J Nat Med (2008) 62:396–402 DOI 10.1007/s11418-008-0258-8

ORIGINAL PAPER

Antidiarrhoeal activity of the standardised extract of Cinnamomum tamala in experimental rats

Chandana Venkateswara Rao Æ Madhavan Vijayakumar Æ K. Sairam Æ Vikas Kumar

Received: 22 October 2007 / Accepted: 17 April 2008 / Published online: 21 May 2008 Ó The Japanese Society of Pharmacognosy and Springer 2008

Abstract The present study was designed to investigate Introduction the antidiarrhoeal potential of 50% ethanolic extract of Cinnamomum tamala on experimentally induced castor oil Cinnamomum tamala Nees et Eberm (Family Lauraceae) is diarrhoea, gastric emptying of phenol red meal, gastroin- also known as Tejpat. It is distributed in the tropical and testinal transit of charcoal meal and in vitro mast cell north-western Himalayas, Nepal, Bangladesh and Myan- degranulation activity. C. tamala extract (25, 50 and 100 mar. Tejpat leaves are used in Indian cookery and as bay mg/kg, orally) produced a dose-dependent reduction in the leaves in Europe [1]. The leaf of this tree is a spice with a total amount of faecal matter in castor oil-induced diar- clove-like taste and a faintly pepper-like odour and is rhoea. The mean distance travelled by charcoal meal at 50 mainly used for flavouring food. Germacrene A, a-gur- and 100 mg/kg of extract showed a significant reduction in junene, 1-8-cineole, p-cymene, methyl eugenol and the secretion of gastrointestinal fluid accumulation by eugenol acetate have been reported in C. tamala. The use 32.5–65.0%. The Na+ and K+ concentrations on castor oil- of the plant in the traditional literature is well documented induced fluid accumulation showed a greater inhibitory as a remedy for various ailments, such as anorexia, bladder effect on Na+ levels than on K+ concentrations. C. tamala disorders, dryness of mouth, coryza, diarrhoea, nausea, significantly reduced the lipid peroxidation (P\0.001) and spermatorhea [1] and hypoglycemic, stimulant and carmi- increased the catalase (P \ 0.01) activity in comparison to native properties [2]. However, limited scientific the castor oil-induced groups. C. tamala leaf extract did not investigations have been done to verify these claims. The show any significant effect at a higher dose (15 mg/ml) on use of medicinal plants that possess antidiarrhoeal activi- mast cell degranulation. However, the extract in the dose of ties have been explored as one of the major measures that 5 and 10 mg/ml conferred significant mast cell protective could benefit in combating widespread diarrhoeal infec- action (P\0.001). The percentage of eugenol in extract is tions, especially in third world countries. In light of the 3.8% w/w, and total tannin is 247.5 mg/g. The result above information on the antidiarrhoeal plants, we exam- indicates the Indian spice C. tamala is useful for diarrhoea. ined the effect of C. tamala leaf extract on the antidiarrhoeal property with defined active components Keywords Cinnamomum tamala Á Antidiarrhoeal Á responsible for its free radical scavenging action in Indian spice diarrhoea.

C. V. Rao (&) Á M. Vijayakumar Pharmacognosy and Ethnopharmacology Division, Materials and methods National Botanical Research Institute, Rana Pratap Marg, Post Box No. 436, Lucknow 226 001, India Animals e-mail: [email protected] Male Sprague–Dawley rats (150–175 g) and albino mice K. Sairam Á V. Kumar Department of Pharmaceutics, Institute of Technology, (16–22 g) were obtained from the National Laboratory Banarus Hindu University, Varanasi 221005, India Animal Centre (NLAC), Lucknow, and housed three to a 123 J Nat Med (2008) 62:396–402 397 cage for the duration of the study. Animals were provided supernatant was measured at 730 nm. The total tannin with standard rodent pellet diet (Amrut, India) and were content was expressed as tannic acid equivalents in milli- maintained in a temperature- and humidity-controlled grams per gram sample. environment on a 12-h dark/light cycle. The food was withdrawn 24 h before the experiment, but water was Effect on normal defecation allowed ad libitum. All studies were performed in accor- dance with the guide for the care and use of laboratory Five groups of six mice each were placed individually in animals, as adopted and promulgated by the Institutional separate cages with filter papers at the bottom. The doses of Animal Care Committee, CPCSEA, India (Reg. No. 222/ the extracts of C. tamala (25, 50 and 100 mg/kg) were 2000/CPCSEA). administered orally to different groups. The non-specific antidiarrhoeal reference drug diphenoxylate HCl (5 mg/kg) Plant material and preparation of standardised extracts and physiological saline (10 mg/kg) were administered to two groups and later served as controls [4]. The total Dried leaves of C. tamala (Family: Lauraceae) were pro- amount of faecal matter in each group was assessed every cured from the local market of Lucknow, India. The plant hour for the next 4 h. Percent reductions in the total amount material was identified and authenticated by Dr Kaushal of faeces in the treated groups were obtained by compari- Kumar, taxonomist, and the voucher specimen number son with control animals. NBR-370 is deposited in the departmental museum. The leaves were powdered and passed through a 10-mesh sieve. Gastric emptying of a phenol red meal The coarsely powdered material was exhaustively extrac- ted thrice with 50% aqueous ethanol. The extracts were In the gastric emptying assay, a test meal (1.5 ml/rat) filtered, pooled and concentrated at reduced temperature consisting of a solution of 50 mg phenol red in 100 ml (5°C) on a rotary evaporator (Buchi, USA) and then aqueous methyl cellulose (1.5%) was administered by freeze–dried (FreezoneÒ 4.5, Labconco, USA) at high gavage through an oro-gastric polyethylene catheter to rats vacuum (133 9 10-3 m bar) and at temperature -40 ± 2°C fasted and with free access to water for 24 h. The first (yield 15.2%, w/w). The extract so obtained was dissolved group received 0.5% physiological saline (10 ml/kg), while in 10% methanol in water and extracted successively with the second and third groups were given the C. tamala n-hexane, chloroform, ethyl acetate, butanol and water. extract (50 and 100 mg/kg) 1 h prior to test meal by oral The lyophilised extract yields are 7.8, 5.5, 7.2, 8.1 and intubation in a constant volume. The rats were killed 60 9.4% w/w, respectively. Keeping in view the % yield and min after the test meal, because at this time point maximal activity, the 50% aqueous ethanolic extract took up further differences were expected between control and treated detailed antidiarrhoeal activity. The high-performance thin groups. The stomach was then exposed by laparotomy, layer chromatography (HPTLC) studies of the 50% EtOH quickly ligated at the pylorus and the cardia and removed. extract of C. tamala was carried on pre-coated silica gel The stomach and its contents were homogenised with 100 plates (Merck 60 F 254) as the stationary phase and tolu- ml of NaOH 0.1 N. The suspension was allowed to settle ene:ethyl acetate (93:7) as the mobile phase. The extract for 1 h at room temperature, and 5 ml of the supernatant was spotted using a Camag Linomat IV spotter. These was added to 0.5 ml of 20% w/v trichloroacetic acid and plates were observed at UV light at 260 nm and were then centrifuged at 1,8009g for 20 min. The supernatant scanned on TLC scanner III using winCAT software, and was mixed with 4 ml of 0.5 N NaOH and the absorbance of eugenol content was estimated. the sample read at wavelength of 560 nm. Phenol red recovered from animals killed immediately after the Determination of total tannins administration of the test meal was used as standard (0% emptying). Percent gastric emptying was calculated Contents of total tannins were determined using the method according to the following formula: gastric emptying (%) = of Folin-Ciocalteu reaction using tannic acid as standard (1 - absorbance of test sample/absorbance of standard) 9 [3]. Initially, the lyophilised 50% aqueous EtOH extract of 100 [5]. C. tamala (5 mg) was dissolved in 5 ml of ethanol:water (50:50, v/v), and the extract solution (0.5 ml) was mixed Effect on in vitro mesentric mast cell degranulation with 500 ll of 50% Folin-Ciocalteu reagent. The mixture was then allowed to stand for a 2–5-min period followed by The mesentery along with small pieces of jejunum and the addition of 1.0 ml of 20% sodium carbonate. After 10 ileum portion of small intestine (expecting the distal most 2 min incubation at room temperature, the mixture was cm, which showed maximum variation in the degree of centrifuged for 5 min (1,0009g), and absorbance of the spontaneous degranulation) was taken out and placed in a 123 398 J Nat Med (2008) 62:396–402 petri dish containing oxygenated Ringer Locke’s solution percentage of that length were evaluated for each animal,

(NaCl 9.0, KCl 0.42, CaCl2 0.24, NaHCO3 0.5 and glu- and group means were compared and expressed as per- cose 1 g/l in double-distilled water; pH 7.4) at 37.0 ± centage inhibition [9]. 0.5°C. In vitro tissue was transferred to C. tamala leaf extract (5, 10, 15 mg/ml) for 30 min and stained supra- Castor oil-induced fluid accumulation vitally [6]. Briefly, tissue was immersed in 0.1% toludine and Na+ and K+ secretion blue in 4% formaldehyde in saline for 15–20 min. The tissue was next transferred and kept in acetone (two The rats fasted for 24 h, but free access to water was changes) for 10 min and then kept in xylene for 2 min randomized and allocated to four groups of six rats each. and then mounted on slides. Before mounting, excess Group I (control) was administered physiological saline (10 pieces of fat were trimmed off the edges of the mesentry. ml/kg), group II was administered castor oil only (2 ml), The tissue mesentry was stretched with the help of a and groups III and IV were administered 50 and 100 mg/kg needle. Usually five to six pieces of mesentery from each of C. tamala extract, respectively, 1 h prior to castor oil animal were used for each concentration of C. tamala leaf administration. After 30 min the rats were killed by cer- extract. From these pieces, five microscopic fields were vical dislocation and exsanguinated; the small intestine was selected at random under 1009 magnification at widely ligated both at the pyloric sphincter and at the ileocaecal separated areas of the mesentery. In each field the first ten junctions. The entire small intestine was dissected out, its mast cells were examined using 4309 magnification contents were expelled into a graduated measuring cylin- starting from the left-hand side of the field and proceed- der, the volume of the contents was recorded, and the fluid ing clockwise. Each cell was scored as either ‘‘disrupted’’ samples were analysed for Na+ and K+ concentrations or ‘‘not disrupted.’’ In each concentration 100–150 mast using flame photometer (ElicoÒ CL361, India) [10, 11]. cells were examined, and average percent disruption was calculated. Estimation of lipid peroxidation (LPO)

Castor oil-induced diarrhoea A volume of the content in castor oil-induced fluid accu- mulation (0.2 ml) was transferred to a vial and was mixed The rats fasted for 24 h were randomly allocated to five with 0.2 ml of a 8.1% (w/v) sodium dodecyl sulphate groups of six animals each. Group I received 0.5% physio- solution, 1.5 ml of a 20% acetic acid solution (adjusted to logical saline (10 ml/kg); groups II, III and IV received orally pH 3.5 with NaOH) and 1.5 ml of a 0.8% (w/v) solution of the C. tamala extract (25, 50 and 100 mg/kg), respectively. thiobarbituric acid (TBA), and the final volume was Groups V and VI received diphenoxylate (5 mg/kg) and adjusted to 4 ml with distilled water. Each vial was tightly yohimbine (1 mg/kg), and 10 min later, 100 mg of C. tamala capped and heated in a boiling water bath for 60 min. The extract was given orally to both groups. After 60 min each vials were then cooled under running water. Equal vol- animal was given with 2 ml of castor oil by oro-gastric umes of tissue blank or test samples and 10% polyethylene catheter and placed in a separate cage and trichloroacetic acid were transferred into a centrifuge tube observed for 4 h defecation. Transparent plastic dishes were and centrifuged at 1,0009g for 10 min. The absorbance of placed beneath each cage, and the characteristic diarrhoeal the supernatant fraction was measured at 532 nm. Control droppings were noted [7, 8]. experiment was processed using the same experimental procedure except the TBA solution was replaced with Small intestinal transit distilled water [12]. 1,1,3,3-Tetraethoxypropan was used as standard for calibration of the curve and is expressed as Animals were divided into four groups of six rats each, and nmol/mg protein. each animal was given orally 1 ml of charcoal meal (5% activated charcoal suspended in physiological saline) 60 Catalase activity min after an oral dose of drugs or vehicle. Group I was administered with physiological saline (10 ml/kg), and Briefly, the assay mixture consisted of 1.95 ml phosphate animals in groups II and III received C. tamala extract (50 buffer (0.05 M, pH 7.0), 1.0 ml hydrogen peroxide (0.019 and 100 mg/kg). Group IV received atropine sulphate (0.1 M) and 0.05 ml homogenate in a final volume of 3.0 ml. mg/kg) as standard drug. After 30 min animals were killed Changes in absorbance were recorded at 240 nm. One unit by cervical dislocation, and the intestine was removed of (U) catalase was defined as the amount of enzyme without stretching and placed lengthwise on moist filter required to decompose 1 lmol of H2O2 per min at 25°C paper. The length of the intestine (pyloric sphincter to and pH 7. Results are expressed as units (U) of catalase caecum) and the distance travelled by the charcoal as a activity/mg protein [13]. 123 J Nat Med (2008) 62:396–402 399

Superoxide dismutase (SOD) activity changes of acute diarrhoea as indictaed by the increase in lipid bodies in mast cells [17]. The assay consisted of EDTA 0.1 mM, sodium carbonate 50 and 96 mM of nitro blue tetrazolium, the inhibition of Gastric emptying of a phenol red meal nicotinamide adenine dinucleotide (reduced)-phenazine methosulphate–nitrobluetetrazolium reaction system [14, The percentage gastric emptying of C. tamala leaf extract 15]. One unit of the enzyme is equivalent to 50% inhibition at 50 and 100 mg/kg showed 68.0 ± 3.5 and 61.0 ± 3.2 as in the formazan formation in 1 min at room temperature with that of control rats 72.0 ± 3.8 (Fig. 2). Gastric emp- (25 ± 2°C), and the results have been expressed as units tying of a phenol red meal was studied as an index for the (U) of SOD activity/mg protein. functionality of the fundus. The fundus by means of its tonic contractions increases intragastric pressure, leading to Statistical analysis a gastro duodenal pressure gradient, which is important in gastric emptying of liquids [18]. The percentage gastric All the data are presented as mean ± SEM, and one-way emptying of C. tamala leaf extract did not show significant analysis of variance (ANOVA) and Newman–Keuls Mul- activity with that of control rats. tiple Comparision Test were applied for determining the statistical significance between different groups. Castor oil-induced diarrhoea

The effect of C. tamala extract at the dose levels of 25, 50 Results and discussion and 100 mg/kg caused a dose-dependent decrease in the amount of faecal matter from 49, 36 and 24, respectively. Standardization of 50% ethanolic extract However, percent reduction in total number of faecal of C. tamala leaf

20 The preliminary HPTLC studies revealed the well-resolved 18 peaks of 50% EtOH C. tamala. The spots of the chromato- 16 gram were visualised at 254 nm with a 400 K filter at 0.02, 14 a 0.05, 0.22, 0.33, 0.41, 0.45, 0.57, 0.76 and 0.82 Rf. The 12 a a percentage of eugenol in 50% EtOH extract is 3.8% w/w and 10 247.5 mg/g of total tannins. 8 6 Evaluation of the effect on normal defecation

percentage degranulation 4 2 The extract of C. tamala leafs at 25 mg/kg had no effect; 50 0 and 100 mg/kg doses inhibited defecation by 100% in the Control C tamala 5mg C tamala 10mg C tamala 15mg initial 2 h on normal defecation in mice. The activity was Fig. 1 Effect of C. tamala on mast cell degranulation. aP \ 0.001 reduced to 40.0 and 73.0%, respectively, at the higher compared to respective control doses in the third hour.

80 Effect on in vitro mesentric mast cell degranulation 70

Cinnamomum tamala leaf extract did not show any sig- 60 nificant effect at a higher dose (15 mg/ml) on mast cell degranulation. However, the extract in the dose of 5 and 10 50 mg/ml conferred significant mast cell protective action 40 (Fig. 1). Mast cells are found in all areas of gastrointestinal 30 tract, in the mucosa and in the other layers of the gut wall. The strong association between degranulating mucosal 20

mast cells and diarrhoea onset suggested a direct causal Percentage of gastric emptying 10 relationship [16]. C. tamala leaf extract showed significant activity on mast cell degranulation in a dose-dependant 0 Control C tamala 50 C tamala 100 manner. It is likely that arachidonate metabolities and cytokines from mast cells are playing a key role in vascular Fig. 2 Effect of C.tamala extract on gastric emptying in rats 123 400 J Nat Med (2008) 62:396–402 matter ranges from 26.5 to 61.3%. Diphenoxylate, a stan- caused a dose-dependent decrease in the intestinal pro- dard antidiarrhoeal drug, in the presence of the extract (100 pulsion from 51.3 to 35.2%, which is equivalent to 33.9 mg/kg) inhibited by 67.7%, and in the presence of and 54.6% intestinal propulsive inhibition relative to con- yohimbine, the antidiarrhoeal effect of the extract was trol. The extract appears to be as inhibitory as atropine, a reduced to 11.3%. The total amount of faecal matter of non-specific muscarinic blocker, and reduces to a greater diphenoxylate and yohimbine in the presence of C. tamala extent the intestinal propulsion to 63.5% (P \ 0.001). is 20 and 55, respectively (Table 1). The action of castor Antispasmodic property is deduced from the fact that the oil as a diarrhoea inductor has been largely studied, and it extract caused a decrease in propulsive movement of the is known that its most active component is ricinoleic acid, charcoal meal through the small intestine. The highest which produces an irritating activity in the small intestine. inhibition of gut motility was, however, obtained with the Prostaglandins contribute to the patho-physiological func- antimuscarinic drug atropine sulphate [1, 21]. These tions of the gastrointestinal tract and act on the local observations demonstrate the inhibitory effect of C. tamala electrical and mechanical activities of the ileal circular extract on castor oil-induced diarrhoea, intraluminal fluid muscles [19]. Castor oil increases peristaltic activity and accumulation and peristaltic activity in small intestine. produces permeability changes in the intestinal mucosal membrane to electrolytes and waters [20]. Castor oil-induced fluid accumulation and Na+ and K+ secretion Small intestinal transit Cinnamomum tamala at 50 and 100 mg/kg showed a sig- As shown in Table 2, the charcoal meal travelled 77.7% of nificant and dose-dependent reduction in the intestinal fluid the total length of the small intestine in control rats. The accumulation by 32.5 to 65.0%. The intestinal fluids of all extract of C. tamala at the dose range of 50 and 100 mg/kg the groups were subjected to estimation of Na+ and K+ concentrations, and we observed a greater inhibitory effect + + Table 1 C. tamala on Na levels than on the K (Table 3). The physiological Effect of extract on castor oil-induced diarrhoea + in rats K concentration in the intestinal fluid was not significantly different as compared to the control group. On the other Treatment (mg/kg) Total amount of faecal matter Reduction (%) hand, Na+ concentration in the castor oil-induced intestinal Control 18 – fluid was reduced significantly (P \ 0.05) and dose Castor oil control 62 – dependently (50 and 100 mg/kg), and the inhibition was + + C. tamala 25 49 26.5 19.9 and 23.7% in Na and 6.2 and 15.4% in K , respec- + + C. tamala 50 36 42.0 tively. Membrane-bound enzyme Na - and K -ATPase C. tamala 100 24 61.3 have been related to sodium and potassium transport in the + Diphenoxylate 100 20 67.7 intestine [22]. When there is a decrease in Na and + Yohimbine 100 55 11.3 K -ATPase in diarrhoeal conditions relating to an inter- ruption in the normal water and electrolyte absorption, Values are presented as mean values of six rats in each group. Per- centage reduction as compared to castor oil control diarrhoea results. Therefore, the decrease of water together with Na+ accumulation might have an effect on the activity of Na+ and K+-ATPase [23]. The C. tamala extract thus Table 2 Effect of C. tamala extract on charcoal meal-stimulated stimulates the reabsorption of intestinal fluids in the small gastrointestinal transit in rats Treatment (mg/kg) Mean distance travelled Reduction (%) Table 3 C. tamala by charcoal [as % total Effect of extract on castor oil-induced fluid length of small intestine accumulation in rats (cm)] Treatment Intestinal Na+ (mmol/l) K+ (mmol/l) (mg/kg) fluid (ml) Control 24.6 ± 2.3 – Castor oil control 77.7 ± 4.2x – Control 1.2 ± 0.1 122.5 ± 10.8 4.8 ± 0.4 C. tamala 50 51.3 ± 3.8a 33.9 Castor oil control 4.0 ± 0.2y 162.6 ± 11.1x 6.6 ± 0.7 C. tamala 100 35.2 ± 3.1a 54.6 C. tamala 50 2.7 ± 0.2c 130.3 ± 9.6a 6.2 ± 0.5 Atropine sulphate 0.1 28.5 ± 2.6a 63.5 C. tamala 100 1.4 ± 0.1c 104.9 ± 10.1b 5.2 ± 0.4 Values are mean ± SEM for six rats. Percentage reduction as com- Values are mean ± SEM for six rats pared to castor oil control x P \ 0.01, y P \ 0.001 as compared to control x P \ 0.001 as compared to control a P \ 0.05, b P \ 0.01 and c P \ 0.001 as compared to castor oil a P \ 0.001 as compared to castor oil control control

123 J Nat Med (2008) 62:396–402 401 and large intestine and does not have any electrolyte Conclusion toxicity. Gut function is controlled by both the enteric nervous Effect on lipid peroxidation, catalase and SOD system and central nervous system. The mast cells play an important role in control of intestinal movements and It is generally accepted that reactive oxygen species-med- secretions. Our results suggested that C. tamala can iated peroxidation of lipid structures in the tissues results in increase the absorption of water and electrolytes from the extensive subcellular damage and plays a major role in the gastrointestinal tract since the extract decreased the small pathogenesis of gastrointestinal disorders [24]. The intes- intestinal transit and tannins as major principles. These tinal fluids in the castor oil group (0.55 ± 0.02) showed a findings justify that the Indian spice C. tamala is useful in significant increase in lipid peroxidation compared to the treatment of diarrhoea; because of this, it may be an control (0.31 ± 0.01). However, C. tamala extract at 50 important ingredient in the majority of Indian food and 100 mg/kg significantly reduced the LPO to 0.44 ± preparations. 0.02 and 0.32 ± 0.02 (P \ 0.001), whereas SOD levels were significantly increased in the castor oil group Acknowledgments This study was supported by grants from the compared to the control from 134.2 ± 9.3 to 197.6 ± 12.4 Department of Science and Technology, Ministry of Science and Technology, New Delhi. Authors are highly thankful to Dr. M. P. (P \ 0.01), and CAT showed a significant decrease from Dubey, Retired Scientist and Head, Department of Pharmacology, 48.6 ± 3.3 to 24.1 ± 1.7 (P \ 0.001). Treatment with Central Drug Research Institute, Lucknow, India, for his critical C. tamala at 50 and 100 mg/kg increased the catalase advice during the course of study. Authors are also thankful to the activity to 36.7 ± 2.2 (P \ 0.001) and 46.7 ± 3.0 Director of NBRI for providing necessary facilities. (P \ 0.01), respectively, with insignificant effect on SOD compared with control (Fig. 3). The increase in the thio- References barbituric acid reactive substances value, the index of tissue lipid peroxidation, in the castor oil-induced fluid 1. Showkat RM, Ali M, Kapoor R (2004) Chemical composition of accumulation was significantly inhibited by C. tamala. essential oil of Cinnamomum tamala Nees et Eberm. Leaves. Superoxide and hydroxyl radicals are major reactive Flavour Fragr J 19:112–114 2. Sharma SR, Dwivedi SK, Swarup D (1996) Hypoglycaemic and oxygen radicals contributing to injury in the stomach. Our hypolipidemic effects of Cinnamomum tamala Nees leaves. studies have accredited an increase in the level of mito- Indian J Exp Biol 34:372–374 chondrial SOD activity, which correlates well with an 3. Kujala TS, Loponen JM, Klika KD, Pihlaja K (2000) Phenolics increase in castor oil-induced fluid accumulation in rats. and betacyanins in red beetroot (Beta vulgaris) root: distribution and effect of cold storage on the content of total phenolics and The increased SOD level indicates increased production of - - three individual compounds. J Agric Food Chem 48:5338–5342 O2 within the tissue as elevated O2 level is thought to 4. Melo L, Thomas G, Mukherjee R (1988) Antidiarrhoeal activity increase the concentration of cellular SOD [25]. C. tamala of bisnordihydrotoxiferine isolated from root bark of Strychonus significantly and dose dependently reduced the LPO and trinervis (Vell.) Mart. J Pharm Pharmacol 40:79–82 5. Scarpignato C, Capovilla T, Bertaccini G (1980) Action of SOD levels and increased the catalase activity in compar- caerulein on gastric emptying of the conscious rat. Arch Int ison to the castor oil-induced groups. Pharmacodyn 246:286–294 6. Norton S (1954) Quantitative determination of mast cell frag- mentation of compound 48/80. Br J Pharmacol 9:494–497 7. Awouters F, Niemegeers CJE, Lenaerts FM, Janssen PAJ (1978) 250 y 0.6 Delay of castor oil-induced diarrhoea in rats; a new way to evaluate the prostaglandin synthesis. J Pharm Pharmacol 30:41– x b 0.5 200 45 0.4 8. Nwodo OFC, Alumanah EO (1991) Studies on Abrus precatori- b 150 ous seed II: antidiarrhoeal activity. J Ethnopharmacol 31:395– 0.3 398 100 0.2 9. Lutterodt GD (1989) Inhibition of gastrointestinal release of a acetylocholine by quercetine as a possible mode of action of 50 b y 0.1 Psidium guajava leaf extracts in the treatment of acute diarrhoeal disease. J Ethnopharmacol 25:235–247 0 0 (SOD CAT) & units/mg of protein

(LPO) nmols of MDA/mgof protein 10. Robert A, Nezamis JE, Lancaster C, Hanchar AI, Klepper MS Control Castor oil C tamala 50 C tamala 100 (1976) Enteropooling assay: a test for diarrhoea produced by SOD CAT LPO prostaglandins. Prostaglandins 11:809–814 11. Di Carlo G, Mascolo N, Izzo A, Capasso F, Autore G (1994) Fig. 3 Effect of C. tamala on mucosal lipid peroxidation (LPO), Effect of quercetin on gastrointestinal tract in rats and mice. superoxide dismutase (SOD) and catalase (CAT) levels in castor oil- Phytother Res 8:42–45 induced fluid accumulation in rats. xP\0.01, yP\0.001 as compared 12. Jamall IS, Smith JC (1985) Effects of cadmium on glutathione to control. aP\0.01 and bP\0.001 as compared to castor oil control peroxidase, superoxidase dismutase and lipid peroxidation in the 123 402 J Nat Med (2008) 62:396–402

rat heart: a possible mechanism of cadmium cardiotoxicity. 20. Bruton LL (1985) Agent affecting gastrointestinal water flux and Toxicol Appl Pharmacol 80:33–42 motility, digestants and bile acids. In: Gillman AG, Rall TW, 13. Claiborne A (1985) Catalase activity. In: Greenwald RA (ed) Nies AS, Tayloy P (eds) The pharmacological basis of thera- CRS handbook of methods for oxygen radical research. CRC peutics. McGraw-Hill, New York, p 914 Press, Boca Raton, pp 283–284 21. Rao Ch V, Amresh Kartik R, Irfan A, Rawat AKS, Pushpangadan 14. Nishikimi M, Rao NA, Yagi K (1972) The occurrence of P (2003) Protective effect of Aegle marmelos Correa fruit in superoxide anion in the reaction of reduced PMS and molecular gastrointestinal dysfunction in rats. Pharm Biol 41:558–563 oxygen. Biochem Biophys Res Commun 46:849–854 22. Shoba FG, Thomas M (2001) Study of antidiarrhoeal activity of 15. Kakkar P, Das B, Viswanathan PN (1984) Modified spectro- four medicinal plants in castor oil induced diarrhoea. J Ethno- photometric assay of SOD. Ind J Biochem Biophys 2:130–132 pharmacol 76:73–76 16. Brandt EB, Strait RT, Hershko D, Wang Q (2003) Mast cells are 23. Manonmani S, William S, Subramanian S, Govindaswamy S required for experimental oral allergen-induced diarrhea. J Clin (1991) Biochemical studies on the antidiarrhoeal effect of Invest 112:1666–1677 Cauvery-100, an ayurvedic formulation in rats. Biochem Int 17. Pulimood MD, Anna B, Minnie M, Mathan MD (1998) Quanti- 24:701–708 tative and ultrastructural analysis of rectal mucosal mast cells in 24. Halliwell B, Gutteridge JMC, Cross CE (1992) Free radicals, acute infectious diarrhea. Dig Dis Sci 43:2111–2116 antioxidants and human diseases: where are we now. J Lab Clin 18. Smits GJM, Romain A (1996) Influence of aging on gastric Med 119:598–620 emptying of liquids, small intestine transit, and fecal output in 25. Fridovich I (1986) Biological effects of superoxide radical. Arch rats. Exp Gerontol 31:589–596 Biochem Biophys 247:1–11 19. Sanders KM (1984) Evidence that prostaglandins are local reg- ulatory agents in canine ileal circular muscles. Am J Physiol 246:G361–G366

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