International Journal of Pharmaceutical Sciences
INT.J.PH.SCI.,SEP-DEC, 2010;2(3):680-686
ISSN 0975-4725
Original Research Manuscript
Date of Submission: 29-06-2010
Date of Acceptance: 20-09-2010
Evaluation of iridoid glycosides from leave of Barleria Prionitis as an Anti-diarrhoeal
Activity: an Ethnopharmacological study

Sunil K. Jaiswal3*, Mukesh K. Dubey2, Ajay K. Verma3, Sanjib Das2, M. Vijaykumar1, Chandana V. Rao1
1 Pharmacognosy & Ethnopharmacology Div., National Botanical Research Institute (CSIR), Rana Pratap Marg, P.O. Box 436, Lucknow 226001, Uttar Pradesh, India. 2 Dept. of Pharmaceutical sciences, Dibrugarh University, Dibrugarh-786004, Assam, India. 3 Rameshwaram Institutes of Technology & Management, Lucknow, Uttar Pradesh, India. ABSTRACT
Barleria prionitis L. (Family Acanthaceae; commonly known as Vajradanti), is an annual shrub, 1–3 feet high, found throughout tropical Asia and in South Africa. In indigenous system of medicine in India, the aerial parts are used in gastrointestinal disorders. The present study was undertaken in order to evaluate the antidiarrhoeal activity of chromatographic fractions B of butanol extract of leave of B.prionitis using several experimental models in Wistar albino mice/Rats. Leaves of B.prionitis were subjected to fractionate in several non polar solvent in which butnol fraction of B.prionitis Linn. led us to the identification of active fraction (iridoid glycoside). The bio active fraction has been prove that it contain three major iridoid glycosides namely acetyl barlerin (6, 8-di-o-acetylshanzhiside methyl ester), barlein (8-o-acetylshanzhiside methyl ester) and shanshiside methyl ester. These iridoids glycoside was subjected to anti diarrhoeal activity against validated experimental models like Castor oil induced diarrhea, gastrointestinal motility tests & PGE2-induced enteropooling. The extract inhibited castor oil induced diarrhoea and PGE2 induced enteropooling in rats; it also reduced gastrointestinal motility after charcoal meal administration. The obtained data demonstrated the excellent anti-diarrhoeal activity of B.prionitis and thus have great potential as a source for natural health products. Keywords: anti-diarrhoeal activity; castor oil; Barleria prionitis; Gastrointestinal motility.
movement, wet stool and abdominal pain [2]. It is a Diarrhoea is a public health problem in developing leading cause of malnutrition and death among children countries. Acute diarrhoea is the leading cause of in the developing countries of the world today [3]. Many morbidity and mortality amongst children in developing governments and international organizations are trying countries [1]. Many rural dwellers in the world depend to control this disease but the rate of incidence is still largely on medicinal herbs for the treatment of high, about 7.1 million per year [4]. Many synthetic diarrhoeal conditions because these herbs are readily chemicals like diphenoxylate, loperamide and antibiotics are available for the treatment of diarrhoea but they have component of traditional medicine practice. Diarrhoea is some side effects. The natural drugs are used as characterized by increased frequency of bowel antidiarrhoeal drugs, which are not always free from Int.J.Ph.Sci.,May-August 2010;2(3):
adverse effects [5]. Therefore, the search for safe and The freshly collected leaves (5 kg) dried under the more effective agents has continued to be an important control conditions and powdered materials was area of active research. Since ancient times, diarrhoea percolated with petroleum ether for 3 days to remove has been treated orally with several medicinal plants or fatty substances, the marc was further exhaustively their extracts based on folklore medicine extracted with of 50% ethanol for 4 days (3 X 5L). The Barleria prionitis L. (Family Acanthaceae; commonly extract was separated by filtration and concentrated on known as Vajradanti), is an annual shrub, 1–3 feet high, rotavapour (Buchi, USA) and then freeze-dried found throughout tropical Asia and in South Africa. In (Freezone® 4.5, Labconco, USA) at high vacuum (133 x indigenous system of medicine in India, the aerial parts 10-3 m Bar) and low temperature (–40 ± 2 ºC) to obtain are used in fever, toothache, inflammation & gastrointestinal disorders; bark in whooping cough as an Fractionation of B.prionitis leaves with different
expectorant; the whole plant and especially the roots are solvents
used as tonic and diuretic [6-7]. Leaves, stem and root of The extract was fractionated successively with n-hexane, B. prionitis possess antibacterial and anti-inflammatory chloroform and n-butanol with the help of separating activities [8-9]. The flower of Barleria prionitis possess funnel for 4 times each, to obtain n-hexane fraction, anti-inflammatory and anti nociceptive activity [10]. Iridoid enriched fraction of aerial parts (leaves and stems) was reported for hepatoprotective activity in Preliminary phytochemical screening
various acute and chronic animal models [11]. An aerial The BPE was analyzed for the presence of part was reported for barlerinoside, shanzhiside methyl phytochemicals viz. alkaloids, carbohydrate, glycosides, 6-O-trans-p-coumaroyl-8-O-acetylshanzhiside protein and amino acids, flavonoids, polyphenols, resins, saponins, steroids and triterpenoids by qualitative methoxydiderroside and lupulinoside [12]. Despite the popular use of this species as a medicinal Isolation procedure of compound(s) from B. prionitis
plant, there are no data about the pharmacological effect On the basis of % yield and review on phytoconstituents, of B. prionitis on the gastrointestinal system. The aim of n-Butanaol fraction 50gm was subjected to silica gel the present study was to evaluate the potential Anti column chromatography (silica gel 0.5 kg) and eluted diarrhoeal activity of butanolic extract of B. prionitis with 2.5L each of 100% chloroform, 25% ethyl acetate leaves (BPE) on different experimental models of in chloroform, 50% ethyl acetate in chloroform, 75% ethyl acetate in chloroform, 100% ethyl acetate, 2% methanol in ethyl acetate, 5% methanol in ethyl acetate, MATERIAL AND METHOD
10% methanol in ethyl acetate, 20% methanol in ethyl Plant material
acetate, 30% methanol in ethyl acetate, 40% methanol in The B. prionitis was collected from Botanical Garden of ethyl acetate, 50% methanol in ethyl acetate, 100% National Botanical Research Institute (NBRI), Lucknow, methanol. The fractions were checked on TLC plate and India in the month of January. The plant material was identified and authenticated and the voucher specimen Preparative TLC was performed in silica gel 60 F254 number NBR-354 is deposited in the departmental preparative TLC plates (20 × 20 cm with 4 × 20 cm concentration zone, 0.5 mm layer thickness and Preparation of extract
fluorescence at 254 nm) (Merck) pre-heated at 105 ± 5 Int.J.Ph.Sci.,May-August 2010;2(3):
ºC for 30 min for the separation of compounds from The method reported by with modifications, has been fraction B (436 mg). A relatively large amount of used in the present study [15]. Rats of either sex (210–235 fraction (50 mg/ml) was applied on the plates with a g) were fasted for 18 h; they were then divided into five TLC sample applicator. The plates (×5) were developed groups of five individuals. The fraction B of butanol with the solvent ethyl acetate: Methanol (8:2). extract of B.prionitis was administered orally at doses of Test animals
25, 50 and 100 mg/kg by gavage as suspension to the Sprague-Dawley rats (150-175g) were procured from the first three groups of animals. The fourth group received animal house of Central Drug Research Institute, loperamide (3 mg/kg) orally as suspension (positive Lucknow. They were kept in the departmental animal control). The fifth group, which served as the blank, was house at temperature 26  2 C, relative humidity 44 - administered with aqueous acacia suspension. After 60 56%, light and dark cycles of 10 and 14 h respectively min of treatment, the animals of each group received for one week before and during the experiments. 1ml of castor oil orally, by gavage, and the consistency Animals were provided with standard rodent pellet diet of faecal material and the frequency of defecation were (Dayal, India) and the food was withdrawn 18-24 h noted up to 4 h in the transparent plastic dishes placed before the experiment though water was allowed ad libitum. All the studies were performed in accordance Gastrointestinal motility tests
with the guidelines for the care and use of laboratory Rats were fasted for 18 h and then placed in five cages animals, as adopted and promulgated by the Institutional containing five individuals in each cage. Each animal Animal Care Committee, CPCSEA, India (Reg. No. was administered orally with 1ml of charcoal meal (5% deactivated charcoal in 10% aqueous tragacanth), Acute toxicity
followed by oral administration of fraction B suspension Different doses (25–500 mg/kg, p.o.) of BPE were to three groups of animals in doses of 25, 50 and 100 administered to groups of rats and observed continuously mg/kg. The fourth group received atropine (0.1 mg/kg, for 1 h and then at half-hourly intervals for 4 h, for any i.p.), the standard drug for comparison and the fifth gross behavior changes further up to 72 h followed 14 group was treated with aqueous acacia suspension (vehicle control). Thirty minutes later, each animal was Evaluation of the effect on normal defecation
sacrificed and the intestinal distance moved by the Five groups of six mice each were placed individually in charcoal meal from the pylorus was cut, measured, and separate cages with filter papers at the bottom. The doses expressed as a percentage of the distance from the (25, 50 and 100 mg dry extract per kg body mass) of fraction B were administered orally to different groups. PGE2-induced enteropooling
In this method, rats were deprived of food and water for diphenoxylate HCl (5.0 mg/ kg, p.o.) and aqueous acacia 18 h and placed in five cages, with five animals per cage. suspension 5 ml/kg were administered to two groups and The first three groups were treated with 25, 50 and 100 they later served as controls [14]. The total number of mg/kg doses of fraction B. The fourth group was treated faecal droppings in each group was assessed every hour with 1ml of a 5% (v/v) ethanol in normal saline (i.p.) and for the next 4 h. Percent reduction in the total number of then it was treated with aqueous acacia suspension, faeces in the treated groups was obtained by comparison which served as vehicle control. Immediately after the extract administration PGE2 (Astra Zeneca, India) was Castor oil-induced diarrhoea in Rats
administered orally to each rat (100ìg/kg) in the first Int.J.Ph.Sci.,May-August 2010;2(3):
three groups. The fifth group was treated with PGE2 kg–1 caused a dose dependent decrease in the total (100ìg/kg) as well as with aqueous acacia suspension faecal matter (15.0 and 58.3%). Loperamide, a standard and served as the PGE2 control group. After 30 min antidiarrhoeal drug, inhibited the diarrhoea by 66.6% following administration of PGE2, each rat was (Table 1). The fraction B at doses of 25 and 100 mg/kg sacrificed and the whole length of the intestine from the decreased the propulsion of charcoal meal through the pylorus to the caecum was dissected out, its content gastrointestinal tract, as compared with the control group collected in a test tube, and the volume measured [17]. (p<0.05 - p<0.001). Atropine (0.1 mg/kg) reduced the motility of the intestine to a greater extent (p < 0.001) STATISTICAL ANALYSIS
(Table 2). The fraction B significantly inhibited PGE2 The values were represented as mean ± S.E.M. for six induced enteropooling in rats in higher dose levels rats. Analysis of variance (ANOVA) test was followed compared with PGE2 treatment (p<0.001) (Table 3). by individual comparison by Newman–Keuls test using PGE2 induced a significant increase in the fluid volume Prism Pad software for the determination of level of of the rat intestine when compared with control animals, significance. The p-values of <0.05 being considered DISCUSSION AND CONCLUSIONS
In the present study, the fraction B of butanol extract of The extractive value of B.prionitis leave in n-Hexane B.prionitis exhibited significant anti-diarrhoeal activity (7.5%), Chloroform (15.35%) in butanol (29.2%) and in against castor oil induced diarrhoea in rats. The fraction aq. Portion (55.10%). The preliminary phytochemical B had a similar activity as loperamide, when tested at 50 studies on the BPE demonstrate the presence of and 100 mg/kg and inhibited the frequency of faecal alkaloids, flavonoids, glycosides, tannins, saponins, droppings. Castor oil releases ricinoleic acid which steroids and triterpenoids. On chemical analysis, fraction induces changes in mucosal fluid and electrolyte B was found to be a mixture of iridoid glucosides, which transport that results in a hypersecretory response and on TLC (silica gel) gives three spots of Rf 0.44, 0.64, diarrhoea [18-19]. The experimental studies in rats 0.69. One major spot of Compound B2 (229 mg) with Rf demonstrated a significant increase in the portal venous 0.44 was further confirmed by Co TLC with a reference PGE2 concentration following oral administration of standard shanzhiside methyl ester (iridoid glucoside) castor oil [20]. Ricinoleic acid markedly increased the (Fig.2). Further chemical profile showed mainly three PGE2 content in the gut lumen and also caused an iridoid glucosides, i.e. acetyl barlerin (6, 8-di-o- increase of the net secretion of the water and electrolytes into the small intestine [21]. Inhibitors of prostaglandin acetylshanzhiside methyl ester) and shanshiside methyl biosynthesis delayed castor oil induced diarrhoea [15]. ester. In the acute toxicity study, no deaths were The extract appears to act on all parts of the intestine. observed during the period at the doses tested. Thus, it reduced the intestinal propulsive movement in In the present investigation, the fraction B of butanol the charcoal meal treated model; at 100 mg/kg fraction B showed activity similar to that of atropine. The fraction antidiarrhoeal activity in various validated models in B at different dose levels 50 and 100 mg/kg significantly rats. Castor oil produced characteristic semisolid inhibited the PGE2 induced intestinal fluid accumulation diarrhoea droppings in all animals of the control group. (enteropooling). These observations tend to suggest that The effect of the fraction B at the dose of 25–100 mg the fraction B at different dose levels 50 and 100 mg/kg Int.J.Ph.Sci.,May-August 2010;2(3):
reduced diarrhoea by inhibiting gastrointestinal motility ACKNOWLEDGEMENT
The authors are thankful to the Head, Dept. of The present results indicate that the fraction B of butanol extract of B.prionitis possesses significant anti- Dibrugarh and Director NBRI for providing necessary diarrhoeal activity due to its inhibitory effect both on gastrointestinal propulsion and fluid secretion. The inhibitory effect of the extract justifies the use of the plant as a non-specific antidiarrhoeal agent in folk Table1. Effect of fraction B of butanol extract of B.prionitis on castor oil induced diarrhoea in rats
Total no. of
(mg kg–1) fecal droppings
* Values are presented as mean values of six rats in each group. Table 2. Effect of fraction B of butanol extract of B.prionitis on charcoal-induced gut transit changes
Distance traveled
Inhibition (%)
(mg kg–1) by charcoal meal (%)
Values are expressed as mean + S.E.M. (n=6). ap<0.05, bp<0.001 compared to respective control group. Table 3. Effect of fraction B of butanol extract of B.prionitis on PGE2-induced enteropooling
Volume of intestinal fluid (ml) Inhibition (%)
B.prionitis (100mg kg–1) Values are expressed as mean + S.E.M. (n=6). a p<0.001 compared with respect to ethanol in saline treatment. b p<0.05 compared with respect to ethanol in saline treatment. x p<0.001 compared with respect to PGE2 treatment. Int.J.Ph.Sci.,May-August 2010;2(3):
Fig. 1 Isolation procedure of compound(s) from butanol fraction of B. prionitis
Fig.2 HPTLC densitometric scan of butanol fraction from the B.prionitis leaves extract at 320 nm
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