Therapeutic Efficacy of Bidens pilosa L. var. radiata and Galinsoga parviflora Cav. in Experimentally Induced Diarrhoea in Mice

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1 3 Therapeutic Efficacy of Bidens pilosa L. var. radiata and Galinsoga parviflora Cav. in Experimentally Induced Diarrhoea in Mice ARUN K. YADAV 1* AND VAREISHANG TANGPU Abstract Bidens pilosa L. var. radiata and Galinsoga parviflora Cav., belonging to the family Asteraceae, are used as a popular folk medicine to treat diarrhoeal disorders in the Naga tribes of northeast India. In the present study the therapeutic efficacy of B. pilosa and G. parviflora was investigated employing experimentally induced diarrhoea in mice. The 800 mg/kg dose of methanol extracts of both plants showed significant (p < 0.001) inhibitor activity against castor oil-induced diarrhoea and PGE 2 -induced intrafluid accumulation. Both plant extracts also showed a significant (p < 0.001) reduction in the gastrointestinal motility in charcoal meal test. In the acute toxicity tests, mice treated with an 800 mg/kg dose of plant extracts did not show any significant change in SGOT, SGPT, cholesterol, and total proteins levels compared with controls. The observed results could explain their use as antidiarrhoeal agents in traditional medicine. Key words :Antidiarrhoeal activity, Bidens pilosa, Castor-oil induced diarrhoea, Galinsoga parviflora, India, Prostaglandins Introduction Diarrhoea is recognized worldwide as one of the most important and serious public health problems, especially in the developing countries of the world, due to its potential cause of morbidity and mortality especially in infants and children (Kosek et al., 2003). It is estimated that global distribution of diarrhoea accounts for more than 5-8 million deaths each year in children of less than 5 years old (Fauci et al., 1998). In India alone, it is estimated that 1. Department of Zoology, North-Eastern Hill University, Shillong , India. * Corresponding author : akynehu@hotmail.com

2 36 RPMP Vol. 23 Phytopharmacology & Therapeutic Values V around 500 million episodes of diarrhoea occur every year with an incidence of 7.9 per million children per year (Mata, 1983). In order to combat the problems of diarrhoea globally, especially in the developing countries of the world, the WHO has provided a special emphasis on the use of traditional folklore medicines in the control and management of diarrhoea (Anonymous, 1979). Ethnomedicine is an integral part of the traditional practices in developing countries of the world, and medicinal plants or plant-derived products play a dominant role in traditional medicines (Githiori et al., 2005). Bidens pilosa L. var. radiata (Asteraceae), locally known as Napnar, is a tall erect herb (Fig 1). It is found distributed in tropical and subtropical regions. This herb is used as a popular folk medicine to treat diarrhoeal disorders and wound healing in the Naga tribes of northeast India. The plant has been reported to possess anti-inflammatory and antiallergic (Horiuchi & Seyama, 2006), hepatoprotective (Chin et al., 1996), antihyperglycemic (Ubillas et al., 2000), antiulcerogenic (Tan et al., 2000), antimicrobial (Khan et al., 2001) and antihypertensive (Dimo et al., 2002) properties. Fig 1. Bidens pilosa; whole plant and leaves Galinsoga parviflora Cav. (Asteraceae), locally known as Japan-khavu, is a weed (Fig 2) found in cultivated and vacant places in the Himalaya at the altitude of ft. Its leaf and inflorescence decoction is used as a remedy for diarrhoeal disorders in the folklore medicine practice of Naga tribes. Barring a single report on its insecticidal activity (Macedo et al., 1997), no information is available in the literature pertaining to its biological activity and/or chemical constituents. This study reports the therapeutic efficacy of leaf extract of B. pilosa and leaf and inflorescence extract of G. parviflora in experimentally induced diarrhoea in albino mice.

3 Therapeutic Efficacy of Bidens pilosa L. var. radiata 37 Fig 2. alinsoga parviflora; whole plant and leaves and inflorescence Materials and Methods Preparation of plant extracts The leaves of B. pilosa and leaves and inflorescence of G. parviflora were collected from various places in Manipur and duly authenticated by a Plant Taxonomist. The plant materials were air-dried under shade and pulverized into powder. Known amount of the powdered materials were extracted with methanol in a Soxhlet apparatus (Yadav et al., 1992). The final recovery of extracts was made with the help of a rotatory evaporator. The percentage yields (w/w) of the final crude extracts were 2.54% (B. pilosa) and 3.30% (G. parviflora). These extracts were stored + 4 C for further experiments. Drugs and chemicals Loperamide (Lopax, Axar Pharmaceuticals, Baroda) was tested as a standard reference drug. Castor oil (S.D. Fine, Mumbai) and Prostaglandin E 2 (Sigma Aldrich Chemical Pvt. Ltd., U.S.A.) as diarrhoea-inducing agents, Activated Charcoal (E. Merck, India) as an intestinal transit marker, and Gum Acacia (S.D. Fine Chem, Bulsar) and Tragacanth Powder (Central Drug House Pvt. Ltd., Bombay) as suspension agents, were used in this study. Experimental animals Experiments were performed on 6 to 8 week-old albino mice (20-30 g). They were housed in polyacrylic cages and fed with standard rodent pellet diet and given water ad libitum. Proper care was taken to protect the welfare of animals. Acute toxicity tests The mice were divided into 13 groups of 6 animals each. Group I served as the control (0.5 ml of 2% gum acacia suspension, p.o); groups II-XIII received 100, 200, 400, 800, 1600 and 3200 mg/kg, p.o. dose of B. pilosa and G. parviflora

4 38 RPMP Vol. 23 Phytopharmacology & Therapeutic Values V extracts, respectively. The animals were observed for mortality and general signs of toxicity pertaining to food and water intake for 72 h. The median lethal dose (LD 50 ) was calculated from the number of dead animals observed within 72 h post-treatment with the extracts, using SPSS software (SPSS Inc. Chicago, IL, U. S. A.). The serum biochemical profile of animals was studied 24 h post-administration of 800 mg/kg dose of extracts (the dose that showed the maximum antidiarrhoeal activity). The levels of serum glutamicoxaloacetic transaminase (SGOT; EC ) serum glutamic-pyruvic transaminase (SGPT; EC ), cholesterol and total protein were estimated as per the methods of Allain et al. (1974); Henry et al. (1974) using a semiautomated biochemical analyzer (Bayer). Antidiarrhoeal experiments Castor- oil induced diarrhoea The methods of Robert et al. (1976) were followed. Overnight-fasted mice were divided into 10 groups of 6 animals each, and diarrhoea was induced by administering 0.5 ml of castor oil orally to mice. Group I served as the control (0.5 ml of 2% gum acacia suspension, p.o.); groups II-IX received 100, 200, 400, and 800 mg/kg, p.o. dose of B. pilosa and G. parviflora extracts, respectively (the extract dosages were selected to match with the doses actually used in practice by people); group X received the standard drug, loperamide (0.5 mg/kg, p.o.). After 1 h of treatment, all the animals were challenged with 0.5 ml/mouse, p.o, dose of castor oil and placed separately over clean filter papers inside cages. The filter papers were inspected for the presence of diarrhoeal droppings at hourly intervals for a period of 4 h. the total number of diarrhoeal episodes was counted group-wise. At hour 4, its absence was considered as a protection from diarrhoea. PGE 2 -induced enteropooling The method of Robert et al. (1976) was applied. Overnight fasted mice were divided into 11 groups of 6 animals each. Group I was given 2% gum acacia and kept as a control; group II served as a vehicle control and received 2% gum acacia + PGE 2 (0.5 ml of 100 µg/kg, i.p.); Groups III-X received 100, 200, 400, and 800 mg/kg p.o. of B. pilosa and G. parviflora extracts, respectively; group XI received loperamide. Immediately afterwards, diarrhoea was induced by 0.5 ml of 100 µg /kg, i.p. dose of PGE 2. After 30 min, the animals were sacrificed, small intestine was removed, and intestinal contents were collected and measured in a syringe. The percentage inhibition in intestinal fluid was determined by comparing the values with vehicle control. Gastrointestinal transit The animals were fasted for 16 h and allowed free access to water. They were divided randomly into groups of six mice each. Group I received 2% gum acacia to serve as control; groups II-IX were given p.o. the different

5 Therapeutic Efficacy of Bidens pilosa L. var. radiata 39 doses of both plant extracts; group X received loperamide (5 mg/kg, p.o.). The animals were given 0.5 ml of marker (3% charcoal suspension in 2% gum acacia) 5 min after treatment. The animals were sacrificed 30 min later and the abdomen was opened. The distance traveled by charcoal meal from the pylorus was measured and expressed as percentage of the total length of the intestine from pylorus to caecum (Akah & Offiah, 1992). Statistical analysis The data were analyzed statistically and are represented as mean ± standard error of mean (SEM). The significance of the difference between the means was determined by the Student's t-test, and p < 0.05 was accepted as significant. Results Castor oil-induced diarrhoea The extracts significantly reduced the number of diarrhoeal episodes in a dose-dependent manner when compared with the untreated control. At 800 mg/kg dose, B. pilosa showed 94.23% and G. parviflora 80.74% reduction in the number of diarrhoeal episodes, whereas loperamide (5 mg/kg) offered 96.08% protection. In terms of protection from diarrhoea at 4 h, the 800 mg/ kg dose of B. pilosa extract protected 3 out of 6 animals and, G. parviflora protected 2 out of 6 animals. Loperamide (5 mg/kg) protected 5 out of 6 mice from diarrhoea (Table 1). Table 1. Effect of the methanol extracts of Bidens pilosa leaves and Galinsoga parviflora leaves and inflorescence on castor-oil induced diarrhoea in mice Treatment No. of wet % No. of animals % (mg/kg, p.o.) faeces at 0-4h Reduction devoid of wet Protection faeces at 4 h (n=6) Control (2% gum acacia) 8.67 ± / B. pilosa extract ± 0.42 b / ± 0.49 c / ± 0.54 c / ± 0.34 c / G. parviflora extract ± 0.56 a / ± 0.56 c / ± 0.48 c / ± 0.67 c / Loperamide ± 0.17 c / * Data represent mean ± SEM, (n = 6). a, b, c p < 0.02, p < 0.01 and p < respectively, versus control, Student's t-test

6 40 RPMP Vol. 23 Phytopharmacology & Therapeutic Values V PGE 2 -induced enteropooling The plant extracts reduced the intestinal fluid accumulation induced by PGE 2 in a dose-dependant manner (Table 2). At 800 mg/kg dose, B. pilosa showed a greater reduction (26.15%) than G. parviflora extract (16.25%) compared with vehicle control. The reduction the intestinal fluid accumulation by loperamide at its 5 mg/kg dose was 28.62%. Table 2. Effect of the methanol extracts of Bidens pilosa leaves and Galinsoga parviflora leaves and inflorescence on PGE 2 -induced enteropooling in mice PGE2 induced enteropooling Volume (ml) of the small intestine Treatment fluid per 100 g mouse* % (mg/kg, p.o.) Reduction Normal control 1.37 ± (2% gum acacia) Vehicle control 2.83 ± (2% gum acacia + PGE2) B. pilosa extract ± ± 0.01 a ± 0.12 a ± 0.11 b G. parviflora extract Loperamide ± ± ± ± ± 0.10 c * Data represent mean ± SEM, (n = 6). a, b, c p< 0.02, p< 0.01, and p< respectively, versus vehicle control, Student's t-test Acute toxicity The oral administration of extracts of both plants up to a dose of 3200 mg/kg neither showed any mortality nor any adverse signs with regard to food and water uptake in the animals up to 72 h post-treatment of plant extracts. The LD 50 of extracts was recorded as mg/kg, p.o. for B. pilosa and mg/kg, p.o. for G. parviflora. Small intestinal transit The distance traveled by the marker in the extract-treated groups showed significant difference compared with control (p < 0.05 to p < 0.001). The intestinal transit of charcoal meal was 71.42% in the control group, but at 800 mg/kg dose was 32.67% in B. pilosa and 48.53% in G. parviflora. The percent transit of marker was 30.41% in the case of 5 mg/kg dose of loperamide (Table 3).

7 Therapeutic Efficacy of Bidens pilosa L. var. radiata 41 Table 3. Effect of the methanol extracts of Bidens pilosa leaves and Galinsoga parviflora leaves and inflorescence on gastrointestinal transit in mice Distance traveled by charcoal Treatment Marker as % of total length of % (mg/kg, p.o.) small intestine* inhibition Control ± (2% gum acacia) B. pilosa extract ± 1.47 a ± 1.55 c ± 5.88 b ± 1.42 d G. parviflora extract ± ± 1.63 b ± 6.49 b ± 4.59 c Loperamide ± 2.97 d * Data represent mean ± SEM, (n = 6). a, b, c, d p< 0.05, p< 0.02, p< 0.01, and p< respectively, versus control, Student's t-test Table 4. Effect of methanol extracts of B. pilosa leaves and G. parviflora leaves and inflorescence on SGOT, SGPT, cholesterol, and total protein levels of experimental mice a Treatment group SGOT SGPT Cholesterol Total protein (mg/kg, p.o.) (U/L) (U/L) (mg/dl) (g/dl) Control ± ± ± ± 0.54 (2% gum acacia) B. pilosa ± ± ± ± 0.18 G. parviflora ± ± ± ± 0.22 a Data represent mean ± SEM, (n = 6). The serum collected from the normal control mice yielded U/L of SGOT, U/L of SGPT, mg/dl of cholesterol and 6.73 g/dl of total protein (Table 4). There was no significant difference in the levels of these parameters in the plant extract treated groups of animals, except a slightly higher rise observed in the level of SGPT for B. pilosa extract (800 mg/kg dose) in comparison with the control.

8 42 RPMP Vol. 23 Phytopharmacology & Therapeutic Values V Discussion This study was aimed at evaluating the therapeutic efficacy of two ethnomedicinal plants, namely Bidens pilosa and Galinsoga parviflora which are used as a popular folk medicine in the treatment of diarrhoeal diseases by Naga tribes in northeast India. One of the main features of small intestine is to absorb and secrete materials. Diarrhoea results from an imbalance between the absorptive and secretive mechanisms in the intestinal tract, accompanied by intestinal hurry, which results in an excess loss of fluid through the faeces (Yegnanarayan & Shroti, 1982). Clinically, in some cases of diarrhoea, the secretory component predominates, whereas other diarrhoeas are characterized by hypermotility. In severe the diarrhoea may culminate in to mortality of incumbent due to loss of fluids and electrolytes from body. Several studies have validated the use of antidiarrhoeal medicinal plants by investigating the biological activity of extracts of such plants, which have antispasmodic effects, delay intestinal transit, suppress gut motility, stimulate water adsorbtion, or reduce the intraluminal fluid accumulation and/or electrolyte secretion (Tangpu & Yadav, 2004; Akah et al., 1999; Otshudi et al., 2001; Yadav & Tangpu, 2006). Some of these experimental models were therefore employed to evaluate the antidiarrhoeal potentials of B. pilosa and G. parviflora extracts in the present study. It emerged from the present study that extracts of both plants had demonstrable antidiarrhoeal efficacy in one or other experimental models, though their effects were more pronounced at their higher doses (800 mg/ kg). Castor oil is metabolized into ricinoleic acid by the action of lipase, which in turn irritates and causes inflammation in the intestinal mucosa, resulting in release of prostaglandins. The prostaglandins thus released stimulate the motility and secretion in the small intestines (Palombo, 2006). The therapeutic effect of Loperamide is believed to be due to its antimotility and antisecretory properties (Pierce et al., 1971). In the castor-oil induced diarrhoea studies the results showed that B. pilosa leaf and inflorescence extract showed better protection from diarrhoea in the animals as compared with G. parviflora leaf extract and so was the case in PGE 2 -induced enteropooling. There could be two mechanisms behind the aforementioned action of plant extracts. The extracts may bring out the above action either through their proabsorbtive property that promotes faster fluid absorbtion in the intestine or they work through an antisecretory mechanism. With respect to B. pilosa, both the hypotheses gains from the fact that in one study by Alvarez et al. (1999) it was found that the ethanol extract of B. pilosa possesses gastric antisecretory property, whereas in another study it was noticed that ethyl caffeate, a natural phenolic compound isolated from B. pilosa, suppresses the potent proinflammatory mediators, namely NO and prostaglandinds E 2 (Chiang et al., 2005). Nitric oxide plays an important role in intestinal fluid and electrolyte secretion in the intestine (Izzo et al., 1998). Inhibition of nitric oxide synthesis seems to block the laxative action

9 Therapeutic Efficacy of Bidens pilosa L. var. radiata 43 of diarrhoeal agents (Izzo et al., 1994). It therefore emerges that any such factors may contribute to the observed therapeutic action of B. pilosa extract. It may be mentioned here that B. pilosa has been found to possess several flavonoids (Wang et al., 1997), and these flavonoids have been reported to show anti-inflammatory activity in laboratory animal studies (Geissberger & Sequin, 1991). It is widely understood that of the numerous phytochemicals (such as alkaloids, tannins, flavonoids and terpenes) present in active extracts, tannins and flavonoids are thought to be responsible for antidiarrhoeal activity by increasing colonic water and electrolyte reabsorption (Palombo, 2006). It is also worth mentioning here that in a screening of Zulu medicinal plants for prostaglandin synthesis inhibitors, B. pilosa was found to be possessing one of the highest activities for inhibiting cyclooxygenase (a key enzyme of the prostaglandin synthesis pathway that leads to proinflammatory prostaglandins); such inhibition was observed to be associated with its flavonoid components (Jager et al., 1996). The reduction of gastrointestinal motility is one of the mechanisms by which many antidiarrhoeal agents can act (Pierce et al., 1971). It was observed that the both the extracts suppress the propulsion of charcoal marker in a dose-dependent manner. In the present study the percentage inhibition of charcoal marker by 800 mg/kg dose of B. pilosa extract was observed to be almost comparable to Loperamide. This finding suggests that B. pilosa extract has the ability to influence the peristaltic movement of intestine indicating thereby the presence of an intestinal antimotility activity in it. The mode of action of loperamide is believed due to its direct effect on the circular and longitudinal muscles of the intestinal wall. A decrease in the motility of gut muscles increases the amount of time substances stay in the intestine (Li et al., 2005). This allows for more water to be absorbed out of the water. We therefore presume that the reduction in the intestinal propulsive movement in charcoal meal model may be due presence of similar antispasmodic properties of plant extracts. These results which showed ability of plant extracts to suppress the production and accumulation of wet faeces and an inhibitory effect on gastrointestinal motility met the standard criterion to prove their efficacy as antidiarrhoeal agents. In several instances it has been found that extracts and/or active ingredients of folk medicinal plants are consumed without considering their potential toxicity to the users. In the current study, the B. pilosa and G. parviflora extracts up to a dose of 3200 mg/kg did not cause any mortality or adverse signs with respect to food and water intake in the animals. Further, the mice treated with an 800 mg/kg dose of plant extracts did not show any significant change in SGOT, SGPT, cholesterol, and total proteins levels compared with controls. These findings suggest that these plant extracts may be considered as non-toxic in nature. The nontoxic effect of B. pilosa leaf extract, in particular, further gains support from the findings of a related study by Chin et al. (1996), where the leaf extract of B. pilosa was reported to significantly reduce the increase in liver enzymes SGOT and SGPT caused

10 44 RPMP Vol. 23 Phytopharmacology & Therapeutic Values V due to acute hepatic lesions induced by carbon tetrachloride and acetaminophen in rats. In conclusion, this study provides support that B. pilosa and G. parviflora possess antidiarrhoeal properties, which may have therapeutic benefits in humans suffering from diarrhoeal disorders. Acknowledgements Fellowship by CSIR, New Delhi to V. Tangpu and partial financial assistance under the DRS-IV Programme of UGC in the Department of Zoology, NEHU are acknowledged. References Akah, P.A., C.N. Aguwa and R.U. Agu Studies on the antidiarrhoeal properties of Pentaclethra macrophylla leaf extracts, Phytotherapy Research 13: Akah, P.A. and V.N. Offiah Gastrointestinal effects of Allamanda cathartica leaf extracts, International J. Pharmacognosy 30: Allain, C.C., L.S. Poon, C.S. Chan, W. Richmond and P.C. Fu Enzymatic determination of total cholesterol, Clinical Chemistry 20: Alvarez, A., F. Pomar, M.A. Sevilla and M.J. Montero Gastric antisecretory and antiulcer activities of an ethanolic extract of Bidens pilosa L. var. radiata Schult, Bip. J. Ethnopharmacology 67: Anonymous Diarrhoeal disease control programme, Weekly Epidemiological Record 16: 121. Chiang, Y.M., C.P. Leo, Y.P. Chen, S.Y. Wang, N.S. Yang, Y.H. Kuo and L.F. Shyur Ethyl caffeate suppresses NF-kB activation and its downstream inflammatory mediators, inos, COX-2 and PGE 2 in vitro or in mouse skin, British J. Pharmacology 146: Chin, H.W., C.C. Lin and K.S. Tang The hepatoprotective effects of Taiwan folk medicine ham-hong-chho in rats, American J. Chinese Medicines 24: Dimo, T., S.V. Rakotonirina, P.V. Tan, J. Azay, E. Dongo and G. Cros Leaf methanol extract of Bidens pilosa prevents and attenuates the hypertension induced by highfructose diet in Wistar rats, J. Ethnopharmacology 83: Fauci, A.S., E. Bravnwold, K. Isselpacher, J.D. Wilson, J.B. Martin, D.L. Kasper, S.L. Hauser and D.L. Longo Harrison's Principles of Internal Medicine. McGraw Hill Company, New York, U. S. A., pp Geissberger, P. and U. Sequin Constituents of Bidens pilosa L.: Do the components found so far explain the use of this plant in traditional medicine? Acta Tropica 48: Githiori, J.B., J. Hoglund and P.J. Waller Ethnoveterinary plant preparations as livestock dewormers: Practices, popular beliefs, pitfalls and prospects for the future, Animal Health Research Review 6: Henry, R.J., D.C. Cannon and J.W. Winkelman Clinical Chemistry: Principles and Techniques. 2nd Edition, Harper & Row Publisher, New York, U. S. A., p Horiuchi, M. and L. Seyama Antiinflammatory and antiallergic activity of Bidens pilosa L. var. radiata Scherff, J. Health Science 52: Izzo, A.A., T.S. Gaginella, N. Mascolo and F. Capasso Nitric oxide as mediator of the laxative action of magnesium sulphate, British J. Pharmacology 113:

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