced kidney stones and uric acid derived<br />
kidney stones have been differentiated by performing<br />
principal component analysis (PCA) with the mass<br />
spectral raw data, which were recorded under either<br />
positive or negative ion detection mode. Since neither<br />
time consuming steps for separation of the sample nor<br />
expensive MS n instrument for molecular structure<br />
identification is required for differentiation of the<br />
melamine-induced kidney stone samples, the results<br />
suggest that the ESI-MS-based method reported here can<br />
be particularly useful for rapid differentiation of kidney<br />
stones at the molecular level.<br />
METHODOLOGY<br />
Materials and sample preparation<br />
The human kidney stone samples for 6 patients were provided by<br />
the Hospital of Gannan Medical University. As shown in Table 1, the<br />
patients for sample 1 to 4 are little children (not more than 7 years<br />
old) who are possible victims of melamine tainted milk event (Xin<br />
and Stone, 2008) that happened in China. There is no clear<br />
evidence showing that the powder milks consumed by these<br />
patients were contaminated with melamine. As recorded in the<br />
clinical data sheet, patient 3, a 7 year old boy, had consumed<br />
powdered milk for the longest time. The preliminary work performed<br />
in a Canada lab using secondary ion mass spectrometry (Sodhi et<br />
al., 2010) also shows that melamine indeed exists in the calculus<br />
from the patient 3. Patients 5 and 6 are adults, who did not consume<br />
any milk products. Therefore, the stone samples from patients 5 and<br />
6 were used as the reference samples. The kidney stone samples (1<br />
to 5 mg) were dissolved in 10 ml acetic acid/methanol solution (1:4,<br />
v/v) and ultrasonicated for 20 min (10 W power) to assist dissolution.<br />
The solution was then diluted 1000 times with methanol/water (1:1,<br />
v/v) solvent for the ESI-MS analysis. Chemicals, such as methanol<br />
(analytical reagent (AR) grade) and acetic acid (AR grade) were<br />
bought from Chinese Chemical Reagent Co. Ltd. (Shanghai, China).<br />
ESI-LTQ mass spectrometer<br />
The experiments were carried out using a commercial available<br />
linear ion trap mass spectrometer (LTQ-XL, Finnigan, San Jose, CA)
2020 Afr. J. Pharm. Pharmacol.<br />
Figure 1. Mass spectrum recorded using positive ESI-MS from<br />
different kidney stone solution samples. 1 to 6#: the stone from<br />
patient 1 to 6, respectively.<br />
installed with an ESI source. The ESI source and the LTQ mass<br />
spectrometer were set to work under positive/negative ion detection<br />
mode. The nebulizing gas (N2) pressure was 1 MPa, the ESI high<br />
voltage was +4.5 kV/-3.5kV, and the temperature of the ion<br />
introduction capillary was 350°C. Other parameters were set at<br />
default values of the instrument and no further optimization of the<br />
ESI-MS was performed.<br />
Data acquisition and analysis<br />
The full scan mass spectra were recorded for an average acquisition<br />
time of 30 s. Collision induced dissociation (CID) was performed<br />
with 20 to 37% collision energy (CE). The parent ions were isolated<br />
with a mass window width of 1.5 Da, and the mass<br />
spectrometry/mass spectrometry (MS/MS) spectra were collected<br />
for a recording time of more than 30 s if necessary. Principal<br />
component analysis (PCA) of the mass spectral fingerprint data was<br />
performed in Matlab (version 7.0, Mathworks Inc., Natick, U.S.A.).<br />
The mass spectral data were exported to Microsoft Excel and the<br />
data were arranged using the mass to charge (m/z) values as<br />
independent variables and using the relative abundance of the full<br />
scan mass fingerprint (MS 1 ) as the dependent variables. The whole<br />
mass spectra data were treated as a matrix X, in which the rows and<br />
the columns corresponded to sample cases and m/z value variables,<br />
respectively. All the mass spectral data expressed by the relative<br />
abundance were directly used for the PCA. The ‘princomp’ function<br />
included in the ‘Matlab Toolbox’ was used for the PCA processing.<br />
When the PCA was completed, the scores of the first three principal<br />
components (PCs) were exported to new spreadsheets, and then<br />
Matlab was used to present the results of statistical analysis for<br />
better visualization.<br />
RESULTS AND DISCUSSION<br />
ESI-MS analysis<br />
Generally, different types of kidney stones vary in<br />
chemical compositions, but most of the kidney stones are<br />
composed of calcium oxalate, calcium phosphate and<br />
magnesium ammonium phosphate (Moe, 2006; Millman<br />
et al., 1982). Dissolved in acetic acid/methanol solution,<br />
the corresponding metal salts, such as calcium acetic,<br />
magnesium acetic, etc., were formed. ESI-MS provides<br />
information-rich spectra and many species can be<br />
simultaneously detected. Note that the solid kidney stones<br />
must be dissolved into liquid form prior to ESI-MS<br />
measurements. In order to avoid the contamination of the<br />
ESI-MS instrument, the stone solutions were diluted 1000<br />
times with methanol/water (1:1, v/v) solvent before they<br />
were electrosprayed for further MS analysis.<br />
The mass spectra of the six samples were recorded<br />
under the positive ion detection mode. As shown in Figure<br />
1, a set of peaks, such as m/z 127, 135, 177, 279, 301<br />
and 377 appeared in all the six samples. It was known<br />
that the peaks at m/z 279 and 301 were the protonated<br />
dibutyl phthalate (DBP) (Hu et al., 2010) and [DBP+Na] + ,<br />
respectively, due to the DBP used as plasticizers in the<br />
instrument system. Although, the signal of m/z 127 was<br />
recorded in all the six samples, the intensity of m/z 127<br />
detected in the sample from patients 1, 5 and 6 was in<br />
an order of magnitude less than those detected from<br />
the others. Furthermore, in the CID experiments, the<br />
precursor ions at m/z 127 detected from the sample 3<br />
generated ions of m/z 43 (CH3N2 + ), 85 (C2H5N4 + ) and 110<br />
(C3H4N5 + ) as the major fragments (Figure 2a) by the loss<br />
of C2H4N4, CH2N2 and NH3, respectively. In the MS 3<br />
experiment, a characteristic fragment of m/z 43 (CH3N2 + )<br />
was successively produced from the fragmentation of m/z<br />
85 (Figure 2b) or 110 (Figure 2c). These fragmentation<br />
patterns match with those obtained with authentic<br />
melamine samples and are in accordance with the<br />
fragmentation pathways of protonated melamine<br />
observed in previous studies (Zhu et al., 2009).<br />
Consequently, the peak at m/z 127 detected from sample<br />
3 was confirmed to the protonated melamine molecule<br />
(M+H) + . However, these characteristic fragments could<br />
not be produced from the other stones (samples 1, 2, 4, 5<br />
and 6) (Figure 3), indicating that only sample 3 is the<br />
melamine-induced kidney stone among these stones.<br />
Upon negative ion detection mode, calcium and<br />
magnesium acetate salts formed the corresponding<br />
deprotonated ions, such as (CaAC2)AC - (m/z 217),
Figure 2. Mass spectra recorded by ESI-MS. (a) MS/MS spectrum of protonated melamine<br />
(m/z 127) in sample 3; (b) MS/MS/MS spectrum of the ionic fragments (m/z 85) produced<br />
from protonated melamine (m/z 127) in sample 3; (c) MS/MS/MS spectrum of the ionic<br />
fragments (m/z 110) produced from protonated melamine (m/z 127) in sample 3; (d) MS/MS<br />
spectrum of deprotonated uric acid (m/z 167) in sample 2.<br />
Figure 3. MS/MS spectra of precursor ions (m/z 127) and MS/MS/MS spectra of ionic<br />
fragments (m/z 85) produced from m/z 127 from different kidney stone solution samples. 1 to<br />
6#: the stone from patient 1 to 6, respectively.<br />
Zhou et al. 2021
2022 Afr. J. Pharm. Pharmacol.<br />
Figure 4. Upon the negative ion detection mode, ESI mass<br />
spectrum recorded from the kidney stone solution samples. 1 to<br />
6#: the stone from patient 1 to 6, respectively.<br />
(CaAC2)2AC - (m/z 375), (CaAC2)3AC - (m/z 533),<br />
(MgAC2)AC - (m/z 201), (NaAC)AC - (m/z 141) and<br />
(KAC)2AC - (m/z 255), as shown in the Figure 4. It is<br />
noteworthy that significant abundant peaks at m/z 167 is<br />
present only in sample 2, while other MS peaks for<br />
sample 2 are much lower than those in other samples,<br />
suggesting that sample 2 might have different chemical<br />
contents. Upon CID, the precursor ions of m/z 167<br />
generated the abundant fragments of m/z 124 (C4H2N3O2<br />
by the loss of CHNO (Figure 2d). These characteristic<br />
fragments were in good agreement with previous work of<br />
uric acid (Dai et al., 2007). Furthermore, CID data also<br />
matched with those recorded using authentic uric acid<br />
compound under the same experimental conditions.<br />
Therefore, the ions at m/z 167 in the samples were<br />
assigned to be the deprotonated uric acid. In comparison<br />
with other samples, abundance of m/z 167 in sample 2<br />
suggests that the content of uric acid in kidney stone<br />
samples 2 should be much higher than others.<br />
Accordingly, the cause of the kidney stone for baby girl<br />
(patient 2) could be diagnosed as uric acid.<br />
In a related study, the same groups of samples were<br />
investigated using time-of-flight secondary ion mass<br />
- )<br />
spectrometry (ToF-SIMS) (Sodhi et al., 2010). Polished<br />
cross-sections of the samples were obtained for recording<br />
SIMS mass spectra in both high spatial and high mass<br />
resolution modes. The motivation was to distinguish<br />
between different stone types using the distribution<br />
patterns of the N-containing species obtained in the<br />
ToF-SIMS experimental data. Although, some interesting<br />
data were obtained, the SIMS technique was not able to<br />
detect melamine molecules from the melamine-induced<br />
stone sample. The results obtained using ESI-MS in this<br />
work differentiated melamine-induced kidney stones from<br />
the uric acid-based kidney stones, showing that ESI-MS<br />
provides complementary information for the ToF-SIMS<br />
results. The combination of these two techniques is<br />
promising for the understanding of the formation<br />
mechanism of the kidney stones. For the clinic diagnosis<br />
purpose, however, ESI-MS is of obvious advantages,<br />
including simple operation, low cost instrument and fast<br />
analysis speed, especially when PCA is applied to<br />
process the experimental data (demonstrated<br />
subsequently).<br />
Principal component analysis (PCA)<br />
As mentioned earlier, the melamine and uric acid induced<br />
human kidney stones can be differentiated under the<br />
positive and/or negative mode of ESI-MS/MS experiments.<br />
However, the sensitive CID measurements require an<br />
instrument of advanced tandem MS capability. This<br />
requirement demands expensive mass spectrometers. As<br />
shown in the full scan mass spectra, more signals rather<br />
than the melamine itself (m/z 127) were differentiable in<br />
the mass spectral fingerprints. Once the differences are<br />
clearly visualized, the melamine-induced kidney stones<br />
can be reliably diagnosed without resorting to tandem MS<br />
experiments, featured on site applications, using simple<br />
and low cost portable mass spectrometers. Therefore,<br />
PCA, a powerful tool for data compression and<br />
information extraction (Jackson, 1980; Moore, 1981;<br />
Tipping and Bishop, 1999) was employed to process the<br />
ESI-MS data for differentiation of the samples.<br />
As a result, a PCA score plot of six types of stone<br />
samples are shown in Figure 5, among which a1 and a2<br />
correspond to the data points of full ESI mass spectrum<br />
recorded upon the positive ion detection mode. Note that,<br />
there are 21 data points generated for the groups of all six<br />
patients, using 21 pieces of the stone samples from the<br />
same patient. As shown in Figure 5a1 and a2, a total of<br />
126 data points were explained by the score graphs of<br />
PC1-PC2 and PC2-PC3. 91.9% of the total variations<br />
were represented and the percentages of variance exp-<br />
lained by PC1, PC2 and PC3 were 54.73, 25.20 and 11.97%,<br />
respectively. Except for samples 4 and 6 which can not be<br />
distinguished from each other, others were all differentia-<br />
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<br />
positive ESI; a2: score plot PC2-PC3 based on positive ESI; b1: score plot PC1-PC2 under negative ESI ion<br />
mode; b2: score plot PC2-PC3 under negative ESI ion mode. c1: 3D-score plot PC1-PC2 under positive ESI<br />
c2: 3-D score plot under negative ESI.<br />
(uric acid induced stones) and 3 (melamine-induced<br />
stones) has been achieved. On the other hand, the<br />
distribution of signs in each cluster from samples 4 to 6 is<br />
restricted in a narrow scope. Thus, the PCs show strong<br />
ability for differentiation of melamine and uric acid induced<br />
stones from the other types of kidney stones. Although, a<br />
few data points of sample 5 were mixed together with<br />
samples 4 and 6 in the PC1 and PC2 plane (Figure 5a1),<br />
all the data points of sample 5 were completely separated<br />
from the other samples in PC3, as shown in Figure 5a2.<br />
However, upon the negative ion detection mode, the six<br />
different types of kidney stones were differentiated from<br />
each other in the PC1 and PC2 plane (Figure 5b1), and<br />
this could be clearly visualized in the corresponding 3-D<br />
space (Figure 5c2). Therefore, negative ESI-MS is<br />
proposed for differentiation of the kidney stone samples<br />
without CID experiments, although the melamine induced<br />
stones have been differentiated from the others under the<br />
positive ion mode.<br />
As shown in the clinical data (Table 1), the different<br />
X-ray imaging properties (both the stone location and<br />
transparency) of sample 1 from sample 5 might explain<br />
the separation of samples 1 and 5 in the PCA score plots.<br />
Zhou et al. 2023<br />
In both Figure 5b1 and 5b2, 1.5-year history of powdered<br />
milk intake was most likely to be the only factor for<br />
differentiating sample 4 from sample 6 in the PCA score<br />
plots, since the stones in patients 4 and 6 were both<br />
located in the left kidney pelvis and were all radiopaque in<br />
the X-ray imaging studied in the clinical examination.<br />
Although, the stones in patient 5 were also radiopaque,<br />
the location of the stone 5 was at the right ureter instead<br />
of the left kidney pelvis. This could be the important factor<br />
for differentiating sample 5 from either sample 4 or<br />
sample 6. Similar to sample 5, the stone in sample 1 was<br />
also located in the ureter (the left ureter), but it was<br />
radiolucent in the X-ray imaging study, indicating that the<br />
chemical composition of sample 1 should be different from<br />
the opaque samples. In the PCA score plots, sample 2<br />
was located remarkably far from the sample 3, because<br />
sample 2 was uric acid induced kidney stones, which<br />
should be chemically different from the melamine induced<br />
kidney stones. Conclusively, the location and imaging<br />
property of kidney stones are two of the most important<br />
factors used to distinguish these kidney stones and<br />
they play important roles in differentiating different types<br />
of kidney stones after performing PCA.
2024 Afr. J. Pharm. Pharmacol.<br />
Figure 6. PCA loading results for the PCs based on the negative<br />
ESI.<br />
Figure 6 presents the PCA loading plots of the 126 data<br />
points upon negative ion detection. The major differential<br />
peaks (that is, peaks of significant abundances in the PCA<br />
loading plots, which correspond to the ions that<br />
contributed most to differentiation of the samples) shown<br />
in the PCA loading plots, could potentially be useful as<br />
molecular markers for differentiating different kidney<br />
stones. For example, the most predominant ions in the<br />
loading of PC2, which corresponded to the deprotonated<br />
uric acid (m/z 167), could serve as the most important<br />
factor for differentiating the uric acid induced kidney stone<br />
(patient 2) from the others. For this reason, typical<br />
differential peaks at m/z 167, 255, 265, 279, 281, 282, 311,<br />
337, 346, 353, 375 and 397 were selected as molecular<br />
markers, because they were outstanding in the PCA<br />
loading plots. Structural identification of all these<br />
differential peaks is theoretically possible using tandem<br />
mass spectrometry experiments, but it is beyond the<br />
scope of this study. Also, Figure 7 gives the loading plots<br />
of 126 data points under positive ion detection model. It is<br />
clear that the peaks at m/z 135, 139, 149, 177, 191, 195,<br />
198, 203, 207, 261, 285, 301, 377, 391 and 413<br />
contributed most to differentiation of these samples, and<br />
could be regarded as molecular markers for differentiating<br />
different kidney stones. The absence of the ion at m/z 127<br />
in the load plots suggests that the ion at m/z 127 is not the<br />
important variable in the differentiation of these samples<br />
in the full ESI scan mass spectra, although the<br />
confirmation of melamine-contained sample requires the<br />
MS/MS of the ion at 127 and MS 3 of the ionic fragments at<br />
m/z 85 produced from the m/z 127. This is because, the<br />
Figure 7. PCA loading results for the PCs based on the<br />
positive ESI.<br />
ion at m/z 127 can be observed in all the six samples in<br />
the full ESI scan experiments, due to disturbance of<br />
impurities.<br />
In summary, the full mass spectra could be collected in<br />
a few seconds using ESI mass spectrometry with minimal<br />
sample pretreatment, indicating that ESI-MS affords high<br />
analysis speed. Sufficient information used for<br />
differentiation of different kidney stones could be provided,<br />
even though the stone liquid solutions were diluted 1000<br />
times before introducing into the ESI ionization,<br />
demonstrating high sensitivity of the ESI-MS. Good<br />
results for differentiation of melamine and uric acid<br />
induced kidney stones from the others whenever the<br />
positive or negative ion mode was used, to a certain<br />
degree, and they showed the stability and reproducibility<br />
of the ESI. Therefore, ESI-MS-based method proposed in<br />
this study is indeed a practical technique for rapid<br />
differentiation of kidney stones, with its high analysis<br />
speed, high sensitivity and good reproducibility.<br />
Conclusions<br />
An ESI-MS-based method was applied for fast detection<br />
of the melamine-induced and uric acid derived human<br />
kidney stone samples with minimal sample pretreatment.<br />
Using the tandem mass spectrometry, specific<br />
compounds, such as melamine and uric acid have been<br />
identified with high confidence. On the other hand, without<br />
the need of tandem MS capability, the ESI-MS combined<br />
with PCA is an analytical tool for differentiation of different<br />
types of human kidney stones, and is especially useful for
the negative ESI-MS mass spectral fingerprints in the<br />
analysis and detection of melamine-induced kidney<br />
stones. Melamine, a basic compound, could not be<br />
detected in the negative ESI mass spectra. However, it<br />
has been experimentally demonstrated that many other<br />
chemicals detected from the melamine-induced kidney<br />
stone samples were different from those detected from<br />
other types of kidney stone samples. These findings are<br />
the sound bases at molecular level for differentiation of<br />
the melamine-induced kidney stones from the others,<br />
particularly in the negative ion detection mode, and they<br />
provide useful hints for further study on the formation<br />
mechanisms of melamine-induced kidney stones in the<br />
pathological research.<br />
ACKNOWLEDGEMENT<br />
This work was jointly supported by the National<br />
Instrumentation Program (No. 2011YQ170067) and the<br />
Major Grant of Natural Science Foundation of Jiangxi<br />
Province (No. 2010GZH002).<br />
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complicated with acute renal failure in infants and young children.<br />
Chin. Med. J., 122: 245-251.<br />
Tipping ME, Bishop CM (1999). Probabilistic principal component<br />
analysis. J. R. Stat. Soc. B., 61: 611-622.<br />
Wen JG, Yang HJ, Wang Y, Wang GX (2009). The clinical analysis of<br />
urolithiasis in 165 infants and children with history of feeding<br />
melamine contaminated milk powder. J. Urol., 5: S33.<br />
Xin H, Stone R (2008). Tainted milk scandal Chinese probe unmasks<br />
high-tech adulteration with Melamine. Science, 322: 1310-1311.<br />
Yang SP, Chen HW, Yang YL, Hu B, Zhang X, Zhou YF, Zang LL, Gu HW<br />
(2009). Imaging melamine in egg samples by surface desorption<br />
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Chin. J. Anal. Chem., 37: 315-318.<br />
Yang SP, Ding JH, Zheng J, Hu B, Li JQ, Chen HW, Zhou ZQ, Qiao XL<br />
(2009). Detection of melamine in milk products by surface desorption
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Yang SP, Hu B, Li JQ, Han J, Zhang X, Chen HW, Liu Q, Liu QJ, Zheng J<br />
(2009). Surface desorption atmospheric pressure chemical ionization<br />
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Zhang L, Wu LL, Wang YP, Liu AM, Zou CC, Zhao ZY (2009).<br />
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African Journal of Pharmacy and Pharmacology Vol. 5(17), pp. 2027-2034, 8 November, 2011<br />
Available online at http://www.academicjournals.org/AJPP<br />
DOI: 10.5897/AJPP11.522<br />
ISSN 1996-0816 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Radical scavenging and antioxidant activity of Hibiscus<br />
rosasinensis extract<br />
Rajesh Mandade 1 *, S. A. Sreenivas 2 , D. M. Sakarkar 3 and Avijit Choudhury 4<br />
1 Pharmacology Department,S.N. Institute of Pharmacy, Pusad, India.<br />
2 Guru Nanak Institute of Pharmacy,Ibrahimpattnam, Hyderabad (A.P.), India.<br />
3 S.N. Institute of Pharmacy, Pusad, India.<br />
4 Shree Dhanvantary Pharmaceutical Analysis and Research Centre, Kim, Surat, Gujarat, India.<br />
Accepted 17 October, 2011<br />
Free radicals induce numerous diseases by lipid peroxidation, protein peroxidation and DNA damage. It<br />
has been reported that numerous plant extracts have antioxidant activities to scavenge free radicals. In<br />
the present study, the antioxidant properties of crude extract of Hibiscus rosasinensis were examined,<br />
using different in vitro analytical methodologies, such as total antioxidant activity determination by<br />
ferric thiocyanate, hydrogen peroxide scavenging, 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH)<br />
scavenging, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS + radical cation) radical cation<br />
scavenging activity and superoxide anion radical scavenging by riboflavin-methionine-illuminate<br />
system. Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and α-tocopherol were used<br />
as the reference antioxidant radical scavenger compounds. The crude extract inhibited 94.58%<br />
peroxidation of linoleic acid emulsion at 20 µg/ml concentration, while the standard antioxidants BHA,<br />
BHT and α-tocopherol indicated an inhibition of 93.75, 96.66 and 83.33% at 60 µg/ml concentration,<br />
respectively. The hydrogen peroxide radical, DPPH radical, ABTS + radical cation(s) and superoxide<br />
anion radical scavenging activities of crude extract were also compared to BHA, BHT and α-tocopherol<br />
as references antioxidant compounds. The present study shows that the crude extract is an effective<br />
natural antioxidant component.<br />
Key words: Hibiscus rosasinensis, antioxidant activity, free radical scavenging activity.<br />
INTRODUCTION<br />
Antioxidants help living organisms to deal with oxidative<br />
stress, caused by free radical damage. Free radicals are<br />
chemical species which contains one or more unpaired<br />
electrons due to which they are highly unstable and<br />
cause damage to other molecules by extracting electrons<br />
from them in order to attain stability. Reactive oxygen<br />
species (ROS) formed in vivo, such as superoxide anion,<br />
hydroxyl radical and hydrogen peroxide, are highly reactive<br />
and potentially damaging transient chemical species.<br />
ROS are continuously produced in the human body, as<br />
they are essential for energy supply, detoxification, chemi-<br />
cal signaling and immune function. ROS are regulated<br />
by endogenous superoxide dismutase, glutathione<br />
*Corresponding author. E-mail: raj_mandade@rediffmail.com.<br />
Tel: +917233249795 or +918888673088. Fax: 07233247308.<br />
peroxidase and catalase, but due to over-production of<br />
reactive species induced by exposure to external oxidant<br />
substances or a failure in the defense mechanisms,<br />
damage to cell structures, DNA, lipids and proteins<br />
(Valko et al., 2006) occur which increases risk of more<br />
than 30 different disease processes (Aruoma, 1998).<br />
Hibiscus rosasinensis Linn (Malvaceae) is a glabrous<br />
shrub widely cultivated in the tropics as an ornamental<br />
plant and has several forms with varying colors of<br />
flowers. In medicine, however the red flowered variety is<br />
preferred (Adhirajan et al., 2003). The leaves and flowers<br />
are observed to be promoters of hair growth and aid in<br />
healing of ulcers (Jadhav et al., 2009). Aerial part of H.<br />
rosasinensis has calcium channel blocking action (Gilani<br />
et al., 2005). Recent reports have also shown antiammonemic<br />
(Essa and Subramanian., 2007), antidiabetic<br />
(Venkatesh et al., 2008), hypolipidemic (Kumar<br />
et al., 2009), post-coital anti-fertility, cardio protective and
2028 Afr. J. Pharm. Pharmacol.<br />
wound healing activities (Gauthaman et al., 2006, Nayak<br />
et al., 2007).<br />
The free radical neutralizing property of several plants<br />
was reported by previous studies. The extracts from a<br />
number of medicinal plants which are known to have<br />
some biologically active principles are used in ayurvedic<br />
preparations and these extracts are prepared in bulk for<br />
commercial purpose. In the present study, the antioxidant<br />
property of 80% aqueous-ethanol crude extract of H.<br />
rosasinensis was examined by different in vitro analytical<br />
methodologies, such as total antioxidant activity<br />
determination by ferric thiocyanate, hydrogen peroxide<br />
scavenging, 1,1-diphenyl-2-picryl-hydrazyl free radical<br />
(DPPH) scavenging, 2,2′-azino-bis(3-ethylbenzthiazoline-<br />
6-sulfonic acid) (ABTS +<br />
radical cation) radical<br />
scavenging activity and superoxide anion radical<br />
scavenging by riboflavin-methionine-illuminate system.<br />
MATERIAL AND METHODS<br />
Plant<br />
Aerial part of H. rosasinensis was collected from the botanical<br />
garden of S. N. Institute of Pharmacy, Pusad, India. Identification<br />
and authentication of the samples was done by using standard<br />
botanical monographs. They were further confirmed at the<br />
Department of Botany, R.S.T.M University Nagpur, India.<br />
Preparation of crude extract<br />
The plant material was cleaned off adulterants, shade dried and<br />
was coarsely grounded. The powdered material (1 kg) was soaked<br />
in 80% aqueous-ethanol for 3 days with occasional shaking. It was<br />
filtered through a muslin cloth and then through a filter paper. This<br />
procedure was repeated thrice and the combined filtrate was<br />
evaporated on a rotary evaporator under reduced pressure (-760<br />
mmHg) to a thick, semi-solid mass of dark brown color, that is, the<br />
crude extract with a yield of approximately 10% (Gilani et al., 2005).<br />
Phytochemical screening<br />
The preliminary phytochemical studies (Adhirajan et al., 2003) were<br />
conducted for the aforementioned crude extracts of H. rosasinensis<br />
to find out the presence of sterols, carbohydrates and glycosides,<br />
tannins and flavonoid using standard analytical procedures (Gupta<br />
et al., 2009) (Table 1).<br />
Estimation of total phenolic compounds<br />
Total phenol<br />
The total phenolic content in the crude extract of H. rosasinensis<br />
was determined spectrophotometrically with Folin-Ciocalteu reagent<br />
using the modified method of Wolfe et al. (2003). An aliquot of the<br />
crude extract (0.5 ml) was mixed with 2.5 ml of 10% Folin-Ciocalteu<br />
reagent and 2 ml of Na2CO3 (75% w/v). The resulting mixture was<br />
vortexed for 15 s and incubated at 40°C for 30 min for colour<br />
development. The absorbance of the samples was measured at<br />
765 nm using spectrophotometer at UV/visible light. Total phenolic<br />
content was expressed as mg/g tannic acid equivalent from the<br />
calibration curve using the equation:<br />
Y = 0.1216x, R2 = 0.936512<br />
Where x was the absorbance and Y was the tannic acid equivalent<br />
(mg/g).<br />
The experiment was conducted in triplicate and the results are<br />
reported as mean ± SD values.<br />
Total flavonoid<br />
The method of Ordon et al. (2006) was used to estimate the total<br />
flavonoid contents of the crude extract solution based on the<br />
formation of a complex flavonoid-aluminium. A volume of 0.5 ml of<br />
2% AlCl3 ethanol solution was added to 0.5 ml of crude extract<br />
solution. After 1 h of incubation at the room temperature, the<br />
absorbance was measured at 420 nm using UV-VIS<br />
spectrophotometer. All determinations were done in triplicate and<br />
values were calculated from calibration curve obtained from<br />
quercetin using the equation:<br />
Y = 0.0255x, R2 = 0.9812<br />
where x was the absorbance and Y the quercetin equivalent (mg/g).<br />
Total flavonols<br />
Total flavonol content was determined by adopting the procedure<br />
described by Kumaran and Karunakaran (2007). The reacting<br />
mixture consisted of 2.0 ml of the sample, 2.0 ml of AlCl3 prepared<br />
in ethanol and 3.0 ml of (50 g/L) sodium acetate solution. The<br />
absorption at 440 nm was read after 2.5 h at 20°C. Total flavonoid<br />
content was calculated as quercetin (mg/g) equivalent from the<br />
calibration curve using the equation:<br />
Y= 0.0255x, R2 = 0.9812<br />
where x was the absorbance and Y the quercetin equivalent (mg/g).<br />
Total proanthocyandins<br />
Total proanthocyandins was determined based on Sun et al. (1998)<br />
procedure. 3 ml of vanillin-methanol (4% v/v) and 1.5 ml of<br />
hydrochloric acid was added to 0.5 ml of 1 mg/ml crude extract<br />
solution and then vortexed. The absorbance of resulting mixture<br />
was measured at 500 nm after 15 min at room temperature. Total<br />
proanthocyandin content was expressed as catechin equivalents<br />
(mg/g) using the following equation from the calibration curve:<br />
Y = 0.5825x, R2 = 0.9277<br />
where x was the absorbance and Y the catechin equivalent (mg/g).<br />
Total antioxidant activity determination by ferric thiocyanate<br />
method (FTC)<br />
The total antioxidant activity of the crude extract and standard<br />
antioxidants (BHA, BHT and α-tocopherol) was determined<br />
according to the ferric thiocyanate method (Mitsuda et al., 1996) as<br />
described by Gulcin (2006b). For stock solutions, 10 mg of the<br />
extract was dissolved in 10 ml distillate water. Then, the solution<br />
which contains 20 µg/ml concentration of the extract solution in<br />
2.5 ml of sodium phosphate buffer (0.04 M, pH 7.0) was added to<br />
2.5 ml of linoleic acid emulsion in sodium phosphate buffer (0.04 M,<br />
pH 7.0). Therefore, 5 ml of the linoleic acid emulsion was prepared<br />
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).<br />
On the other hand, 5 ml of control was composed of 2.5 ml of<br />
linoleic acid emulsion and 2.5 ml sodium phosphate buffer (0.04 M,<br />
pH 7.0). The mixed solution (5 ml) was incubated at 37°C in<br />
polyethylene flask. The peroxide level was determined by reading<br />
the absorbance at 500 nm in a spectrophotometer after reaction<br />
with FeCl2 (3.5%) and thiocyanate (30%) at intervals during<br />
incubation. During the linoleic acid peroxidation, peroxides are formed<br />
and that leads to the oxidation of Fe 2+ –Fe 3+ . The latter ions form a<br />
complex with ammonium thiocyanate and this complex has a<br />
maximum absorbance at 500 nm. This step was repeated every<br />
5 h. The percentage inhibition values were calculated at this point<br />
(30 h). High absorbance indicates high linoleic acid emulsion<br />
peroxidation (Table 2). The solutions without the extract were used<br />
as blank samples. Total antioxidant activity determination was<br />
performed in triplicate. The inhibition percentage of lipid peroxidation in<br />
linoleic acid emulsion was calculated by following equation:<br />
Inhibition of lipid peroxidation (%)=100-(As/Ac×100)<br />
AC is the absorbance of control reaction which contains only linoleic<br />
acid emulsion and sodium phosphate buffer and AS is the<br />
absorbance in the presence of sample crude extract or standard<br />
compounds (Gulcin, 2006a, b).<br />
Hydrogen peroxide scavenging activity<br />
The hydrogen peroxide scavenging assay was carried out following<br />
the procedure of Ruch et al. (1989). For this aim, a solution of H2O2<br />
(43 mM) was prepared in phosphate buffer (0.1 M, pH 7.4). Crude<br />
extract at 20 µg/ml concentration in 3.4 ml phosphate buffer was<br />
added to 0.6 ml of H2O2 solution (0.6 ml, 43 mM). The absorbance<br />
value of the reaction mixture was recorded at 230 nm. Blank<br />
solution contains sodium phosphate buffer without H2O2. The<br />
concentration of hydrogen peroxide (mM) in the assay medium was<br />
determined using a standard curve (r 2 : 0.9895):<br />
Absorbance = 0.038 × [H2O2] + 0.4397<br />
The percentage of H2O2 scavenging of crude extract and standard<br />
compounds were calculated using the following equation:<br />
H2O2 scavenging effect (%) = (1 – As/Ac) × 100<br />
where AC is the absorbance of the control and AS is the absorbance<br />
in the presence of the sample extract or standards (Gulcin, 2006b;<br />
Elmastas et al., 2005).<br />
DPPH free radical scavenging activity<br />
The DPPH free radical scavenging activity of crude extract was<br />
determined according to the method described by Gulcin (2006b)<br />
with slight modifications. In its radical form, DPPH absorbs at<br />
517 nm, but upon reduction by an antioxidant or a radical species<br />
its absorption decreases and thereby the bleaching rate of a stable<br />
free radical, DPPH in the presence of the sample is monitored at a<br />
characteristic wavelength.<br />
Briefly, 0.1 mM solution of DPPH was prepared in ethanol and<br />
0.5 ml of this solution was added 1.5 ml of the extract solution in<br />
ethanol at different concentrations (20 to 60 µg/ml). These solutions<br />
were vortexes thoroughly and incubated in dark, half hour later, the<br />
absorbance was measured at 517 nm against blank samples.<br />
Mandade et al. 2029<br />
Lower absorbance of the reaction mixture indicates higher DPPH<br />
free radical scavenging activity. A standard curve was prepared<br />
using different concentrations of DPPH. The DPPH concentration<br />
scavenging capacity was expressed as mM in the reaction medium<br />
and calculated from the calibration curve determined by linear<br />
regression (r 2 : 0.9845):<br />
Absorbance = 9.692 × [DPPH] + 0.215<br />
The capability to scavenge the DPPH radical was calculated using<br />
the following equation:<br />
DPPH scavenging effect (%) = (1- As/Ac) × 100<br />
where AC is the absorbance of the control which contains DPPH<br />
solution and AS is the absorbance in the presence of the extracts<br />
(Gulcin et al., 2004c; Elmastas et al., 2006).<br />
ABTS + radical cation(s) decolorization assay<br />
The spectrophotometric analysis of ABTS + radical cation(s)<br />
scavenging activity was determined according to Re et al. (1999)<br />
method. This method is based on the ability of antioxidants to<br />
quench the long-lived ABTS radical cation, a blue/green<br />
chromophore with characteristic absorption at 734 nm, in<br />
comparison to that of BHA, BHT and α-tocopherol. The ABTS + was<br />
produced by reacting 2 mM ABTS + radical cation(s) in H2O with<br />
2.45 mM potassium persulfate (K2S2O8), stored in the dark at room<br />
temperature for 4 h. Before usage, the ABTS + solution was diluted<br />
to get an absorbance of 0.750 ± 0.025 at 734 nm with sodium<br />
phosphate buffer (0.1 M, pH 7.4). Then, 1 ml of ABTS + solution was<br />
added 3 ml of the extract solution in ethanol at different<br />
concentrations (20 to 60 µg/ml).<br />
After 30 min, percentage inhibition at 734 nm was calculated for<br />
each concentration, relative to a blank absorbance. Solvent blanks<br />
were run in each assay. The extent of decolorization is calculated<br />
as percentage reduction of absorbance. For preparation of a<br />
standard curve, different concentrations of ABTS + radical cation(s)<br />
were used. The ABTS + radical cation(s) concentration (mM) in the<br />
reaction medium was calculated from the following calibration<br />
curve, determined by linear regression (r 2 : 0.9841):<br />
Absorbance = 4.6788 × [ABTS + ] + 0.199<br />
The scavenging capability of ABTS + radical was calculated using<br />
the following equation:<br />
ABTS scavenging effect (%) = (1- As/Ac) × 100<br />
where AC is the initial concentration of the ABTS + radical cation(s)<br />
and AS is absorbance of the remaining concentration of ABTS +<br />
radical cation(s) in the presence of the extract.<br />
Superoxide anion radical scavenging activity<br />
Superoxide radicals were generated by the method of Beauchamp<br />
and Fridovich (1971) described by Zhishen et al. (1999) with slight<br />
modification. Superoxide radicals are generated in riboflavin,<br />
methionine, illuminate and assayed by the reduction of nitroblue<br />
tetrazolium (NBT) to form blue formazan (NBT 2+ ). All solutions were<br />
prepared in 0.05 M phosphate buffer (pH 7.8). The photo-induced<br />
reactions were performed using fluorescent lamps (20 W). The<br />
concentration of the extract in the reaction mixture was 20 µg/ml.
2030 Afr. J. Pharm. Pharmacol.<br />
Absorbance (500 ηm)<br />
Table 1. Preliminary phytochemical screening.<br />
Chemical constituent Crude extract<br />
Alkaloids -<br />
Sterols +<br />
Carbohydrates and glycosides +<br />
Fixed oil and fats -<br />
Tannins and phenolic +<br />
Proteins +<br />
Triterpenoids +<br />
Flavonoids +<br />
3<br />
2<br />
1<br />
0<br />
0 20 40 60 80<br />
Incubation Time (h)<br />
Control<br />
α-Tocopherol<br />
BHA<br />
20 µg/ml<br />
40 µg/ml<br />
60 µg/ml<br />
Figure 1. Total ferric reducing power (FRAP) of different<br />
concentrations (20 to 60 µg/ml) of extract of H. rosasinensis and<br />
reference antioxidants; BHA, BHT and α-tocopherol. Total ferric<br />
reducing power determined according to the ferric ions (Fe 3+ )ferrous<br />
ions (Fe 2+ ) transformation. The reducing power was<br />
estimated based on the absorbance reading at 700 nm with a<br />
spectrophotometer. Values are expressed as mean ± standard<br />
deviation of three replicate determinations.<br />
The total volume of the reactant mixture was 3 ml and the<br />
concentrations of the riboflavin, methionine and NBT was 1.33 × 10 -<br />
5 , 4.46 × 10 -5 and 8.15 × 10 -8 M, respectively. The reactant was<br />
illuminated at 25°C for 40 min. The photochemically reduced<br />
riboflavin generated O2 . This reduced NBT to form blue formazan.<br />
The unilluminated reaction mixture was used as a blank. The<br />
absorbance was measured at 560 nm. The extract was added to<br />
the reaction mixture, in which O2 was scavenged, thereby inhibiting<br />
the NBT reduction. Decreased absorbance of the reaction mixture<br />
indicates increased superoxide anion scavenging activity. The<br />
inhibition percentage of superoxide anion generation was<br />
calculated by using the following formula:<br />
O2 .- scavenging effect (%) = (1- As/Ac) × 100<br />
where AC is the absorbance of the l-ascorbic acid and AS is the<br />
absorbance of the extract or standards (Gulcin et al., 2003, 2004b).<br />
RESULTS AND DISCUSSION<br />
A wide variety of in vitro methods have been set up to<br />
assess radical scavenging ability and antioxidant activity.<br />
Antioxidant capacity is widely used as a parameter for<br />
medicinal bioactive components. Different artificial free<br />
radical species, such as ABTS + radical cation, DPPH<br />
radical, as well as biological radicals or H2O2 scavenging<br />
activity have been used.<br />
Polyphenol are the major plant compounds with high<br />
level of antioxidant activity. This activity could be due to<br />
their ability to adsorb, neutralize and to quench free<br />
radicals (Duh et al., 1999). Their ability as free radical<br />
scavenger could also be attributed to their redox<br />
properties, presence of conjugated ring structures and<br />
carboxylic group which have been reported to inhibit lipid<br />
peroxidation (Rice-Evans et al., 1995).<br />
In the present study, it was found that the crude extract<br />
of H. rosasinensis contains high level of phenol content<br />
that might account for the strong activity observed<br />
against ABTS + radical cation(s) and H2O2 radicals. This<br />
scavenging activity may be due to the presence of<br />
hydroxyl groups attached to the aromatic ring structures<br />
and thus, help to quench the radicals (Vinson et al.,<br />
1998). On the other hand, the activity depicted in DPPH<br />
and superoxide anion may be as a result of the content of<br />
flavonoid which has been reported to possess high<br />
antioxidant activity.<br />
Total antioxidant activity determination in linoleic<br />
acid emulsion system by ferric thiocyanate method<br />
Lipid peroxidation contains a series of free radicalmediated<br />
chain reaction processes and is also<br />
associated with several types of biological damage. The<br />
total antioxidant activity of the crude extract, BHA, BHT<br />
and α-tocopherol in the linoleic acid system was<br />
determined by the ferric thiocyanate method. This<br />
method measures the amount of peroxide produced<br />
during the initial stages of oxidation, which is the primary<br />
product of lipid oxidation.<br />
Crude extract showed effective antioxidant activity in<br />
this system. The effect of different concentration (20 to<br />
60 µg/ml) of the extract on lipid peroxidation of linoleic<br />
acid emulsion is as shown in Figure 1.<br />
At these concentrations of the extract caused 94.58,<br />
95.00 and 95.88% lipid peroxidation inhibition of linoleic<br />
acid emulsion. Their activities are greater than 60 µg/ml<br />
concentration of BHA (93.75%), α-tocopherol (83.33%),<br />
but close to BHT (96.66%). Consequently, these results<br />
clearly indicate that the extract has an effective and<br />
powerful antioxidant activity by ferric thiocyanate method.<br />
Hydrogen peroxide scavenging activity<br />
Hydrogen peroxide has strong oxidizing properties. It can
Table 2. Polyphenol contents of crude extract of H. rosasinensis.<br />
Antioxidant compound Total antioxidant compound<br />
Phenol contents 49.44 mg/g tannic acid equivalent<br />
Proanthocyandins 9.15 mg/g catechin equivalent<br />
Flavonols 5.5 mg/g quercetin equivalent<br />
Flavonoids 4.8 mg/g quercetin equivalent<br />
Table 3. Ability of crude extract to scavenge hydrogen peroxide when compared with BHA,<br />
BHT and α-tocopherol.<br />
H2O2 scavenging activity (%) Superoxide scavenging activity (%)<br />
BHA 38.2 ± 2.8 76.4 ± 5.3<br />
BHT 36.3 ± 3.2 72.2 ± 6.4<br />
α-Tocopherol 41.2 ± 2.7 24.1 ± 3.2<br />
Extract 51.9 ± 4.5 68.2 ± 3.4<br />
be formed in vivo by many oxidizing enzymes, such as<br />
superoxide dismutase and can cross cellular membranes<br />
and may slowly oxidize a number of intracellular<br />
compounds. The ability of crude extract to scavenge<br />
hydrogen peroxide when compared with BHA, BHT and<br />
α-tocopherol is as shown in Table 3.<br />
Hydrogen peroxide scavenging activity of the extract at<br />
the used concentration of 20 µg/ml was found to be<br />
51.9 ± 4.5%. On the other hand, BHA, BHT and αtocopherol<br />
exhibited 38.2 ± 2.8, 36.3 ± 3.2 and<br />
41.2 ± 2.7% hydrogen peroxide scavenging activity at the<br />
same concentration, respectively. These results showed<br />
that the extract had an effective hydrogen peroxide<br />
scavenging activity. At the aforementioned concentration,<br />
the hydrogen peroxide scavenging effect of the extract<br />
and three standard compounds decreased in the order of<br />
the extract > α-tocopherol > BHA > BHT. Hydrogen<br />
peroxide itself is not very reactive; however, it can<br />
sometimes be toxic to cell, because it may give rise to<br />
hydroxyl radical in the cells.<br />
Radical scavenging activity<br />
Radical scavenging activities are very important due to<br />
the deleterious role of free radicals in foods and in<br />
biological systems. Diverse methods are currently used<br />
to assess the antioxidant activity of plant phenolic<br />
compounds. Chemical assays are based on the ability to<br />
scavenge synthetic free radicals, using a variety of<br />
radical-generating systems and methods for detection of<br />
the oxidation end-point. ABTS + radical cation(s) or DPPH<br />
radical scavenging methods are common<br />
spectrophotometric procedures for determining the<br />
antioxidant capacities of components.<br />
These chromogens (the violet DPPH radical and the<br />
blue green ABTS + radical cation(s) are easy to use,<br />
Mandade et al. 2031<br />
have a high sensitivity and allow for rapid analysis of the<br />
antioxidant activity of a large number of samples. These<br />
assays have been applied to determine the antioxidant<br />
activity of pure components (Awika et al., 2003, Yu et al.,<br />
2002; van den Berg et al., 2000b). DPPH has been<br />
widely used to evaluate the free radical scavenging<br />
effectiveness of various antioxidant substances (Ozcelik<br />
et al., 2003). DPPH is usually used as a reagent to<br />
evaluate free radical scavenging activity of antioxidants.<br />
DPPH is a stable free radical and accepts an electron<br />
or hydrogen radical to become a stable diamagnetic<br />
molecule (Soares et al., 1997). In the DPPH assay, the<br />
antioxidants are able to reduce the stable radical DPPH<br />
to non-radical form, DPPH-H. The purple colored<br />
alcoholic solution of DPPH radicals changes color to<br />
yellow in the presence of a hydrogen-donating<br />
antioxidant which could be measured at 517 nm.<br />
Figure 2 illustrates a significant decrease (P < 0.01) in<br />
the concentration of DPPH radical due to the scavenging<br />
ability of the extract and standards. BHA, BHT and αtocopherol<br />
were used as references. The scavenging<br />
effect of the extract and standards on the DPPH radical<br />
decreased in the order of BHA > Extract > BHT > αtocopherol<br />
which were 86.63, 84.01, 71.54 and 59.55%,<br />
at the concentration of 60 µg/m, respectively. DPPH free<br />
radical scavenging activity of the extract also increased<br />
with an increasing concentration (r 2 : 8246).<br />
Generation of the ABTS + radical cation(s) forms the<br />
basis of one of the spectrophotometric methods that have<br />
been applied to the measurement of the total antioxidant<br />
activity of pure substances solutions, aqueous mixtures<br />
and beverages (Miller, 1996). A more appropriate format<br />
for the assay is a decolorization technique, in that the<br />
radical is generated directly in a stable form prior to<br />
reaction with putative antioxidants. The improved<br />
technique for the generation of ABTS + described here<br />
involves the direct production of the blue/green ABTS +
2032 Afr. J. Pharm. Pharmacol.<br />
Absorbance (517 ηηm)<br />
3<br />
2<br />
1<br />
0<br />
0 20 40 60 80<br />
Concentration µg/ml (µg/ml)<br />
Extract<br />
BHT<br />
BHA<br />
α-Tocopherol<br />
Figure 2. DPPH free radical scavenging activity of different concentrations (20 60<br />
60 µg/ml) of extract of H. rosasinensis and reference antioxidants; BHA, BHT and<br />
α-tocopherol.<br />
Absorbance (734 ηηm)<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0.0<br />
0 20 40 60 80<br />
Concentration µg/ml (µg/ml)<br />
Extract<br />
BHA<br />
BHT<br />
α-Tocopherol<br />
Figure 3. ABTS free radical scavenging activity of different concentrations (20 to<br />
60 µg/ml) of extract of H. rosasinensis and reference antioxidants; BHA, BHT and<br />
α-tocopherol.<br />
chromophore through the reaction between ABTS +<br />
radical cation(s) and potassium persulfate.<br />
All the tested compounds exhibited affectual radical<br />
cation scavenging activity. As seen in Figure 3, the<br />
extract had effective ABTS + radical scavenging activity in<br />
a concentration-dependent manner (20 to 60 µg/ml).<br />
There is a significant decrease (P < 0.01) in all the<br />
concentration of ABTS + due to the scavenging capacity<br />
of all the extract concentrations. Also, the scavenging<br />
effect of the extract and standards on the ABTS + radical<br />
cation(s) decreased in the order: BHA > BHT > Extract<br />
> α-tocopherol, which were 97.33, 97.06, 89.2 and<br />
73.33% at the concentration of 60 µg/ml, respectively.<br />
Superoxide anion are a precursor to active free radicals<br />
and plays an important role in the formation of other<br />
ROS, such as hydrogen peroxide, hydroxyl radical and<br />
singlet oxygen, which induce oxidative damage in lipids,<br />
proteins and DNA (Pietta, 2000). Superoxide radical is<br />
normally formed first, and its effects can be magnified,<br />
because it produces other kinds of free radicals and<br />
oxidizing agents. Superoxide anions have the potential of<br />
reacting with biological macromolecules and have been<br />
implicated in several pathophysiological processes due to<br />
its transformation into more reactive species, such as<br />
hydroxyl radical that initiate lipid peroxidation. Also,<br />
superoxide has been observed to directly initiate lipid<br />
peroxidation (Wickens, 2001). In addition, it has been<br />
reported that antioxidant properties of some flavonoids<br />
are effective mainly via scavenging of superoxide anion<br />
radical. Superoxide anion derived from dissolved oxygen
iboflavin/methionine/illuminate system and reduces NBT<br />
in this system. In this method, superoxide anion reduces<br />
the yellow dye (NBT 2+ ) to produce the blue formazan<br />
which is measured spectrophotometrically at 560 nm.<br />
Antioxidants are able to inhibit the blue NBT formation<br />
(Parejo et al., 2002) and the decrease of absorbance at<br />
560 nm indicates the consumption of superoxide anion in<br />
the reaction mixture. Table 3 shows the inhibition<br />
percentage of superoxide radical generation by 20 µg/ml<br />
concentration of crude extract and antioxidant standards.<br />
The inhibition of superoxide radical generation results of<br />
extract and standards were found to be similar<br />
statistically. As shown in Table 3, the percentage<br />
inhibition of superoxide anion radical generation by<br />
20 µg/ml concentration of the extract was found as<br />
68.2 ± 3.4 %.<br />
On the other hand, at the same concentration, BHA,<br />
BHT and α-tocopherol exhibited 76.4 ± 5.3, 72.2 ± 6.4<br />
and 24.1 ± 3.2% superoxide anion radical scavenging<br />
activity, respectively. According to these results, crude<br />
extract had similar superoxide anion radical scavenging<br />
activity to BHA and BHT; however, it had higher<br />
superoxide anion radical scavenging activity than αtocopherol.<br />
Conclusion<br />
According to data obtained from the present study, 80%<br />
aqueous-ethanol crude extract of H. rosasinensis extract<br />
was found to be an effective antioxidant in different in<br />
vitro assays, including total antioxidant activity<br />
determination by ferric thiocyanate, DPPH radical,<br />
ABTS + radical cation(s) radical, superoxide anion radical<br />
scavenging and hydrogen peroxide scavenging when it is<br />
compared to standard antioxidant compounds, such as<br />
BHA, BHT and α-tocopherol.<br />
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African Journal of Pharmacy and Pharmacology Vol. 5(17), pp. 2035-2041, 8 November, 2011<br />
Available online at http://www.academicjournals.org/AJPP<br />
DOI: 10.5897/AJPP11.511<br />
ISSN 1996-0816 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Therapeutic monitoring of isoniazid, rifampicin,<br />
ethambutol and pyrazinamide serum levels in the<br />
treatment of active pulmonary tuberculosis and<br />
determinants of their serum concentrations<br />
Servet Kayhan 1,2 * and Alper Akgüneş 1,3<br />
1 Chest Diseases and Thoracic Surgery Hospital, Samsun, Turkey.<br />
2 Department of Pulmonary Disease And Tuberculosis, Ministry of Health, Chest Diseases and Thoracic Surgery<br />
Hospital, Samsun, Turkey.<br />
3 Department of Microbiology, Ministry of Health, Chest Diseases and Thoracic Surgery Hospital, Samsun, Turkey.<br />
Accepted 12 October, 2011<br />
Inadequate serum levels of antimycobacterial drugs have been associated with treatment failure,<br />
relapse and acquired drug resistance as well as high concentrations of these drugs may cause<br />
intolerance and toxic effects. We objected in this study to determine serum concentrations of antituberculosis<br />
drugs and to know the determinants of their concentrations. Venous blood samples was<br />
obtained 2 and 6 h after drug ingestion, and serum levels of drugs were analysed using high<br />
performance liquid chromatography. Among 49 enrolled active pulmonary tuberculosis patients, the<br />
prevalances of a low concentration of isoniazid, rifampicin, ethambutol and pyrazinamide were 28.6,<br />
75.5, 18.4 and 20.4%, respectively. 2 h ısoniazid (INH) concentration was found to be associated with<br />
sex (p = 0.005), correlated with body mass index (r = -0.390) and associated with drug dose (mg/kg) (p =<br />
0.000). By Independent samples t-test analysis, low 2 h rifampicin concentration was found to be<br />
associated with sex (p = 0.000) and smoking cigarette (p = 0.004). In conclusion, the results of this<br />
study have shown that, low 2 h serum INH and rifampicin (RIF) concentration are common and It may be<br />
necessary to optimise drug doses by therapeutic drug monitoring.<br />
Key words: Therapeutic drug monitoring, tuberculosis, isoniazid, rifampicin, ethambutol, pyrazinamide.<br />
INTRODUCTION<br />
The World Health Organisation estimated the global<br />
burden of tuberculosis(TB) in 2010 as around 14 million<br />
prevalance and 2.38 million deaths from this curable<br />
infectious disease (WHO, 2010). Despite directly<br />
observed therapy (DOT) in TB control programs,<br />
treatment failure, relapse, acquired drug resistance,<br />
increasing in number of multidrug resistant cases and<br />
drug toxicities remain ongoing complications in some TB<br />
patients. Serum concentrations of anti-tuberculosis drugs<br />
have been associated with many factors such as<br />
malabsorption, smoking status, comorbidity of diabetes<br />
mellitus and HIV, alcohol consumption,<br />
hypoalbuminaemia, calculated creatinine clearance by<br />
*Corresponding author. E-mail: servet-kayhan@hotmail.com.tr.<br />
Cockcroft-Gault equation for EMB (Cockroft and Gault,<br />
1976), liver and kidney dysfunctions, low dose per<br />
kilogram of body weight, changes in drug formulation,<br />
age and gender (Um et al., 2007; Mcllleron et al., 2006;<br />
Narita et al., 2001). Low serum concentrations of antituberculosis<br />
drugs has been reported as a reason for<br />
treatment failure, relapse and acquired drug resistance in<br />
previous studies (Heysell et al., 2010; Mehta et al., 2001).<br />
Low serum levels can be a consequence of<br />
malabsorption, inaccurate dosing, altered metabolism, or<br />
drug interactions, but in most instances low serum levels<br />
can be readily corrected with dose adjustment. TDM is<br />
currently recommended in TB treatment guidelines as<br />
optional (Peloquin, 2002; Blumberg et al., 2003).<br />
Although certain patients infected with HIV and thus<br />
prone to malabsorption, are at higher risk for low drug<br />
levels, studies of TDM that included patients responding
2036 Afr. J. Pharm. Pharmacol.<br />
well to anti-TB medications found lower than expected<br />
drug levels of isoniazid and rifampin in many patients with<br />
adequate clinical response (Mcllleron et al., 2006; Narita<br />
et al., 2001; Holland et al., 2009; Holdiness, 1984). In the<br />
present study, we measured serum concentrations of<br />
anti-tuberculosis drugs (INH, RIF, EMB and PZA) using<br />
HPLC method at 2 and 6 h after drug ingestion. The aims<br />
of the present study were to determine the prevalance of<br />
low serum concentrations of antituberculosis drugs and to<br />
identify the determinants of the serum concentrations of<br />
these drugs in TB patients.<br />
MATERIALS AND METHODS<br />
Study subjects and patients<br />
The present study was performed at a tuberculosis referral center,<br />
Samsun (Turkey) Chest Diseases and Thoracic Surgery Hospital<br />
and all the patients were informed about the study and they gave<br />
their consent to this investigation. 49 adult patients enrolled in the<br />
study between January 2011 to June 2011. All the patients were<br />
bacteriologically confirmed as active pulmonary tuberculosis. Study<br />
subjects had to have received daily standart anti-tuberculosis<br />
drugs: Isoniazid (INH) (daily oral 300 mg), rifampicin (RIF) (daily<br />
oral 600 mg), EMB (weight adjusted daily oral 1000 or 1500 mg)<br />
and PZA (weight adjusted daily oral 1500 or 2000 mg) for at least 7<br />
days before serum drug level measurements. The same trade<br />
marks of drugs were used in all study subjects and the antituberculosis<br />
drugs were single drug products rather than fixed-dose<br />
combinations. Patients with any of the following were excluded from<br />
the study: HIV co-infection, diabetes mellitus, a history of diarrhea,<br />
hepatic, gastrointestinal or renal disease, regular alcohol<br />
consumption and any medication that could affect drug serum<br />
concentrations (e.g. antacids and theophylline) .<br />
Sample preparation<br />
The specific drugs were administered to patients under fasting<br />
conditions and directly observed therapy by the nurses. Venous<br />
blood samples were obtained in an EDTA tube, 2 h after drug<br />
ingestion to estimate peak serum levels of the four drugs and<br />
second samples were taken after 6 h to rule out the possibility of<br />
delayed absorption. After collection, blood samples were<br />
centrifuged at 4000 rpm for a period of 15 min immediately. Plasma<br />
was frozen after centrifugation and stored at -20°C until analysis<br />
and transferred to the reference laboratory on dry ice according to<br />
published recommendations (Holdiness, 1984).<br />
High performance liquid chromatography (HPLC)<br />
Samples were deproteinized by trichloroacetic acid for INH and by<br />
acetonitrile for EMB, RIF and PZA. Solutions were injected to HPLC<br />
system with an LC-20AT pump and 20AC-HT autosampler<br />
(Shimadzu, Japan); results were analysed by using reversed phase<br />
technique, SPD-20A UVdetector and a VP-ODS(150 × 3.9 mm)<br />
Nova-pak C18 4 um (Waters) column (Moussa et al., 2002). The<br />
gradient elution created using 10 mmol potasium hidrojen<br />
phosphate (pH 6.24). Mobile phase A was 10 mmol potasium<br />
hydrogen phosphate and mobile phase B was acetonitrile for INH,<br />
RIF and PZA analysis. Samples were eluted at a flow rate of 0.8<br />
ml/min. EMB mobile phase consisted methanol and deionized water<br />
(70/30) and flow rate was 1,0 ml/min. The limit of quantitation<br />
values in this system for INH, RIF, EMB and PZA were 2.0, 1.5, 1.4<br />
and 1.6 µg/ml, respectively.<br />
Reference serum levels of anti-tuberculosis drugs<br />
INH, RIF, EMB and PZA reach peak serum concentrations<br />
approximately 2 h after ingestion and may delay in malabsorption.<br />
Using published reference ranges, low 2 h drug levels were defined<br />
as follows: INH < 3 µg/ml (300 mg/day); RIF < 8 µg/ml (600<br />
mg/day); EMB < 2 µg/ml (weight adjusted daily dose; 25 mg/kg) and<br />
PZA < 20 µg/ml (weight adjusted daily oral dose; 25 mg/kg). The<br />
patients were treated with oral and standart dose of drugs.<br />
Therapeutic serum levels for INH, RIF, EMB and PZA were 3 to 6, 8<br />
to 24, 2 to 6 and 20 to 50 µg/ml, respectively (Heysell et al., 2010;<br />
Peloquin, 2002).<br />
Statistical analysis of determinants<br />
Statistical analysis was performed using SPSS 15.0 for Windows<br />
(SPSS Inc., Chicago, IL, USA). Variabbles are represented as<br />
mean ± SD (standart deviation) when normally distributed and as<br />
medians (range) in other cases. The potential determinants were<br />
age, sex, smoking status, serum albumin, heamoglobin and drug<br />
dose per kilogram of body weight for all drugs, calculated creatinine<br />
clearance (CCr) for EMB, as it requires renal elimination.<br />
Independent samples T-test was used to compare drug plasma<br />
levels with sex and smoking behaviour variables. The other<br />
variables were statistically analysed with Pearson correlation.<br />
Statistical significance was accepted at P
Kayhan and Akgüneş 2037<br />
Table 1. Comparison of median serum concentration at 2 h after medication administration as estimate of peak serum concentration<br />
levels and expected range, therapeutic drug monitoring.<br />
Medication Median serum concentration (µg/ml) Expected serum concentration range (µg/ml)<br />
Isoniazid (daily 300 mg) 3.83 ± 2.09 3-6<br />
Rifampicin (daily 600 mg) 6.13 ± 4.27 8-24<br />
Ethambutol (weight adjusted) 3.68 ± 2.41 2-6<br />
Pyrazinamide (weight adjusted) 32.20 ± 16.96 20-50<br />
Table 2. Study variables and their correlation with drug serum concentrations (µg/ml).<br />
Variable<br />
Means± SD or<br />
n<br />
Test used<br />
Age (years) 41.51±16.90 PC<br />
Analysis INH<br />
result<br />
p = 0.160<br />
r = -0.204<br />
Analysis RIF<br />
result<br />
p = 0.193<br />
r = -0.189<br />
Analysis<br />
EMB result<br />
p = 0.726<br />
r = 0.051<br />
Analysis<br />
PZA result<br />
p = 0.106<br />
r = -0.234<br />
Sex (males/females) 31/18 IST p = 0.005 p = 0.000 p = 0.570 p = 0.002<br />
Body mass index (kg/m 2 ) 22.50±4.34 PC<br />
p = 0.006<br />
r = 0.390<br />
p = 0.935<br />
r = -0.012<br />
p = 0.317<br />
r = 0.146<br />
p = 0.139<br />
r = -0.214<br />
Smoking status smoker/ nonsmoker IST p = 0.532 p = 0.004 p = 0.551 p = 0.391<br />
'Drug dose(mg/kg)<br />
Isoniazid 4.49±1.14<br />
Rifampicin 9.92±2.21<br />
Ethambutol 24.16±5.15<br />
Pyrazinamide 28.98±8.65<br />
Serum Albumin (g/dl) 3.87±0.54 PC<br />
Heamoglobin 12.60±1.51 PC<br />
PC<br />
p = 0.000<br />
r = 0.487<br />
p = 0.303<br />
r = -0.150<br />
p = 0.303<br />
r = -0.150<br />
p = 0.039<br />
r = 0.295<br />
p = 0.734<br />
r = -0.012<br />
p = 0.083<br />
r = -0250<br />
Creatinine clearence* 118.73±37.08 PC - -<br />
p = 0.827<br />
r = -0.155<br />
p = 0.889<br />
r = 0.020<br />
p = 0.581<br />
r = 0.081<br />
p = 0.816<br />
r = 0.034<br />
p = 0.005<br />
r = 0.395<br />
p = 0.550<br />
r = -0.087<br />
p = 0.005<br />
r = -0.203<br />
*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<br />
creatinine) used for ethambutol; PC = Pearson correlation; IST = Independent samples T-test.<br />
significantly associated with drug dose (mg/kg) (p =<br />
0.000) and there was a correlation between these two<br />
variables (r = 0.487). 2 h INH concentration was not<br />
associated with age, serum albumin, smoking status and<br />
heamoglobin levels (Table 2).<br />
Serum RIF concentration<br />
The mean serum concentration of RIF was 6.13 ± 4.27<br />
µg/ml and the mean RIF dose was 9.92 ± 2.21 mg/kg<br />
(Tables 1 and 2; Figure 3). The prevalance of a low 2 h<br />
RIF concentration was 75.5% (37/49) in this study (Figure<br />
1) . RIF concentrations did not reach to therapeutic levels<br />
at 6 h in these patients. 2 h RIF concentration showed<br />
difference according to sex (p = 0.000). The mean RIF<br />
concentration of males (4.23 µg/ml) was lower than<br />
females (9.41 µg/ml). By Independent samples T-test<br />
analysis, 2 h RIF concentration was found to be<br />
associated with cigarette smoking (p = 0.004). The mean<br />
serum RIF level of smokers (4.50 µg/ml) was lower than<br />
non-smokers (7.50 µg/ml). 2 h RIF serum concentration<br />
was not associated with the other variables (age, body<br />
mass index, serum albumin, drug dose (mg/kg) and<br />
-
2038 Afr. J. Pharm. Pharmacol.<br />
PYRAZINAMIDE<br />
ETHAMBUTOL<br />
RIFAMPICIN<br />
ISONIAZID<br />
20.4%<br />
18.4%<br />
28.6%<br />
75.5%<br />
61.2%<br />
65.3%<br />
55.1%<br />
18.4%<br />
16.3%<br />
24.5%<br />
16.3%<br />
0 100<br />
Figure 1. Results of serum concentration 2 h after oral drug administration levels<br />
of antituberculosis medications . Frequencies(%) are reported for low, within<br />
target (therapeutic), and high levels corresponding to levels below, within, or<br />
above the expected range for each medication.<br />
Figure 2. Isoniazid(INH) serum levels of each patient (low
heamoglobin) (Table 2).<br />
Serum EMB concentration<br />
Figure 4. Ethambutol (EMB) serum levels of each patient < 2 µg/ml ; 18.4% (9/49) , n = 49.<br />
The mean serum concentration of EMB was 3.68 ± 2.41<br />
µg/ml and the mean EMB dose was 24.16 ±5.15 mg/kg<br />
(Tables 1 and 2). The prevalance of a low 2 h EMB<br />
concentration was 18.4% (9/49) (Figures 1 and 4). A<br />
delayed absortion was observed in seven of nine low 2 h<br />
EMB concentrations and they reached to therapeutic<br />
serum levels at 6 h measurements. 2 h EMB<br />
concentration was not found to be associated with any of<br />
variables including calculated creatinine clearence in the<br />
present study (Table 2).<br />
Serum PZA concentration<br />
The mean serum concentration of PZA was 32.20 ±<br />
16.96 µg/ml and the mean PZA dose was 28.98 ± 8.65<br />
mg/kg (Tables 1 and 2; Figure 5). The prevalance of a<br />
low 2 h PZA concentration was 20.4% (10/49) (Figure<br />
1). There was a difference between 2 h PZA<br />
concentration and sex variable (p = 0.002). The mean<br />
concentration of males (26.57 µg/ml) was lower than<br />
females (41.88 µg/ml). Drug dose (mg/kg) was found to<br />
be statistically significant in serum PZA concentration.<br />
There was a correlation between 2 h serum PZA<br />
concentration and drug dose (p = 0.005 and r = 0.395)<br />
(Table 2).<br />
Treatment outcomes<br />
<strong>Complete</strong> outcomes were available for all patients. Fourty<br />
nine patients succesfully completed treatment. No patient<br />
Kayhan and Akgüneş 2039<br />
had died during therapy and follow up. There was not any<br />
treatment failure among the study cases. Median time to<br />
completion of therapy among all 49 patients was 7.3<br />
months (inter quartille range [IQR] 6 to 9 months). We did<br />
not achieve a correlation between low antimycobacterial<br />
drug levels and the time to completion of therapy. There<br />
were no reports of relapse of infection over a median of 5<br />
months (IQR 3 to 8 months) from the conclusion of<br />
treatment. No patient had documented acquisition of<br />
medication resistance in follow up TB cultures while on<br />
treatment.<br />
The 2 h value after ingestion of tuberculosis drugs is<br />
the peak serum concentration of drugs in normal<br />
patterns. Should the 2 and 6 h values be roughly the<br />
same, perhaps somewhat below the expected ranges, or<br />
should the 6 h levels be higher than the 2 h, delayed<br />
absorption is most likely. In these situations, it is possible<br />
that the actual peak occurred between the two intervals.<br />
It is recommended to take the drugs under fasting<br />
positions, especially for isoniazid and rifampicin.<br />
Malabsorption is most likely if both 2 and 6 h serum drug<br />
levels below the expected ranges, and consideration<br />
should be given higher doses (Peloquin, 2002, Peloquin<br />
et al., 1993). Second blood samples were taken after 6 h<br />
to rule out the possibility of delayed absorption secondary<br />
to poor gastric emptying in this study. We observed some<br />
delayed absorptions and therapeutic drug levels after 6 h<br />
in EMB (14.28%, 7/49), PZA (4.08%, 2/49), INH (2.04%,<br />
1/49) and RIF (0%) respectively. The frequency of<br />
delayed absorption has been rare in other cohorts for<br />
which 2 and 6 h measurements were performed (Holland<br />
et al., 2009).<br />
According to previous studies, the prevalences of low<br />
concentrations of anti-tuberculosis drugs showed wide<br />
variations in TB patients, that is, 2 to 48% for INH, 5 to<br />
78% for RIF and 2 to 41% for EMB (Mcllleron et al., 2006;<br />
Mehta et al., 2001; Peloquin et al., 1996; Holland et al.,<br />
2009; Chang et al., 2008; Fahimi et al., 2011). According
2040 Afr. J. Pharm. Pharmacol.<br />
Figure 5. Pyrazinamide(PZA) serum levels of each patients < 20 µg/ml 20.4% (10/49) n = 49.<br />
to previous studies, serum concentrations of INH are<br />
related to many factors, that is, age, sex, a prior history of<br />
TB, serum haemoglobin levels, laxative use, HIV<br />
infection, fixed-drug combinations formulation, fasting<br />
and weight-adjusted dose (Heysell et al., 2010; Mehta et<br />
al., 2001; Peloquin, 2002; Kimerling et al., 1998). In our<br />
study prevalance of low concentration of INH was 28.6%.<br />
Sex, body mass index and dose of drug (mg/kg) were<br />
effective factors in serum INH levels in this study.<br />
Previous studies revealed that weight-adjusted dose<br />
and a higher serum albumin level were associated with a<br />
higher RIF concentration (Heysell et al., 2010; Mehta et<br />
al., 2001). Patients with diabetes were at significantly<br />
increased risk of having a low rifampin level. Diabetes<br />
was significantly associated with slow response in a<br />
study population, and, among persons with a slow<br />
response with diabetes, 2 h levels of rifampsin were<br />
significantly more likely to be below than the expected<br />
ranges. Hyperglycemia can decrease gastric hydrochloric<br />
acid secretion, which results in a higher gastric pH and<br />
reduced rifampin absorption (Heysell et al., 2010). In this<br />
study, patients enrolled to study were normoglycemic but<br />
serum RIF levels were low in 75.5% of patients. Sex (to<br />
be male) and smoking cigarette were found to be<br />
associated variables in low RIF serum concentrations in<br />
our study. Diabetic patients are at greater risk for incident<br />
TB and are more likely to have poor TB treatment<br />
outcomes, which may partially be explained by<br />
inadequate pharmacotherapy (Jeon and Murray, 2008).<br />
Dose-titration studies of rifampisin confirm a continuously<br />
increasing response of early bactericidal activity by<br />
measurement of sputum colony counts with<br />
corresponding increase in rifampicin dose (Sirgel et al.,<br />
2005; Diacon et al., 2007). As a result a second drug<br />
dose adjustment has to be done to prevent from slow<br />
response to therapy especially in patients with low RIF<br />
serum levels.<br />
A TDM study from South Korea reported low 2 h EMB<br />
concentration ratio as 22.4% and an association between<br />
EMB concentration and calculated creatinine clearance<br />
(Mcllleron et al., 2006). We observed delayed EMB<br />
absortion in seven of nine patients with low 2 h EMB<br />
concentrations. 2 h EMB concentration was not found to<br />
be associated with any of variables including calculated<br />
creatinine clearence in our study. We have found the<br />
prevalance of a low 2 h PZA concentration as 20.4%. The<br />
mean concentration of males was lower than females.<br />
Another result of the study is a correlation between 2 h<br />
serum PZA concentration and drug dose. These<br />
variables have accordance with previous studies (Um et<br />
al., 2007; Mcllleron et al., 2006; Tappero et al., 2005).<br />
Although an existence of high prevalance of low<br />
antimycobacterial drug concentrations, treatment<br />
outcomes included succesfull therapy results in the<br />
present study. Early diagnosis, performing a directly<br />
observing therapy in hospital, absence of extensive<br />
disease and drug resistance may play role in this<br />
condition. This study may support that all four antituberculosis<br />
drugs should be dosed as mg/kg, smoking is<br />
a negative factor in therapeutic RIF serum<br />
concentrations. This study does not have an analysis<br />
about TDM and slow response to therapy with clinical<br />
outcomes in long periods of TB treatment. Another<br />
limitation of present study is not having results of second<br />
TDM after dose adjustment in low serum drug<br />
concentrations. Thus further studies are required to
determine the nature of these conditions.<br />
Conclusıon<br />
A low serum concentration of at least one<br />
antituberculosis drug was found in about all of the<br />
patients included in this study. Several risk factors of drug<br />
concentration variation were also identified. It is clear<br />
that, therapeutic drug monitoring is necessary to optimise<br />
drug doses in tuberculosis treatment.<br />
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UPCOMING CONFERENCES<br />
American Association of Pharmaceutical Scientists Annual meeting and<br />
Exposition (AAPS), Chicago, USA, 14 Oct 2012<br />
International Conference on Pharmacy and Pharmacology, Bangkok,<br />
Thailand, 26 Dec 2012
Conferences and Advert<br />
October 2012<br />
American Association of Pharmaceutical Scientists Annual meeting and Exposition<br />
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International Conference on Pharmacy and Pharmacology, Bangkok, Thailand, 26<br />
Dec 2012
African Journal of<br />
Pharmacy and<br />
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