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In vitro evaluation of Bacopa monniera on anti-Helicobacter pylori activity and accumulation of prostaglandins.

Summary

Bacopa monniera is an Indian tratidional medicine widely used to improve intellectual functions. Earlier, we had reported the prophylactic and curative effects of standardized extract of Bacopa monniera (BME) in various gastric ulcer models. The effect was due to augmentation of the defensive mucosal factors like increase in mucin secretion, life span of mucosal cells and gastric antioxidant effect rather than on the offensive acid-pepsin secretion. The present study includes evaluation of standardized BME (bacoside A content--35.5 [+ or -] 0.9) on other contributing factors towards ulcerogenesis. BME in the dose of 1000 [micro]g/ml showed anti-Helicobacter pylori activity in vitroI and in the dose of 10 [micro]g/ml increased in vitro of prostanoids (PGE and PG[I.sub.2]) in human colonic mucosal incubates. It may be concluded that these factors may contribute to antiulcerogenic activity of BME.

Key words: Bacopa monniera, bacoside-A, antiulcerogenic, gastric mucosal offensive and defensive factors, Helicobacter pylori, prostaglandins

* Introduction

Bacopa monniera Wettst. (syn. Herpestis monniera L.; Scrophulariaceae), finds wide mention in Ayurveda, the ancient medicine of India as a medhya rasayana, a class of plant drugs used to promote mental health and improve memory and intellect (Udupa and Singh, 1995). Commonly called as 'Brahmi', it has been reported to posses several neuropsychopharmacological properties (Dhavan and Singh, 1996; Bhattacharya and Ghosal, 1998; Vohora et al. 2000) and antioxidant effect (Bhattacharya et al. 2000; Sairam et al. 2001). Bacopa monniera (BM) contains, apart from other saponins (Rastogi et al. 1994; Garai et al. 1996a; Garai et al. 1996b), bacoside A, which has been reported to be the major chemical moiety responsible for pharmacological effects of the drug (Bhattacharya and Ghosal, 1998; Sairam et al. 2001). Other new saponins have also been reported, but their activities are relatively unexplored (Mahato et al. 2000). In earlier studies, the juice (Rao et al. 2000) and standardized methanolic extract (Sairam et al. 2001) of fresh whole plants of BM was reported to have significant ulcer protective and healing effects in different experimental models. Ulcers are thought to be due to imbalances in offensive and defensive gastric mucosal factors (Goel and Bhattacharya, 1991). The effect was attributed to enhancement of defensive mucosal factors like mucin secretion, life span of mucosal cells and anti-oxidant effects rather than on the offensive mucosal factors like acid and pepsin secretion. In continuation the present study analysis the effect of standardized extract of BM on other important contributing factors towards ulcerogenesis.

H. pylori is an ancient infection present in human stomach for thousand of years (Blaser, 1999) and has been recognized as an important offensive factor in genesis of gastric ulcers among humans. Although there is no direct evidence to relate eradication of H. pylori to decline of peptic ulcer over the last decades, the relative risk of peptic ulcer with H. pylori remains as high as for non-steroidal antiinflammatory drugs (NSAIDs) (Kuruta and Nogawa, 1997). Even though there have been advances in eradication of H. pylori in peptic ulcer patients using multiple therapies, there are several reports on the limitations of such treatments. Resistance of H. pylori to antimicrobials (Megraud, 1999) and poor patient compliance due to multiple doses (Buring et al. 1999) are reported to be major causes for failure of anti-ulcer therapy. This necessitates use of newer antibacterials and reduction of doses during therapy. BME has been reported to possess anti-ulcer properties and its potential anti-H, pylori activity would be of importance in overcoming the limitations of multiple therapies. Prostaglandins (PGs), especially PGE and prostacyclins (PG[I.sub.2]) are another important defensive factor, whose role in the protection of gastric mucosa is well documented (Robert, 1979; Wallace and Whittle, 1985; Lutnicki et al. 2001). Hence, estimation of both PGs would enhance our understanding of the antiulcerogenic activity of BME. PGs were estimated from human colonic incubates as it has been reported that both gastric and colonic mucosal incubated are similar in PGs composition (Goel et al. 1990).

Thus, the present study includes in vitro evaluation of anti-H, pylori activity and prostanoid (PGE and PG[I.sub.2]) synthesis in human colonic mucosal incubates in relation to BME treatment.

* Materials and Methods

Extraction and standardization of Bacopa monniera Whole plant of cultivated variety of Bacopa monniera (Ayurvedic Gardens, Banaras Hindu University) were collected in the month of march and was identified with the standard sample preserved in the department of Dravyaguna, Institute of Medical Sciences, Varanasi.

The fresh whole plants of Bacopa monniera were size reduced and were macerated with methanol for 7 days. The extract was filtered, vacuum dried and stored in a refrigerator until further use. The yield was 1.2%. The methanolic extract was subjected to HPTLC (CAMAG TLC evaluation software; CATS 3.16; Scanner III) are described earlier (Bhattacharya and Ghosal, 1999; Sairam et al. 2001) for estimation of bacoside A. The solvent systems used were n-Butanol:Acetic acid :water (4:1:1) and ethyl acetate: acetic acid: formic acid :water (100:11:11:27); staining reagent-2,4-dinitrophenyl-hydrazine; reflectance spectra: [lambda] max 278 nm. The percentage of bacoside A was 35.5 [+ or -] 0.9.

Experimental methods

* Evaluation of in vitro H. pylori activity: The NCTC 12822 strain of Helicobacter pylori was used. It was cultured on brain heart infusion agar supplemented with 5% horse blood and incubated at 37 [degrees]C under microaerophilic conditions (5% [O.sub.2], 10% C[O.sub.2], 80% N[O.sub.2]) (Wagner et al. 1992; Goel et al. 2001).

--Preparation of test solutions: 50 mg of BME was dissolved in 5 ml (10 mg/ml) of distilled water and was sterilized by passing through Minisart N filter. Bismuth subcitrate (BSC, Brocades House, Surrey) was prepared in the dilution of 10 mg/ml and was sterilized by autoclaving (MIC 5 [micro]g/ml). Using sterile water, dilusions of 0.32, 1.6, 8, 40, 200, 1000 [micro]g/ml of BME and BSC were prepared and used for the experiments.

--Detection of inhibitory activity: The experiment was done using a 96 well plate technique. To each test well 30 [micro]l of BME or BSC, 250 [micro]l of growth medium and 20 [micro]l of NCTC were added in order. H. pylori was reconstituted aseptically introducing a loopoful of the organism in 100 ml of sterile Ringer's solution. 100% growth controls contained 30 [micro]l of solvent; 250 [micro]l of growth medium and 20 [micro]l of NCTC and 0% control contained 30 [micro]l of solvent, 250 [micro]l of growth medium and 20 [micro]l of Ringer solution. The inner 10 x 6 wells were used. The plates were then incubated in microaerophilic atmosphere at 37 [degrees]C hr, and examined for growth on surface of each well.

* In vitro prostanoids study: Human colonic tissues were taken from surgical specimens removed for benign and malignant disease, at least 5 cm from any microscopically detected lesion. It was transported to the laboratory in 154 mM NaCl at ambient temperature within 30 min of removal and on arrival it was transferred to ice-cold phosphate buffer saline pH 7.4 PBS. The mucosa/submucosa was carefully removed from the underlying muscle, while the tissue was bathed in PBS. As far as we know, patients had not recently taken drugs affecting eicosanoid synthesis. The mucosa and submucosa was cut into pieces of about 4 [mm.sup.2] and washed with PBS, which was then drained off. Pieces of 100 mg of mucosa were carefully weighed and preincubated in PBS (1 ml, 4 [degrees]C, for 30 min) which was then drained off, and the tissues were transferred to tubes containing 1 ml of either normal saline (NS) or DW. Test samples were incubated with BME dissolved in distilled water. BME was used in the concentrations of 0.1 to 10 [micro]g/ml. After further incubation at 37 [degrees]C for 30 min, the fluid, which contained the released eicosanoids, was removed and stored at -20 [degrees]C until assayed (Tavares et al. 1987; Goel et al. 1990). Radioimmunoassay method was based on that of Jaffe and Behrman (1974), using suitable dilution's of antisera and titrated standards. Assay sensitivities were 10 pg, and the intrainter-assays coefficients of variation were 6-9% and 10-12%, depending on the eicosanoid measured. All assays were in duplicate. Tritiated prostanoids were purchased from Radiochemical Center (Amersham, UK). PGE antiserum was obtained from ICN (High Wycombe, UK). This antiserum does not distinguish between PG[E.sub.1] and PG[E.sub.2], the measurements are expressed as PGE and PG[I.sub.2] antisera were from Wellcome (Beckenham, UK). Cross-reactions were previously reported. Results are expressed as mean [+ or -] SEM, and analyzed by Student's t test for paired data (2-tailed).

* Results

In vitro evaluation of bismuth subcitrate, a known H. pylori growth inhibitor and BME in different dilutions showed that both the drugs in the concentration of 1000 [micro]g/ml inhibited the growth of H. pylori to significant level (Table 1).

The amounts of prostaglandin E (PGE) and prostacycline (PG[I.sub.2]) accumulating in the control incubates of human colonic mucosal pieces (ng/g Wet weight tissue for 30 minutes) were PGE 38.7 [+ or -] 12.8; 6-keto PG[F.sub.1[alpha]] (a metabolic of PG[I.sub.2]) 73.1 [+ or -] 15.2 (n = 6). Indomethacin in PBS 0.1 to 1 [micro]g/ml caused a concentration related reduction of both PGE (64.6% to 89.6% reduction, P < 0.01) and PG[I.sub.2] (80.8% to 90.0% reduction, P < 0.01), while BME at 10 [micro]g/ml showed increase of both PGE (40.8% increase, P, 0.05) and PG[I.sub.2] (50.5% increase, P < 0.2) accumulation indicating enhancement PGs synthesis by BME (Table 2).

* Discussion

In vitro studies with H. pylori showed significant inhibition at the concentration of 1000 [micro]g/ml of BME. H. pylori has now been accepted if not the cause, at least as a facilitating factor in ulcerogenesis. Although further studies are needed to understand the mechanism of anti-H, pylori activity of BME, there is a possibility that its reported antioxidant activity (Sairum et al. 2001) may be involved, as H. pylori infection has been reported to induce reactive oxygen species (Davies et al. 1994). It is well documented that free radicals have a decisive role in ulcerogenesis. Further there are reports of compromised immunity and success of immunization in preventing H. pylori infection (Tosi et al. 1992). BM is classified as a rasayana in Ayurveda, which are thought to increase generalized immunity, similar to modern classification of adaptogens. This may also be an important contributing factor, which needs to be further studied. Even through effective antimicrobial therapy is available to eradicate H. pylori infection, current therapies require patients to ingest multiple agents several times a day for at least one week (Soll, 1996), leading to non-compliance of treatment schedules. Further treatment can be accompanied by nausea, diarrhea, abdominal pain and pseudomembranois colitis (Rauws, 1993). There are also reports of altered physiological functions by anti-ulcer drugs in H. pylori positive patients (Hoist et al. 1992; Gillen et al. 1999). An anti-ulcer agent having anti-H. pylori activity can have better compliance among patients and can also decrease incidences of drug interactions.

Prostaglandins have long been known to afford protection to the gastric mucosa. They do so by inhibiting acid secretion and increasing mucosal defenses (Robert, 1979). PGs are reported to restore mucosal defense, and thereby prevent damage by several irritants and influence repair of gastric ulcers (Wallace, 2001). PGs are reported to act through endogenous prostaglandin (EP) receptors and the subtype EP-3 in the stomach and duodenum has been reported to be responsible for bicarbonate secretion (Takeuchi et al. 1999a, b). There are several reports on the contributing role of PGs in the antiulcerogenic activity of many drugs. Rebamipide a mucoprotective agent is reported to induce COX-2 and thereby increase PG release (Sun et al. 2000). Lansoprazole has been reported to protect against ethanol-induced damage by increased accumulation of PGs rather than reduction of acid secretion (Blandizzi et al. 1999). Similarly, BME was also reported to have no significant effects on acid secretion and in the present study, BME significantly increased both PGE and PG[I.sub.2] accumulation by human colonic incubates in vitro. There are many reported uses of PG analogs, which are as effective as many widely used anti-ulcer drugs (Wu et al. 1998). Although correlation of in vitro accumulation of PGs by human colonic mucosa to antiulcerogenic activity of rat models of gastric ulcer reported earlier may be contentious, the study nevertheless proves the inducing effect of BME on PG synthesis.

Thus, the present study shows that BME has in vitro anti-H, pylori activity and causes an increase in the accumulation of PGs by human mucosal colonic incubates, which could account for its anti-ulcerogenic activity. Further, in vivo studies would substantiate the anti-H, pylori activity and inducement of PG accumulation by BME.
Table 1. In vitro effect of bismuth subcitrate (BSC) and
BME on growth of H. pylori.

Test No. of wells % P Value
substance showing Inhibition (Chi
([micro]g/ml) inhibition square test)

BSC
 200 2/8 25% NS
 1000 6/8 75% <0.01
BME
 200 0/8 0% NS
 1000 6/8 75% <0.01

Table 2. Effect of BME on the accumulation of human
colonic mucosal prostaglandins E (PGE) and [I.sub.2],
(data are mean [+ or -] SEM, 6 experiments in each group).

Treatment Colonic mucosa (ng/g wet tissue, 30 min)
([micro]g/ml)
 PGE PG[I.sub.2]

Control 38.7 [+ or -] 12.8 73.1 [+ or -] 15.2
 (1 % methanol)
Indomethacin
 0.1 13.7 [+ or -] 5.4 (b) 14.0 [+ or -] 4.9 (b)
 1.0 7.0 [+ or -] 4.2 (b) 8.8 [+ or -] 5.8 (c)
 10.0 4.0 [+ or -] 1.5 (b) 3.7 [+ or -] 2.0 (b)
BME
 0.1 29.5 [+ or -] 5.2 59.0 [+ or -] 10.6
 1.0 37.7 [+ or -] 8.2 76.0 [+ or -] 20.4
 10.0 54.5 [+ or -] 6.0 (a) 110.0 [+ or -] 30.1

(a) P < 0.05, (b) P < 0.01, (c) P < 0.001.


Acknowledgements

RKG is thankful to Indian Council of Medical Research for grant-in aid and The Royal Society, London and INSA, New Delhi for scientist exchange programme. The authors are thankful to, Indian Herbs Ltd. (IH), Saharanpur, India, for technical facilities provided for chemical standardization of the extract.

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* Address

Prof. R. K. Goel, Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi--221005, India

Tel.: (0542) 316739; Fax: (0542) 367567; e-mail: rkgoel@banaras.ernet.in

R. K. Goel (1), K. Sairam (1), M. Dora Babu (1), I. A. Tavares (2) and A. Raman (3)

(1) Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India

(2) Department of surgery, King's college, school of Medicine and Dentistry, The Rayne Institute, London, U.K.

(3) Department of Pharmacy, GKT school of Medicine and Dentistry, King's Kollege, London, U.K.
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Author:Goel, R.K.; Sairam, K.; Babu, M. Dora; Tavares, I.A.; Raman, A.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:9INDI
Date:Jul 1, 2003
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