Free Radical Scavenging and Lipid Peroxidation Inhibition by Terpenes Isolated from Red Mushroom (Ganoderma lucidum (Fr.) P.

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1 Human Journals Research Article May 2015 Vol.:3, Issue:2 All rights are reserved by Hamid Bakshi et al. Free Radical Scavenging and Lipid Peroxidation Inhibition by Terpenes Isolated from Red Mushroom (Ganoderma lucidum (Fr.) P. Karst) Keywords: Antioxidant, DPPH assay, FRAP assay, Ganoderma lucidum, Terpenes ABSTRACT Hamid Bakshi 1 *, Smitha Sam 2, Saud Iqbal 1, Sajid Hussain 5, Muhammad Zubair Chaudhry 3, Jackson Achankunju 1, Syed Shahid Ali 1, 4 1. Department of Food Science and Human Nutrition, College of Applied Science, ASharqiyah University, Ibra-400, Sultanate of OMAN. 2. Department of Research, Jawaharlal Nehru Cancer Hospital and Research Center, P.B No 32, Idgah Hills, Bhopal , Madhya Pradesh, INDIA. 3. Ibra Polyclinic, Safala, Ibra-413, sultanate of OMAN 4. Institute of Environmental Toxicology, School of Environmental Science. Louisiana State University, Baton Rouge, LA-70803, USA. 5. Department of Mechanical Engineering, The Petroleum Institute, P.O.Box 2533, Abu Dhabi, The United Arab Emirates Submission: 5 May 2015 Accepted: 9 May 2015 Published: 25 May 2015 Ganoderma lucidum (Fr) P. Karst is considered a panacea for several disease conditions. The major constituents of this mushroom are polysaccharides and terpenoids. The reactive oxygen species (ROS) induced cellular damage has been involved in several diseases, which shows significance of antioxidants in disease management. We examined the antioxidant activity of terpenes isolated from red mushroom (Ganoderma lucidum) fruiting bodies in Wister rats in vitro. Free radical scavenging and lipid peroxidation were evaluated by, FRAP (Ferric reducing antioxidant power) assay, DPPH (1.1-diphenyl hydrazyl) assay and hydroxyl radical scavenging activity. Lipid peroxidation was induced by Fe 2+ ascorbate. The total terpenes isolated from Ganoderma lucidum showed potential DPPH radical scavenging capacity of 50.7% which is equivalent to 0.019% of trolox and ferric reducing ability of 0.356% of trolox in concentration dependent manner. The IC 50 value of terpenes in inhibiting lipid peroxidation was 177±6.7 whereas the IC 50 value for hydroxyl radical scavenging was 225±3.8. It was considered statically significant (P<0.001). Our preclinical studies clearly indicate that the terpenes isolated from Ganoderma lucidum possess significant in vitro antioxidant activity. The potent antioxidant activity of Ganoderma terpenes might be responsible for many biological properties of this mushroom and its therapeutic potential.

2 INTRODUCTION One of the important processes developed by organisms to live in an anaerobic environment is the mechanism for defense against oxygen, hydroxyl radicals and superoxide anions induced damage 1, 2. Oxidation of unsaturated lipids in biomembranes has been involved in a vast range of diseases including aging and cancer 3, 4, Rheumatoid arthritis, cardiovascular problems and diabetes 5-7. The components that have ability to scavenge free radicals, are of greater importance in preventing disease processes. Free radicals are involved in the activity of some xenobiotics also. The varieties of antioxidants are usually involved in the inactivation of reactive species in vivo models 8. The absence of antioxidant defense may cause oxidative stress which would be associated with the large number of disorders. The human body has inherent mechanisms to reduce the free radical induced injury by the action of enzymes such as, catalase, glutathione peroxidase superoxide dismutase etc. and by non enzymatic means involving ascorbic acid, Vitamin E, etc 9,10. These natural protective mechanisms may not be sufficient always. Several methods have been developed to calculate potential of antioxidant agents. Ganoderma lucidum (Fr) P. Karst commonly known as red mushroom is popular for its medicinal properties. Ganoderma is a rich source of bitter triterpenes. Currently more than 140 triterpenoids are reported in Ganoderma lucidum alone 11. The vast majority are ganoderic acids and lucidenic acids 12. The biological activities reported for oxygenated triterpenes include bitterness, hepato-protection and inhibition of hepatoma cells, antihistamine release, angiotensin converting enzyme, cholesterol absorption, cholesterol biosynthesis and inhibition of platelet aggregation 13. However it has been reported that some of the medicinal and physiological properties of Ganoderma lucidum are strain dependent 14. The immunomodulating activity of Gonoderma lucidium indicated its potential for cancer prevention and its employment as alone or in combination with chemotherapy and radiotherapy 11, 15. The Ganoderma lucidum occurring in south India has been reported to exhibit potential antioxidant activity 16, 17. In the present study, we investigated the in vitro antioxidant activity of terpenes which is one of the major chemical constituents of Ganoderma lucidum. 108

3 MATERIALS AND METHODS Animals Animals selected were Wister male albino, 150±25g from our rat s colony. The rats were kept under standardized and water ad- libitum. The experiment was carried out according to the guidelines of our Institutional Animal Ethics Committee. Chemicals Acetic acid, Methanol, Anhydrous Sodium Sulphate, Sulphuric acid, Ferrous sulphate (FeSO 4 ), Hydrochloric acid, Potassium chloride (KCl), Ferrous ammonium sulphate (FeSO 4 (NH 2 ) 2 SO 4 7H 2 0), Sodium dodecyl sulphate (SDS), Thiobarbituric acid (TBA), Pyridine, Butanol, Ethylene Diamine Tetra Acetic acid (EDTA), Hydrogen peroxide (H ), Potassium dihydrogen phosphate (KH 2 PO 4 ), Potassium hydroxide (KOH), Sodium chloride, Acetic anhydride and Formalin were purchased from Merck India Ltd, Mumbai. Trolox,1.1-diphenyl-2-picryl hydrazyl (DPPH), 6- hydroxy-2, 5, 7, 8 tetra methyl chroman 2-carboxylic acid (Trolox), 2, 4, 6-tripyridyl-s-triazine (TPTZ) and carrageenan were purchased from Sigma Chemical Co. USA. Chloroform, Anisaldehyde, Ferric chloride (FeCl 3 ), Tris-HCl, Ascorbic acid and Deoxyribose were purchased from Sisco Research Laboratories Pvt. Ltd, Mumbai, India. Isolation of total terpenes The fruiting bodies of Ganoderma lucidum were collected from Kerala, South India. The type specimen was deposited in the herbarium of our laboratory, under voucher number (HERB. JNCHRC. 2119). The sporocarps were cut into small pieces, dried at C for 48 hours and powdered. 400,00 g sample of the powdered material was extracted in 4 batches of g each with chloroform using a Soxhlet apparatus for 8-10 hours. The extraction was repeated again for the same period of time. The chloroform extracts were combined and dried with anhydrous sodium sulphate. The extract was then concentrated at low temperature under vacuum and the solvent was finally evaporated completely. The residue thus obtained was named as total terpene fraction, (2.00 g). The terpene fraction was further purified by column chromatography. The sample was loaded on a silica gel column and eluted with petroleum ether, then chloroform and finally with chloroform: methanol (90:10) mixture. The fractions were analyzed by TLC on silica gel G using chloroform: methanol (90:10) solvent system. The terpene fraction was detected by spraying with anasaldehyde-sulphuric acid and antimony trichloride reagents. The terpenes were 109

4 combined and again analyzed by TLC to determine the purity of the compound. Pure terpenoid fraction was used for the experiments. DETERMINATION OF IN VITRO ANTIOXIDANT ACTIVITY DPPH radical scavenging assay In this method, a commercially available and stable free radical (DPPH +, 2, 2-diphenyl-1- picrylhydrazil), soluble in methanol, was used [21]. DPPH in its radical form has an absorption peak at 515 nm, which disappeared on reduction by an antioxidant compound. An aliquot (37.50 µl) of terpenes was added to 1.5mL of freshly prepared DPPH solution (0.25g/L in methanol). Absorbance was measured at 515 nm, 20 minutes after the reaction. DPPH radical scavenged was calculated from the calibration curve of %DPPH scavenged versus concentration of the standard antioxidant (Trolox) 18. Ferric reducing antioxidant power (FRAP) assay The ferric reducing ability was measured at low ph (Benzie and Strain, 1996; Pulido et al., 2000). The stock solution of 10mM 2, 4, 6-tripyridyl-s-triazine (TPTZ), 40mM HCl, 20mM FeCl 3.6H 2 O, and 0.3 M acetate buffer (ph 3.6) were prepared. The FRAP reagent was mixed with 90µL water and 10µL test sample/methanol/distilled water/ standard antioxidant solution. The reaction mixture was then incubated at 37 0 C for 30 min, and absorbance was recorded at 595 nm. An intense blue colored complex was formed when ferric tripyridyl triazine (Fe 3+ TPTZ) complex was reduced to the ferrous (Fe 2+ ) form and the absorption at 595 nm was recorded. The calibration curve was plotted with absorbance at 595 nm versus concentration of FeSO 4 which in turn was plotted against concentrations of standard antioxidant (Trolox) 18. Inhibition of lipid peroxidation The reaction mixture contained 0.1 ml of rat liver homogenate (25 %, w/v) in Tris- HCl buffer (20mM, ph 7); FeSO 4 (NH 4 ) 2 SO 4. 6H 2 O (0.16mM); ascorbate (0.06 mm); and various concentrations of the terpenes from Ganoderma lucidum in a final volume of 0.5 ml (19).The reaction mixture was incubated for 1 hour at 37 C. After the incubation period, 0.4 ml was removed and treated with 0.2 ml SDS (8.1 %), 1.5 ml thiobarbituric acid (0.8%) and 1.5mL acetic acid (20%, ph 3.5). The total volume was made upto 4 ml with distilled water and then kept in a water bath at C for 1 hour. After cooling, 1.0 ml of distilled water and 5.0 ml 110

5 of n-butanol and pyridine mixture (15:1) were added to the reaction mixture, shaken vigorously and centrifuged at 4000 rpm for 10 minutes. The n-butanol-pyridine layer was removed and its absorbance at 532 nm was read. Inhibition of lipid peroxidation was determined by comparing the optical density of the treatments with that of control 2. Hydroxyl radical scavenging activity The reaction mixture contained Deoxyribose (2.8 mm); FeCl 3 (0.1 mm); KH 2 PO 4 -KOH buffer (20mM, ph 7.4); EDTA (0.1 mm); H 2 O 2 (1mM); Ascorbic acid (0.1mM) and various concentration of terpenes of G. lucidum in a final volume of 1.0 ml. The reaction mixture was incubated at 37 C for 1 hour. The 2-Thiobarbituric Acid-Reactive Substances (TBARS) formed was estimated by thiobarbituric acid method of (19). The hydroxyl radical scavenging activity was determined by comparing absorbance of control with that of treatments 2. Absorbance (test sample) - Absorbance (control) % Inhibition of TBARS = x 100 Statistical analysis Absorbance (control) All results are expressed as by mean ± SD. Experimental data was statistically analyzed using one-way analysis of variance (ANOVA) (using Graph Pad Instant software package). When found significant, the groups were further analyzed by Bonferonni s multiple comparison test. P Values less than were considered significant. RESULTS DPPH radical scavenging assay The total terpenes isolated from the fruiting bodies of G. lucidum showed potent DPPH radical scavenging capacity. 0.1% of the total terpenes showed 29.36% DPPH radical scavenging activity, equivalent to 0.011% of trolox, 0.5% showed 44.52% DPPH radical scavenging activity equivalent to 0.017% of trolox and 1.0% showed 50.07% DPPH radical scavenging activity equivalent to 0.019% of trolox. This indicated the significant DPPH radical scavenging activity of terpenes isolated from this mushroom (Figure 4). 111

6 Ferric Reducing Antioxidant Power (FRAP) assay The terpenes isolated from the fruiting bodies of G. lucidum showed ferric reducing ability. The extract at a concentration of 0.1% showed ferric reducing ability equivalent to 0.058% of trolox, for 0.5% ferric reducing ability was equivalent to 0.212% of trolox and for 1.0% ferric reducing ability was equivalent to 0.356% of trolox. This indicated the significant ferric reducing ability of the terpenes isolated from this mushroom (Figure 5). Inhibition of lipid peroxidation The terpenes isolated from the fruiting bodies of G. lucidum were effective in inhibiting the lipid peroxidation induced by Fe 2+ -ascorbate system in rat liver homogenate. The IC 50 value of terpenoids in inhibiting lipid peroxidation was 177 ± 6.7. This indicated the significant lipid peroxidation inhibiting activity of total terpenes isolated from this mushroom (Table 1). Hydroxyl radical scavenging activity The terpenes isolated from the fruiting bodies of Ganoderma lucidum showed significant scavenging activity of hydroxyl radical generated from Fe 2+ ascorbate EDTA H 2 O 2 system. The IC 50 value of terpenes for hydroxyl radical scavenging was 225 ± 3.8 (Table 1). The hydroxyl radical scavenging activity was found to increase in a concentration dependent manner. DISCUSSION For thousands of years, mushrooms have been attributed to many pharmacological properties. The fruiting bodies of G. lucidum are considered as a panacea and are extensively used for treatment of a number of disease conditions. Mushrooms and their extracts are sold as nutritional supplements and good for health. Mushroom is traditionally employed in folk medicine and also in many other systems of medicines. The major marker compounds present in the fruiting bodies of the red mushroom (G. lucidum) are polysaccharides and terpenes 11. These components have been reported to have significant pharmacological activities. The present investigation reveals that the terpenes isolated from this mushroom possess profound free radical scavenging property. The DPPH assay and FRAP assay of terpenes support this conclusion. The Ganoderma terpene 112

7 fraction also possesses significant lipid peroxidation inhibiting and hydroxyl radical scavenging activities. In the pathophysiology of a large number of human diseases including cancer, reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide are produced as by-products 20. Therefore, the elimination or inhibition of reactive oxygen species and their cellular function is assumed as an important approach to decrease the risk of cancer. During this investigation, antioxidant activity of the terpenes is tested at various levels of antioxidant action. The inactivation of non-enzymatic antioxidants occurs by its reaction with proantioxidants. In FRAP assay, a blue colored complex of Fe3+ TPTZ is formed by Fe3+ by antioxidants and this antioxidant capability can be considered as its reducing ability. The chain propagation and initiation reactions are suppressed by antioxidants, due to its ability to scavenge free radicals. In the DPPH assay, the depolarization of DPPH radical is evaluated by antioxidants 21. In our study we showed that the terpenes of Ganoderma lucidum have strong ferric reducing and DPPH radical scavenging activities. The lipids of biomembranes are particularly prone to lipid peroxidation. Under pathophysiological conditions, the aldehydes formed during lipid peroxidation act as bioactive molecules. The conjugated dienes produced as first product of lipid peroxidation, form lipid hydro peroxides by oxidation that further form aldehydes which then bind with TBA to produce thiobarbutric acid maloneldehyde adduct 22. Our results showed that terpenes of Ganoderma lucidum are capable of preventing lipid peroxidation that might lead to membrane damage. Trace amount of elements like ferrous ions, bind with peroxide in iron-catalyzed Fenton reaction to form hydroxyl radicals. A hydroxyl radical breaks bonds in proteins and carbohydrates, causes biomembrane disruptions and lipid peroxidation 23. Our results marked that terpenes of G. lucidum have significant hydroxyl radical scavenging activity. In normal biological function, free radical scavenging activity has significance as it is implicated in various pathological events 24 reported the antioxidant activity of the extract of red mushroom (Ganoderma lucidum ). Results of our study indicated the ability of Ganoderma terpenes to suppress formation of free radicals (FRAP assay) and also to scavenge the free radicals (DPPH) 113

8 assay). Natural diet or crude drugs rich in antioxidants are of significance to protect or prevent tissue damage and disease management. Since Ganoderma terpenes possess profound antioxidant activity, a part of the medicinal properties of this mushroom can be attributed to its terpene components. The results of the present investigation support this conclusion. ACKNOWLEDGEMENT Authors are grateful to the Vice Chancellor of the A Sharqiyah University for providing facilities and humble support. Conflict of Interest The authors declare that there are no competing conflicts of interest. REFERENCES 1. Halliwell B., and Gutteridge J.M.C. Free radicals in Biology and Medicine. (1 st eds.), Clarendon Press, Oxford, pp Ajith T.A., and Janardhanan K.K. Antioxidant and antihepatotoxic activities of Phellinus rimosus (Berk) Pilat. Journal of Ethanopharmacology.2002 ; 81: Lunec J. Free radicals ; their involvement in disease processes. Annals of Clinical Biochemistry. 1990;27: Aviva Romm, Lisa Ganora, David Hoffmann, Eric Yarnell, Kathy Abascal, and Mitch Coven.Fundamental Principles of Herbal Medicines, Botanical Medicine for Women's Health (First edition), pages Blake D.R., Merry P., Unsworth J., Kidd B.L., Outhwaite J.M., Ballard R., Morris C.J., Grey L., and Lunec J. Hypoxic reperfusion injuiry in the inflamed human joint. Lancet.1989; 1: Baynes J. Perspectives in diabetics: role of oxidative stress in development of complications in diabetes. Diabetes. 1991; 40: Sudheesh, N.P., T.A. Ajith, V. Ramnath, and K.K. Janardhanan. Theraputic potential of Ganoderma lucidum (Fr.) P. Karst. against the declined antioxidant status in the mitochondria of post-mitotic tissues of aged mice. Clinical nutrition. (Article in press) John R. Underwood, Mark Chivers, Thi Thuong Dang, and Paul V. Licciardi. Stimulation of tetanus toxoidspecific immune responses by a traditional Chinese herbal medicine. Vaccine ;27(47): Kavita Shakya, Renu Mishra, and Smitha Sam.Inhibitory effect of Glycorrhyza glabra Linn. extract on deoxyribose degradation. Biosci. Biotech Res. Comm. 2008; 1(1): Thulasi G.Pillai, Mathew John, and Gifty Sara Thomas. Prevention of cisplatin induced nephrotoxicity by terpenes isolated from Ganoderma lucidum occurring in Southern Parts of India, Experimental and Toxicologic Pathology (Article in press) Gao Y., and Zhou S. Cancer prevention and treatment by Ganoderma, a mushroom with medicinal properties. Food Reviews International. 2003; 19: Nishitoba T., Sato H., Kasai T., Kawagishi H., and Sakamura S., New bitter C 27 and C 20 terpenoids from the fungus G.lucidum. Agri. Biol. Chemistry. 1984; 48: Shiao M, Lee K.R, Lin L.J, and Wang C.T. Natural products and biological activities of the Chinese medicinal fungus G.lucidum in Food phytochemicals II, Teas, Spices and herbs, Am. Chemical Society. Pp

9 14. Nishitoba T., Sato H., Shirasu, S., and Sakamura S.,. Evidence of strain specific terpenoids pattern of G.lucidum. Agri. Biol.Chemistry. 1986;50: Jin Yi Wong, and Fook Yee Chye. Antioxidant properties of selected tropical wild edible mushrooms Journal of Food Composition and Analysis. 2009; 22(4): Jones S, and Janardhanan K.K. Antioxidant and Antitumor Activity of Ganoderma lucidum (Curt.;Fr.) P. Karst.- Reishi (Aphyllophoromycetideae) from South India. Int.J.Med. Mushroom. 2000; 2: Russell,R.,M. Paterson.Ganoderma A therapeutic fungal biofactory Phytochemistry. 2006; 67(18): Lakshmi B., Tilak J.C., Adhikari S., Devasagayam T.P.A and Janardhanan KK. Evaluation of antioxidant activity of selected Indian mushrooms. Pharmaceutical Biology. 2004; 42: Ohkawa H., Ohishi W, and Yagi K. Assay for peroxidase in animal tissue by thiobarbituric acid reaction, Biochemistry. 1979; 95: Pinc , J.J. Free radicals and antioxidatants in human diseases. In analysis of free radicals in biological systems. Birkhauser Verlang, Berlin, pp Aquino R., Morelli S., Lauro M.R., Abdo S., Saija A., and Tomaino A. Phenolic constituents and antioxidant activity of an extract of Anthurium versicolor leaves. J.Natl.Pro. 2001; 64: Girotti A.W. Photodynamic lipid peroxidation in biological systems. Photochemistry and Photobiolog. 1990; 51: Cuzzocrea S., Riley D.P., Caputi A.P., and Salvemini D. The American Society of Pharmacology and Experimental Therapeutics. Pharmacol.Rev. 2001; 53: Ajith,T.A., N.P. Sudheesh, D. Roshny, G. Abishek, and K.K. Janardhanan. Effect of Ganoderma lucidum on the activities of mitochondrial dehydrogenases and complex I and II of electron transport chain in the brain of aged rats. Experimental Gerontology. 2009; 44(3): Min Yang, Xiaoming Wang, Shuhong Guan, Jiameng Xia, Jianghao Sun, Hui Guo, and De-an Guo. Analysis of Triterpenoids in Ganoderma lucidum using Liquid Chromatography Coupled with Electrospray Ionization Mass Spectrometry, Journal of the American Society for Mass Spectrometry. 2007; 18(5): FIGURE LEGENDS: Figure 1: A typical Ganoderma basidiocarp Figure 2: Chemical structure of some Ganoderic acids Figure 3: Major Fragmentation pathways of some triterpenoids Figure 4: DPPH radical scavenging activity of terpenoids isolated from Ganoderma lucidum. Figure 5: Ferric reducing antioxidant capacity of terpenoids isolated from Ganoderma lucidum. TABLE LEGENDS: Table1. Hydroxyl radical scavenging and lipid peroxidation inhibiting activity of the terpenes isolated from Ganoderma lucidum (Inhibition %) 115

10 Figure 1: A typical Ganoderma basidiocarp Figure 2: Chemical structure of some Ganoderic acids 116

11 Figure 3: Major fragmentation pathways of some triterpenoids (25) 117

12 Concentration of trolox (%) Figure 4: DPPH radical scavenging activity of terpenoids isolated from Ganoderma lucidum % terpenes 0.5% terpenes 1% terpenes Concentration of 1 terpenes (%) Figure 5: Ferric reducing antioxidant capacity of terpenoids isolated from Ganoderma lucidum 118

13 Table 1: Hydroxyl radical scavenging and lipid peroxidation inhibiting activity of the terpenes isolated from Ganoderma lucidum (Inhibition %). Activities Terpenes (IC 50 ) (µg/ml) Catechin Standard (µg/ml) Hydroxyl radical scavenging activity 225 ± 3.8 * 848 ± 20.0 Lipid Peroxidation Inhibiting activity 177 ± 6.7 * 416 ± 28.6 Values are mean ± S.D, n = 3. *(P<0.001) 119