BioMed Research Year: 2014; Volume: 1; Issue: 1 Article ID: PM14 08; Pages: 1-7 The Open Access Publisher

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Research Article Antipyretic Activities of Methanol Extract of the sinense J.D. Zhao, L.W. Hsu & X.Q. Zhang on Albino Rats-An Experimental Study

Raja Naika and Ashok Chittaragi*

Department of Post Graduate Studies and Research in Applied Botany, Laboratory, Bio-Science Complex,

Jnana Sahyadri, Kuvempu University, Shankaraghatta-577451, Shimoga (District) Karnataka, INDIA

Correspondence should be addressed to Ashok Chittaragi

Received 25 July 2014; Accepted 30 July 2014; Published 12 August 2014

Copyright: © 2014 Ashok Chittaragi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract Aim: Antipyretic effect of the methanol extract of Ganoderma sinense against Brewer’s yeast induced pyrexia model in albino rats of both sexes was investigated. Methods: Pyrexia was induced by subcutaneously injecting 20% w/v Brewer's yeast suspension (20ml/kg) in the back below the nape of the neck of animals. Twenty four hours after the injection, the rectal temperature of each rat was measured. The temperature was measured at 30, 60, 90 and 120 min. after drug administration. Paracetamol (150mg/kg p. o.) was used as standard drug. The group received methanol extract 300mg/kg showed significant decrease in rectal temperature from 39.42± 0.24 to 35.50± 0.34 as compared with the group received standard drug. All experimental values are given as means ± standard deviation (SD). Statistical significance was determined by one-way variance analysis (ANOVA). Differences at P < 0.05 were considered to be significant. Results: The statistically processed results support the conclusion, that the methanol extract of Ganoderma sinense (300mg/kg) possesses dose dependent significant antipyretic activity. Conclusion: From the study it was concluded that Wild Mushroom of Ganoderma sinense possess significant antipyretic activity.

Key words: Ganoderma sinense, Methanol extract, Paracetamol, Antipyretic activity.

Introduction prevention and treatment of various types of Ganoderma is a genus of polypore mushrooms diseases, such as cancer, hepatopathy, arthritis, which grow on wood and include about 80 species, hypertension, neurasthenia and chronic hepatitis many from tropical regions [1]. Ganoderma has [2-4]. been used in traditional Asian medicines for the

BMR Phytomedicine www.bmrjournals.com ISSN 2349-4611 Among the genus, two species, Ganoderma sinense methanol extract of Ganoderma sinense mushroom and are the key species for the in antipyretic activity. production of medicinal materials because of their extensive use in traditional Asian medicines and Material and Methods their potential in bioremediation; they are a very important genus economically [5]. The practice of The present study was conducted on adult Albino herbal medicine dates back to the very earliest rat (150-200g) of either sex was procured from the period of known human history. There is evidence central animal house, National College of Pharmacy, of herbs having been used in the treatment of Shimoga district, Karnataka, India. The acute oral diseases and for revitalizing body system in almost toxicity study was carried out as per the guidelines all ancient civilization. Ayurveda, the Science of Life, set by Organization for Economic Co-operation and has provided a rationale basis for treatment of Development (OECD), revised draft guidelines 423, various ailments. Pain, inflammation and fever are received from Committee for the Purpose of Control very common complications in human beings [6]. and Supervision of Experiments on Animals It is the body’s natural defense to create an (CPCSEA), Ministry of Social Justice and environment where infectious agent or damaged Empowerment, Government of India. tissue cannot survive. Most of the antipyretic drugs The rats were acclimatized to laboratory condition inhibit Cox-2 expression to reduce the elevated for 5 days before commencement of the body temperature by inhibiting prostaglandin E2 experiment. The animals (six per cage) were (PGE2) biosynthesis. Moreover these synthetic maintained under standard laboratory conditions agents irreversibly inhibit Cox-2 with high (light period of 12 h/day and at room temperature), selectivity but are toxic to the hepatic cells, with access to commercial pellet diets and water ad glomeruli, cortex of brain and heart muscles, libitum. Food was withdrawn 12 hrs before and whereas the natural Cox-2 inhibitors have lower during the experimental hours. After the approval selectivity with fewer side effects. A natural by the Institutional Animal Ethical Committee, an antipyretic agent with reduced or no toxicity is experimental study was undertaken according to therefore essential. The demand for herbal their rules and regulations of the Committee for the medicines is increasing rapidly due to their fewer Purpose of Control and Supervision of Experiments side effects. Further as health care costs continue to on Animals. escalate, the attraction for low cost remedies has stimulated consumers to re-evaluate the potential Collection of mushroom and authentication of alternatives [7-10]. The Ganoderma sinense was collected from forest Recent studies show that the triterpenoids or GAs region which is located in Agumbe, Thirthahalli (T), from G. sinense and G. lucidum have various Shimoga (D), Karnataka, India, during the month of biological functions such as cytotoxicity to several June to August 2013. The G. sinense of mushroom cancer cells in vitro, or inhibition of tumor invasion was picked from the litter and decaying wood in-vitro and in-vivo, inhibition of human surface, with help of forceps and then they were immunodeficiency virus (HIV)-1 protease, cleaned and air dried in an oven at 40˚C for 48h. inhibition of eukaryotic DNA polymerases, dried mushroom samples were powdered inhibition of cholesterol synthesis and absorption, mechanically for further use. Identification was regulation of osteoclastogenesis and inhibition of done by comparing their morphological, anatomical U46619-induced platelet aggregation [11-19]. and physiological characteristics with the help of An extensive search of the literature reveals no standard literatures [20, 21]. The voucher specimen reports on the antipyretic activity of the (KUABARN-74) has been deposited at the mushrooms. Thus, present investigation was herbarium of mycology laboratory, Department of planned to find out the therapeutic level of P. G. Studies and Research in Applied Botany,

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BMR Phytomedicine www.bmrjournals.com ISSN 2349-4611 Kuvempu University, Jnana Sahyadri, Shimoga cause marked pain and distress should be avoided. district (Karnataka) for future reference. The method enables a judgment with respect to classifying the test substances to one of the series of

Chemicals toxicity classes defined by fixed LD50 cut-off values. Yeast was purchased from a local market. The temperature in the experimental animal room Paracetamol tablets 500mg were obtained from was kept 25±3˚C. Lighting was artificial, the Remidex Pharma Pvt. Ltd. sequence of 12 hours light, 12 hours dark followed. For feeding, conventional laboratory diet was used Extraction of mushroom with water ad libitum. The animals were randomly The air dried powdered mushroom (500g) was selected and kept in their cages for at least 5 days extracted with 90% methanol in a Soxhlet prior to dosing to allow for acclimatization to the apparatus at 60°C. The extract was filtered and laboratory conditions. Emulsion was prepared concentrated to dryness at room temperature to using 1% Tween 80 as surfactant and the avoid decomposition of natural metabolites. The concentrations were prepared according to yield (18.36%) was used for the experimental 1.5ml/100g of body weight. studies [22, 8, 23-28]. The extracts were administered in a single dose by gavages using a stomach tube, after fasting the Animals animals for 3-4 hours. In each step, six animals For this study healthy Wistar Albino rats (150- were used, starting dose of methanol extract of 200g) were procured from the central animal mushrooms was 100mg/kg body weight, then 200, house, National College of Pharmacy, Shimoga 500, 1000, 1500, 2000 and 3000. Animals were district, Karnataka, India was used. Rats were observed initially after dosing at least once during maintained under controlled condition at the first 30 minutes, periodically during the first 24 temperature in the experimental animal room was hours. In all cases death was observed within first kept 25±3˚C and humidity 55%. Lighting was 24 hours. Additional observations like changes in artificial, the sequence of 12 hours light, 12 hours skin, fur, eyes, mucous membrane, respiratory, dark followed. For feeding, conventional laboratory circulatory and autonomic and central nervous diet was used with water ad libitum. The animals systems and somatomotor activity and behavior were randomly selected and kept in their cages for pattern. Attention was also given to observations of at least 5 days prior to dosing to allow for tremors and convulsions. Further the animals were acclimatization to the laboratory conditions. The under investigation up to a period of one week [29- experiments and procedure used in this study were 32]. approved by the Ethical Committee of the National The LD50 value obtained for methanol extracts of College of Pharmacy, Shimoga district, Karnataka, mushrooms extracts was 3000mg/kg body weight, India. Therefore 1/10th weight of maximum tolerated dose were found to be 300mg/kg body weight of Acute oral toxicity study mushroom extracts of G. sinense. However, The acute oral toxicity of mushroom extract of 100mg/kg, 200mg/kg and 300mg/kg body weight Ganoderma sinense was carried out as per the were chosen for the pharmacological study. guidelines set by Organization for Economic Co- operation and Development (OECD), revised draft Antipyretic activity guidelines 423, received from Committee for the Antipyretic activity of alcoholic extracts of the Purpose of Control and Supervision of Experiments whole fruiting body of mushroom of G. sinense was on Animals (CPCSEA), Ministry of Social Justice and studied by Brewer’s yeast induced pyrexia method. Empowerment, Government of India. Principle of the method depends upon Evaluation of antipyretic activity: administration of lethal doses and doses which 3 BMR Journals| bmrjournals.com

BMR Phytomedicine www.bmrjournals.com ISSN 2349-4611 Hyperthermia was induced in mice by s.c. injection Statistical Analysis: of 20ml/kg of a 20% aqueous suspension of The results of statistical analysis for animal brewer's yeast in the back below the nape of the experiment were expressed as means ± standard neck. The animals were then fasted for the duration deviation (SD). Statistical significance was of the experiment (approximately 24 hrs), water determined by one-way variance analysis (ANOVA). was made available ad lib. Control temperatures The results obtained were compared with the were taken 24 hrs after the yeast injection to vehicle control group. The results were considered determine the pyretic response to yeast [33, 34]. significant at p<0.05. Pyrexia was measured by Digital thermometer. Temperature measured 1 hr prior to drug Results administration in fevered animals served as a pre- drug control. Rectal temperature of each rat was In acute toxicity study, it was found that all the animals measured using a digital thermometer after 24 hrs were safe at a dose of 3000mg/kg body weight and of administration of yeast suspension. Rats those there was no abnormal behavior. The 1/10th and 1/5th shown an increase in temperature of at least 0.7°C tolerated dose i.e. 200mg/kg body weight and used for the experiments. After it animals were 300mg/kg body weight were selected as a therapeutic divided into five groups (5 groups) and each group dose for antipyretic studies. In the present study the containing six animals. Group-I acted as control antipyretic effect of various concentrations of methanol which received vehicle (distilled water 5ml/kg-1 extract, with that of Paracetamol at different times are b.w., p.o.). Group-II, III, IV and V acted as study compared. This anti-pyretic effect appears to be dose groups. Group-II received standard drug dependent as 100mg/kg b.w. and 200mg/kg b.w. dose (Paracetamol 150mg/kg b.w., p.o.) was given 24 hrs did not produce any pyrexia lowering effect but at the after the yeast injection, p.o. and Group-III, IV and V 300mg/kg b.w. dose showed (Table-1) the anti-pyretic were treated with graded doses of methanol extract effect on and after 90 min, comparable to that of 100, 200 and 300 mg/kg b.w., per oral respectively. standard drug (Paracetamol) and was then sustained The rectal temperature was recorded at 30, 60, 90, during the whole observation period (Figure-1). This and 120 min after its administration [34]. may be due to ineffective content of flavanoids in extract dose (100 and 200mg/kg b.w.).

Table 1: Antipyretic activity of methanol extract of Ganoderma sinense on rats

Drug Dose Temperature °C±SEM (mg/kg) Initial Pyretic 30 min 60 min 90 min 120 min oral Normal vehicle 1 ml 37.4±0.09 37.33±0.29 37.89±0.16 37.80±0.07 37.93±0.04 37.78±0.12

Standard 150 37.2±0.05 39.22±0.34 39.34±0.27 39.66±0.50 39.68±0.30 39.75±0.55 (Paracetamol) Methanol 100 37.3±0.29 39.45±0.19 38.88±0.43 38.58±0.29* 38.27±0.21* 38.31±0.24* extract 200 37.4±0.06 39.24±0.34 36.64±0.34* 36.04±0.53* 36.17±0.13* 36.51±0.21* 300 37.3±0.05 39.42±0.24 35.05±0.40* 35.62±0.28* 35.41±0.40* 35.50±0.34*

Values are mean± SEM (n=6), *p<0.05, **p<0.01 (p value as compared to control group)

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Figure 1: Effect of the methanol extract of Ganoderma sinense on yeast-induced pyrexia in rats

Normal vehicle 1ml 40 39 Standard 38 (Paracetamol) C) 37 150mg/kg 36 Methanol extract 100mg/kg 35

Body Temp (0º Temp Body 34 Methanol extract 33 200mg/kg 32 Methanol extract Initial Pyretic 30 min 60 min 90 min 120 min 300mg/kg Time (min)

Discussion The present study reveals that the methanol extract of Ganoderma sinense causes a significant In general non steroidal anti inflammatory drugs antipyretic effect in yeast provoked elevation of produce their antipyretic action, through inhibition body temperature. In the cases, the ethanol extract of prostaglandin synthesis within the hypothalamus caused a significant lowering of body temperature, [35]. Therefore it appears that antipyretic action of with the effect being comparable to that of methanol extract of Ganoderma sinense may be Paracetamol. Thus the present pharmacological related to the inhibition of prostaglandin synthesis evidence provides support for the folkfore claim as in hypothalamus. Fever may be a result of infection an antipyretic agent. Flavonoids are known to or one of the sequels of tissue damage, target prostoglandins which are involved in the late inflammation, graft infection or other diseases phase of acute inflammation, pyrexia and pain states. Regulation of body temperature requires a perception. Flavonoids reduce lipid peroxidation by delicate balance between the production and loss of preventing or slowing the onset of cell necrosis and heat and the hypothalamus regulates the set point by increasing the vascularity. Hence the presence of at which body temperature is maintained. In fever flavanoids in the methanol extract of Ganoderma this set point is elevated and drugs like Paracetamol sinense may be contributory to its antipyretic don’t influence body temperature when it is activity [37]. elevated by factors such as exercise or increase in ambient temperature [36]. Yeast-induced pyrexia is called pathogenic fever and it’s a etiology involves Conclusion production of prostaglandins. The effect of the In the present pharmacological evaluation the drugs may be due to inhibition of prostaglandin methanol extract of Ganoderma sinense mushroom synthesis [34]. was extensively investigated for its antipyretic

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BMR Phytomedicine www.bmrjournals.com ISSN 2349-4611 activity against Brewer’s yeast induced pyrexia of Pharma and Pharmaceutical Science 2005, model in rats. At the end of our study, a strong 8(3): 558-64. conclusion can be drawn that, the methanol extract 8. Khan A, Baki MA, Alim Al-Bari MA, Hasan S, of Ganoderma sinense possess Antipyretic activity Mosaddik MA, Rahman MM, et al : Antipyretic Activity of Roots of Laportea crenulata Gaud in more or less depending on the dose levels. The Rabbit. Res J Med Med Sci 2007, 2: 58-61. methanol extract of Ganoderma sinense has 9. Valarmathi R, Rajendran A, Akilandeswari S, antipyretic effect supporting the ethno Senthamarai R: Study on Antipyretic Activity of a pharmacological use as antipyretics. The effect may Mollugo pentaphylla Linn in Albino Mice. be explored in the use of the plant in the International Journal of Pharm. Tech Research management of some other diseases. 2010, 2: 2388-2390. 10. Jaiswal A, Sutar N, Garai R, Pati MK, Kumar A: Acknowledgment Antipyretic Activity of Platycldus orientalis leaves extract. International Journal of Applied Authors are thankful to Dr. I. J. Kuppasth (Principal, and Pharmaceutical Technology 2011, 2(1): National College of Pharmacy, Shimoga, Karnataka, 175-178. India) for providing us a platform for this research 11. Chen YS, Ming SS, Cheng TW: Differential effects work for his valuable guidance and precious time. of ganodermic acid S on the thromboxane A2- signaling pathways in human platelets. Biochem. References Pharmacol 1999, 58: 587-595. 1. Kirk PM, Cannon PF, Minter DW, Stalpers JA: 12. Mizushina Y, Takahashi N, Hanashima L, Koshino Dictionary of the Fungi. 10th ed. Wallingford: H, Esumi Y, Uzawa J, Sugawara F, Sakaguchi K: CABI 2008, p. 272. Lucidenic acid O and lactone, new terpene 2. Liu GQ, Zhang KC: Mechanisms of the anticancer inhibitors of eukaryotic DNA polymerases from action of Ganoderma lucidum (Leyss. ex. Fr.) a basidiomycete, Ganoderma lucidum. Bioorg. Karst: A new understanding. J. Integr. Plant Biol Med. Chem 1999, 7: 2047-2052. 2005, 47: 129-135. 13. Min BS, Gao JJ, Nakamura N, Hattori M: 3. Shiao MS: Natural products of the medicinal Triterpenes from the spores of Ganoderma Ganoderma lucidum: Occurrence, lucidum and their cytotoxicity against meth-A biological activities, and pharmacological and LLC tumor cells. Chem. Pharm. Bull 2000, functions. Chem. Rec 2003, 3: 172-180. 48: 1026-1033. 4. Weng CJ, Yen GC: The in-vitro and in-vivo 14. Kimura Y, Taniguchi M, Baba K: Antitumor and experimental evidences disclose the antimetastatic effects on liver of triterpenoid chemopreventive effects of Ganoderma lucidum fractions of Ganoderma Mechanism of action and on cancer invasion and metastasis. Clin. Exp. isolation of an active substance. Anticancer Res Metastasis 2010, 27: 361-369. 2002, 22: 3309-3318. 5. Liu GQ, Wang XL, Zhang YG, Wu YH, Han WJ and 15. Miyamoto I, Liu J, Shimizu K, Sato M, Kukita A, Zhang HY: Promotion of ganoderic acid Kukita T, Kondo R: Regulation of production in Ganoderma sinense by the addtion osteoclastogenesis by ganoderic acid DM of an ether extract from Eupolyphaga sinensis, a isolated from Ganoderma lucidum. Eur. J. medicinal insect. African Journal of Pharmacol 2009, 602: 1-7. Biotechnology 2010, 9(37): 6129-6134. 16. Sato N, Zhang Q, Ma CM, Hattori M: Anti-human 6. Rathore MK, Sharma MC, Goyal M, Singh GK, immunodeficiency virus-1 protease activity of Gupta S: Antipyretic activities of ethanolic new lanostane type triterpenoids from extract of the whole plant of Fagonia Ganoderma sinense. Chem. Pharm. Bull. (Tokyo) schweinfurthii Hadidi on albino rats-An 2009b, 57: 1076-1080. experimental study. Spatula DD 2012, 2(3): 17. Chen NH, Liu JW, Zhong JJ: Ganoderic acid T 159-164. inhibits tumor invasion in-vitro and in-vivo 7. Chattopadhyay D, Arunachalam G: Antipyretic through inhibition of MMP expression. Activity of Alstonia macrophylla Wall ex A. DC: Pharmacol. Rep 2010, 62: 150-163. An Ethnomedicine of Andaman Islands. Journal

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