Indian Journal of Natural Products and Resources Vol. 3(4), December 2012, pp. 547-550

Screening of intestinal transit time of Euphorbia fusiformis Buch.- Ham. ex D. Don in Swiss albino mice

B K Ashok1*, Savitha D Bhat2 and B Ravishankar3 1Pharmacology Laboratory, Institute for Post Graduate Teaching & Research in Ayurveda, Gujarat Ayurved University, Jamnagar-361 008, Gujarat, 2Department of Dravyaguna, Muniyal Institute of Ayurveda Medical Sciences, , Karnatka, India 3Director, SDM Research Centre for Ayurveda and Allied Sciences, Kuthpady, -574 118,

Received 29 August 2011; Accepted 28 August 2012

Effect of Euphorbia fusiformis Buch.-Ham. ex. D. Don root powder on intestinal transit time was carried out after standardizing the kaolin expulsion test by administering 40% kaolin to overnight fasted Swiss albino mice. Evaluation on intestinal transit time was carried out by using this standard procedure by administering the drug at 130 mg/kg and 260 mg/kg doses. E. fusiformis root powder significantly shortened the duration required for the expulsion of kaolin in dose dependant manner. The study shows that the root powder of E. fusiformis possesses significant intestinal motility enhancing effect, the exact mechanism of which needs to be elucidated.

Keywords: Albino mice, Euphorbia fusiformis, Intestinal transit, Kaolin, Purgative, Virechana drug, Vishanika. IPC code; Int. cl. (2011.01)  A61K 36/47, A61K 125/00, A61P 1/10 Introduction Buch.-Ham. ex D. Don (Euphorbiaceae) Plant based medicaments had served, from the Dolichandrone falcata Seem (Bignoniaceae) and earliest period of human civilization, as the most Ervatamia heyneana T. Cooke (Apocynaceae). important therapeutic weapon available to man to fight Ashok et al (2005) critically analysed macroscopical, various human and animal diseases. Herbal drug microscopical, phytochemical and pharmacological therapy is the most trusted system of medicine in aspects and suggested6 that E. fusiformis is the countries like India where people strongly believe in possible source of classical Vishanika. Since it is the Ayurveda as herbs are the part of rural Indian life style. important potent classically described Virechana Euphorbia fusiformis Buch.-Ham. ex D. Don drug, it is expected to have purgative action which is (Family: Euphorbiaceae) is a rare medicinal plant suppose to decrease the intestinal transit time. Hence, found in Tropical Himalaya up to 450 m from the present study was undertaken to find out its action Garhwal to Nepal and Wet Bengal. This plant is also on intestinal transit time in experimental animals. rarely found in Konkan region and Deccan Hills1-4. In Gujarat state it is found in Dang, Rajpippala and Materials and Methods Chotaudaipur regions5. The Bhagats (Folk physicians) Test drug of Dangs region use this drug in the name of Ghate The tuberous roots of E. fusiformis (Plate 1) were for treatment of various abdominal disorders collected from Waghai forest, Gujarat in fully especially for tumors of abdomen. matured condition and the material was authenticated Vishanika is one of the important classical by qualified taxonomist of our institute. A voucher Mulini Virechana (roots with purgative action) specimen has been preserved in the herbarium drug of Ayurveda employed for purgation therapy. attached to the institute. The tuberous roots were There are many plants which are used in the name made into slices and shade dried for 12 days and then of Vishanika, viz. Vallaris solanacea O. Ktze. pulverized to fine powder (mesh no 80) and stored in (Apocynaceae), Cryptolepis buchnani Roem. & airtight container for experimental purposes. Schult. (Asclepidaeceae), Euphorbia fusiformis Animals —————— *Correspondent author: Swiss albino mice of either sex of 8 weeks old E-mail: [email protected]; Phone: 09429333816 (Mob.) weighing between 26 ± 4 g were procured from the 548 INDIAN J NAT PROD RESOUR, DECEMBER 2012

Plate 1  Euphorbia fusiformis: a-Plant, b-Tuberous root animal house attached to pharmacology lab, IPGT & been instilled into the stomach or intestinal RA, Gujarat Ayurved University, Jamnagar, Gujarat. lumen of conscious animal to travel along the They were housed in large spacious polypropylene length of small intestine8. However many limitations cages and fed with Amrut brand rat pellet feed of these methods like difficulty in screening, supplied by Pranav Agro Industries and tap water expensive and in almost all these methods animals given ad libitum. The animals were acclimatized for are need to be killed. By considering these at least one week in lab condition before aspects, a simple non-invasive method called commencement of the experiment in standard kaolin expulsion test was standardized prior to the laboratory conditions 12 ± 01 hour day and night proper experiment. rhythm, maintained at 25 ± 3°C and 40 to 60% Six Swiss albino mice of either sex were kept on humidity. Institutional Animal Ethics Committee had fasting for 12 h before the commencement of approved the experimental protocol (Approval experiment, but drinking water was provided. number; IAEC 04-05/01/MSc.01) and the care of Next day, 40% kaolin solution was administered to animals was taken as per the CPCSEA guidelines. the fasted animals with the help of oral catheter. The animals were placed in a transparent arena and Dose selection and schedule were carefully observed for the beginning of The dose selection was done on the basis of body the kaolin expulsion which begins in the form surface area ratio by referring to the standard table of 7 of white coloured faecal pellets (Plate 2). The Paget and Barnes . On this basis the mouse dose was time required for the appearance of white coloured found to be 130 mg/kg. The test drug was suspended pellets was recorded and it was found to be in distilled water with suitable concentration 354.66 ± 6.88 minutes (n=6). This standardized depending upon body weight of animals and method was used for the screening of test drug administered orally to overnight fasted animals with on intestinal transit time. the help of oral catheter. The study was carried out at The selected animals were divided in to three two dose levels, viz. TED (Therapeutically Equivalent groups. First group contained 8 animals and served as Dose (130 mg/kg) and TED × 02 (260 mg/kg). control. The second and third groups contained

Experimental design 6 animals each and were administered with test drugs Standardization of Kaolin expulsion model to screen intestinal at the dose of 130 and 260 mg/kg, respectively. transit time One hour later 40% kaolin solution was Intestinal transit is usually quantified by measuring administered with the help of oral catheter and the movement of charcoal, dyes like phenol red meal, was observed for the beginning of the kaolin radio-opaque pellets or radioactive markers that have expulsion as described above. ASHOK et al: SCREENING OF INTESTINAL TRANSIT TIME OF EUPHORBIA FUSIFORMIS 549

newer drugs in treatment of clinically known conditions of motility disorders such as acute intestinal ileus, chronic intestinal pseudoobstruction and generalized disorders of motility. In this experiment to assess the action of test drug on the intestinal motility, latency of onset of kaolin expulsion in faecal matter was selected as a parameter. As it was difficult to assess in vivo movement of the drug, it was thought useful to administer a marker, which causes colour change of faecal matter and doesn’t alter the effect of drug. Kaolin is a native aluminium silicate and has traditionally been used internally to control diarrhoea. It is reported that kaolin is insoluble and is not absorbed into the bloodstream. Instead it acts locally in the intestines, where it absorbs toxins and relieves 9 mild diarrhoea . Therefore, it is not generally Plate 2  Rat faecal pellets: N-Normal coloured pellets, W- associated with toxicity. The kaolin used as a marker Whiite coloured pellets in this study in much lower dose (40% kaolin, 0.1 ml administered for 20 g mouse) than that of therapeutic  Table 1 Effect of test drug on intestinal transit time human dose (26.2 g for every 6 hours) to avoid any Kaolin pellet expulsion Group % Change significant action on intestine. Further, kaolin was time (Minutes) administered one hour after drug administration to Control (n=8) 362.60 ± 10.30 - avoid possible interaction with test drug. While Low dose (n=6) 250.00 ± 12.67* 32.0 ↓ observing for the expulsion of pellets by keeping the High dose (n=6) 220.00 ± 16.14* 40.0 ↓ animals in transparent arena, they were carefully Data : Mean ± SEM; ↓- Decrease *P < 0.001 (Compared with observed for apparent toxic symptoms if any due to control group) kaolin as well as symptoms of Virechana like uneasiness, etc. which may likely to be produced by Statistical analysis test drug. Conversely, all the animals were healthy Student’s t test for unpaired data has been used with normal activities throughout the experiment. for analyzing the data generated during the study. P value less than 0.05 was considered as statistically It is well known fact that, it is not an easy task to significant. prove the Virechana action of a drug in experimental animal model because of its broader meaning. It may Results and Discussion be the reason why attempts were not made by Data pertaining to the effect of test drug on intestinal researchers to provide experimental basis for motility (Table 1) shows that administration of test Virechana effect of drug. As explained by drug at graded doses decreased intestinal transit time in Dalhanacharya, tikshna guna of Virechana Dravya is dose dependant manner. The observed decrease in responsible to break the mala and helps in its quick intestinal transit time is found to be statistically excretion. In quick excretion, there is all the chance of significant in comparison to control group. increase in intestinal motility, based on these criteria Measurement of intestinal transit is a widely used the present study was designed to evaluate the effect technique for assessing the actions and mechanisms of test drug on intestinal motility. by which compounds influence intestinal motility. Administration of E. fusiformis root powder The evaluation of intestinal motility is helpful in, significantly shortened the duration required for the determining the therapeutic potential of new drugs in expulsion of kaolin in dose dependant manner. motility disorders, determining alteration in motility The mechanism of observed effect may be due to secondary to physiological or pharmacological stimuli interference with local stimulant effect on motility or and evaluating the effect of pathological condition on acceleration of gastric emptying. The neural intestinal transit. It is also helpful in development of regulation of gastric motility involves stimulation by 550 INDIAN J NAT PROD RESOUR, DECEMBER 2012

cholinergic neurons inhibition by adrenergic neurons. 2 Cooke Theodore, Flora of the Presidency of Bombay, Antagonist of D and 5-HT receptors as well as Reprint Edn, Botanical Survey of India, Calcutta, 1997, Vol. 2 3 2, p. 562. agonists of 5-HT4 receptors can stimulate gastric 10-12 3 Raju K, Natarajan S, Karuppusamy S and Manian S, New motility . Some of the drugs increase the motility of ethnobotanical report on Euphorbia fusiformis from Chitteri intestine by modifying the fluid dynamics of the Hills of Tamil Nadu, India, J Swamy Bot Club, 2004, 21, 79-80. mucosal wall and may cause fluid accumulation in 4 Britto S J, Soosai Raj S, Natarajan D and Ravi Paul S, lumen. In the light of the above it can be suggested that Euphorbia fusiformis Buch.-Ham. a new record for Tamil the test drug may enhance the intestinal motility by Nadu, J Econ Taxon Bot, 2002, 26(2), 469-471. 5 Bedi S J, Ethnobotany of the Ratan Mahal Hills Gujarat, cholinergic stimulation or stimulation of 5-HT4 Econ Bot, 1978, 32, 278-284. receptors; it is also possible that it may be antagonizing 6 Ashok B K, Pharmacognostic and phyto-pharmacological the effect of sympathetic system. Another probable investigations on Euphorbia fusiformis Buch.-Ham. mechanism is stimulation of the enteric nervous (Euphorbiaceae) – A putative source of classical Vishanika, system. It may not be affecting the fluid dynamics M.Sc. dissertation, Gujarat Ayurved University, Jamnagar, Gujarat, India, 2005. because the test drug did not change the consistency of 7 Paget G E and Barnes J M, Evaluation of drug activities, the expelled fecal matter to significant extent. In: Pharmacometrics, by D R Lawrence and A L Bacharach It has been reported that the tuber of E. fusiformis (Eds), Academic Press, New York, 1964, Vol. 1, p. 161. contains phytoconstituents like diterpene lactone 8 Peddireddy M K R, In vivo methods for evaluation of drugs caudicifolin, methylellagic acid and euphol13,14. It is for the treatment of gastrointestinal motility disorders, Indian J Pharm Educ Res 44 possible that one or the combination of these , 2010, (1), 42-48. 9 Berardi R R, Kroon L A and McDermott J H, Handbook of phytoconsituents may be responsible for the observed Nonprescription Drugs, 15th Edn, Washington, DC: American effect. However, further detailed study is needed to Pharmacists Association, 2006, pp. 340, 357, 358, 769. identify the component responsible for this effect and 10 Kiso T, Ito H, Miyata K, Kamato T, Naitoh Y, Iwaoka K and to elucidate the probable mechanism(s) involved. Yamaguchi T, A novel 5-HT3 receptor agonist, YM- 31636, increases gastrointestinal motility without increasing Conclusion abdominal pain, Eur J Pharmacol, 2001, 431, 35-41. 11 Nagakura Y, Naitoh Y, Kamato T, Yamano M and Miyata K, The root powder of E. fusiformis possesses Compounds possessing 5-HT3 receptor antagonistic activity significant intestinal motility enhancing effect, inhibit intestinal propulsion in mice, Eur J Pharmacol, indicating towards some of the working mechanisms 1996, 311, 67-72. of a Virechana drug as described in Ayurveda. Based 12 Sternini C, Patierno S, Selmer I S and Kirchgessner A, on the results obtained detailed study can be designed The opioid system in the gastrointestinal tract, Neurogastroenterol Motil, 2004, 16, 3-16. for future experiments. 13 Khanna N M, Chemical examination of E. acaulis Roxb., Indian J Pharm, 1954, 16, 110-111. References 14 Rastogi Ram and Mehrotra B N, Compendium of Indian 1 Gamble J S, Flora of Presidency of Madras, Published under Medicinal Plants, Central Drug Research Institute Lucknow authority of the secretary of State of London, 1921, Vol. 2, and National Institute of Science Communication, p. 1272. New Delhi, 1999, Vol. 4, p. 308; Vol. 5, p. 351.