|Year : 2011 | Volume
| Issue : 6 | Page : 689-693
Antidiarrheal potential of standardized extract of Rhododendron arboreum Smith flowers in experimental animals
Neeraj Verma1, Anil P Singh2, Amresh Gupta1, PK Sahu3, Ch V Rao2
1 Department of Pharmacology, Goel Institute of Pharmacy & Sciences, Lucknow, India
2 Department of Pharmacology, Pharmacognosy and Ethnopharmacology Division, National Botanical Research Institute (Council of Scientific and Industrial Research), Rana Pratap Marg, Lucknow, Uttar Pradesh, India
3 Department of Pharmacology, School of Pharmaceutical Sciences, Siksha 'O' Anusandhan University, Kalinga Nagar, Ghatikia, Bhubaneswar, Orissa, India
|Date of Submission||05-Apr-2011|
|Date of Decision||15-Aug-2011|
|Date of Acceptance||02-Sep-2011|
|Date of Web Publication||14-Nov-2011|
Department of Pharmacology, Goel Institute of Pharmacy & Sciences, Lucknow
Source of Support: Indian Council of Medical Research (ICMR)
and Department of Science and Technology
(DST), New Delhi, Conflict of Interest: None
Objective : To investigate standardized ethyl acetate fraction of Rhododendron arboreum (EFRA) flowers for antidiarrheal activity in experimental animals.
Materials and Methods : A simple sensitive high performance thin layer chromatography (HPTLC) method was used for the determination of hyperin in EFRA. The standardized fraction was investigated for castor oil, magnesium sulfate-induced diarrhea, measurement of gastrointestinal transit using charcoal and castor oil-induced enteropooling.
Results : The concentration of hyperin in flowers of R. arboreum was found to be 0.148% by HPTLC. Oral administration of EFRA at 100, 200 and 400 mg/kg exhibited dose-dependent and significant (P<0.05-0.001) antidiarrheal potential in castor oil and magnesium sulfate-induced diarrhea. EFRA at doses of 100, 200 and 400 mg/kg also produced significant (P<0.05-0.001) dose-dependent reduction in propulsive movement in castor oil-induced gastrointestinal transit using charcoal meal in rats. EFRA was found to possess an antienteropooling in castor oil-induced experimental animals by reducing both weight and volume of intestinal content significantly.
Conclusion : These findings demonstrate that standardized ethyl acetate fraction of R. arboreum flowers has potent antidiarrheal activity thus justifying its traditional use in diarrhea and have great potential as a source for natural health products.
Keywords: Antidiarrheal, enteropooling, gastrointestinal motility, high performance thin layer chromatography, Rhododendron arboreum
|How to cite this article:|
Verma N, Singh AP, Gupta A, Sahu P K, Rao CV. Antidiarrheal potential of standardized extract of Rhododendron arboreum Smith flowers in experimental animals. Indian J Pharmacol 2011;43:689-93
|How to cite this URL:|
Verma N, Singh AP, Gupta A, Sahu P K, Rao CV. Antidiarrheal potential of standardized extract of Rhododendron arboreum Smith flowers in experimental animals. Indian J Pharmacol [serial online] 2011 [cited 2021 Oct 27];43:689-93. Available from: https://www.ijp-online.com/text.asp?2011/43/6/689/89827
| » Introduction|| |
Diarrheal diseases are one of the leading causes of childhood morbidity and mortality in developing countries. An estimated 1000 million episodes occur each year in children under 5 years of age. Diarrhea causes an estimated 5 million deaths in children less than 4 years of age per year.  Incidence of diarrheal diseases still remains high despite intervention of government agencies and international organization to halt the trend. Many synthetic drugs like diphenoxylate, loperamide and antibiotics are available for the treatment of diarrhea but they have some side effects.  The World Health Organization (WHO) encourages studies for the treatment and prevention of diarrheal diseases based on traditional medical practices.  A number of medicinal plants have been used traditionally in the management of diarrheal diseases, and one of such medicinal plant is Rhododendron arboreum Sm. (Ericaceae), a small evergreen tree and distributed in temperate Himalaya from Kashmir to Bhutan. Flowers are 2.5-5-cm long deep red or pale pink crowded in large rounded corymbs.  The flowers of the plant are used traditionally in far-west Nepal as remedy for diarrhea and dysentery but have not been investigated for its antidiarrheal activity.  In continuation of our studies the ethyl acetate fraction of R. arboreum showed significant anti-inflammatory and antinociceptive activity and previously isolated one compound from an ethyl acetate fraction was hyperin.  Therefore, the present study was undertaken to evaluate the antidiarrheal activity of ethyl acetate fraction of R. arboreum in experimentally induced diarrhea in animal models and standardization of isolated compound (hyperin) using high performance thin layer chromatography (HPTLC).
| » Materials and Methods|| |
The flowers of R. arboreum were collected in the month of March, 2009, from Rudraprayag, Uttaranchal and authenticated by central facility at National Botanical Research Institute, Lucknow. A voucher specimen (NBRI/CIF/83/2009) was deposited in institute for future reference.
All chemicals were of analytical grade. Castor oil, magnesium sulfate, charcoal meal (SD Fine chemicals, Mumbai); loperamide, the standard drug (Micro labs, Bangalore), atropine sulphate (Sigma Aldrich), acacia powder (gum acacia) was purchased from Qualigens fine chemicals.
Extraction and Fractionation
The air-dried powdered flowers (200 g) were extracted with ethanol (50%) using Soxhlet apparatus, which was then evaporated in a Rotavapour and afford the ethanol extract of flowers. The extract thus obtained was partitioned with organic solvents afford the n-hexane, chloroform, ethyl acetate and n-butanol fractions. The HPTLC standardized extract was tested and showed presence of alkaloids, carbohydrates, saponins, flavanoids, anthocyanins and tannins. 
Reagents used were from Merck (Germany) and standard hyperin was procured from Sigma-Aldrich (Germany). The ethyl acetate fraction was concentrated under vacuum, redissolved in methanol, filtered and finally made up to 100 ml with methanol.
Chromatography was performed in commercially available Merck HPTLC precoated silica gel 60 GF 254 (5 x 10 cm) plates. Methanol solution of sample 9 μl (Ms) into track 1 and standard concentration of hyperin 3μl (S1 a ) and 6μl (S1 b ) into track 2 and track 3 on the plate were applied to 6-mm-wide bands positioned from 8 mm from the bottom using Camag Linomat V, which is microprocessor-controlled and programmable with nitrogen flow providing a delivery speed of 150 nl/s from an application syringe. All three tracks were scanned at 366 nm on HPTLC instrument. For quantitative estimation of hyperin in R. arboreum flowers, a comparison spectra was determined at 360 nm.
Wistar albino rats (150-200 g) and Swiss albino mice (15-30 g) of either sex were kept in the departmental animal house of National Botanical Research Institute, Lucknow at 25±2°C and relative humidity 50 ± 5%, light and dark cycles of 10 and 14 hours, respectively, for 1 week before and during the experiments. All experiments were performed according to guidelines of Institutional Animal Ethics Committee, CPCSEA, India (Reg. No.222/2000/CPCSEA).
Castor Oil-Induced Diarrhea
A total of 30 albino rats were divided into five groups of six animals in each. All rats were fasted for 18 hours and received castor oil at a dose of 1 ml/animal orally (p.o.) using orogastric cannula for induction of diarrhea.  Thirty minutes after castor oil administration, rats of group I (control) received 1.0 ml/100 g of 0.9% NaCl in distilled water (normal saline) and rats of groups II, III and IV received 100, 200 and 400 mg/kg EFRA p.o. and group V received standard drug, loperamide (3 mg/kg p.o.), respectively. The animals were placed separately in metabolic cages over white clean Whatman filter paper, which was changed every hour. The severity of diarrhea was assessed each hour for 4 hours. The total number of diarrhea feces of the control group was considered 100%.
% inhibition= (Control- Test) ×100/Control
Magnesium Sulfate-Induced Diarrhea
Swiss albino mice (15-30 g) were divided into five groups of six animals each. Diarrhea was induced by oral administration of magnesium sulfate at a dose of 2 g/kg in all groups.  Thirty minutes after magnesium sulfate administration, mice of group I (control) received 1.0 ml/100 g of 0.9% NaCl in distilled water (normal saline) and mice of groups II, III and IV received 100, 200 and 400 mg/kg EFRA p.o. and group V received the standard drug, loperamide (3 mg/kg orally), respectively. After this administration, the animals were placed separately in metabolic cages with filter paper, which was changed every hour. The severity of diarrhea was assessed each hour for 4 hours. The total number of diarrhea feces of the control group was considered 100%. The results were expressed as a percentage of inhibition of diarrhea compared with control.
Measurement of Gastrointestinal Transit Time Using Charcoal
The adult rats selected without sex discrimination were fasted for 18 hours and divided into five groups of six animals each. Castor oil (1 ml) was administered orally to the animals. One hour later, Group I (control) was administered 1.0 ml/100 g of 0.9% NaCl in distilled water (normal saline) and rats of groups II, III and IV received 100, 200 and 400 mg/kg EFRA p.o. and group V received standard drug, atropine sulfate at a dose of 5 mg/kg through oral route, respectively. After 30 min of the administration, 1 ml of charcoal meal (10% suspension in 5% gum acacia) as a marker diet was given orally to rats in each group. The rats were sacrificed by ether (20% v/v) anesthesia and small intestine was carefully separated from mesentery avoiding being stretched. For each animal, gastrointestinal transit was calculated as percentage distance travelled by charcoal meal to the total length of intestine. The inhibitory effect of EFRA on gastrointestinal transit was calculated relative to respective group. 
Castor oil-induced enteropooling
Castor oil-induced enteropooling was determined by the method of Robert et al.  The adult rats selected without sex discrimination were fasted for 18 hours and divided into five groups of six animals each. Castor oil (1 ml) was administered orally to these animals. One hour later, Group I received 1 ml/100 g of normal saline solution and rats of groups II, III and IV received 100, 200, and 400 mg/kg EFRA p.o. and group V received standard drug, loperamide (3 mg/kg orally) respectively. After 2 hours of treatment, the rats were sacrificed by ether anesthesia. The edges of the intestine from pylorus to ceacum were tied with thread and the intestine was removed and weighed. Intestinal content was collected by milking into a graduated tube and the volume was measured. The intestine was reweighed and differences between full and empty intestines were calculated.
All the data were presented as mean±S.E.M. and analysed by using one-way ANOVA followed by Student's t-test for the possible significant identification between the various groups. A value of P<0.05 was considered statistically significant.
| » Results|| |
Detection and Quantification of Hyperin
Following sample application, plates were developed in a Camag twin trough chamber and the bands were separated using butanol:methanol:water (2:7:1) as a mobile phase [Figure 1]. After development, the layers were dried with a dryer and hyperin was simultaneously quantified using Camag TLC scanner model 3 equipped with Camag Wincats IV software [Figure 2]. The concentration of hyperin in R. arboreum flowers was found to be 0.148% with R f value 0.72.
Castor Oil-Induced Diarrhea
The EFRA flowers was found to be effective against castor oil-induced diarrhea on experimental rats at various doses of 100, 200 and 400 mg/kg body weight as compared to control [Table 1]. Standard drug loperamide significantly (P<0.001) inhibited diarrheal feces by 79.72% and total weight of feces by 86.8%. EFRA significantly (P<0.05-0.01) inhibited number of diarrheal feces by 24.48-71.07% in a dose-dependent manner from 100 to 400 mg/kg.
|Table 1: Effect of ethyl acetate fraction of Rhododendron arboreum Smith fl owers (EFRA) at different dose levels on castor oil-induced diarrhea in rats|
Click here to view
Magnesium Sulfate-Induced Diarrhea
EFRA caused a significant (P<0.05-0.001) reduction in number of diarrheal feces by 24.55% at a dose of 100 mg/kg, 56.28% at a dose of 200 mg/kg and 73.65% at a dose of 400 mg/kg body weight. Standard drug loperamide cause significant (P<0.001) reduction in diarrheal feces by 89.22% at a dose of 3 mg/kg body weight [Table 2].
|Table 2: Effect of ethyl acetate fraction of Rhododendron arboreum Smith fl owers (EFRA) at different dose levels on magnesium sulfateinduced diarrhea in mice|
Click here to view
Measurement of Gastrointestinal Transit Using Charcoal Meal
Administration of EFRA showed a significant (P<0.05-0.001) dose-dependent reduction of gastrointestinal transit in rats [Table 3]. EFRA showed significant decrease in gastrointestinal transit and percent inhibition 91.36%, 72.14% and 66.03% at dose levels of 100, 200, and 400 mg/kg. The gastrointestinal transit using charcoal meal under the same experimental condition with atropine sulfate (P<0.001) showed 42.13% inhibition in gastrointestinal transit.
|Table 3: Effect of ethyl acetate fraction of Rhododendron arboreum Smith flowers (EFRA) at different dose levels on gastrointestinal transit using charcoal meal in rats.|
Click here to view
Castor oil-induced enteropooling
The ethyl acetate fraction of R. arboreum flowers (EFRA) was found to possess an antienteropooling in castor oil-induced experimental animals. EFRA significantly (P<0.05-0.001) inhibited the intestinal content by 2.64 ± 0.37 to 1.43 ± 0.29 dose dependently from 100 to 400 mg/kg. The standard drug, loperamide at a dose of 3 mg/kg also significantly (P<0.001) reduced intestinal content by 1.28 ± 0.67. The weight of intestinal content was also significantly (P<0.05-0.001) decreased by 3.87 ± 0.45 to 2.21 ± 0.67 at dose levels of 100-400 mg/kg [Table 4].
|Table 4: Effect of ethyl acetate fraction of Rhododendron arboreum Smith flowers (EFRA) at different dose levels on castor oil-induced enteropooling in rats|
Click here to view
| » Discussion and Conclusions|| |
Diarrhoea is the frequent passage of liquid feces and it involves both an increase in the motility of the gastrointestinal tract, along with increased secretion and decreased absorption of fluid, and thus a loss of electrolytes (particularly sodium) and water.  Hence to restore personal comfort and convenience, many patients require antidiarrheal therapy and treatment is carried out to achieve, among other objectives, increased resistance to flow (segmental contraction, decreased propulsion and peristalsis) and increased mucosal absorption or decreased secretion. , In this context, the investigations of the antidiarrheal effect of R. arboreum in this study comprised evaluation of its effect on castor oil-induced diarrhea and magnesium-induced diarrhea. Its effect on gastrointestinal transit using charcoal meal and castor oil-induced enteropooling was also investigated with reference to actions of drugs like atropine sulfate in reducing gastrointestinal transit.
Castor oil, a very effective laxative, is hydrolyzed in the upper small intestine to ricinoleic acid,  which can stimulate fluid secretion, inhibit water and electrolyte absorption, reduce active Na + and K + absorption, and decrease Na + , K + -ATPase in the small intestine and colon. , Castor oil also increases the peristaltic activity and produces permeability changes in the intestinal mucosal membrane to electrolytes and water. Furthermore, ricinoleic acid can also lead to the release of endogenous prostaglandins,  which play an important role in the modulation of GIT, stimulate motility and secretion, and cause diarrhea.  In our study, the results showed that EFRA could, in a dose-dependent manner, reduce castor oil-induced diarrhea as well as the number of diarrheal feces and total weight of feces, which could be taken as antidiarrheal activities. Loperamide is one of the most efficacious and widely employed antidiarrheal drug. Loperamide effectively antagonized the diarrhea induced by castor oil,  prostaglandin  or cholera toxin.  The therapeutic effect of loperamide is believed to be due to its antimotility and antisecretory activity. 
Magnesium sulfate has been reported to induce diarrhea by increasing the volume of intestinal content through prevention of reabsorption of water. It has been demonstrated that it promotes the release of cholecystokinin from the duodenal mucosa, which increases the secretion and motility of small intestine and thereby prevents the reabsorption of sodium, chloride and water.  The ethyl acetate fraction of R. arboreum flowers was also found to reduce magnesium sulfate-induced diarrhea significantly which could be due to increased absorption of water and electrolytes.
The EFRA exhibited significant antidiarrheal activity on gastrointestinal transit using charcoal meal in rats. Hypermotility characterizes forms of diarrhea where the secretory component is not the causative factor.  The EFRA suppressed the propulsive movement or gastrointestinal transit of charcoal meal which clearly indicates that extract may be capable of reducing the frequency of stools in diarrheal conditions. The extract inhibits gastrointestinal motility in diarrhea through anticholinergic effect. Anticholinergic agents are known to inhibit gastrointestinal hypermotility. Castor oil-induced gastrointestinal hypermotility has been suggested to be indirectly mediated by cholinergic system since it is inhibited by atropine sulfate, a known anticholinergic agent. 
EFRA was found to possess an antienteropooling in castor oil-induced experimental animals by reducing both weight and volume of intestinal content. These effects are direct consequences of reduced water and electrolytes secretion in small intestine, suggest that extract may enhance water and electrolyte absorption from intestinal lumen. Phytochemical screening revealed the presence of numerous constituents such as flavonoids, saponins, tannins, phytosterols, reducing sugars and phenolic compounds. Antidiarrheal properties of medicinal plants were found to be due to tannins, flavonoids, alkaloids, saponins, reducing sugar, sterol and terpenes. , Hence tannins, reducing sugars and sterols may be responsible for mechanism of antidiarrheal activity of EFRA. These provide a scientific basis for the potential use of EFRA in GI disorders such as diarrhea. In conclusion, the data obtained in this study suggest that the ethyl acetate fraction of R. arboreum flowers has antidiarrheal activity thus justifying its traditional use in diarrhea. Further studies are needed to identify the exact mechanisms and chemical compounds that are responsible for these pharmacological actions.
| » Acknowledgment|| |
This study was supported in part by grants from the Indian Council of Medical Research (ICMR) and Department of Science and Technology (DST), New Delhi. Thanks are also extended to Mrs. Neelam Mishra, Department of Chemistry, School of Pharmaceutical Sciences, Siksha 'O' Anusandhan University, Bhubaneswar, Orissa for her technical assistance rendered in the course of this research work.
| » References|| |
|1.||Carlos CC, Saniel MC. Etiology and epidemiology of diarrhoeal, Philipp. J Microbiol Infect Dis 1990;19:51-3. |
|2.||Maiti A, Dewanjee S, Mandal SC. In vivo evaluation of antidiarrhoeal activity of the seed of Swietenia macrophylla King (Meliaceae). Trop J Pharm Res 2007;2:711-6. |
|3.||Atta AH, Mouneir SM. Antidiarrhoeal activity of some Egyptian medicinal plant extracts. J Ethnopharmacol 2004;92:303-9. |
|4.||Kirtikar KR, Basu BD. Indian Medicinal Plants, 2 nd ed. Dehradun: International Book Publisher; 2005. p. 2020-1. |
|5.||Ripu KM, Uprety Y, Burlakoti C, Chowdhary CL, Bussmann RW. Indigenous Use and Ethnopharmacology of Medicinal Plants in Far-west Nepal. Ethnobot Res Appl 2009;7:5-28. |
|6.||Verma N, Singh AP, Amresh G, Sahu PK, Rao Ch V. Anti-inflammatory and anti-nociceptive activity of Rhododendron arboreum. J Pharm Res 2010;3:1376-80. |
|7.||Doherty SS. Inhibition of arachidonic acid release, mechanism by which glucocorticoids inhibit endotoxin-induced diarrhoea. Br J Pharmacol 1981;73:549-54. |
|8.||Inayathulla, Shariff WR, Karigar AA, Sikarwar MS. Evaluation of Antidiarrhoeal activity of Crataeva nurvala root bark in experimental animals. Int J Pharm Pharm Sci 2010;2:158-61. |
|9.||Robert A, Nezamis JF, Lancaster C, Hanchar AJ, Klepper MS. Enteropooling assay: A test for diarrhoea produced by prostaglandins. Prostaglandins 1976;11:809-28. |
|10.||Rang HP, Dale MM, Ritter JM, Moore PK. The gastrointestinal tract. Pharmacology, 5 th ed. Edinburgh: Churchill Livingstone; 2003. p. 367-79. |
|11.||Burks TF, Gastrointestinal drugs. In: Kist K, editor. Human Pharmacology: Molecular to Clinical. London: Wolfe Publishing Ltd; 1991. p. 789-11. |
|12.||Akindele AJ, Adeyemi OO. Evaluation of the antidiarrhoeal activity of Byrsocarpus coccineus. J Ethnopharmacol 2006;108:20-5. |
|13.||Mascolo N, Izzo AA, Barbato F, Capasso F. Inhibitors of nitric oxide synthetase prevents castor-oil-induced diarrhoea in the rat. Br J Pharmacol 1993;108:861-4. |
|14.||Ammon HV, Thomas PJ, Phillips S. Effect of the oleic and ricinolic acid on net jejuna water and electrolyte movement. J Clin Invest 1974;53:374-9. |
|15.||Gaginella TS, Stewart JJ, Olsen A, Bass P. Action of ricinoleic acid and structurally related fatty acid on the gastrointestinal tract II. Effect on water and electrolyte absorption in vitro. J Pharmacol Exp Ther 1975;195:355-6. |
|16.||Cohen MM. The effect of cathartics on prostaglandin synthesis by rat gastrointestinal tract. Prostaglandins Leukot Med 1982;8:389-97. |
|17.||Sanders KM. Evidence that prostaglandins are local regulatory agents in canine ileal circular muscles. Am J Physiol 1984;246:G361-71. |
|18.||Niemegeers CL, Lenaerts FM, Janseen PA. loperamide (R-18553), A novel type of antidiarrhoeal agent. Part 1: In vitro oral pharmacology and acute toxicity. Comparison with morphine, codeine, diphenoxylate and difinoxine. Arzneimittelforschung 1974;24:1633-6. |
|19.||Karim SM, Adaikan PG. The effect of loperamide on prostaglandin- diarrhoea in rats and man. Prostaglandins 1977;13;321-31. |
|20.||Farack UM, Kantz U, Loescke K. Loperamide reduces the intestinal secretion but not the mucosal cAMP accumulation induced by choleratoxin. Naunyn Schmiedebergs Arch Pharmacol 1981;317:178-9. |
|21.||Couper IM. Opioid action on the intestine: The importance of the intestinal mucosa. Life Sci 1987;41:917-25. |
|22.||Galvez J, Zarzuelo A, Crespo ME, Lorente MD, Ocete MA, Jimenez J. Antidiarrhoeal activity of Euphorbia hirta extract and isolation of an active flavonoids constituent. Planta Med 1993;59:333-6. |
|23.||Chitme HR, Chandra R, Kaushik S. Studies on anti-diarrhoeal activity of Calotropis gigantean in experimental animals. J Pharmacol Pharm Sci 2004;7:70-5. |
|24.||Brown JH, Taylor P. Muscarinic receptor agonists and antagonist. In: Hardman JG, Limbird LE, editor. Goodman and Gilman's the Pharmacological Basis of Therapeutics. 9 th ed. New York: MacGraw Hill; 1996. p.141-6. |
|25.||Longanga Otshudi A, Vercruysse A, Foriers A. Contribution to the ethnobotanical, phytochemical and pharmacological studies of traditionally used medicinal plant in the treatment of dysentery and diarrhoea in Lomela area, Democratic Republic of Congo (DRC). J Ethnopharmacol 2000;71:411-23. |
|26.||Galvez J, Zarzuelo A, Crespo ME. Antidiarrhoeic activity of Scleroarya birrea bark extract and its active tannin constituent in rats. Phytother Res 1991;5:276-8. |
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]