Comparison of Ovarial Malondialdehyde (MDA) Level between Endometriosis Rat Given with and without Curcumine Supplementation

Chronic inflamation at endometriosis causing oxidati ve stree at granulosa cell which cause unbalance bet ween free radical and anti oxidant. This cause the chains effect continuously and cause the destruction of cell membrane and the last effect is unstable and the fragmentation producing MDA. The role of curcumin a s antioxidant can decrease overall MDA titer has no t been studied. The

of this study was to investigate the effec t of curcumin to ovarial MDA in endometriosis rat. This was eksperimental laboratory trial with population female 40 rat ( Rattus novergicus strain Winstar) which met the inclusion criteria t he rats were randomnized and with 20 sample in each group. To bec ome endometriotic the rats were, we injected with cycl osporin A and Ethynil Estradiol intra musculary also injection in endomet rial tissue of human benign ovarial tumor in human into intrapertitoneal rat. Treatment group was given curcumine suplementation 24 mg (240 g/kg) every day in 14 days, another grou p given placebo. All rats were operated to get the ovarial tissue and check th e titer MDA was examined with spectrofotometry. There were difference in ovarial MDA titer at in endometriosis rat which giv en with dan without curcumine suplementation.(p< 0,00 01). In conclusion, MDA titer at endometriosis rats with curcumin supplementation is lower than placebo. (MOG 2012;20:30-34)

endometriosis, curcumin, MDA Correspondence: Bernadetta Caroline Panjaitan, Department of Obstetrics and Gynecology, Faculty of Medicine, Airlangga University, Dr. Soetomo Hospital, Surabaya, [email protected]

Endometriosis is a gynecology abnormality which known as the growth of endometrial tissue outside t he uterine cavum. This abnormality is often found in reproductive age. Most of them come with symptom of pain and infetility. Endometriosis therapies recent ly are medical therapy, surgery and combination of both. Medical therapy recently with hormonal therapy like GnRHagonis, progestin, danazolor oral contraception . Hormonal therapu is the most effective in reducing pain at endometriosis. Commonly to get the optimal effec t, these drugs given in 3-6 months, with side effect t he cease of ovulation progress and hipoestrogen. This can cause endometriosis patient cannot pregnant while i n therapy Chronic inflammation at endometriosis causes oxidat ive stress at granulosa cell which cause unbalance betw een free radical and anti oxidant. This leads to chains effect continuously and cause the destruction of cell mem- brane and the last effect is unstable and the fragm ent- ation producing MDA. One of the alternative therapy recently is herbal therapy from plantation which is Panjaitan et al. : Comparison of Ovarial Malondialdehyde (MDA) Level 31 Curcuma longa and has been used for many centuries in South East Asia. Many active ingredients in curcumi n have different effect which are anti inflammation, anti oxidant, anti proliferative, anti angiogenesis. Phe nolic and methoxy group in the structure of curcumin is t he basic things which function is anti oxidant. Until know the role of curcumin as antioxidant that can decrea se the overall MDA titer, has not been studied. Ideal therapy is to supress the target cell as effe ctive, safety, cheap and with little side effect and witho ut hypoestrogenic effect. Curcumin has not been used i n human to cure endometriosis, which connected to infertility and in human experimental to know the e ffect of curcumin still constraint with ethic. So in this studying, we use endometriosis type rat. The aim is to prove the MDA titer in ovarian rat can decreasing w ith curcumin in endometriosis type rat compared with control group. With group fenolic in the structure of curcumin is the basic things of the effect as an antioxidant to clean the free radical directly and undirectly by activate the GSH reductase enzyme whi ch it role to increasing the supply of GSH. In this experiment, the concept which shows that curcumin b y oral can prevent perioxidation lipid membran which has been caused by oxidative stress. The damage of granulosa cell membrane causes interference in folliculogenesis. The effect of antioxidant curcumi n can decrease the oxidative stress and lipid membrane pe r- oxidation. In the future, curcumin can be the alter native therapy to cure endometriosis, particularly which r elated to infertility.

Research was conducted in Embriology Laboratory, Faculty of Veterinary Medicine, Airlangga Universit y, in March 2012. Sample was taken from two groups of rats: group A and B, injected by cyclosporin and go t injected by human endometrial tissue intraperitonea lly, then injected by estradiol at day 1 and 5, start fr om day 14, group A got curcumin by sondage for 14 days, as if group B got placebo per sondage for 14 days. The amount of sample got 19 with federer formulla. Inclusion criteria for this research are: female ra t, (Rattus novergicus strains wistar), 3 months, weight 100-150 grams, virgin, health. Exclusion criteria: have been used by others research. All materials in this study was endometriosis type rat ovarial. Tissue was put in reaction tube. Ovarial tissue is sepparated and homogenizing in homogenizer with buffer contain 1,5 % chloride potasium to get 1:10 (b/v) fully homogeniz ed. Supernatan (MDA) was mixed with 1 ml TBA reagent, measure with spectrophotometry at 541nmol. All experimental animals were taken from experiment al animal unit in Faculty of Veterinary Medicine, Airlangga University. We use female rat ( Rattus novergicus ) with age less than three months and weight 100-150 grams, chosen by inclusion and exclusoin criteria. Rat will be in adaptation time for 1 week s in clean nest, enough air, light, food and drink and homogenize. All rats were divided to 2 groups, with each group consisted 19 rats. Group A and B were endometriosis type rats. Group A received curcumin, group B only placebo. To make the endometriosis typ e rat is from endometrial biopsy which injected by cyclosporin and estrogen. The injection of cyclospo rin in day 1 intramusculary to group A and B, each 0.2 ml with disposable syringe 1 ml. Injection with endome trial biopsy at day 1 intraperitonealy to 2 groups A and B, each rat injected by 0.7 ml. Injection with disposa ble syringe 1 ml, with needle size 16 so the endometria l tissue can enter. Injection at day 1 and 5 intramus culary for group A and B, each rat injected by 0,05 ml, in jected with disposable syringe 1 ml, treatment with curcum in supplementation given in day 14 to group A. Group B was given with placebo. Treatment for two groups un til day 27 (14 days). Sample was taken at day 28, and analyzed by spectrofotometry to measure the MDA tit er in the ovary. All data in this research were recorded in specific form. Data analysis was done with software SPSS (Software Package for Social Science). Conduct normality test with Kolmogorov Smirnov test. If the data were distributed normally, continued with t test 2 free samples. If data not normally distributed, continue d with Mann-Whitney test. The confidence interval at this research is 0.05.

AND DISCUSSION After tested by normality test with distribution Kolmogorov-Smirnovone sample in variable which is rat weight placebo group, the result was p = 0.431 (before treatment) and p = 0.381 (after treatment) also with curcumin got p = 0.141 (before treatment) and p = 0.411 (after treatment). because p > 0.05 so the tw o groups are normally distributed (Table 1). Because normally distributed, then tested with t te st two sample and get p 0.05) show there a re no difference at weight change in two groups and two group can be categorize as homogenize. Because the weight change is variable so weight is not put at n ext analysis (Table 2). In this research ovarial MDA ti ter Majalah Obstetri & Ginekologi Vol. 20 No. 1 Januari – April 2012 : 30-34 32 can be measure with spectrofotometry with kit OXIte k TBARS produced by Zepto Metrix Corporation. The principle of this test was based on the measurement by spectrophotometry from pink, which produced by Thiobarbituric Acid (TBA) reaction with MDA, concentration of TBARS titer calculated by absorban cy calculation from MDA-TBA curve. The evaluation by spectrophotometry conduct in laboratory unit, consultation and training at Faculty of Veterinary Medicine, Airlangga University. Normality variable MDA ovarial titer was tested with Kolmogorof-Smirno v one sample, which p = 0.972 (p > 0.05) at placebo group and p = 0.792 (p > 0.05) at curcumin groups which means this two group were normally distributed (Tab le 3). Because of it normally distributed, stastically test which use is parametric test with t test two sampl e. T test two sample result was p = <0.0001 (p < 0.05) means there is differece between ovarial MDA averag e between curcumin group and placebo group. (Table 4) On day 14 the treatment of curcumin and placebo started. We did not do confirmation examination abo ut the incident of endometriosis histopatologically in our research design, but as a result we found that the variation coefficient was 12.7% for the placebo gro up and 22.2% for the curcumin group that shows MDA variable measurement. Those narrow variation coefficient value shows that there might be other factors that can affect the re sult but it can be ignored. Success rate of incidence of endometriosis is based on previous studies conducte d byAwwad (1999) 96%, Vika (2006) 87.5%, Kuswojo (2009) 89.5% and Sa’adi (2010) 80%. So we make a correction of the sample to anticipate the possibil ity of failure occurrence of endometriosis by adding the sample to remain unfulfilled. Comparison of ovarian MDA levels in the placebo group and curcumin Transcription factor NF-Kß known role in the pathogenesis of endometriosis, which stimulate the process of adhesion, invasion, angiogenesis, inflam - mation, cell proliferation and inhibits apoptosis endometriosis. The role of classical pathway activa tion of NF-kß is a natural immune response that stimulat es inflammation and maintain endometriosis lesions. 10 Activation of transcription factor NF-kß also assoc iated with increased ROS. Oxidative stress is believed to contribute negative ly to the number of reproductive processes including folikulogenesis and endometriosis (Behrman, 2001). Suspected a link between oxidative stress and infer tility. Women with endometriosis often show an increase in macrophage activity which may result in excessive R OS in the peritoneal environment. 3 Increased oxidative stress and ROS concentration in peritoneal fluid an d serum has also been shown in unexplained infertilit y, tubal factor infertility and endometriosis. 8,3 Polak et al study the levels of MDA which is a marker of oxidat ive stress and also the enzymes catalase and SOD levels in endometrial tissue and blood on the woman's inferti lity, the results of these studies found that MDA levels were significantly higher and levels of the enzymes cata lase and SOD were significantly lower in women with endometriosis and also in unexplained infertility compared with the levels of MDA in the control. 8 Curcumin has potent antioxidant capabilities. Curcu min inhibits lipid peroxidation induced by iron. 11 the formation of ROS and oxidation of ferrous ions. 11 The ability of curcumin in removing free radicals espec ially with regard to O2-and OH 11 may be due to the phenolic group of the donor H atom. 2,6 Phenolic and methoxy groups on the phenyl ring and 1,3-diketon system se ems to be an important structure that contributes to th is effect. Another fact is that put forward in the antioxidant activity increases when the phenolic and methoxy groups are in the ortho position. In our study, the mean levels of MDA in the rat mod el of endometriosis curcumin group was lower than the placebo group. Based on the free two-sample t test found significant differences between the two group s. Difference in results is likely due to the success of curcumin in inhibiting the process of apoptosis tha t occurs, while the mechanism may be through multiple pathways including inhibition of activation of the transcription factor NF-kß with various results, suppression of inflammatory activity through the suppression of TNF-α directly or through its antioxidant effects. Singh and Aggarwal (1995) have shown that curcumin can inhibit the activation of the transcri ption factor NF-kß with emphasis on the target before the Ikß- a phosphorylation. Research of Jobin (1999) have al so proved the same.Curcumin was shown to reduce the negative impact of endometriosis patients with immu ne defects caused by anti-TNF- α , anti-NF-kß, anti- oxidants, anti-JNK and anti-caspase activation path way. Curcumin can suppress NF-kß pathway and gene target of NF-kß cytokines. Panjaitan et al. : Comparison of Ovarial Malondialdehyde (MDA) Level 33 Table 1. Normality tes rat weight in curcumin dan placebo Group Mean SD P price Information Placebo Before 131.0 13.3 0.431 Normal After 137.5 10.2 0.381 Normal Curcumin Before 135.5 16.7 0.141 Normal After 148.0 14.4 0.411 Normal Note : p > 0.05 is normally distributed Table 2. Homogenicity test result in rat weight fot group curcumin and placebo Group Before After delta P Placebo 131 + 13.3 137.5 + 10.2 6.5 + 12.7 0.033 Curcumin 135.5 + 16.7 148 + 14.4 12.5 + 10.7 0.05 is not meaningfull (homogen) Table 3. Normality test result for MDA ovarial Group Mean SD p Info Placebo Curcumin 0.63 0.18 0.08 0.04 0.972 0.792 Normal Normal Note: p > 0.05 is normally distributed Table 4 T test two sample result from free MDA ovar ial titer group p Info Placebo curcumin MDA titer (nmol/ml) 0.63 + 0.08 0.18 + 0.04 < 0.0001 Difference Meaningfull Note: p < 0.05 is meaningfull difference With inhibition of apoptosis that occurs will cause the number of granulosa cells that survived became more so that proteins and hormones are secreted also higher . With more number of granulosa cells that survived t he process of abnormal apoptosis, it is expected to in crease fertility in patients with endometriosis.

Ovarian MDA level is lower in group receiving supplementation of curcumin.

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