Studies on Antipyretic-Analgesic and Ulcerogenic Activity of Polyherbal...

[Full title: Studies on Antipyretic-Analgesic and Ulcerogenic Activity of Polyherbal Preparation in Rats and Mice]

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Life International Journal of Pharmacology ISSN 1811-7775 Life science alert ansinet Asian Network for Scientific Information

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International Jounal of Pharmacology 4 (2): 88-94, 2008 ISSN 1811-7775 2008 Asian Network for Scientific Information Studies on Antipyretic-Analgesic and Ulcerogenic Activity of Polyherbal Preparation in Rats and Mice ¡M. Gupta, 'B.P. Shaw and A. Mukerjee 'Institute of Post Graduate Ayurvedic Education and Research, 294/3/1, APC Road, Kolkata-700009, India Division of Pharmaceutical Technology, Department of Chemical Technology, Calcutta University, 92, APC Road, Kolkata-700009, India Abstract: The aqueous extract of polyherbal Ayurvedic preparation PD-10 (from the rools of Hemidesmus indicus R. Br. (Asclepiadaceae), Rubia cordifolia L. (Rubiaceae), Cissampelos pareira L. (Menispermaccac): fruits of Terminalia chebula Retz. (Combretaceae). Emblica officinalis Gacitn. (Euphorbiaceae), Terminalia bellirica Roxh. (Combretaceae), Vitis vinifera L. (Vilaceae). Grewia asiatica T.. (Tiliaceae), Salvadora persica L. (Salvadoraceae) and granules of Saccharum officinarum L. (Poaceae)) was investigated for antipyretic property. The extract caused significant (p<0.05) antipyretic activity induced pyrexia by using Brewer's yeast in rats. The evaluation of analgesic activity of PD-10 using acetic acid induced writhing model, hot plate method and tail immersion methods in mice revealed very significant (p<0.01) analgesic activity. The ulcerogenicity effect of PD-10 studied at different dosages by Barrel's method in rats showed significantly lesser ulcer effect even at very high dosage as compared to that of aspirin. These data confirm the antipyretic, analgesic and ulcerogenic properties of the polyherbal Ayurvedic preparation (PD-10) as cminciated in the traditional texts. Key words: Polyherbal preparation, antipyretic, analgesic, anti-ulcerogenic INTRODUCTION Antipyretic-analgesics and more particularly the nonsteroidals account for the largest class of medication in demand worldwide. However, use of most of these formulations has been associated with gastrointestinal renal, hepatic, central nervous system and dermatological effects (Simon, 1995; Suleyman et al., 2007). On the other hand, traditional Indian systems of medicine, such as Ayurveda are based on holistic treatment of diseases. primarily relying on natural herbal drugs. Ayurvedic literatures like Charak Samhita provide a rich text for herbal drugs that are available till today. This indicated the possibility to explore into the literatures and concepts of holistic Ayurvedic medication systems for some antipyretic-analgesic preparations with lesser untoward effects. According to Ayurveda, pyrexia originates from a combination of indigestion, seasonal variations and significant alterations in daily routine. Charak has defined the ten antipyretic drugs in Jwarhar Mahakashay Sulra. Sthana of Charak Samhita (Shastri, 1988). Most Ayurvedic preparations are polyherbal in nature to take care of the multiple components of disease conditions. This Jwarhar Mahakashay group of antipyretic drugs includes Sariva (Temidesmus indicus R. Br.), Manjistha (Rubia cordifolia L.), Palha (Cissampelos pareira L.). Haritaki (Terminalia chebula Retz.). Amala (Emblica officinalis Gaertn.), Vibhitak (Terminalia bellirica Roxb.), Draksha (Vitis vinifera L.), Parushak (Grewia asiatica L.), Peelu (Salvadora persica L.) and Sharkara (Saccharum officinarum L.) plants. These medicinal plants have been in traditional usage for treatment of pyrexia as detailed in ancient Ayurvedic texts like Charak Samhita and Susrula Sambila. Many of these medicinal plants have been individually reported to exhibit diverse pharmacological actions such as anti-inflammatory, analgesic, hepato-protective, antimicrobial and antiulcer properties as shown in Table 1 (Nijveldt et al., 2001; Anoop and Jagadeesan, 2003; Perianayagam et al., 2001; Ledon al., 2003; Kasture et al., 2001; Saito et al., 1998; Monforte et al., 2001 Anand et al., 1994; Valsaraj et al., 1997; Asmawi et al., 1993; Gulati et al., 1995). Analysis of the et Corresponding Author: Dr. Mradu Gupta, Department of Dravyaguna, Institute of Post Graduate Ayurvedic Education and Research, 294/3/1, APC Road, Kolkata-700009, India Tel: 91-33-24614461, 91-9433665125 88

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Int. J. Pharmacol., 4 (2): 88-94, 2008 Table 1: Pharmacological properties and chemical constituents of Jwarhar Mahakashay drugs Pharmacological properties reported Anti-inflammatory, Anti-ulcer Tumour-inhibitor Anti-inflammatory, anti-bacterial Name Sariva Scientific namie Family Hemidesmus indicus R. Br. Asclepiadaccac Parts used Rools Sharkara l'atha Saccharum officinarum L. Cissampelos pareira L. Poaceae Granules Anti-inflammatory, analgesic Menispennaccac Roots Manjistha Rubia cordifolia L. Rubiaceae Fruits Draksha Vitis viutera.. Vitaceae Truits Anti-ulcer, hepatoprotective Peelu Salvadora persica L. Salvadoraceae Fruits Anti-ulcer, anti-microbial l'arus hak Grewia asiatica L. Tiliaccac Fruits Haritaki Terminalia chebnda Retz. Combretaceae Fruits Anti-microbial, purgative Vibhitak Terminalia bellirica Roxb. Combretaceae Truits ITepato-protective, anti-histaminic Amala Emblica officinalis Gaertn Euphorbiaceae Fruits Anti-ulcer, anti-inflammatory, hepato-protective chemical constituents of these plants revealed the presence of tannins, triterpenoids, phenols, glycosides, sucrose, glucose, flavonoids and flavonidic glycosides as shown in Table 1 (Sharma et al., 2001; Kirtikar and Basu, 1984, Baltenweck-Guyot et al., 2000; Foo et al., 1998; Kundu and Mahato, 1993). Ilowever, there was no scientific evaluation of the antipyretic-analgesic effect of this traditional polyherbal Ayurvedic preparation till now even though it was observed to be inhibiting the pyretic ignition mechanism and was clinically effective. The current research intends to systematically evaluate the antipyretic and analgesic activity as well as the acule gastrointestinal toxicity of such a polyherbal preparation made following Ayurvedic literature experiments. MATERIALS AND METHODS 1I1 rodent Preparation of polyherbal extract (PD-10): The roots of Sariva (Hemidesmus indicus R. Br.), Manjistha (Rubia cordifolia L.) and Patha (Cissampelos pareira L.), fruits of Haritaki (Terminalia chebula Retz), Amala (Emblica officinalis Gaertn.), Vibhitak (Terminalia bellirica Roxb.), Draksha (Vitis vinifera L.), Parushak (Grewia asiatica L.) and Peelu (Salvadora persica L.) and Sharkara granules (Saccharum officinarum L.) were obtained from the Apothecary department of the Institute of Post Graduate Ayurvedic Education and Research. Kolkata during May 2006. These were authenticated and identified by the Department of Ethnobotany, Botanical Survey of India, Shibpur, Howrah and a voucher specimen was deposited in the herbarium before their utilization (Table 1). Plant parts were shade dried and coarsely powdered up to 10 mesh size. Equal portions by weight of all ingredients were homogenously mixed and subjected to soxhlet extraction in refluxing distilled water. The Anti-malarial, anti-ulcer Active chemical constituents Flavonoids, biterpenoids, tannin, phytosterol. SS-sitosterol Flavonoids, glucosides, glycans, glucose, sucrose, phenols Alkaloids, berberin, hayatinc, cissanpareine Glycosides, anthraquinone, mibiadin, triterpenes Flavonoids, glucose, tinctose, glycosides, polyphictiols Tannin, salvadorin, glucose, trictose Flavonoids, tannin, glucose, glycosides Tanin, triterpenes, chebulinic acid, glycosides Gallic acid, glycosides, chebulagic acid, triterpenoids Vitamin C, elagic acid, polyphenol, glucose, phyllemhin extraction was continued for 48 h using distilled water four times by weight of the crude drug mixture. The aqueous extract was filtered through calico cloth and was further concentrated to solid under reduced pressure over water bath in a rotary evaporator. The extract yield from 300 g of plant powder mixture was 75 g. This warhar Mahakashay preparation was termed PD-10 for all systematic evaluation studies and preserved at the Division of Pharmaceutical Technology Laboratory. Department of Chemical Technology, Calculla University, Kolkata, where the experimental studies were carried out. during 2006-07. Experimental animals: Colony bred Swiss albino mice (20-25 g) and Wister rats (120-130 g) obtained from the Indian Institute of Chemical Biology (LLC.B.), Kolkata were used for this study. They were housed in polypropylene cages in the well-ventilated animal house facility of the Department of Chemical Technology, Calculla University, Kolkata. The animals were maintained with standard pellet diet and potable water ad libitum. They were then divided in seven groups of six each for the five different test drug (PD-10) dosage groups, the standard (Aspirin/Morphine sulphate) group and the control (saline) group. For all pharmacological evaluations including toxicity studies, prior approval was obtained from the Animal Ethics Committee under the faculty of Chemical Technology of Calcutta. University (Reg. No. 506/01/a/CPCSEA DL 31.10.2001). Test drugs: Aspirin and Morphine Sulphate procured from M's Dey's Medical Stores, Kolkata were used as standard drugs and PD-10 was used in different oral dosage for comparison of efficacy. Normal saline has served as the vehicle control. 89

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Int. J. Pharmacol., 4 (2): 88-94, 2008 Phytochemical analysis: Standard phytochemical test was used in screening the extract for different constituents. Briefly, FeCl, test was used to characterize for tannins and salicylates, Dragendorf's reaction and Mayer's test was used for alkaloids and Fehling's test was used for reducing sugars. Similarly, IIC1-Magnesium test was used for flavonoids while frothing test was deployed for saponins and the Liebermann Buchard reaction was used for delecting the presence of triterpenoids and steroids (Sawadogo et al., 2006b; Furniss et al., 1989). Toxicity studies: Acute toxicity was estimated following the graphical method of Litchfield and Wilcoxon (1945) in mice. Different doses for test drug PD-10 extract (1000, 2000, 4000, 6000 and 8000 mg kg) were administered p.o. to 5 groups of 6 Swiss albino mice each. The Control group received normal saline (5 ml. kg po.). Animals were under observation for 72 h, during which their behavioral patterns were noted and signs of toxicity recorded. All deaths occurring during the first 24 h were meticulously noted. Antipyretic activity studies on PD-10: The antipyrelie activity was evaluated in Brewer's yeast induced pyretic model in rats (Hajare et al., 2000; Ghosh, 1984). All the Wister rats were fasted for 12 h water ad libitum and then divided into seven homogenous groups. Saline (0.5 mL) was administered orally to the control group, Aspirin (100 mg kg) p.o. was given to the standard group while The five drug treated groups were administered with 100 200, 300, 400 and 500 mg kg 1 bxxly wl of the test drug PD-10 p. o. respectively. The rectal temperatures of all the animals were noted down at the beginning of the experiment. Each animal was given the prescribed drug p.o. and was immediately injected with 10 mL kg of 15% w/ yeast solution in water subcutaneously to induce fever. The rectal temperature was thereafter noted in degrees Centigrade at the end of 1, 2, 3, 4 and 24 h. Analgesic activity studies on PD-10: The central analgesic activity against thermal stimulus was recorded. in mice following hot plate method as well as tail immersion method (Hajare et al., 2000; Ghosh, 1984). Morphine sulphate (2.5 mg kg, i.m.) was used as a standard drug. The test drug PD-10 dissolved in water was administered p.o. in doses of 100, 200, 300, 4100 and 500 mg kg body wt to different drug groups 1 h before applying the thermal stimulus, which was maintained at 55±0.2°C. The latency in hind paw licking and clear tail withdrawal were recorded as responses after 10, 30 arid 60 min of drug administration in the hot plate method and tail immersion methods respectively. Maximum reaction time of observation was about 60 sec throughout to avoid tissue damage. 90 The peripheral analgesic activity of PD-10 was evaluated in acetic acid induced writhing experiments in mice. The abdominal constriction writhings resulting from intraperitoneal injection of acetic acid (10 ml kg of 0.6% v/v glacial acetic acid solution in water) were recorded according to standard procedure (Veerappan et al., 2005; Nwalor and Okwunsaba, 2003). The animals were divided into seven groups of 6 mice each. The test drug PD-10 dissolved in water was administered i.p. in doses of 100, 200, 300, 400 and 500 mg kg body wt to the five test drug groups, while saline was administered to the control group Aspirin (100 mg kg po.) was used as the standard drug for comparison Acetic acid solution was administered after 30 min and number of writhings counted in each animal for 15 min. Percentage inhibition response was calculated as the reduction in the number of abdominal constrictions between control group and test drug treated groups as a percentage of the number of wriths observed in case of the control group. Assessment for ulcerogenic effect: Acute ulcerogenicity was evaluated in Wister rats divided in 6 groups, housed in individual cages and fasted for 24 h prior to administration of the test drug or aspirin (Shay et al.. 1945). Four drug-treated groups were administered with the experimental drug PD-10 in po. doses of 200, 500,1000 and 1500 mg kg, respectively. Aspirin was administered at 200 mg kgp.o. dose. Animals were euthanited after 12 h of drug treatment, alxlomen was opened and the stomach was cut open along the greater curvature (Rifal-uz-Zaman et al., 2006). The stomach content was cleaned and washed with saline. The mucosal surface was examined by two independent observers. The degree of ulceration was graded from zero to five (0-5) depending on the size and severity of ulcers (Barret et al., 1953). Statistical analysis: All values were expressed as mean SEM. Analysis of variance was performed by ANOVA procedures (Pipkin and Livingstone, 1981). Differences were considered statistically significant at p0.05 and very significant at p<0.01. RESULTS Phytochemical analysis: Phytochemical analysis of the PD-10 extrael. revealed the presence of famins, reducing sugars, flavonoids, glycosides and salicylates. Toxicity study: Detailed studies revealed very low toxicity a ni mortality even al very high dosage of 8000 mg kg b.wt. during acute toxicity tests. During initial period (up to 1h), the higher test dose (6000 and

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Decrease in rectal temperature (°C) 1.5 Int. J. Pharmacol., 4 (2): 88-94, 2008 -PD-10 400 mg kg Table 2: Average reaction time in hot plate method Control (saline) Aspirin 100 mg kg¯ ± PD-10 500 mg kg PD-10 100 mg kg PD-10 200 mg kg -*-PD-10 300 mg kg L 1.0- 0.5Group Dose (mg kg p.o. Control 10 min 5.75010.438 30 min 5.43310.235 60 min 6.33310.329 (saline) Morphine 29 PD-10 100.0 9.18310.291* 5.883±0.386 11.15010.111* 11.01710.297 5.983±0.224 6.833±0.312 PD-10 200.0 6.350±0.231° 6.517±0.221° 7.533±0.267° PD-10 300.0 7.23310.300h 7.43310.288 9.73310.297 PD-10 400.0 9.333+0.274 9.700+0.376* 10.533+0.229 PD-10 500.0 10.25010.217 11.76710.126 12.38310.368 Significance relative to control: a: p<0.001, b: p<0.01, c: p<0.1 (ANOVA test; Values represent mean-SEM (n Table 3: Average reaction time in tail immersion method 0.0Dose 2 h 3h 4Th Group Control (mg kg )p.o. 10 min 5.083+0.384 30 min 5.183+0.368 60 min 5.133+0.384 (saline) Morphine 2.5 PD-10 100.0 -0.5PD-10 200.0 PD-10 300.0 9.917±0.280 10.117±0.256" 10.817±0.412" 5.78310.138 5.81710.128 5.98310./190 6.017±0.187 6.300±0.188 6.600±0.153° 6.433±0.263 6.950±0.543 7.067±0.599° PD-10 400.0 8.517+0.289 9.100+0.310 9.500+0.350PD-10 500.0 10.650±0.281* 11.233±0.336 11.367±0.331- -1.0Significance relative to control: a: p<0.001, b: p<0.01, c: p<0.1 (ANOVA test; Values represent mean-SEM (n ) Table 1: Effects of PD-10 and aspirin on writhing induced by acetic acid in mice -1.5 Fig. 1: Decrease in average rectal temperature w.r.t. initial temperature. Vertical bars are mean±SEM (n = 6) 8000 mg kg) treated animals showed some symptoms of decreased motor activity and respiratory distress. The animals recovered thereafter and no mortality was recorded. Antipyretic activity of PD-10 in rats: The Antipyrelic effect for p.o. administration of PD-10 was dose dependent and the response at 100 and 500 mg kg¯ dosages was more pronounced compared to that of 100 mg kgp.o. aspirin (Fig. 1). Marked antipyretic response for PD-10 at 100 and 500 mg kg p.o. dosages was observed (p<0.05) even al. 24 h of treatment. The polyherbal preparation PD-10 has proved to be significantly antipyretic in Brewer's Yeast pyrexia model. Analgesic activity in mice: Table 2 and 3 shows the analgesic activity due to thermal stimulus following hot plate analgesia and tail immersion methods in mice. The central analgesic activity was found to be comparatively weaker at lower dosages of the test drug as compared to The standard drug (morphine sulphate). However, the nociceptive response at 400 and 500 mg kg dosage of PD-10 (p<0.01) was quite comparable to that of morphine sulphate. Group Control (salinc) Dose (mg kg ) ip. No. of writhings Aspirin PD-10 5.33±0.42 3.67+0.216 PD-10 PD-10 Inhibition (%) - 100 31.3 100 4.17+0.17 21.9 200 3.83±0.31 28.1 300 3.6710.21h 31.3 400 3.3310.216 37.5 500 2.67+0.21° 50.0 PD-10 PD-10 Significance relative to control: a: p<0.001, b: p<0.01, c: p<0.1 (ANOVA test); Values represent mean-STM (n = 6) Table 5: Effects of PD-10 and aspirin on ulceration in rats Group Control (saline) Aspirin PD-10 PD-10 PD-10 PD-10 Dose (img kg po. 200 200 500 1000 1500 Score for degree of ulceration 1.3310.21 3.00±0.20 1.6710.21 1.83±0.31 2.17±0.31 2.50 0.22 Significance relative to control: a: p<0.001, b: p<0.01, c: p<0.1 (ANOVA test); Values represent mean-STM (n = 6) Peripheral analgesia response due to oral doses of PD-10 was very significant (p<0.01) at 300, 400 and 500 mg kg p.o. dosage and was more pronounced than aspirin p.o. dosage of 100 mg kg (Table 1). Significant dose dependant inhibition of control wriths were observed at 100, 200, 300, 100 and 500 mg kg p.o. doses of PD-10 and compared well in range with aspirin p.o. dosage of 100 mg kg¯¯ Acute ulcerogenicity in rats: Results of acute ulcerogenicity were studied in rats and the results were shown in Table 5. PD-10 exhibited almost negligible 91

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Int. J. Pharmacol., 4 (2): 88-94, 2008 ulcerogenicity at dosage of 200 and 500 mg kg-1. Ulcerogenic responses due to 1000 and 1500 mg kg p.o. dosage of PD-10 were also markedly low as compared to aspirin dosage of 200 mg kg¯¹. DISCUSSION The polyherbal Ayurvedic preparation PD-10 exhibited marked antipyretic effect in yeast pyrexia model and the response is dose-dependent. Oral doses of PD-10 at 300, 100 and 500 mg kg produced significant antipyretic effect in test animals. Fever is known to be associated with the production of prostaglandins in the hypothalamus (Okokon et al., 2008). Yeast pyrexia is generally associated with prostaglandin's responses in later hours (1 h) of yeast treatment, while the initial (1 to 4 h) pyrexia response is associated with a number of factors including the presence of histamine and bradikinin. Marked and sustained antipyretic response due to PD-10 is perhaps indicative of a prostaglandin receptor mediated response (Bennell and Plum, 1996). The central analgesic effect of PD-10 was found to be weaker at lower dosage (100, 200 and 300 mg kg ) as compared to the standard drug, morphine sulphate. However, the central analgesic activity of PD-10 was quite pronounced and noticeable at higher doses (400 ankl 500 mg kg) and comparable to the standard drug. The results of both hot plate method and tail immersion method indicate very significant (p<0.01) central analgesic activity in PD-10. The thermal test is sensitive to strong analgesics (Ibironke and Ajiboye, 2007) and the test drug response was dose-dependent, especially at higher. ranges PD-10 inhibited acetic acid induced writhing in mice (p<0.01). Percentage reduction in normal acetic acid writhings was highly pronounced at 300, 400 and 500 mg kg po. doses of lest drug (31.3, 37.5 and 50.0%) when compared with pure compound aspirin (31.3%). The abdominal contractions (writhings) were directly related to peripheral responses due to prostaglandins. Therefore, PD-10 components exhibited pronounced inhibitory responses either in synthesis, release or receptor reactions in prostaglandin mediated effects. Nociceptive response due to the test drug, although dose dependent was pronounced at higher dosages and comparable to that of morphine (2.5 mg kg 'im.). The ability of the extract to inhibit acetic acid-induced writhing in mice. (a model of visceral pain) indicated that it could be useful in the management of visceral pain (bironke and Ajiboye, 2007). Components of PD-10, therefore, definitely possess some central analgesic responses. Increased body temperature and pain are two major signs of the body against inflammation (Meli et al., 2001). A drug with anti-inflammatory activity usually also exhibits anti-pyretic and analgesic properties. The best examples would be the Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) which possess anti-inflammatory, analgesic and antipyretic properties (Perianayagam et al., 2004; Buffum and Buffum, 2000). Pronounced antipyretic action due to PD-10 possibly mediated through inhibition of prostaglandin production especially since the extract. has been shown to exhibit analgesic and antiinflammatory activities. Preliminary phytochemical screening of the extract indicated the presence of tannins, reducing sugars, flavonoids, glycosides and salicylates. Of these, flavonoids are well known for their ability to inhibit pain perception (Sawadogo et al., 2006a) and to exhibit antiinflammatory properties due to their inhibitory effects on enzymes involved in the production of the chemical mediator of inflammation (Oweyele et al., 2005). Flavonoids and its related compounds also exhibit inhibition of arachidonic acid peroxidation, which results in reduction of prostaglandin levels thus reducing the fever (Baumann et al., 1980). Since flavonoids exhibit several biological effects such as anti-inflammatory, antimicrobial, anti-hepato-toxic and anti-ulcer activities (Narayana et al., 2001; Nijveldt et al., 2001), it is likely that The antipyretic action of PD-10 preparation is primarily related to the presence of flavonoids. PD-10 has proved to be very low ulcerogenic even in high p.o. doses of 1000 and 1500 mg kg as compared to p. o. aspirin dosage of 200 mg kg, validating its efficacy as a polyherbal therapeutic compound having minimal adverse side-effects as compared to standard nonsteroidal antipyretic analgesic drugs. The low ulcerogenicity of the test drug could be primarily on account of the anti-ulcerogenic activity exhibited by many of its constituent plants as shown in Table 1 (Anoop and Jagadeesan, 2003; Saito et al., 1998; Monforte et al., 2001). Antipyretic-analgesic action for PD-10 could be attributed to the presence of lavonoids in the constituent. herbs, since flavonoids normally exhibit antipyretic, analgesic and anti-inflammatory properties. The preparation also proved to be much less ulcerogenic as compared to the standard Non steroidal anti inflammatory (NSAIDS) drugs. The extract PD-10 therefore continues to be used with significant success as an antipyretic compound in traditional clinical practice in several parts of India and the specific attributes ascribed to it in ancient Ayurvedic literature is thus justified and revaluated. 92

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