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Anti-ulcer & antioxidant activities of Hedranthera barteri {(Hook F.) Pichon} with possible involvement of [H.sup.+], [K.sup.+] ATPase inhibitory activity.

Acid, peptic activity and a collapse of mucosal defense mechanism have been implicated in the genesis of gastro-duodenal ulcers. Efforts are being made to find a suitable agent for the treatment of peptic ulcer disease (1). The extracts and compounds from medicinal plants and other natural products have become the widely acceptable source of therapeutic agents for the treatment of peptic ulcers.

Hedranthera barteri (HB) {Apocynaceae} is a shrub found in damp situations of the closed-forest in South Nigeria, Ghana, North/West Cameroon, Congo Brazzaville and other parts of the world. It has large white tubular flowers, with fragrant scent and contains white latex that does not coagulate. HB is used as medicinal infusion for children as a laxative (2); the leaf decoction for dizziness (2); the fruit for treating gonorrhoea, as a femifuge and the exudates from the leaf for treating painful tumor and inflammation and to prevent miscarriage in women (3). The plant has been reported to be rich in alkaloids especially amataine and vobstusine (4) and the alkaloids from the root of the plant have antibacterial properties (5). Its leaf extract has been reported to have anti-inflammatory, anti-malarial and anti-bacterial activities; in vitro (6) and in vivo studies reported its anti-nociceptive and anti-inflammatory potentials (7). Beta-sitosterol was suggested to be partly responsible for its anti-inflammatory pain relief (8). Despite its long years of use as a vermifuge and laxative; there is no information on its biological activity to support its anti-ulcer properties or any other effect on gastrointestinal system.

Reactive oxygen species have been implicated in the aetiology and pathophysiology of gastrointestinal inflammation and gastric ulcers (9). Hence, there is a need for agents to minimize and repair free radical-induced damage. The antioxidants play a key role in these defense mechanisms. Formulations with potent antioxidant actions have been reported to be effective in the cytoprotection and/or healing in the experimentally induced peptic ulcers (10).

The present study investigated the anti-oxidant and anti-ulcer properties of the dichloromethane fraction of H. barteri root (DMHBR) extract using various experimentally-induced gastric and duodenal ulcer models.

Material & Methods

Animals: Sprague Dawley rats weighing 140-160 g and guinea pigs weighing 320-350 g were obtained from National Animal Laboratory Centre of Central Drug Research Institute (CDRI), Lucknow. Animals were kept in raised mesh bottom cages to prevent coprophagy and kept in environmentally controlled rooms (25 [+ or -] 2[degrees]C, 12 h light and dark cycle), with free access to water. Animals were fed with standard laboratory food pellets (Hind Lever, Chandigarh, India) ad libitum. Animals were deprived of food for 24 h before subjecting them to ulcerogens and were randomly allocated to different experimental groups. Six rats/guinea pigs were used in each group. Experimental protocols were approved by the institutional ethical committee following guidelines of CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals), which complies with international norms of INSA (Indian National Science Academy).

Drugs and chemicals: All chemicals used were of analytical grade and were purchased from Sigma Chemical Co. (St. Louis, MO, USA) except otherwise stated.

Preparation of extract: The roots of H. barteri were purchased from the Herbarium Department, Forest Research Institute of Nigeria (FRIN), Ibadan, Nigeria with authenticated voucher specimen (FHI-108375). The roots of HB were dried under shade during the harmattan season in Nigeria (November-December) and 2 kg of the dried root powder were percolated with hexane for 48 h, after which the hexane fraction was filtered off through a separate funnel and the solvent was removed at 40[degrees]C under reduced pressure in a rotavapor. The marc was dried under shade and subjected to further extraction with the dichloromethane. The same procedure was employed to extract the dichloromethane fraction. The yields for hexane and dichloromethane extracts were 13.1 and 18.7 per cent respectively. The extracts were prepared as suspensions using 2.5 per cent tween 20/distilled water, and were administered 45 min before each experimental model.

Treatment schedule: All animals were deprived of food for 16 h before ulcerogens exposure and were divided into three groups, (n=6 in each group).

(i) Control group of animals were treated with vehicle 2.5 per cent tween.

(ii) Graded doses of dichloromethane of HB root (50, 100 and 200 mg/kg, po) were tested against cold restraint ulcer (CRU) model to identify the effective dose and selected for further studies in other ulcer models.

(iii) Experimental group was treated with standard anti-ulcer drugs such as omeprazole (Omz, 10 mg/kg, po) in CRU, aspirin (AS), pyloric ligation (PL), histamine induced duodenal ulcer (HA) and sucralfate (500 mg/kg, po) in alcohol (AL) induced ulcer model.

Phytochemical screening: Preliminary phytochemical screening of the powdered root was performed for the presence of alkaloids, cardenolides, flavonoids and saponins (11).

Acute toxicity and extract dose selection: The rats were housed individually in suspended, stainless steel, wire-mesh cages at the animal house, Department of Physiology, University of Ibadan, Nigeria, and maintained at the standard laboratory conditions. They were fasted overnight prior to DMHBR administration and the animal rooms were illuminated with fluorescent light on an approximate 12 h light/ dark cycle. Six animals received a single oral dose of 2000 mg/kg, body weight of DMHBR. After the administration of DMHBR, food was withheld for 4 h. The rats were observed individually during the first 60 min after dosing, periodically during the first 24 h and daily thereafter for a period of 14 days. There were observations from the cage side once daily for changes in the eyes and nasal mucous, changes in furs disposition, heart rate, salivation, pilo-erection, urinary incontinence, defecation, drowsiness, gait, tremors and convulsion. Mortality, if any, was determined over a period of 2 wk and the Organization for Economic Cooperation and Development (OECD) guidelines were followed for dose fixing in biological evaluation (12).

Anti-ulcer studies

Cold restraint stress (CRU) induced gastric ulcer model in rats: The rats were subjected to cold-stress paradigm (13) after 45 min of treatment of DMHBR (50, 100 and 200 mg/kg, po) and omeprazole (10 mg/kg, po). All animals were immobilized in a restraint cages, kept at 4[degrees]C in an environmental chamber for 2 h and sacrificed thereafter. The stomach was cut along the lesser curvature and ulcers were scored with the help of magnascope.

Alcohol (AL) induced gastric ulcer model in rats: Gastric ulcer was induced in rats by administering absolute alcohol (1 ml/200g, body, 1 h) (14). DMHBR (100 mg/kg, po) and sucralfate (500 mg/kg, po) were administered 45 min before alcohol treatment. After 1h, the animals were sacrificed and stomachs were excised to observe the gastric lesions and were measured using Biovis image analyzer software (Expert Vision Lab Pvt. Ltd., Mumbai).

Aspirin-induced (ASP) gastric ulcer model in rats: Aspirin at a dose of 150 mg/kg, po was administered to induce ulcer after 45 min of treatment of DMHBR (100 mg/kg, po) and omeprazole (10 mg/kg, po). The animals were sacrificed 5 h after aspirin treatment (15) and the stomach was dissected out and the lesion was scored.

Pyloric ligation-induced (PL) gastric ulcer model in rats: This was done (16) by ligating the pyloric end of stomach of rats under chloral hydrate anaesthesia (300 mg/kg, ip). After 45 min of DMHBR and omeprazole administration, the pyloric end of the stomach was ligated and the abdomen was stitched. After 4 h of surgery, rats were sacrificed and the gastric juice was collected for the estimation of free and total acids; mucin and peptic activity.

Histamine-induced (HST) duodenal ulcer model in guinea pigs: Duodenal ulcers were induced by intraperitoneal administration of histamine acid phosphate at a dose of 0.25 mg/kg at every 30 min interval for 4 h after 45 min of DMHBR and omeprazole treatments (17). Promethazine hydrochloride at a dose of 2.5 mg/kg, ip was injected to each animal, 15 min prior to the commencement of histamine administration, in order to protect the rats from histamine toxicity. The rats were sacrificed after 30 min of the last dose of histamine and ulcers were scored.

Measurement of ulcer index: Ulcers were scored with the help of magnascope under 5X magnification using the ulcer scoring criteria (18). The following scoring system was used to grade the incidence and severity of the lesions: (i) shedding of epithelium = 10; (ii) petechial and frank haemorrhages = 20; (iii) one or two ulcers = 30; (iv) more than two ulcers = 40; (v) perforated ulcers = 50. Length of haemorrhagic band is measured in AL model using Biovis Image Analysis Software (BIAS). Percentage protection index is calculated as follows:

% protection = ([U.sub.c]--[U.sub.t]) x 100 / [U.sub.c]

Where [U.sub.c] = ulcer index in control group; Ut = ulcer index in treated group.

Gastric secretion analysis: The free acidity, total acidity, peptic activity and dissolved mucous substances of gastric juice were measured using Topfer's reagent and phenolphthalein as indicators respectively (19) and titrated with 0.01N NaOH, and expressed in terms of [micro]eq./ ml. Peptic activity was determined by measuring the amount of liberated tyrosine by the action of pepsin on haemoglobin as substrate (20) and expressed in terms of U/ml. Mucin level in gastric juice was quantified with a fluorometric assay (21) and expressed as [micro]g of mucin/ml of gastric juice.

Preparation of gastric microsomes: The H+, K+-ATPase containing gastric microsomes were isolated from non-stimulated rat stomach (22) at 4[degrees]C. Gastric mucosal layer was scrapped and homogenized in a buffer containing 125 mM mannitol, 40 mM sucrose, 1mM EDTA, 5mM piperazine-1, 4-Bis (2-ethanesulphonic acid) (PIPES), pH 6.7 using Teflon pestle. The homogenate was centrifuged at 14,500 g for 10 min. The resulting supernatant was centrifuged at 100,000 g for 45 min. The microsomal pellets were re-suspended in homogenization buffer and layered on the top of gradients sucrose solutions (33, 27 and 21%), dissolved in the homogenization buffer. Thereafter, this was ultracentrifuged at 100,000 g on a SW 28 swinging rotor (Beckmann Coulter Inc., USA) for 2 h. The microsomes fraction present on the top of 21 per cent sucrose layer was harvested. Protein concentration was immediately measured through Bradford method (23).

Assay of H+, K+-ATPase activity: For the enzyme assay, gastric microsomes, incubated with or without different concentrations of DMHBR as well as standard drug OMZ for 10 min at 37[degrees]C, were added to an assay buffer containing 150 mM KCl, 10 mM PIPES, 1 mM MgS[O.sub.4], 5 mM Mg-ATP, 1 mM EGTA and 0.1 mM ouabain, at pH 7.2 and 10 [micro]g/ml valinomycin, 2.5 [micro]g/ ml oligomycin. The reaction was carried out at 37[degrees]C for 20 min and was stopped by adding 10 per cent ice-cold trichloroacetic acid. After centrifugation (2000 g for 1 min), inorganic phosphate release was determined from the resulting supernatant spectrophotometrically at 310 nm wavelength (24) and expressed as [micro]M/h/mg protein.

Antioxidant assays

Scavenging/inhibitory activity coefficient: The scavenging or percentage inhibitory activity of DMHBR in each assay was calculated from:

% Inhibition = ([A.sub.0]--[A.sub.1]) x 100 / [A.sub.0]

where [A.sub.0] = absorbance of the control (without extract), and [A.sub.1] = absorbance of the treated (with DMHBR). The [IC.sub.50] value of the DMHBR was extrapolated from the standard linear regression curve.

Free radical scavenging activity

DPPH photometric assay: The free radical scavenging activity of DMHBR was estimated, in vitro, by 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) scavenging activity (25). The absorbance at 517 nm was measured with spectrophotometer. The [IC.sub.50] value of DMHBR was compared with the standard ascorbic acid.

Nitric oxide scavenging activity assay: Nitric oxide radical scavenging activity was determined according to Garrat's method (26). The absorbance at 540 nm was measured with a spectrophotometer. The [IC.sub.50] value of DMHBR was compared with that of the curcumin.

Hydroxyl radical scavenging activity assay: The hydroxyl radical scavenging activity of DMHBR was determined by studying the competition between deoxyribose and DMHBR for hydroxyl radical generated by Fenton's reaction (27). The absorbance was read at 532 nm. The reference compound used was

DMSO.

Superoxide anion scavenging activity assay: The scavenging activity of the DMHBR on superoxide anion radicals was measured by Liu's method (28). The absorbance was measured at 560 nm, spectrophotometrically. The [IC.sub.50] value of DMHBR was compared with that of the ascorbic acid.

Statistical analysis: All values are expressed as mean [+ or -] SEM. Data of ulcer index was analyzed by non-parametric ANOVA while one-way analysis of variance (ANOVA) was used for statistical comparison of other results, followed by Newman-Keul's multiple comparison test. Differences between means were considered significantly different when P was <0.05 using Graph-Pad Prism version 3.00 for Windows (GraphPad Software, San Diego, California, USA).

Results

Acute toxicity and DMHBR dose selection: In the estimation of the [LD.sub.50] of the extract according to the OECD guidelines so as to fix the dose for biological evaluation, the dose came under class four values with no significant signs of acute toxicity or death at 2 g/kg, and 200 mg/kg was taken as the effective dose. The initial biological evaluation was carried out at doses of 50, 100 and 200 mg/kg body weight.

Anti-ulcer effects of DMHBR against acute gastric ulcer models: The DMHBR exhibited significant anti-ulcer effects against induced gastric and duodenal ulcers. From the pilot study conducted using CRU model, graded doses of DMHBR (50, 100 and 200 mg/kg, po) showed percentage protection of 22.60, 63.30 (P<0.01) and 36.10 per cent (P<0.05) respectively whereas omeprazole (10 mg/kg, po) showed a protection of 78.25 per cent (P<0.01) with reference to the control group. Based on these results, 100 mg/kg was taken as the effective dose and was chosen for further studies. In PL, ASP and HST induced gastric ulcer model, DMHBR (100 mg/kg, po) showed protection index of 58.50 (P<0.01), 52.70 (P<0.01) and 75.00 per cent (P<0.01) respectively, whereas omeprazole (10 mg/kg, po) exhibited 69.42 (P<0.01), 57.70 (P<0.01) and 70.79 per cent (P<0.01) protection respectively. Pre-treatment of rats with DMHBR (100 mg/kg, po) also exerted 60.40 per cent (P<0.01) protection against gastric mucosal damage induced by AL compared with sucralfate (500 mg/kg, po), the standard drug, which exhibited 64.50 per cent (P<0.05) with reference to the control group (Fig. 1).

Effect of DMHBR on gastric secretion in vivo: The effects of DMHBR on free acidity, total acidity, peptic activity and defensive factors, mucin (which play a crucial role in the pathogenesis of gastric ulcers) were studied using gastric juice from PL model. DMHBR (100 mg/kg, po) reduced free acidity, total acidity and peptic activity by 49.4 (P<0.05), 45.8 (P<0.05) and 32.9 per cent respectively, while omeprazole (10 mg/ kg, po) significantly reduced free acidity, total acidity and peptic activity by 67.41 (P<0.01), 52.69 (P<0.01) and 53.2 per cent (P<0.05) respectively, compared with the control group. The same dose of DMHBR and omeprazole (10 mg/kg, po) increased the mucin secretion by 81.6 (P<0.01) and 55.0 per cent (P<0.05) respectively, compared with the control (Table I).

The effects of DMHBR on gastric proton pump (H+, K+-ATPase) activity: The influence of DMHBR and omeprazole on the H+, K+-ATPase isolated from albino rat gastric microsomes as compared with the control group. The incubation of DMHBR (60-100 [micro]g/ml) with the microsomes inhibited the inorganic phosphate release from gastric proton pump activity proportionately from 16.2 per cent (291.62 [micro]M/irig protein) to 64.6 per cent (123.19 [micro]M/mg protein) respectively, with [IC.sub.50] of 89.64 [micro]g/ml. At the same experimental conditions, the proton pump inhibitor omeprazole (10-50 [micro]g/ml) inhibited the enzyme from 12.5 per cent (304.50 [micro]M/mg protein) to 87.5 per cent (87.00 [micro]M/mg protein) respectively, with [IC.sub.50] = 32.26 [micro]g/ml (Fig. 2).

Effect of DMHBR extract on free radical generation, in vitro: The DMHBR exhibited antioxidant activity in DPPH, nitric oxide radical, hydroxyl radical and superoxide anion radical inhibition assay as evidenced by the [IC.sub.50] values (Table II). The [IC.sub.50] values obtained are 397.69, 475.88, 244.22 and 285.20 [micro]g/ml respectively compared to the standard with [IC.sub.50] of 42.5 [micro]g/ml (ascorbic acid), 38.3 [micro]g/ml (curcumin), 24 mM (DMSO) and 18.4 [micro]g/ml (ascorbic acid) respectively.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Discussion

This work has demonstrated the safety of using HB when administered to rats at doses up to 2 g/kg body weight. So, a dose of 200 mg/kg was considered as the effective dose based on the acute toxicity of the Fixed Dose Procedure and extract's dose selection experiment as recommended by the Organization for Economic Cooperation and Development (12).

DMHBR significantly decreased the ulcer index in all the models when compared to the control group of animals. It is reported that CRU is a well accepted model for the induction of gastric ulcer and in this model activation of peripheral sympathetic nerves has been implicated in the induction of ulcers induced by restraint (29). Based on this fact, 200 (extrapolated effective dose), 100 and 50 mg/kg were taken for CRU model. The results revealed significant anti-ulcer property of DMHBR against CRU with the 100 mg/kg comparable to omeprazole (10 mg/kg). This suggested 100 mg/kg DMHBR to be the effective experimental dose in this study. This dose was then taken for further studies in other ulcer models.

The anti-ulcer properties of DMHBR were significant against PL, ASP and HST as compared with omeprazole and against AL as compared with sucralfate, with reference to the control group. PL, CRU and HST are the models that involve more of anti-secretory mechanisms while ASP and AL models are involved more of cytoprotective mechanisms. Accumulation of gastric juice and pepsin which involves in the autodigestion of gastric mucosa is implicated in ulcer incidence in PL model (30). DMHBR (100 mg/kg) reduced the ulcer index in this model with a protection of 58.5 per cent which was comparable with omeprazole, revealing the anti-secretory ability of DMHBR. The analyses of the gastric juice from PL model showed that DMHBR significantly reduced both the free and total acidity but no significant effect on peptic activity of gastric juice was observed. Omeprazole significantly reduced the free and total acidity, and peptic activity more than DMHBR with reference to the control group. The mucin content was significantly higher than that of omeprazole showing its better cytoprotective potentials compared with omeprazole.

DMHBR significantly displayed anti-ulcer properties against AL, ASP and HST models, with its effects against HST, showing better potential than omeprazole, with reference to the control group. Though, histamine is known to be involved in the induction of acid secretion but the activity of DMHBR in protecting the duodenal mucosa in HST model is more of cytoprotection. This corroborates the anti-secretory as well as cytoprotective potentials of DMHBR. Exposure of the gastric mucosa to necrotizing agents like ethanol causes gastric ulcers and induces a major damage of superficial epithelial cells on gastric mucosa (31). Hence, the significantly reduction in the ulcer index produced by DMHBR may be cytoprotective in nature either by stabilizing the integrity of the gastric mucus or by increasing mucus secretion. Ulceration produced by aspirin has been suggested to be attenuated by an H2 antagonist (32), implicating its antisecretory property as well.

The enzyme H+, K+-ATPase is unique to the parietal cells and transports the H+ against a concentration gradient. Proton pump inhibitors are more potent at reducing gastric acid production since that is the final common pathway of all stimulation of acid production. Parietal cells are rich of mitochondria and consume more of ATP, thereby generating inorganic phosphate which acts as indirect measure of proton pump (H+, K+AT-Pase) activity in the formation and transport of H+ for gastric acid formation. Our results showed an inhibitory potential of the DMHBR on the proton pump activities which may be related to the conception that DMHBR has interaction with enzymes at the ATP sites.

Most of the anti-ulcer compounds or extracts are known for their scavenging activities on free radicals in the process of ulcer healing. In vitro studies of DMHBR revealed its anti-oxidant properties, scavenging the free radicals and reactive oxygen species in a dose dependent manner in DPPH, nitric oxide, hydroxyl radical and superoxide anion radical scavenging assays.

The phytochemical screening of the root extract of HB revealed the presence of alkaloids, flavonoids and saponins, amongst other secondary metabolites (12). Any of these secondary metabolites may be responsible for the anti-oxidant and anti-ulcer properties of DMHBR especially amataine and vobstusine characterized from the HB root (4). Likewise, beta-sitosterol from the plant, suggested being partly responsible for the anti-inflammatory pain relief potential of HB, may be involved in its anti-ulcer property (8). Phytochemical investigations of the H. barteri demonstrated the presence of pure compounds namely [beta]-sitosterol, amataine and vobstusine. The [beta]-sitosterol has been reported as anti-ulcerative agents in cold stress and acetic acid induced ulcer models (33).

In conclusion, the present results showed a significant anti-ulcer potential of DMHBR with more of cytoprotective than anti-secretory properties. It exhibited a proton pump inhibition and its anti-ulcer properties may be partly ascribed to its antioxidant activities. Further laboratory work on the separate phytochemical constituents of DMHBR will suggest the actual compound responsible for its anti-ulcer properties, especially, its proton pump inhibitory activity.

Acknowledgment

Authors acknowledge the academy of sciences for the developing world (TWAS), Trieste, Italy and the Council of Scientific & Industrial Research (CSIR), New Delhi for the Post-Doctoral Fellowship given to the first author (SAO) under the collaborative project of both TWAS and CSIR and Indian Council of Medical Research (ICMR), New Delhi for providing financial support to the second author (NS). The research facilities provided by the CDRI, Lucknow, India are acknowledged. Authors thank Shrimati Shibani Sen Gupta for technical assistance.

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Samuel A. Onasanwo ***, Neetu Singh **, Samuel B. Olaleye *, Vaibhav Mishra ** & Gautam Palit **

* Department of Physiology, College of Medicine, University of Ibadan, Ibadan, Nigeria & ** Division of Pharmacology, Central Drug Research Institute (CSIR), Lucknow, Uttar Pradesh, India

Received September 16, 2009

Reprint requests: Dr Gautam Palit, Division of Pharmacology, Central Drug Research Institute, Lucknow 226 001, Uttar Pradesh, India e-mail: gpalitcdri@gmail.com
Table I. Effect of dichloromethane extract of H. barteri root (DMHBR)
extract (100 mg/kg) and omeprazole on free acidity, total acidity,
peptic activity and mucus secretion of gastric juice in pylorus
ligation-induced gastric ulcer model

Group Free acidity
 ([micro]eq./ml)

Control (10 ml/kg) 86.83 [+ or -] 2.45
DMHBR (100 mg/kg) 43.92 [+ or -] 3.31 *
Omeprazole (10 mg/kg) 28.31 [+ or -] 1.24 **

Group Total acidity Peptic activity
 ([micro]eq./ml) (U/ml)

Control (10 ml/kg) 160.7 [+ or -] 0.36 13.57 [+ or -] 0.92
DMHBR (100 mg/kg) 87.08 [+ or -] 8.72 * 9.11 [+ or -] 0.30 *
Omeprazole (10 mg/kg) 76.03 [+ or -] 4.56 ** 6.35 [+ or -] 0.16 *

Group Mucin secretion
 ([micro]g/ml)

Control (10 ml/kg) 93.27 [+ or -] 1.79
DMHBR (100 mg/kg) 502.85 [+ or -] 9.30 **
Omeprazole (10 mg/kg) 207.27 [+ or -] 7.68 **

Values are expressed as mean [+ or -] SEM (n=6) in each group. * P <
0.05 ** < 0.01 compared with the control (ANOVA test)

Table II. In vitro antioxidant activities of dichloromethane extract
of H. barteri root extracts (DMHBR) on different antioxidant
models (n = 6 in each group)

 Scavenging activity of DMHBR (% inhibition) *
Concentration
([micro]g/ml) DPPH Nitric oxide Hydroxyl Superoxide
 radical anion

10 9.89 19.2 14.79 14.44
100 26.7 32.7 41.87 27.94
200 34.92 34.6 49.53 49.66
300 43.79 36.5 57.93 55.47
400 49.49 40.4 66.24 61.4
500 56.58 55.7 74.54 69.37
[IC.sub.50] 397.69 475.88 244.22 285.20
 ([micro]g/ml)

* Values are expressed as percentage mean of 3 replicates
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Author:Onasanwo, Samuel A.; Singh, Neetu; Olaleye, Samuel B.; Mishra, Vaibhav; Palit, Gautam
Publication:Indian Journal of Medical Research
Article Type:Report
Geographic Code:9INDI
Date:Oct 1, 2010
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