{"paper_id":"6d1a1257-1e46-4147-88b6-c6bbc66f901d","body_text":"Majalah Obstetri & Ginekologi Vol. 20 No. 1 Januari – April 2012 : 30-34 \n \n 30  \nComparison of Ovarial Malondialdehyde (MDA) Level between Endometriosis Rat \nGiven with and without Curcumine Supplementation \n \nBernadetta Caroline Panjaitan 1, Ashon Sa’adi 1, Hendy Hendarto 1, Widjiati 2 \n1Department of Obstetrics and Gynecology, Faculty of Medicine, Airlangga University, Dr. Soetomo Hospital, Surabaya \n2Department of Embryology, Faculty of Veterinary Medicine, Airlangga University \n \n \nABSTRAK \n \nPeradangan kronis pada endometriosis menyebabkan st res oksidatif pada sel granulosa yang menyebabkan k etidakseimbangan \nantara oksidan dan anti radikal bebas. Hal ini meny ebabkan efek berantai terus menerus dan menyebabkan  kerusakan membran sel. \nEfek akhirnya adalah ketidakstablian dan fragmentas i produksi MDA. Peran kurkumin sebagai antioksidan yang dapat menurunkan \nMDA titer menyeluruh belum diteliti. Tujuan dari pe nelitian ini adalah untuk mengetahui pengaruh kurku min ke ovarium MDA di \ndalam tikus jenis endometriosis. Penelitian ini mer upakan uji eksperimental dengan populasi 40 tikus ( Rattus novergicus  strain \nWinstar) betina yang memenuhi kriteria inklusi dila kukan randomisasi dengan 20 sampel dalam setiap kel ompok. Untuk \nmendapatkan tikus endometriosis, kami menyuntikkan tikus dengan intra Estradiol cyclosporin A dan Ethy nil musculary juga injeksi \ndalam jaringan endometrium dalam tumor ovarium jina k pada manusia ke dalam tikus intrapertitoneal. Kelo mpok perlakuan diberi \nsuplementasi kurkumin 24 mg (240 g/ kg) setiap hari  dalam 14 hari, kelompok lain diberi plasebo. Setel ah semua tikus dioperasi \nuntuk mendapatkan jaringan ovarium dan memeriksa ti ter MDA diperiksa dengan spektrofotometri. Didapatk an ada perbedaan \ndalam titer ovarium MDA di dalam tikus endometriosi s yang diberikan dan tidak diberikan suplementasi k urkumin (p <0,0001). \nSebagai kesimpulan, titer MDA pada tikus endometrio sis dengan suplementasi kurkumin lebih rendah dari plasebo. (MOG \n2012;20:30-34) \n \nKata kunci: endometriosis, curcumin, MDA  \n \n \nABSTRACT \n \nChronic inflamation at endometriosis causing oxidati ve stree at granulosa cell which cause unbalance bet ween free radical and anti \noxidant. This cause the chains effect continuously and cause the destruction of cell membrane and the last effect is unstable and the \nfragmentation producing MDA. The role of curcumin a s antioxidant can decrease overall MDA titer has no t been studied. The \nobjective of this study was to investigate the effec t of curcumin to ovarial MDA in endometriosis rat. This was eksperimental \nlaboratory trial with population female 40 rat ( Rattus novergicus  strain Winstar) which met the inclusion criteria t he rats were \nrandomnized and with 20 sample in each group. To bec ome endometriotic the rats were, we injected with cycl osporin A and  Ethynil \nEstradiol intra musculary also injection in endomet rial tissue of human benign ovarial tumor in human into intrapertitoneal rat. \nTreatment group was given curcumine suplementation 24 mg (240 g/kg) every day in 14 days, another grou p given placebo. All rats \nwere operated to get the ovarial tissue and check th e titer MDA was examined with spectrofotometry. There  were difference in \novarial MDA titer at in endometriosis rat which giv en with dan without curcumine suplementation.(p< 0,00 01). In conclusion, MDA \ntiter at endometriosis rats with curcumin supplementation is lower than placebo.  (MOG 2012;20:30-34) \n \nKeyword :  endometriosis, curcumin, MDA \n \nCorrespondence:  Bernadetta Caroline Panjaitan, Department of Obstetrics and Gynecology, Faculty of Medicine, Airlangga \nUniversity, Dr. Soetomo Hospital, Surabaya, detta_p@yahoo.com \n \n \nINTRODUCTION \n \nEndometriosis is a gynecology abnormality which \nknown as the growth of endometrial tissue outside t he \nuterine cavum. This abnormality is often found in \nreproductive age. Most of them come with symptom of  \npain and infetility. Endometriosis therapies recent ly are \nmedical therapy, surgery and combination of both. \nMedical therapy recently with hormonal therapy like  \nGnRHagonis, progestin, danazolor oral contraception . \nHormonal therapu is the most effective in reducing pain \nat endometriosis. Commonly to get the optimal effec t, \nthese drugs given in 3-6 months, with side effect t he \ncease of ovulation progress and hipoestrogen. This can \ncause endometriosis patient cannot pregnant while i n \ntherapy \n \nChronic inflammation at endometriosis causes oxidat ive \nstress at granulosa cell which cause unbalance betw een \nfree radical and anti oxidant. This leads to chains  effect \ncontinuously and cause the destruction of cell mem-\nbrane and the last effect is unstable and the fragm ent-\nation producing MDA. One of the alternative therapy  \nrecently is herbal therapy from plantation which is  \n\nPanjaitan et al. : Comparison of Ovarial Malondialdehyde (MDA) Level  \n \n \n31  \nCurcuma longa  and has been used for many centuries in \nSouth East Asia. Many active ingredients in curcumi n \nhave different effect which are anti inflammation, anti \noxidant, anti proliferative, anti angiogenesis. Phe nolic \nand methoxy group in the structure of curcumin is t he \nbasic things which  function is anti oxidant. Until  know \nthe role of curcumin as antioxidant that can decrea se the \noverall MDA titer, has not been studied. \n \nIdeal therapy is to supress the target cell as effe ctive, \nsafety, cheap and with little side effect and witho ut \nhypoestrogenic effect. Curcumin has not been used i n \nhuman to cure endometriosis, which connected to \ninfertility and in human experimental to know the e ffect \nof curcumin still constraint with ethic. So in this  \nstudying, we use endometriosis type rat. The aim is  to \nprove the MDA titer in ovarian rat can decreasing w ith \ncurcumin in endometriosis type rat compared with \ncontrol group. With group fenolic in the structure of \ncurcumin is the basic things of the effect as an \nantioxidant to clean the free radical directly and \nundirectly by activate the GSH reductase enzyme whi ch \nit role to increasing the supply of GSH. In this \nexperiment, the concept which shows that curcumin b y \noral can prevent perioxidation lipid membran which has \nbeen caused by oxidative stress. The damage of \ngranulosa cell membrane causes interference in \nfolliculogenesis. The effect of antioxidant curcumi n can \ndecrease the oxidative stress and lipid membrane pe r-\noxidation. In the future, curcumin can be the alter native \ntherapy to cure endometriosis, particularly which r elated \nto infertility. \n \n \nMATERIALS AND METHODS \n \nResearch was conducted in Embriology Laboratory, \nFaculty of Veterinary Medicine, Airlangga Universit y, \nin March 2012. Sample was taken from two groups of \nrats: group A and B, injected by cyclosporin and go t \ninjected by human endometrial tissue intraperitonea lly, \nthen injected by estradiol at day 1 and 5, start fr om day \n14, group A got curcumin by sondage for 14 days, as  if \ngroup B got placebo per sondage for 14 days. The \namount of sample got 19 with federer formulla.  \n \nInclusion criteria for this research are: female ra t, \n(Rattus novergicus  strains wistar), 3 months, weight \n100-150 grams, virgin, health. Exclusion criteria: have \nbeen used by others research. All materials in this  study \nwas endometriosis type rat ovarial. Tissue was put in \nreaction tube. Ovarial tissue is sepparated and \nhomogenizing in homogenizer with buffer contain 1,5 % \nchloride potasium to get 1:10 (b/v) fully homogeniz ed. \nSupernatan (MDA) was mixed with 1 ml TBA reagent, \nmeasure with spectrophotometry at 541nmol.  \nAll experimental animals were taken from experiment al \nanimal unit in Faculty of Veterinary Medicine, \nAirlangga University. We use female rat ( Rattus \nnovergicus ) with age less than three months and weight \n100-150 grams, chosen by inclusion and exclusoin \ncriteria. Rat will be in adaptation time for 1 week s in \nclean nest, enough air, light, food and drink and \nhomogenize. All rats were divided to 2 groups, with  \neach group consisted 19 rats. Group A and B were \nendometriosis type rats. Group A received curcumin,  \ngroup B only placebo. To make the endometriosis typ e \nrat is from endometrial biopsy which injected by \ncyclosporin and estrogen. The injection of cyclospo rin \nin day 1 intramusculary to group A and B, each 0.2 ml \nwith disposable syringe 1 ml. Injection with endome trial \nbiopsy at day 1 intraperitonealy to 2 groups A and B, \neach rat injected by 0.7 ml. Injection with disposa ble \nsyringe 1 ml, with needle size 16 so the endometria l \ntissue can enter. Injection at day 1 and 5 intramus culary \nfor group A and B, each rat injected by 0,05 ml, in jected \nwith disposable syringe 1 ml, treatment with curcum in \nsupplementation given in day 14 to group A. Group B  \nwas given with placebo. Treatment for two groups un til \nday 27 (14 days). Sample was taken at day 28, and \nanalyzed by spectrofotometry to measure the MDA tit er \nin the ovary.  \n \nAll data in this research were recorded in specific  form. \nData analysis was done with software SPSS (Software  \nPackage for Social Science). Conduct normality test  \nwith Kolmogorov Smirnov test. If the data were \ndistributed normally, continued with t test 2 free \nsamples. If data not normally distributed, continue d \nwith Mann-Whitney test. The confidence interval at this \nresearch is 0.05. \n \n \nRESULTS AND DISCUSSION \n \nAfter tested by normality test with distribution \nKolmogorov-Smirnovone sample in variable which is \nrat weight placebo group, the result was p = 0.431 \n(before treatment) and p = 0.381 (after treatment) also \nwith curcumin got p = 0.141 (before treatment) and p = \n0.411 (after treatment). because p > 0.05 so the tw o \ngroups are normally distributed (Table 1).  \n \nBecause normally distributed, then tested with t te st two \nsample and get p < 0.05 at two groups so can be \nconcluded even we found increasing weight at curcum in \ngroups more than placebo group, but as stastically with t \ntest 2 sampe, got p = 0.114 (p > 0.05) show there a re no \ndifference at weight change in two groups and two \ngroup can be categorize as homogenize. Because the \nweight change is variable so weight is not put at n ext \nanalysis (Table 2). In this research ovarial MDA ti ter \n\nMajalah Obstetri & Ginekologi Vol. 20 No. 1 Januari – April 2012 : 30-34 \n \n 32  \ncan be measure with spectrofotometry with kit OXIte k \nTBARS produced by Zepto Metrix Corporation. The \nprinciple of this test was based on the measurement  by \nspectrophotometry from pink, which produced by \nThiobarbituric Acid (TBA) reaction with MDA, \nconcentration of TBARS titer calculated by absorban cy \ncalculation from MDA-TBA curve. The evaluation by \nspectrophotometry conduct in laboratory unit, \nconsultation and training at Faculty of Veterinary \nMedicine, Airlangga University. Normality variable \nMDA ovarial titer was tested with Kolmogorof-Smirno v \none sample, which p = 0.972 (p > 0.05) at placebo group \nand p = 0.792 (p > 0.05) at curcumin groups which \nmeans this two group were normally distributed (Tab le \n3). Because of it normally distributed, stastically  test \nwhich use is parametric  test with t test two sampl e. T \ntest two sample result was  p = <0.0001 (p < 0.05) \nmeans there is differece between ovarial MDA averag e \nbetween curcumin group and placebo group. (Table 4) \n \nOn day 14 the treatment of curcumin and placebo \nstarted. We did not do confirmation examination abo ut \nthe incident of endometriosis histopatologically in  our \nresearch design, but as a result we found that the \nvariation coefficient was 12.7% for the placebo gro up \nand 22.2% for the curcumin group that shows MDA \nvariable measurement. \n \nThose narrow variation coefficient value shows that  \nthere might be other factors that can affect the re sult but \nit can be ignored. Success rate of incidence of \nendometriosis is based on previous studies conducte d \nbyAwwad (1999) 96%, Vika (2006) 87.5%, Kuswojo \n(2009) 89.5% and Sa’adi (2010)  80%. So we make a \ncorrection of the sample to anticipate the possibil ity of \nfailure occurrence of endometriosis by adding the \nsample to remain unfulfilled. \n \nComparison of ovarian MDA levels in the \nplacebo group and curcumin \n \nTranscription factor NF-Kß known role in the \npathogenesis of endometriosis, which stimulate the \nprocess of adhesion, invasion, angiogenesis, inflam -\nmation, cell proliferation and inhibits apoptosis \nendometriosis. The role of classical pathway activa tion \nof NF-kß is a natural immune response that stimulat es \ninflammation and maintain endometriosis lesions. 10  \nActivation of transcription factor NF-kß also assoc iated \nwith increased ROS. \n \n \n \n \nOxidative stress is believed to contribute negative ly to \nthe number of reproductive processes including \nfolikulogenesis and endometriosis (Behrman, 2001). \nSuspected a link between oxidative stress and infer tility. \nWomen with endometriosis often show an increase in \nmacrophage activity which may result in excessive R OS \nin the peritoneal environment. 3 Increased oxidative \nstress and ROS concentration in peritoneal fluid an d \nserum has also been shown in unexplained infertilit y, \ntubal factor infertility and endometriosis. 8,3 Polak et al \nstudy the levels of MDA which is a marker of oxidat ive \nstress and also the enzymes catalase and SOD levels  in \nendometrial tissue and blood on the woman's inferti lity, \nthe results of these studies found that MDA levels were \nsignificantly higher and levels of the enzymes cata lase \nand SOD were significantly lower in women with \nendometriosis and also in unexplained infertility \ncompared with the levels of MDA in the control. 8 \n \nCurcumin has potent antioxidant capabilities. Curcu min \ninhibits lipid peroxidation induced by iron. 11  the \nformation of ROS and oxidation of ferrous ions. 11  The \nability of curcumin in removing free radicals espec ially \nwith regard to O2-and OH 11 may be due to the phenolic \ngroup of the donor H atom. 2,6 Phenolic and methoxy \ngroups on the phenyl ring and 1,3-diketon system se ems \nto be an important structure that contributes to th is \neffect. Another fact is that put forward in the antioxidant \nactivity increases when the phenolic and methoxy \ngroups are in the ortho position. \n \nIn our study, the mean levels of MDA in the rat mod el \nof endometriosis curcumin group was lower than the \nplacebo group. Based on the free two-sample t test \nfound significant differences between the two group s. \nDifference in results is likely due to the success of \ncurcumin in inhibiting the process of apoptosis tha t \noccurs, while the mechanism may be through multiple  \npathways including inhibition of activation of the \ntranscription factor NF-kß with various results, \nsuppression of inflammatory activity through the \nsuppression of TNF-α directly or through its antioxidant \neffects. Singh and Aggarwal (1995) have shown that \ncurcumin can inhibit the activation of the transcri ption \nfactor NF-kß with emphasis on the target before the  Ikß-\na phosphorylation. Research of Jobin (1999) have al so \nproved the same.Curcumin was shown to reduce the \nnegative impact of endometriosis patients with immu ne \ndefects caused by anti-TNF-  α , anti-NF-kß, anti-\noxidants, anti-JNK and anti-caspase activation path way. \nCurcumin can suppress NF-kß pathway and gene target  \nof NF-kß cytokines. \n \n \n \n \n\nPanjaitan et al. : Comparison of Ovarial Malondialdehyde (MDA) Level  \n \n \n33  \n \nTable 1. Normality tes rat weight in curcumin dan placebo \n \nGroup Mean SD P price Information \nPlacebo Before 131.0 13.3 0.431 Normal \nAfter 137.5  10.2 0.381 Normal \nCurcumin Before 135.5  16.7 0.141 Normal \nAfter 148.0  14.4 0.411 Normal \n            Note : p > 0.05 is normally distributed \n \nTable 2. Homogenicity test result in rat weight fot group curcumin and placebo \n \nGroup Before After delta  P \nPlacebo 131 +  13.3 137.5 +  10.2 6.5 +  12.7 0.033 \nCurcumin  135.5  +  16.7  148 +  14.4 12.5 +  10.7 <0.0001 \n          Note: p > 0.05 is not meaningfull (homogen) \n \nTable 3. Normality test result for MDA ovarial \n \nGroup Mean SD p Info \nPlacebo \nCurcumin \n0.63  \n0.18  \n0.08 \n0.04 \n0.972 \n0.792 \nNormal \nNormal  \nNote: p > 0.05 is normally distributed \n \nTable 4 T test two sample result from free MDA ovar ial titer \n \n group  \n p \n \nInfo Placebo curcumin \nMDA titer \n(nmol/ml) \n 0.63 + 0.08  0.18 +  \n0.04 \n< \n0.0001 \nDifference \nMeaningfull \nNote: p < 0.05 is meaningfull difference \n \n \nWith inhibition of apoptosis that occurs will cause  the \nnumber of granulosa cells that survived became more  so \nthat proteins and hormones are secreted also higher . \nWith more number of granulosa cells that survived t he \nprocess of abnormal apoptosis, it is expected to in crease \nfertility in patients with endometriosis. \n \n \nCONCLUSION \n \nOvarian MDA level is lower in group receiving \nsupplementation of curcumin.  \n \n \nREFERENCES \n \n1.  Aggarwal S, Ichikawa H, Takada Y, Sandur S, \nShishodia S, Aggarwal BB. 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Ramos RG, Donnes J, Defrere S, Leclercq I, Squiflet  \nJ, Luosse JC, van Langendonckt A. 2007. Nuclear \nfactor kappa B is constitutively activated in \nperitoneal endometris. Mol Hum Reprod 13: 503-\n509. \n11.  Reddy AC, Lokesh BR. 1994. Studies on the \ninhibitory effects of curcumin on the formation of \nROS and Oxidation of ferrous ions. Mol Cell \nBiochem 137: 1-8. \n12.  Reddy AC, Lokesh BR.1996. Effect of Curcuma \nlonga  on iron-induced lipid peroxidation in the rat \nliver. Food Chem Toxicol 32: 279-83. \n13.  Sa’adi A. 2010. Efek curcumin dan progestin (MPA) \nterhadap ekspresi EGF dan luas implan \nendometiosis. Penelitian Konsultan FER \nDepartemen/SMF Kebidanan dan Penyakit \nKandungan FK Unair/RSUD dr Soetomo Surabaya. \n14.  Vika SP, Hendarto H, Suhartono DS, Widjiati. 2006. \nPengaruh pemberian siklosporin A sebagai penurun \njumlah limsofit serum terhadap terjadinya implant \nendometriosis pada mencit. Penelitian \nDepartemen/SMF Kebidanan dan Penyakit \nKandungan FK Unair/ RSUD dr Soetomo Surabaya.","source_license":"CC0","license_restricted":false}