ISSN:2o89- 6o69

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octoberrrs'r.z*2ort . Serpong, WidyaBhaktiPuspiptek Graha Gedung

Anticancer Potential Of Jamur Dewa Extract (AgarieusBlazei Murill) On Cervical Cancer Cells
Farmasi *ffflf""" Aludemi Analis Malang PutraIndonesia

Jalan Barito 5 Malang foiz2I9@Yahoo-co.id

Abstraet Agaricus blazei Murill (ABIO known as jamur dewa and is one plant that can be uied as a drug, such as for diabetes,hypertensionand cancer.Jamur dewa contain compogndsbetaglukan,ergosterol, and terpenoids.These compoundsare potent anticancer.Thus, the anticancerabitity shoutdbe developed this jamur dewa ixfact on cervical cancercells. Cervicalcanceris a tlpe of cancerthat affectswomen And to contributeto the world of education. first. The resultsof this study is expected in the teatnent using traditional medicine. in generalas alternatives society This type of researchinclude experimental.God mushroomextract obtained from the extraction solvent maceration and percolation with ethanol 50%. Concentation used 0 pdml (control), 200 pglml, a00 pg/ml, 600 pglml, 800 pglml, made the preliminary test hematoxilenand eosin and 1000 pg/ml. The o6servations by cell staining is observeacoloration cells undergoing division are characterized method elongaiionandthe numberof cell nuclei. The cytotoxic testusing MTT assay Test kinetics proliferation of cells is doneby using the that ilrilt get the value of IC so. methodwith a different fieatrrent incubationtime than the control cells. MTT ^toy test conductedby acridine-ethylbromide staining (doublestaining). Data Apoptosis to obtain IC so were analyzedusing ANOVA followed by LSD and linear regression values foi cytotoxic test, t-testto test the kinetics of cell proliferation, and to test test preliminary descriptiveanalysisof H & E stainingandapoptosis Reiults showed that mushroomextract gods have acnvity in inhibiting the growth of HeLa cells at 200 p{ml, with IC so 194.44pglml. The extact is also able io reducethe proliferation of HeLa cells and stimulateapoptosis.Tht rerylt1 of this godshavethe ability to HeLa cells showthat mushroomextact asan anticancer sn1dy by inhibiting cell division andpromoteapoptosis. potential,jarnur dewaextrac! cervicalcancercells Keywords: anticancer

Introduction Cervical cancer is a tlpe of first cancer in womenwho sufferedmost in ttre whole world, and in Indonesia servikas cancr most often found. Conventional treatment is generally such as cancerwith surgery, done on diseases chemotherapy and radiotherapy. However, cancer is surgical therapy can not be done specifically in cancer cells that have spread (metastasized), while chemotherapy and radiation treatments can cause major side effects despite chemotherapy treatnoent

capable of stopping growth. Therefore, the search for chemotherapeutic agents ftom nahral ingredientsthat attack the target of its action on genes regulating the growth or proliferation of cells with minimum side effects, very necessaryin the teafrent of cancer. Jamur dewa (Agaricas blazei Murill) is a fungus that can be used as medicine. Jamur dewa is a class of fungi that af,esaprophyte. Based on the results research,jamur dewa containing1,3-D is polysaccharide compounds

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|SSN:zo89-6o69 pGlucan and p l,GD-Glucan(naso,20lO),4fiber 6-8 45Toprotnin" 3845% carbohydrates, Yo, 34o/o fat, vitamins Bl, B.2, niacin et a1.,2006).Jamurdewa also @ruggemann, contain various minerals such as ironn potassiurn, phosphonrs, magnesium"copper, and zinc (Novaes,el a/., seleniuq rnanganese, 2006). From some of the content, there are funotioning as anticancer, among others, p and Glucan1,3-D, 1,6-p-DGlucan,ergosterol, Thesecompourdsinhibit the activity trpnes. of the tumor and cell proliferation (Novaes,et Lavitschka, e/ a/., et a1.,2008; aI.,2OO7;Yl'l.a, 2W7). The purpose of this shrdy to determine the ability of anticancergod rnushroomexfact on cervicalcancercells. Researchmethod Jamur dewa simplicia used were obtained from the CV ASIMAS Lawang,then extracted with ethanol 50%, Culhred HeLa cell line collection of PhysiologyLaboratoryof the UB Schoolof Medicine. Cytotordc Test HeLa cells grown in 96 wells mikroplhte of 5000cellV wells and incubatedfor 48 hoursto get good growth. After that the new medium was replaced with MEM exFact added at (200pg/ml, 400 pg/ml, various concentrations 600 pg/ml, 800 pglml, 1000 pg/ml) with a co. solvent DMSO and incubatedat 37 0 C in S% CO z incubator for 48 hours. At the end of incubation, MEM media and then discarded cell extacts were washedwith PBS.In eachof the wells, added 100 nrl- culture medium and l0 mL prea*si MTf 5 mg/ml. Tues incubated for 4+fhonrsin incubator COzSo/o370C. MTT reageirtreaction was stoppedwith acid isopropanol (HCl 4N and isopropanol; l:4), shake,non a shaker for 10 minutes. Uptake readwith ELISA readerat a wavelengthof 550 nm. Furthermore, live cell % Absorbance of cells with the treatnent - Absorbance of mediacontrol cells o/oLivecells=

serpong,octoberres - ir* ront BhahiPuspiptek Gedung Graha Widya Basedon% living cells, canbe calculated using the price of IC sowith a linear regression equation Khedcs of proliferatlon Testing Cells (doubling time) HeLa cells grown in 96 wells milcroplateof 5000 cells/wells and incubatedfor 48 hours to get good growth. After that the new medium was replaced with MEM extract added at with co-solventDMSO certain concentrations 0 and incubatedat3T C in 5% CO 2 incubator for 0, 24, 48n 72 hours. At the eird of incubation,RPM medium and then discarded cell exfacts were washedwith PBS.In eachof the wells, added 100 mL MEM medium and l0 mL MTT 5 mg / ml. Tuesincutated for 4-6 hours in incubator CO z sYo 3? o C. MTT reagnt reaction was stopped with acid isopropanol (HCl 4N and isopropanol; 1:4), shaken on a shaker for 10 minutes. Uptake readwith ELISA readerat a wavelengthof 550 performedas cytotoxic nm. DAPT calculations test Testing Apoptosis HeLa cells grown on coverslipsinsertedin 96 mikroplate sinks as much as 3xl0 a cells / wells and incubated until 50-60% confluent. for 48 After it is incubated with test compormd hours. Medium was taken, washedwitlt PBS. Coverslip containing the cells is removd placed on the objec glass and add 10 mL lx Working Solution ethidium bromide-acridine orange and then allowed to stand for 5 minutes. Tues immediately observedunder a microscope. Living cells fluorescegreen(with acridine orange) and dead cells fluoresce orange(with ethidiumbromide) Researchresulb The resultsof H & E stainingon contol HeLa cells showedthat cells rmdergoingdivision are shown with an elongatedcell shape and the presence oftwo cell uuclei. tn addition to the with cells with contol cells that are congested condensed cell distribution in trms of pollmomial, membrane tfuough lysis. This condition occun starting concentrationof200 pgml.

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|SSN:zo89-6o69 Percentage of living cells due to the activity of jamur dewa extracts listed in the diagram below
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serpong, ocoi",ert - r"d"or.t Puspiptek Widya Bhakti Graha Gedung inhibiting normal cell growth. Bawh Figure 3 shows &at the death of HeLa cells to jamur of apoptosis dewa extact with the mechanism as indicatedbyfluorescentcells orange/broum, whereas the cells showed greeir fluorescent of cells were alive or not show the mechanism apoptosis stimulate apoptosis. A@au the imagebelow

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Diagram Lo/oaf living HeLa cells to the anticanceractivity of mushroomextractsgod, (1) 0 F.eiml,(2) 200 Fslml, (3) 400 Fg/ml, (4) 600 pg/ml, (5) 800 pslml, (6) 1000pg/ml of There are differences in the perce'lrtage surviving cells was significantly different at all of mushrcom cxtract lcvcls of concentrations gods,exceptin the treatmentof 200 pg/ml and 400 pglml, and 200 pg/ml and 600 pg/ml. analysisofdata can Basedon linear regression be obtained value of IC 5e values obtained 1944.4 pgiml. Test of cell proliferation kinetics s[owed inhibition profiles basedon the confrol of cell division. Treatmentofjamur dewaexftactused was 400 pglml, this is because the visual of inhibition indicatethe presence observations absorbance at a of cell division. Comparison of concentration of 400pg/ml and controls at different incubation time can be indicated by the diagrambelow

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(b) (a) Figure 3: (a) The cell apoptosismechanism a green HeLa without gling (b) Th" cell extractsof fluorescence, HeLa with mushroom god 400 !g/ml gave fluorescenceorange / brown

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Figure 2. Comparisonbetween the conhol absorbancevalue (0 pglml) with 400pg/ml with diffsrent incubation 3= time (l : 0 hours,2= 24 hoursn = 48 hours,4 72 hours) Apoptosis test aims to see ttrat cell death is apoptosis rather than necrosiq which is inhibited so that only cancer cells without
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Discassion level of Jarrur dewa extract at a concentration 200 ug/ml already have the abilrty to inhibit cell division. This is shown in a preliminary test of H&E stainingand cytotoxic test. Jamur dewa extract has the ability to inhibit ceil division because they contain trpn compounds with the inhibitory mechanism amitosis during cell division (Novaes, e/ a/. 2007). King (2000) mentionsthat the terpenes nyew plants (a tlpe of fimgus) provide action thus preveirting binds to tubulin at interphase, depolimerisasitubulin. Setiawati,et aI Q009) also mentions that the continued life of in plants (Gynuraproanmbms(t our.) terpnes Men) Can block the cell cycle in phaseGzlM by stabilizing the threadson the phasc of the mitotic spindlc thus hamperedthe processof mitosis, the next phasewill occur inhibition of cell proliferation. Inhibition of cell proliferation may be thrmgh the mechanism of leaves in the Terpenoidcompounds apoptosis. continuedto inhibit the enzyne topoisomerase lives. In addition to terpenoids,there is also a betaglucan. According to Ching-Han Yu (2008) fungal beta-glucan conteirt of the god with prostatc cancer can be inhibited at concentations of 400 [g/ml to 800 ng/ml

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|SSN:zo89-6o69 with antiproliferasieffect. Beta glucan alsohas sitolitik activity in human tumor cells in vitro (Novaes, et al.,2007). Levitschka" et a1.,2007; (2010) that also says beta glucan Naso, e/ al., has antimutagenactivity. The next compound is ergosterolmay slow tumor growth (Takeshi, et a1.,2007) et a1.,2001;Novaes, of surviving cells The resultsof the percentage level was show by raisrng the conce,lrtration increasing.This ntill be inverselyproportional to test the introduction by using a staining H&E and the observationof HeLa cells under a microscope to test the cytotoxic (Fig.2), namely the rising levels of conce,ntration disfribution of the density of cells decreases, buildup cells decreased, the amount of lysis more and more, and formation membrane of apoptoticbodies more and more. This was due to a death in cancercells still leavesthe results of the cell metabolisrn, causing sediment/colloid so it will be absorbed by spectrophotometry.Jamur dewa extract has growth inhibitory activity of HeLa cells with lcso 194.M trg/ml througb a processof linear regression analysis. This meaxrs that the of 194.44trg/ml of yeastextract concentration has the ability to inhibit the god of HeLa cells to 50%. of the kinetics of cell proliferation Observation performed to determinethe inhibition of cell proliferation that canbe either cell cycle arrest and cell cycle delcy. Incubation time is done differently, namely o clock, 24 hours, 48 hours,and 72 hoursto seethe time spanof the inhibition, by comparing the cells decreased treafinent of fungal extracts god 400 !g/ml The occurrence of and control (0ndml) proliferation is indicative of inhibition of cell compoundsthat have activity kemopreventiv (Meiyanto,et aI., 2M8) Conclusion The conclusion is that the mushroomextract hasthe ability as an anticancergod in cenrical cancer cells at a concentrationof 200 ug/ml, ug/ml with IC 5svalue of 194.44 Achnowledgemenls We speakto the CV Agaricus Sido Makmur Lawang thank's who have cooperatd Sentosa with investigatorsin terms of financing this research

o*oi"r.t* Serpong, "t*r*t Bhakti Puspiptek Graha Widya Gedung

Refenal list Albert, Bnrce; et al. 2008. Molecalar Biologt of the Cell 5 d' New York Garland Scieirce, Taylor & FrancisGroup,LLC Bruggemann,R., Orlandi, JM, & Benati, FJ,. 2006. Antiviral Activity Of Agaricas blazeii Murill ss. Heinem Extract Against In Cell Human And Bovine herpesviruses of Journal Brazilian Culture. Microbiologt, Vol. 37, No. 4, (online), =..., (http//www.scielo.br/scielophp?script accessed August3, 2010) Chan, Y., Chang, T., & Chan, CH 2007. Immunomodulatory Effects Of Agaricus blazei Murill in Balb / cByJ Mice. Joumal of Microbiologt, Immunologl, And Infection, Vol 40: 201-208 Diaka, JK, Hassanhi, M., & Brown, J. 2006. CytoregRinhibits Growth andProliferation of Human Adenocarcinoma Cells Via Induction of Apoptosis. Journal of Vol. 5, No. 1, (Online), ( Carcinogenesrs, accessed http:/lwww.carcinogenesis.com, July 6, 2010) Defen, W. 2008. Klnik Textbookof Oncolagt, 2nd edition. London: Faculty of Medicine Universityof Indonesia Ferreira, et al., 2A10. Compoundsfrom wild mushrooms with antitumor potential. Agents Med Chem.Iwei 10(5): Anticancer (Online). 424-36. Abstract. ${ttp/ / www. Ncbi.ntn.nih.gov / pubmed/20545620, June29, 2011) accessed Firenzuoli,F., Gori, & Lombardo,G. 2007. The Medicinal MushroomAgaricus blazei Review of Literature and Munill: p harm ac*toxic o logic a I Problems. Empoly: Natural Medicine and Deparhrent Hospital, of Intemal Medicine, S. Giuseppe AZUSL 11 Fihiasari, A., Wijayanti, NK & Fitiyah, NQ 2007. Ethanolic extract Proliferative Effects Long Beans In cells T47D. Pharmacon, Vol. 8, no.2: 44-50 d King, Roger JB. 2000. Cancer Biologt, 2 Pre,ntice Hall. Singapore: Peatson, Lavitschk4 RJ, Oliveira, CR, & Mascara"D. 2007.In vitno cytotoxicity And Antioxidant Agaricus Activity Of Extracts Journal of Subrufescens. Afican Biotechnalogt, (Online), Vol. 6 (9), (http://

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ISSN:zo89-6o69 www. Academicjournals. Org / AJB. June30,2010) accessed Meiyanto, E., Susidarti,RA, & Handayani,S. 2008. Ethanolic extacts of betel nuts (Areca catechu L.) Ability to Inhibit Proliferation and Stimulate MCF-? cell apoptosis. Magazines Pharmaceatical Vol. 19,No. l, 12-19 Indonesia, Naso, FCD, Mello, RND, & Bona, S. 2010, Agaricas hlazei llldvrill Effect On The Pulmonary Tissue Of Animals With Diabetes. Sfreptozotocin-Induced (Online), ExperimentalDiabetesResearch, (2010), ( Vol 2010 92 http://www.hindawi.conr/journals/.../543 March8, 2010) 6 , accessible Novaes,MRCB,Novaes, LCG, & Taveira,VC 2007. Nafirral Product From Agaricales Biology, Mushrooms: Medicinal And Properties, Nutritional

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Pharmacological Effects on Cancr. (4): Revista Brasileira de C.ancerologia,53 4r1420 Sstiawati, A., Dkk. 2009. Connect Lives (Gyrura proanmbens(Laur.) Men.) As a chemopreventiveagent. London: Cancer Research Center,Faculty Chemoprevention University of GajahMada. of Pharmacy, Yu, CH, Kana, SF, & Shub, CH 2009.. Inhibitory Nfsghanisms of Agarians blazei Murill on the Growth of ProstateCancerIn Vito And In Vivo. Joumal of Nutritional Biochemestry 20753-764,

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