Bioactive molecules and their Anti-oxidant activity of Agaricus bisporus

February 2013- April 2013, Vol. 3, No. 2, 1222-1228. E- ISSN: 2249 –1929Journal of Chemical, Biological and Physical SciencesAn International Peer Review E-3 Journal of SciencesAvailable online atwww.jcbsc.orgSection B: Biological scienceCODEN (USA): JCBPATResearch ArticleBioactive molecules and their Anti-oxidant activity ofAgaricus bisporusJ.Sankara Rao 1* , K.Ravi Kumar 1 , Venkat Kartheek Vale 1 , Prithvi Prasad Yarlagadda 2,S.Vijaya Saradhi, 21 Division of Molecular Biology, Institute of Biological Sciences, Vijayawada, Krishna district,Andhra Pradesh, India.2 Department of Biotechnology, Koneru Lakshmaiah University, Vaddeswaram, Guntur district,Andhra Pradesh, India.Received: 24 January 2013; Revised: 15 March 2013; Accepted: 25 March 2013Abstract: White button mushroom (species Agaricus bisporus), cultivation increased throughoutthe world during the last few decades. Its popularity increased due to its ease of cultivation, highyield potential and high nutritional value. Mycochemical analysis of Agaricus bisporus extractsrevealed the presence of terpenoids, tannins, steroidal glycosides and carbohydrates. Ethanolicextracts of the mushrooms, screened for possible antioxidant activity by four complementary testsystems, namely DPPH free radical scavenging, β- carotene/linoleic acid systems, total phenoliccompounds and total flavonoid concentration revealed antioxidant activity in Agaricus bisporus.The species are edible and can be used as dietary intake for immune enhancement due to theiranti-oxidant activity.Keywords: Agaricus bisporus, antioxidant activityINTRODUCTIONAgaricus bisporus, white button mushroom is an edible mushroom and is one of the most commonly andwidely consumed mushrooms in the world 1 . Agaricus bisporus only contains 16 IU of vitamin D asergocalciferol (vitamin D2), since they also contain high amounts of ergosterol, by brief exposure to UVlight the ergocalciferol contents rise immensely 2 , 3 . Agaricus bisporus also contains sodium, potassiumand phosphorus, conjugated linoleic acid4 and antioxidants5. Procatechuric acid and pyrocatechol are1222 J. Chem. Bio. Phy. Sci. Sec.B, 2013, Vol.3, No.2, 1222-1228.

Bioactive …J.Sankara Rao et al.found in A. bisporus 6 . In search of new cancer treatments over the past several years, many species ofbasidiomycete mushrooms have been investigated for their anti-cancer properties 7 . Lucas 8 , firstdemonstrated the anti-cancer activity of mushrooms. A 2009 case control study of 2,018 womencorrelated a large decrease of breast cancer incidence in women who consumed mushrooms. Women inthe study who consumed fresh mushrooms daily were 64% less likely to develop breast cancer, whilethose that combined a mushroom diet with regular green tea consumption reduced their risk of breastcancer 9 by nearly 90% . The table mushroom also shown to possess possible immune system enhancingproperties 10 , 11 . Many other anticancer compounds of mushroom origin, including b-D-glucans,heteropolysaccharides, glycoproteins, lectines, and terpenoids have been investigated. Mushroomspossess bioactive molecules that act as antioxidant substances and prevent the oxidative damage withinthe organism. The present study was taken up to assess the antioxidant property A. bisporus.MATERIALS AND METHODSCultures of Agaricus bisporus were procured from Department of Horticulture, Andhra Pradesh andmaintained on 2% agar basidiomycetes synthetic medium (BSM). The composition includes (g l -1 ):glucose (5), K 2 HPO 4 (1), asparagines (0.6) , yeast extract (0.5), KCl(0.5), MgSO 4 .7H 2 O (0.5), FeSO 4(0.1), Mn(CH 3 COO) 2 (0.008), ZnNO 3 (0.003), Ca(NO 3 ) 2 .4 H 2 O (0.006) and CuSO 4 .5 H 2 O (0.003) at pH5.5. These cultures were incubated at 28º C in the dark.β-Carotene, linoleic acid, 1,1-Diphenly-2-picrylhydrazyl (DPPH), butylated hydroxytoluene (BHT),butylated hydroxyanisol (BHA) and α-tocopherol, were purchased from MERCK India. Pyrocatechole,Tween-20, Folin-ciocalteu’s phenol reagent (FCR), sodium carbonate, ethanol, chloroform and the otherchemicals and reagents were purchased from Qualigens, India. All other unlabeled chemicals andreagents used were of analytical grade.Preparation of extracts: Mushrooms cultured in the BSM medium were harvested (150 g) and air driedin an oven at 40 o C. A 50 g dried sample was extracted by stirring with 500 ml of 70% ethanol at 30 o C at150 rpm for 24 h and filtered through Whatman No. 4 filter paper. The residue was re-extracted twice asdescribed above. Combined extracts were rotary evaporated at 40 o C to dryness, redissolved in ethanol(10 mg ml -1 ) and stored at 4 o C for further use.Mycochemical analysis of Agaricus bisporus: The mycochemical constituents from the ethanol extractsof Agaricus bisporus were determined qualitatively by thin layer chromatography (TLC) as given bySofowora 12 , Trease and Evans 13 and Harbone 14 . The ethanol extracts were subjected to TLC separationusing butanol- glacial acetic acid-water (100:10:10) solvent system. The developed plates were thensprayed with vanillin solution for steroid detection. The presence of carbohydrate was detected byspraying with sodium metaperiodate (0.1%) followed by ethanolic benzidine. Tannins were determinedby the presence of brownish green-blue black coloration with 0.1% FeCl 3 Spray. The steroidal glycosideswere determined by Keller-Killani test and Cynogenic glycosides were determined by red colourformation in the picrate paper. Flavonoids were detected under UV light and the bands should appear inyellowish colour.Antioxidant activity Determination: The antioxidant activity was determined by the 2, 2 – diphenylpicryl hydrazyl radical neutralization (DPPH) assay, β- carotene/linoleic acid systems, total phenoliccompounds and total flavonoid concentrations.DPPH Assay: Free radical scavenging capacities of the extracts (Butylated hydroxy toluene, Butylatedhydroxyanisol, α-tocopherol of Agaricus bisporus) were measured by DPPH assay. One thousand1223 J. Chem. Bio. Phy. Sci. Sec. B, 2013, Vol.3, No.2, 1222-1228.

Bioactive …J.Sankara Rao et al.microlitres of various concentrations of the extracts in ethanol were added to 4 ml of 0.004% methanolsolution of DPPH. Absorbance was read by the formula, against a blank at 517 nm. Inhibition of freeradical by DPPH in percent (I %) was calculated in following way. After, 30 min incubation at roomtemperature, absorbance (A) was recorded as (A blank – A sample / A blank) x 100, where A blank isabsorbance of the control reaction (all reagents except the test compound), and A sample is theabsorbance of the test compound. Extract concentration providing 50% inhibition (IC 50 ) was calculatedfrom the plotted graph, inhibition percentage against extract concentration. All the tests were carried outin triplicate 15 , 16 .β-Carotene-linoleic acid Assay: β-Carotene-linoleic acid was determined by measuring the inhibition ofthe volatile organic compounds and the conjugated diene hydroperoxides arising from linoleic acidoxidation. Briefly, a stock solution of β-carotene-linoleic acid mixture was prepared by dissolving 0.5 mgβ-carotene in 1 ml of chloroform (HPLC grade) and adding 25 µl linoleic acid and 200 mg of Tween 20.Chloroform was completely evaporated using a vacuum evaporator and 100 ml distilled water saturatedwith oxygen (30 min 100 ml/min) was added with vigorous shaking. 4 ml of this reaction mixture wasdispensed into test tubes and 200 µl of the extracts, prepared at 2 mg/l concentrations, were added. Theemulsion system was incubated for 2 h at 50 0 C. The same procedure was repeated with syntheticantioxidant, BHT, BHA, α-tocoferol as positive control, and a blank. Absorbance of the mixtures weremeasured at 490 nm and antioxidative capacities of the extracts were compared with those of BHA, α-tocoferol and blank 17 at 0.5 mg/mL. The absorbance was measured until the β-carotene colordisappeared. The antioxidant activity (AA) was calculated as the percent inhibition (R) relative to thecontrol using Equation. : AA = [(R control − R sample ) /R control ] × 100. The extract concentration providing50% inhibition (IC 50 ) was calculated from the plotted graph, inhibition percentage against extractconcentration. All the tests were carried out in triplicate.Total phenolic Content: Total soluble phenolic compounds in the mushroom ethanolic extracts weredetermined with Folin-Ciocalteu reagent and gallic acid as standard 18 . Extract solution (0.1 ml)containing 1mg extract was taken in a volumetric flask, 46 ml distilled water and 1 ml Folin-Ciocalteureagent were added and flask was shaken thoroughly. After 3 min, 3 ml of solution 2% Na 2 CO 3 wasadded and the mixture was allowed to stand for 2 h with intermittent shaking. Absorbance was measuredat 760 nm. The same procedure was repeated to all standard gallic acid solutions (0–1000 mg, 0.1 ml -1 )and standard curve was obtained. Extract concentration providing 50% inhibition (IC 50 ) was calculatedfrom the plotted graph, inhibition percentage against extract concentration. All the tests were carried outin triplicate.Total flavonoid Content: The total flavonoid concentration in mushroom ethanolic extracts wasdetermined by diluting 1 ml extract with 4.3 ml of 80% aqueous ethanol in test tubes containing 0.1 ml of10% aluminum nitrate and 0.1 ml of 1 M aqueous potassium acetate. After 40 min at room temperature,the absorbance was determined spectrophotometrically at 415 nm. Total flavonoid concentration wascalculated by the formula, Absorbance = 0.002108 µg quercetin– 0.01089, using quercetin as standard 19 .Extract concentration providing 50% inhibition (IC 50 ) was calculated from the plotted graph, inhibitionpercentage against extract concentration. All the tests were carried out in triplicate.STATISTICAL ANALYSISAll data are given as Mean ± SD of three measurements. Statistical analysis was performed using MSExcel for Windows. The deviation of each experimental value was considered significant at P

Bioactive …J.Sankara Rao et al.RESULTS AND DISCUSSIONMycochemical analysis of ethanol extracts of Agaricus bisporus showed the presence of steroids, tannins,steroidal glycosides and carbohydrates, but cyanogenic glycosides were absent in the extracts (Table.1).The phenolics and flavanoids reported to possess antioxidant activity 20 were detected Agaricus bisporus.Table.1: Comparative analysis of Mycochemical constituentsMycochemicalSteroids ++Tannins +Steroidal glycosides +Carbohydrates +Cyanogenic glycosides -Flavonoids +(+): Present; (-) : AbsentAgaricus bisporusThe inhibitory concentration (IC 50 ) of P. ostreatus was higher in DPPH assay when compared to that ofP. florida (Table.2). These values for BHT, BHA and α-tocopherol were much lower in this assay aswell. The statistical analysis was significant at P

Bioactive …J.Sankara Rao et al.The statistical analysis was significant at P

Bioactive …J.Sankara Rao et al.REFERENCES1. Cappelli, Alberto . Fungi Europaei:Agaricus. Saronno, Italy: Giovanna Biella.1984,. 123–25.2. S.R.Koyyalamudi,S.C. Jeong,C.H. Song,K.Y. Cho, G. Pang, "Vitamin D2 formation andbioavailability from Agaricus bisporus button mushrooms treated with ultravioletirradiation". Journal of Agricultural and Food Chemistry,2009, 57 (8): 3351–5.3. G.S.Lee,H.S. Byun,K.H. Yoon,J.S. Lee,K.C. Choi,E.B. Jeung, "Dietary calcium and vitamin D2supplementation with enhanced Lentinula edodes improves osteoporosis-like symptoms andinduces duodenal and renal active calcium transport gene expression in mice". European Journalof Nutrition,2009, 48 (2): 75–83.4. S.Chen,S.R. Oh,S. Phung,G. Hur,J.J. Ye, S.L. Kwok, G.E. Shrode, M. Belury, M et al. "Antiaromataseactivity of phytochemicals in white button mushrooms (Agaricus bisporus)". CancerRes. 2006, 66 (24): 12026–34.5. Y.L.Shi,A.E. James,I.F. Benzie,J.A. Buswell, "Mushroom-derived preparations in the preventionof H2O2-induced oxidative damage to cellular DNA". Teratog Carcinog Mutagen ,2002,22 (2):103–11.6. A.Delsignore, F. Romeo, M.Giaccio, "Content of phenolic substances inbasidiomycetes". Mycological Research ,1997,101: 552–6.7. Q.Y.Yang, and S.C.Jong, Medicinal mushrooms in China. Mushr. Sci.1989, 9, 631-643.8. E.H.Lucas, Tumor inhibitors in Boletus edulis and other Holobasidiomycetes. Antibiot.Chemotherapy, 1957,7, 1-4.9. M.Zhang, J. Huang, X. Xie, C.D. Holman, "Dietary intakes of mushrooms and green tea combineto reduce the risk of breast cancer in Chinese women".International Journal ofCancer ,2009,124 (6): 1404–1408.10. Z.Ren,Z. Guo,S.N. Meydani,D. Wu, "White button mushroom enhances maturation of bonemarrow-derived dendritic cells and their antigen presenting function in mice". J.Nutr.2008,138 (3): 544–50.11. D.Wu,M. Pae,Z. Ren,Z. Guo,D. Smith,S.N. Meydani , "Dietary supplementation with whitebutton mushroom enhances natural killer cell activity in C57BL/6 mice". J. Nutr. 2007,137 (6):1472–7.12. A.Sofowora,. Medicinal plants and Traditional medicine in Africa. Spectrum Books. Ltd. Ibadan,Nigeria,1993, 270-289.13. G.Trease and W.C.Evans, Pharmacognosy. 11th edn. Brailliar Tiridel. Can. Macmillianpublishers,1989.14. J.B.Harbone , Phytochemical methods. London. Chapman and Hall. Ltd. 1973,15. M.Burits and F.Bucar, Antioxidant activity of Nigella sativa essential oil. Phytother Res. 2000;14: 323-08.16. M.K.Cuendet,K. Hostettmann and O.Potterat, Iridoid glucosides with free radical scavengingproperties from Fragarea blumei. Helvetica Chimica Acta, 1997, 80:1144-1152.17. A.Dapkevicius,R. Venskutonis,T.A. Van Beek and P.H.Linssen, Antioxidant activity of extractsobtained by different isolation procedures from some aromatic herbs grown in Lithuania. J. Sci.Food and Agric, 1998, 77:140-146.18. K.Slinkard and V.L.Singleton, Total phenol analyses: automation and comparison with manualmethods, American J. Enolo. Viticul, 1977, 28: 49–55.19. Y.K.Park,M.H. Koo,M. Ikegaki and J.L.Contado, Comparison of the flavonoid aglycone contentsof Apis mellifera propolis from various regions of Brazil, Arquivos de Biologiae Techno,1997, 40(1): 97-106.20. R.A.A.Mothana,R. Jansen,W.D. Julich and U.Lindequist, Ganomycin A and B, new antimicrobialfarnesyl hydroquinones from the basidiomycete Ganoderma pfifferi. Journal of NaturalProducts,2000, 63(3): 416-418.1227 J. Chem. Bio. Phy. Sci. Sec. B, 2013, Vol.3, No.2, 1222-1228.

Bioactive …J.Sankara Rao et al.21. P.Jayakumar,N. Jothivel,A. Thimmappa and Paul VI , Physicochemical characterisation of alentic water body from Tamil Nadu with special reference to its pollution status. TheEcoscan.2009, 3(1and2): 59-64.22. Türkoğlua Aziz, Mehmet Emin Duru and Nazime Mercan,Antioxidant and Antimicrobial Activityof Russula delica Fr: An Edidle Wild Mushroom, Eurasian Journal of Analytical Chemistry,2007,2(1).23. J.K.S.Moller,H.L. Madsen,T. Altonen and L.H. Skibsted, Origanum dictamnus as a source ofwater-extractable antioxidants. Food Chem.1999, 64: 215–219.24. T.Hatano,R. Edamatsu,A. Mori,Y. Fujita, and E.Yasuhara, Effect of interaction of tannins withco-existing substances. VI. Effects of tannins and related polyphenols 1989.25. A.Cakir,M.A. Ahmet Yıldırım,M.E. Duru,M. Harmandar and C. Kazaz, Isolation andCharacterization of antioxidant Phenolic Compounds From the Aerial Parts of Hypericumhyssopifolium L. by activity-guided Fractionation., J Ethnophar.2003, 87: 73-83.26. P.D.Duh,Y.Y. Tu and G.C.Yen, Antioxidant activity of water extracts of harn jyurChyrsanthemum morifolium (Ramat). Lebensmittel-Wissenschaft und Technologie1999,32:269–277.27. M.Tanaka,C.W. Kuei,Y. Nagashima and T.Taguchi, Application of antioxidative maillradreaction products from histidine and glucose to sardine products. Nippon Suisan Gakkaishil,1998, 54: 1409-1414.28. A.M.Murcia,M. Martinez-Tome, A.M. Jimenez,A.M. Vera,M. Honrubia and P.Parras ,Antioxidant activity of edible fungi (truffles and mushrooms): Losses during industrialprocessing. Journal of Food Protection,2002, 65, 1614−1622.29. C.A.Rice-Evans,N.J. Miller,Paganga,. Structure-antioxidant activity relationships of flavonoidsand phenolic acids. Free Radic. Biol. Med.,1996, 20: 933-956.30. C.Manach,A. Scalbert,C. Morand,C. Remesy,L. Jimenez, Polyphenols: Food sources andbioavailability. Am. J. Clin. Nutr. ,2004,79: 727747.31. S.Z.Dziedzic and B.J.F.Hudson, Hydroxy isoflavones as antioxidants for edible oils. FoodChemistry ,1993,11: 161-166.32. Ratty AK, Das NP. Effects of flavonoids on non enzymatic lipid peroxidation: structure-activityrelationship. Biochem Med Metab Biol. 1988 Feb;39(1):69-7933. Y.Hanasaki,S. Ogawa,S. Fukui, The correlation between active oxygens scavenging andantioxidative effects of flavonoids. Free Radic. Biol. Med.,1994, 16:845-850.34. K.Nakamura,R. Kawashima,M. Sugiura,T. Kato,A. Nakamura,K. Hatano,S. Nagumo,K.Kubota,H. Fukuda,K. Ito,S. Kojima, Neural substrates for recognition of familiar voices: a PETstudy. Neuropsychologia,2001, 39. 1047– 1054.Corresponding author J.Sankara Rao; Division of Molecular Biology, Institute of BiologicalSciences, Vijayawada, Krishna district, Andhra Pradesh, India – 520 1001228 J. Chem. Bio. Phy. Sci. Sec. B, 2013, Vol.3, No.2, 1222-1228.