Studies on Pongamia pinnata (L.) Pierre leaves: understanding the mechanism(s) of action in infectious diarrhea

Studies on Pongamia pinnata (L.) Pierre leaves: understanding the mechanism(s) of action in infectious diarrhea 1 first-author *E-mail:fmrbom@hathway.com *E-mail:fmrbom@hathway.com fmrbom@hathway.com 1 *E-mail:fmrbom@hathway.com *E-mail:fmrbom@hathway.com fmrbom@hathway.com 2 **E-mail:ngcpr@lawkimindia.com **E-mail:ngcpr@lawkimindia.com ngcpr@lawkimindia.com 3 ***E-mail:rojatkar@dalton.ncl.res.in ***E-mail:rojatkar@dalton.ncl.res.in rojatkar@dalton.ncl.res.in 1 4 corresp *E-mail:fmrbom@hathway.com *E-mail:fmrbom@hathway.com fmrbom@hathway.com 1 corresp ‡Corresponding Author ‡Corresponding Author *E-mail:fmrbom@hathway.com *E-mail:fmrbom@hathway.com fmrbom@hathway.com The Foundation for Medical Research, R.G. Thadani Marg, Worli, Mumbai 400018, India The Foundation for Medical Research, R.G. Thadani Marg, Worli, Mumbai 400018, India Naoroji Godrej Centre for Plant Research, Lawkin Ltd. Campus, Shindewadi, Shirwal, Satara 412801, India Naoroji Godrej Centre for Plant Research, Lawkin Ltd. Campus, Shindewadi, Shirwal, Satara 412801, India The National Chemical Laboratory, Homi Bhabha Road, Pune 411008, India The National Chemical Laboratory, Homi Bhabha Road, Pune 411008, India The Foundation for Research in Community Health, Anand Park, Pune 411007, India The Foundation for Research in Community Health, Anand Park, Pune 411007, India While data are available on the effect of medicinal plants on intestinal motility and their antibacterial action, there is a paucity of information on their mode of action on various aspects of diarrheal pathogenicity, namely colonization to intestinal epithelial cells and production/action of enterotoxins. Crude decoction of dried leaves of Pongamia pinnata was evaluated for its antimicrobial (antibacterial, antigiardial and antirotaviral) effect; and its effect on production and action of enterotoxins (cholera toxin, CT; Escherichia coli labile toxin, LT; and E . coli stable toxin, ST); and adherence of enteropathogenic E . coli and invasion of enteroinvasive E . coli and Shigella flexneri to epithelial cells. The decoction had no antibacterial, antigiardial and antirotaviral activity, but reduced production of CT and bacterial invasion to epithelial cells. The observed results indicated that the crude decoction of P . pinnata has selective antidiarrheal action with efficacy against cholera and enteroinvasive bacterial strains causing bloody diarrheal episodes. Pongamia pinnata Medicinal plants Diarrhea Antidiarrheal agent Enterotoxin Colonization INTRODUCTION 1 Diarrhea occurs worldwide and causes 3.2% of all deaths (WHO, 2004 2004 ). It is the second largest cause of years of productive life lost due to premature mortality and disability (Murray and Lopez, 1997a 1997a ). Even though economic development and progress in health care delivery are expected to catalyze substantial improvements in infectious disease related morbidity and mortality by the year 2020, it is predicted that diarrhea will remain a leading health problem (Murray and Lopez, 1997b 1997b ). Diarrhea is most commonly caused by gastrointestinal infections, which kill around 1.8 million people globally each year, mostly children in developing countries (WHO, 2004 2004 ). The main cause of death from diarrhea is dehydration, which results from loss of electrolytes in diarrheal stools. During the past decade there have been some major improvements regarding the treatment of infectious diarrhea. Oral rehydration therapy has contributed greatly to the reduction of diarrheal mortality rates in children and the elderly. However, the diarrheal attack rate has remained unchanged and this treatment often fails in the high stool output state. Symptomatic therapy with antimotility agents is restricted to non-dehydrated patients without features of systemic infection. These are not indicated in infants and patients with febrile bloody diarrhea. Moreover, there is an increasing threat of drug resistance, side effects of treatment with antibiotics, supra infection when normal flora is eradicated with antimicrobial agents and the possibility of induction of disease producing bacteriophages by antibiotics (Dham, 2003 2003 ). Vaccines have been considered as the most feasible approach to diarrheal management. Various attempts for developing vaccines against diarrhea causing organisms have been made (Cohen et al., 2000 2000 ; Ghose, 1996 1996 ; Klee et al., 1997 1997 ; Martines et al., 1993 1993 ). However, the response to such vaccines in developing countries has not been encouraging (Lagos et al., 1999 1999 ). Considerable technical barriers need to be overcome before continuing clinical evaluation of prospective vaccine candidates. Thus an important niche exists for development of cost effective alternative approaches and medicinal plants may serve to fulfill this niche. Despite the phenomenal growth and success of modern medicine, plant remedies have gained immense popularity over the recent years as cures for treatment of several ailments. With emphasis being on scientific validation, a number of plants have been chemically and biologically evaluated for their acclaimed properties. A number of medicinal plant preparations have been recognized for treating diarrhea, although their mode of action in elimination of organisms causing diarrheal diseases is not well understood. While data are available on the effect of various plants on intestinal motility in experimental models and antibacterial action (Akah et al., 1999 1999 ; Kambu et al., 1990 1990 ; Miranda et al., 1993 1993 ; Tona et al., 1999 1999 ), there is paucity of information on the mode of action of medicinal plants on various aspects of diarrheal pathogenicity. The pathogenesis of infectious diarrhea has been extensively studied. It is caused by a variety of enteric pathogens including bacteria such as enterotoxigenic Escherichia coli (ETEC), enteropathogenic E . coli (EPEC), enteroinvasive E . coli (EIEC), Salmonella , Shigella flexneri , Vibrio cholerae , and Campylobacter jejuni ; viruses such as rotavirus, astrovirus, adenovirus, and calicivirus; and protozoa such as Giardia lamblia , Entamoeba histolytica , and Cryptosporidium parvum (Martines et al., 1993 1993 ; Bhattacharya, 2000 2000 ; Guerrant and Bobak, 1991 1991 ). Mechanisms by which these organisms disrupt intestinal function to cause malabsorption or diarrhea are microbial attachment and localized effacement of the epithelium, production of toxin(s) and direct epithelial cell invasion (Guerrant et al., 1999 1999 ). Adherence is a means of colonizing the appropriate ecological niche. It enables the organism to resist being swept away by mucosal secretions and also helps the organism in subsequent proliferation and colonization of the gut. Adherence can be followed by toxin production or invasion (Ashkenazi and Pickering, 1989 1989 ). Furthermore, bacterial adherence may increase the efficiency of these toxins particularly in the intestinal tract, where proteolytic enzymes may inhibit the long-term effect of toxins. Toxigenic diarrhea is caused by enterotoxins secreted by bacteria (e.g., E . coli stable toxin), some of which act directly on a transmembrane signaling peptide such as membrane bound particulate guanylate cyclase. Toxins such as E . coli haemolysin, listeriolysin, and Streptolysin O alter membrane permeability, while others enzymatically alter specific intra cellular targets (Ashkenazi and Pickering, 1989 1989 ). Invasive diarrhea is caused by damage to intestinal epithelial cells due to invasion and penetration by the organism. Pathogens like Salmonella sp., Shigella sp., Listeria sp., EIEC, and Yersinia sp. (Kagnoff, 2003 2003 ) infect deeper layers of intestinal mucosa, and spread systemically while C . parvum , and Chlamydia trachomatis (Kagnoff, 2003 2003 ) are minimally invasive. These pathogens invade the intestinal epithelium, where they reside and replicate. Leaves of Pongamia pinnata (L.) Pierre (synonym, P . glabra vent) has been known as a remedy for diarrhea (Nadkarni, 1954 1954 ; Warrier et al., 1993 1993 ). Shoba and Thomas ( 2001 2001 ) reported on the effectiveness of P . pinnata in controlling castor oil induced diarrhea. However, there is no information on its effect on infectious forms of diarrhea. Hence we selected P . pinnata for studying its effect on various parameters such as adherence to and invasion of intestinal epithelium and production and action of toxins towards understanding its possible mechanism(s) of action in controlling infectious diarrhea. MATERIALS AND METHODS 1 Plant materials 2 Fresh leaves of P . pinnata were collected from Parinche Valley (near Pune, Maharashtra, India) in November, 2003. The plant was authenticated by Dr. P. Tetali, Naoroji Godrej Centre for Plant Research. A voucher specimen has been deposited at the Botanical Survey of India (Western Circle, Pune, India), under herbarium No. 124677. The leaves were shade dried, hand crushed and stored at 4 °C. All experiments were performed with the same dried material within six months from the date of collection. Preparation of extract 2 The decoction was prepared by boiling 1 g of the shade dried leaves in 16 ml double distilled water till the volume reduced to 4 ml as described in Ayurvedic text (Thakkur, 1976 1976 ). To replicate field conditions, studies were carried out with only the hot water decoction that was freshly prepared every time. The decoction was centrifuged and filtered through a 0.22 μm pore size membrane before use. The yield of the decoction thus obtained was 8.15%±0.42% (w/w) of the starting material. For each experiment, 1%, 5%, and 10% (v/v) concentrations of the decoction in appropriate medium were used. Cell culture and media 2 The human laryngeal cell line, HEp-2, and the embryonic monkey kidney cell line, MA-104, were obtained from the National Centre for Cell Sciences, Pune, India. The cell lines were maintained in Dulbecco’s modified eagle’s medium, DMEM (GibcoBRL, UK) and minimal essential medium, MEM (Himedia, Mumbai, India) respectively, supplemented with 5% fetal calf serum (GibcoBRL, UK), in 60 mm-diameter tissue culture dishes (Tarsons Pvt. Ltd., Kolkata, India). The cells were maintained in logarithmic growth by sub-culturing every 3~4 d. Antibacterial activity 2 The antibacterial activity was determined against six different bacterial strains viz. E . coli B170, E . coli B831-2, E . coli TX1 (all obtained from Centre for Disease Control, Atlanta, USA), E . coli E134 (kindly provided by Dr. J. Nataro, Veterans Affairs Medical Centre, Maryland, USA), V . cholerae El Tor (kindly provided by Dr. S. Calderwood, Massachusetts General Hospital, Boston, USA), S . flexneri M9OT (kindly provided by Dr. P. Sansonetti, Institut Pasteur, France), by minimum inhibitory concentration plate method (Cruickshank et al., 1975 1975 ). Log phase cultures (10 6 cells/ml) were plated onto nutrient agar containing the decoction and the growth was graded on a scale of 0 (no growth) to 4+ (control). Gentamycin (100 μg/ml) was used as the antibiotic control. Antigiardial activity 2 A 24 h culture of Giardia lamblia P1 trophozoites (kindly provided by Dr. P. Das, National Institute of Cholera and Enteric Diseases, Kolkata, India) was incubated with the decoction in Diamond’s TYI-SS medium (constituents procured from local Indian manufacturers), supplemented with bovine serum (Sigma, USA). The number of viable trophozoites after 24 h was counted in a haemocytometer using the vital stain, trypan blue (HiMedia, Mumbai) (Trowell, 1965 1965 ). Metronidazole (100 μg/ml) was used as an antibiotic control. Antirotaviral activity 2 The entry and subsequent survival of rotavirus (kindly provided by Dr. S. Kelkar, National Institute of Virology, Pune, India) in MA-104 cells were assayed by the neutral red uptake assay (Parish and Mullbacher, 1983 1983 ). Briefly, MA-104 cells were grown in 96-well tissue culture plates for 72 h. The cells were then infected with rotavirus in the presence of trypsin (0.4 μg/ml) for 90 min without and with different concentrations of the decoction. The rotavirus was previously treated with trypsin (7 μg/ml) at 37 °C for 30 min. After infection, the decoction and the unadsorbed virus were removed and the culture was further incubated for 72 h. Thereafter, the cells were incubated with 0.03% neutral red dye (Sigma, USA) for 30 min. The intracellular dye was released with 1:1 (v/v) solution of 100 mmol/L acetic acid and ethanol. The released dye was measured at 540 nm (reference 630 nm) in an ELISA plate reader (Thermo Electron Corp., Finland) as an indicator of cell viability. Effect on adherence 2 The effect on the adherence of E . coli strain B170 to epithelial cells was assayed by a method described by Cravioto et al.( 1979 1979 ). Briefly, a 48 h culture of HEp-2 cells was infected with a log phase culture (5×10 7 cells/ml) of E . coli B170 and incubated for 3 h. Non-adherent bacteria were washed off and microcolony formation was observed by toluidine blue staining (0.1%, w/v). HEp-2 cells having ≥5 adherent E . coli cells were counted. The adherence assay was performed using two different protocols. The HEp-2 cells were incubated with different concentrations of the decoction either before infection (pre-incubation) or simultaneously with the infection (competition). Effect on invasiveness 2 The effect on invasion of E . coli E134 and S . flexneri was based on a method described by Vesikari et al.( 1982 1982 ). Briefly, a 48 h culture of HEp-2 cells grown in a 24-well tissue culture plate was infected with log phase culture (10 8 cells/ml) of the bacteria and incubated for 2 h. The culture was further incubated with gentamycin (100 μg/ml) for 3 h. The epithelial cells were then lysed by cold shock and the released bacteria were counted by plating on nutrient agar. The invasive assay was performed using two different protocols. The HEp-2 cells were incubated with different concentrations of the decoction either before infection (pre-incubation) or simultaneously with the infection (competition). Effect on labile toxin (LT) and cholera toxin (CT) 2 An endotoxin, LT, was obtained from E . coli B831-2 by lysing the bacterial cells with polymyxin B sulphate (Sigma, USA). CT was obtained as culture supernatant of V . cholerae , as it is an exotoxin. Both toxins were assayed by the ganglioside monosialic acid-enzyme linked immunosorbent assay (GM1-ELISA) (Svennerholm and Wilkund, 1983 1983 ). Briefly, the toxins were added to ELISA plates pre-coated with 1.5 μmol/ml of GM1 (Sigma, USA). Anti-cholera toxin (Sigma, USA) and peroxidase labelled swine anti-rabbit immunoglobulin (Dako, Denmark) were used as primary and secondary antibodies respectively. Orthophenylene diamine was used as the substrate. The color developed was read at 492 nm in an ELISA plate reader. To study the effect of the decoction on the action of LT and CT, the toxins were assayed with different concentrations of the decoction. To study its effect on their production, the respective bacteria were grown with different concentrations of the decoction and the toxins thus obtained were assayed. Effect on stable toxin (ST) 2 An exotoxin, ST, was obtained as a culture supernatant of E . coli TX1. It was assayed by the method originally described by Gianella ( 1976 1976 ). Briefly, the toxin was inoculated intra-gastrically into 2~3 d old Swiss White suckling mice. Following 3 h incubation at room temperature, the suckling mice were sacrificed and the ratio of gut weight to that of the remaining carcass weight was calculated. Ratio ≥0.083 was considered as positive for ST. To study the effect of the decoction on the action of ST, the toxin was inoculated intra-gastrically with different concentrations of the decoction. To study its effect on the production of ST, the bacterium was grown with different concentrations of the decoction and the toxin thus obtained was inoculated intra-gastrically. The institutional ethical committee and the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) cleared the use of animals in the study. The Foundation for Medical Research (FMR) is registered with CPCSEA (registration No. 424/01/a/CPCSEA, June 20th, 2001). Phytochemical analysis 2 Qualitative phytochemical analyses were carried out with the decoction in order to determine the presence of carbohydrates, glycosides, proteins, amino acids, phytosterols, saponins, flavonoids, alkaloids and tannins (Kokate et al., 1990 1990 ). The leaves, flowers, seeds and stem bark of P . pinnata are known to have karanjin (Malik et al., 1977 1977 ; Meera et al., 2003 2003 ), a furanoflavanoid, which is toxic (Prabhu et al., 2002 2002 ). The levels of this compound in water decoction and acetone extract of the leaves of P . pinnata were detected with high performance liquid chromatography (HPLC) at 254 nm with acetonitrile:water (60:40, v/v) as mobile phase at a flow rate of 1 ml/min in a 12.5 cm Lichrosphere RP-18 column (Shimadzu CLASS-VP V6.10). Statistical analysis 2 Each assay was performed three times and the results were expressed as their mean±standard deviation. The differences in the mean values amongst the treatment groups were analyzed by analysis of variance (ANOVA). Further, the significance of the difference between the means of the test and the control observations were established by Dunnett’s post-test. Statistical analyses were performed using the software Prism 4.0 (GraphPad, Inc.). P ≤0.05 was considered to be statistically significant. The statistical analysis was not applied on the results of the antibacterial and the suckling mouse assays. RESULTS 1 Antimicrobial activity 2 As shown in Table 1 1 , the decoction did not inhibit the growth of any of the six bacterial strains tested. Similarly, the viability of the G . lamblia trophozoites (Fig. 1 1 ) and rotavirus (Fig. 2 2 ) was also not affected by the decoction. The bars represent mean±standard deviation (from three individual experiments) of percentage reduction in viability of trophozoites as compared to respective value of controls (100%); C: Trophozoites incubated without the decoction was used as a control; M: Metronidazole (100 μg/ml) was used as a positive control The bars represent mean±standard deviation (from three individual experiments) of percentage optical density as compared to respective value of controls (100%); C: Uninfected MA-104 cells incubated without the decoction was used as a control; RV: Rotavirus infected MA-104 cells Growth of bacteria graded on a scale of 0 (no growth) to 4+ (control) Bacteria grown on nutrient agar without the decoction 2 Bacteria 5 Growth a a Control b b Gentamycin (100 μg/ml) 1% decoction 5% decoction 10% decoction E . coli B170 4+ 0 4+ 4+ 4+ E . coli B831-2 4+ 0 4+ 4+ 4+ E . coli E134 4+ 0 4+ 4+ 4+ E . coli TX1 4+ 0 4+ 4+ 4+ S . flexneri 4+ 0 4+ 4+ 4+ V . cholerae 4+ 0 4+ 4+ 4+ Effect on colonization to HEp-2 cell line 2 No competitive inhibition of the adherence of E . coli B170 to the epithelial cells was observed when the HEp-2 cells were incubated with the decoction simultaneously with infection. Similarly, no effect of the decoction was observed when the HEp-2 cells were incubated with the decoction prior to infection (Table 2 2 ). This indicates that the decoction did not have any effect on the epithelial cells that could lead to a decrease in adherence. However, the decoction significantly reduced the invasion by both E . coli E134 and S . flexneri of the epithelial cells in both the protocols (Table 2 2 ). Since the reduction was observed in both protocols, it appears that decoction prevents invasion by modification of the host cells. Values represent mean±standard deviation (from three individual experiments) of percentage reduction in colonization as compared to respective value of controls (100%) Analysis of variance HEp-2 cells incubated with the decoction simultaneously with the infection HEp-2 cells incubated with the decoction prior to the infection P < 0.05 by Dunnett’s post-test P < 0.01 by Dunnett’s post-test The negative values indicate that there is an increase in adherence 2 Parameter 2 Organism 3 Reduction in colonization a a (%) 3 ANOVA b b Assay Protocol 1% decoction 5% decoction 10% decoction F df P 2 Adherence Competitive 1 1 E . coli B170 −5.88±2.03 # # −7.23±4.31 # # 21.70±16.30 * * 7.607 3, 8 0.0099 Pre-incubation 2 2 E.coli B170 −0.87±1.83 # # −2.73±4.50 # # 6.22±3.63 4.901 3, 8 0.0321 4 Invasion 2 Competitive 1 1 E . coli E134 56.94±28.4 * * 83.78±13.38 ** ** 73.96±19.57 ** ** 12.370 3, 8 0.0023 S . flexneri 20.02±11.08 ** ** 16.71±28.04 ** ** 64.79±26.88 ** ** 133.000 3, 8 0.0237 2 Pre-incubation 2 2 E . coli E134 78.15±3.74 81.82±12.62 98.33±0.96 * * 5.528 3, 8 <0.0001 S . flexneri 43.79±3.12 ** ** 65.76±3.49 ** ** 43.13±4.65 ** ** 209.100 3, 8 <0.0001 Effect on LT and CT 2 There was significant reduction in the production of CT when V . cholerae was grown in the presence of the decoction (Table 3 3 ) even though the bacteria were not killed. Unlike CT, the decoction did not inhibit the production of LT by E . coli B831-2. The decoction had no effect on the action of CT once the toxin was produced, as there was no competitive inhibition of the binding of toxin to its receptor GM1 in presence of the decoction. Similarly, the action of LT was also not inhibited by the decoction once the toxin was produced. On the contrary, an increase in binding of LT to GM1 was observed. Values represent mean±standard deviation (from three individual experiments) of percentage reduction in optical density as compared to respective value of controls (100%) Analysis of variance Toxin incubated onto GM1 receptor with the decoction Toxin produced by bacteria grown in presence of the decoction P < 0.05, by Dunnett’s post-test P < 0.01 by Dunnett’s post-test The negative values indicate that there is an increase in the action/production of labile toxin 2 Parameter 2 Organism 3 Reduction in optical density a a (%) 3 ANOVA b b Assay Protocol 1% decoction 5% decoction 10% decoction F df P 2 Cholera toxin Action 1 1 V. cholerae 7.41±2.69 11.26±6.49 * * 7.69±3.11 5.483 3, 8 0.0242 Production 2 2 V. cholerae 15.32±13.05 39.04±14.02 * * 23.09±5.21 5.298 3, 8 0.0264 2 Labile toxin Action 1 1 E . coli B 831-2 −31.94±11.46 * * # # −58.24±12.66 ** ** # # −44.16±12.59 ** ** # # 16.450 3, 8 0.0009 Production 2 2 E . coli B 831-2 10.82±15.94 −11.73±21.74 # # 5.19±6.17 1.450 3, 8 0.2991 Effect on ST 2 The decoction was neither able to inhibit the production of ST when E . coli TX1 was grown in the presence of the decoction nor could it control the excessive intestinal secretion due to the binding of ST as was evident from the ratio of the gut weight to that of the carcass weight in the suckling mouse assay (Table 4 4 ). Values represent mean±standard deviation (from three individual experiments) of ratio of gut weight to that of the carcass weight Cholera toxin without the decoction was used as negative control Stable toxin without the decoction was used as positive control Intra-gastric inoculation of the toxin carried out with the decoction Toxin produced by bacteria grown in presence of the decoction All the readings observed are in the positive range (≥0.085) except CT control 2 Protocol 5 Gut weight/carcass weight a a CT control b b ST control c c ST+1% decoction ST+5% decoction ST+10% decoction Action 1 1 0.058±0.006 0.109±0.010 0.111±0.003 0.101±0.010 0.097±0.010 Production 2 2 0.054±0.001 0.109±0.015 0.114±0.009 0.109±0.002 0.110±0.008 Phytochemical analysis 2 Carbohydrates, proteins, saponins, flavonoids and tannins were detected in the decoction. The HPLC profiles of the water decoction and the acetone extract of the leaves of P . pinnata showed presence of negligible (0.367%) amount of karanjin in the water extract (Fig. 3a 3a ) as compared to 14.749% in the acetone extract (Fig. 3b 3b ). A similar form of the water decoction is a common form of administration used in popular medicine. Peak No. Retention time (min) Area (mV·min) Area (%) Height (mV) Height (%) Theoretical plates (a) 1 1.233 1462603 39.548 156412 48.817 524.03 2 1.600 2212725 59.831 161789 50.495 457.48 Karanjin→3 2.675 13556 0.367 1442 0.450 648.40 4 2.925 9404 0.254 765 0.239 89.69 Totals 3698288 100.000 320408 100.000 (b) 1 1.158 285993 1.754 23189 1.477 262.78 2 1.617 759710 4.659 61378 3.911 215.19 3 1.942 930300 5.706 66936 4.265 37.52 4 2.208 2114870 12.971 146250 9.318 398.91 5 2.483 1119520 6.866 118185 7.530 262.53 Karanjin→6 2.708 2404873 14.749 259085 16.507 675.86 7 2.850 1664648 10.209 280128 17.848 211.83 8 2.950 2773810 17.012 315490 20.101 475.23 9 3.083 2616074 16.045 231795 14.769 152.37 10 3.617 449405 2.756 27176 1.732 379.67 11 4.017 427193 2.620 19911 1.269 436.09 12 4.542 201337 1.235 6569 0.419 170.28 13 5.375 172986 1.061 3292 0.210 40.57 14 6.417 30113 0.185 1612 0.103 0.00 15 7.075 129236 0.793 2930 0.187 380.05 16 7.967 161057 0.988 3466 0.221 379.39 17 10.158 63859 0.392 2107 0.134 3881.30 Totals 16304984 100.000 1569499 100.000 DISCUSSION 1 The leaves of P . pinnata , also known as Indian Beech, are known to be digestive, laxative and anti-helminthic, and are good for flatulence, dyspepsia, diarrhea, leprosy, and gonorrhea and cough (Warrier et al., 1993 1993 ). The hot water decoction of the leaves is good for rheumatalgia and for cleaning ulcers and wounds (Warrier et al., 1993 1993 ). On the basis of this utilization profile it is suggested that P . pinnata may have antimicrobial as well as anti-inflammatory action. In this study, we examined the antidiarrheal properties of hot water decoction of dried leaves of P . pinnata against various virulence parameters of infectious diarrhea. The decoction of P . pinnata leaves did not have antibacterial, antigiardial and antirotaviral activity. Though it did not arrest the growth of V . cholerae , it prevented the production of CT, thereby indicating that the decrease in production of the toxin was due to its effect on bacterial metabolism and not due to reduction in bacterial counts. The binding of LT and CT to the GM1 receptor was not inhibited. On the contrary an increase in the binding of LT was noted. It was interesting to see that in spite of CT and LT being structurally, functionally, biologically, and immunologically closely related (Ganguly and Kaur, 1996 1996 ), there was a difference in the binding of these toxin moieties to the receptor in presence of the decoction. The decoction did not affect adherence of E . coli B170, while it was observed that the invasion of both E . coli E 134 and S . flexneri was significantly reduced. Interestingly, the reduction in the invasion of the bacteria to the epithelial cells, and not the adherence, indicates that the decoction affects bacterial entry by acting at a post-adherence stage. Another interesting observation of the biological results is the effectiveness of a 5% dilution of the decoction over the 10% dilution in certain assays as indicated by the reduction in invasion of S . flexneri in the pre-incubation protocol (Table 2 2 ) and suppression of production and action of CT (Table 3 3 ). This probably indicates the synergistic combination of active compound(s) being attained at a slightly lower concentration. Literature shows presence of flavonoids, especially furanoflavonoids, quercetin, amino acids, fatty acids and triterpenoids in P . pinnata (Satyavati et al., 1987 1987 ; Sharma et al., 2001 2001 ). However, none of the known chemical constituents from the plant has been attributed with antidiarrheal activity. Moreover the chemical composition of the water decoction of P . pinnata leaves has not been studied in detail. In our study, the decoction showed presence of carbohydrates, proteins, saponins, tannins and flavonoids. Tannins, flavonoids and saponins in general have been reported to have several pharmacological activities including antidiarrheal activity (Agbor et al., 2004 2004 ; Galvez et al., 1991 1991 ; 1993a 1993a ; 1993b 1993b ; Lutterodt et al., 1999 1999 ; Miranda et al., 1993 1993 ; Mukherjee et al., 1998 1998 ; Oben et al., 2006 2006 ; Longanga Otshudi et al., 2000 2000 ; Venkatesan et al., 2005 2005 ). The antidiarrheal activity of these groups of compounds has been attributed to antimotility and antisecretory effects (Di Carlo et al., 1993 1993 ; Galvez et al., 1993a 1993a ; Oben et al., 2006 2006 ; Rao et al., 1997 1997 ) and antimicrobial action (Lutterodt et al., 1999 1999 ; Miranda et al., 1993 1993 ; Tona et al., 1999 1999 ). Thus, the tannins, flavonoids and saponins present in the decoction may be responsible for the observed activity in the present study. Nevertheless it must be stressed that the decoction did not show any antimicrobial activity. This is probably due to the difference in the individual constituents of the aqueous extract. Most of the studies reporting antimicrobial activity have used crude organic extracts or active principles. In the absence of antimicrobial activity, especially in infectious diarrhea, it is necessary that the study of antidiarrheal activity of a plant should consider the disease pathogenesis. Assaying bacterial virulent features as a marker for demonstrating the antidiarrheal efficacy of a plant had been previously reported by us using indigenous plants namely Cyperus rotundus (Daswani et al., 2001 2001 ), Holarrhena antidysenterica (Daswani et al., 2002 2002 ) and Dalbergia sissoo (Brijesh et al., 2006 2006 ). The study was restricted to the crude extract since it is our belief that the biological activity could be due to the synergistic activity of a combination of two or more active components as discussed above. 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Gurav, the field workers of FRCH, for collection of plant material. 10.1631/jzus.2006.B0665 Project (No. 91283) supported by the Department of Science and Technology, Ministry of Science and Technology, Government of India 2006-03-09 2006-06-02 2006-07-14 2022-01-05 Copyright © 2006, Journal of Zhejiang University Science 2006 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 08 07