Covalently Conjugation of Genistein with Biodegradable Poly L ...

146 P. Sojitra, A. Raval, D. Kothwala, A. Rawal, H. Kotadia, S. Adesharapeanuts [8] and in dietary isoflavones such asbeans and legumes [9]. Phenolic rings inGenistein is are responsible for estrogenreceptor binding [10]. The hydroxyl group at theC-4’ position enhanced the antioxidantproperties of Genistein drug. The structure ofGenistein consisting of two benzyl rings by athree- carbon bridge, which is simplified as C 6-C 3-C 6. It is also classified as a phytoestrogensdue to its weak estrogenic activity in mammaliansystems [11].Genistein is a potential flavonoid whichpossesses anti-thrombotic and antiproliferativeproperties. Data suggest thatGenistein may exert a strong anti-carcinogeniceffect, and that this effect possibly involves aninduction of p21, which inhibits the thresholdkinase activities of Cdks and associated cyclins,leading to a G2/M arrest in the cell cycleprogression [12, 13]. It inhibited collage inducedhuman platelet aggregation in a dosedependentmanner [14,15]. It has been shownto enhance NO production from theendothelium. NO is a potent inhibitor of plateletadhesion, aggregation and thrombosis.Impaired platelet production of NO has beenassociated with acute coronary syndromes.Thus Genistein may affect platelet aggregationvia an NO-dependant signal transductionpathway, a potential mechanisam. Theantitumor effect of Genistein has been reportedthrough the inhibition of protein kinasepathways leading to gene expressionmodification of many proteins, including VEGF.Novel drug-biodegradable polymer conjugatescan serve the purpose of sustained drugdelivery simultaneously with the polymerdegradation period. In the present study,Genistein was conjugated with biodegradablePLLA and was performed as coating on coronarystent.Materials and methodsThe Stainless steel 316L electropolished stents(16mm length) were used in the present study.L-Lactide (Boehringer-Ingelheim, Germany)was purified by recrystallization from dry ethylacetate and dried in vacuo at room temperature.Genistein was obtained from Ren YoungPharmaceutical Co Ltd, China and used withoutfurther purification process. Stannous octoate(Sn-oct, Sigma), Dicyclohexylcarbodiimide(DCC, FLUCA), 4-(dimethyl amino) pyridine(DMAP, FALUCA), dichloromethane (DCM),Dimethyl Sulphoxide (DMSO) and otherchemicals used in the current investigationwere of HPLC grade procured from RanbaxyFine Chemicals Ltd, India.MethodsPLLA was synthesized by the reported method[16]. Briefly, L-lactide (10 g, 0.07 mol) wasmelted at 180 °C for 30min in a nitrogenatmosphere. Stannous octoate (0.1 wt % of L-lactide) was added to the melted L-lactidesolution. The mixture was degassed and stirredat 150 °C for 30 min. The product was dissolvedin chloroform and then precipitated in excessmethanol. The precipitate was filtered and driedovernight under vacuum.A DCC, DMAP chemistry was used to synthesizethe PLLA-Genistein. Genistein C 15H 10O 5(0.02702 gm 1x10 -4 mol) and PLLA (0.1g,2.0x10 -4 mol) was dissolved in the DMSO (50ml), N, N-dimethyl formamide (DMF, 50 ml)respectively. DCC (2x10 -4 mol) and DMAP (2x10 -4mol) were added to drop by drop PLLA solutionin DCM was added to Genistein solution withstirring for 10 min at constant 50°C. The reactionwas further carried out for 18 hours undernitrogen atmosphere. After the couplingreaction, the reaction solution was precipitatedin excess methanol. The precipitate wasdissolved in chloroform and the solution wasprecipitated in excess methanol. After filtering,the precipitate was dried at 35°C for 24 h invacuum to eliminate the residual solvent.HPLC analysis of Genistein were performed onHPLC-LC-2010 AHT [Shimadzu (Asia Pacific)Pvt. Ltd.] consisting of X-Terra RP-18 (250*4.6mm) analytical column having particle size 5µm.The drug content was analyzed using mobilephase consisting of acetonitrile: methanol:water (45:35:20 v/v) at flow rate of 1.2 ml/minand oven temperature 40°C. The retention timefor Genistein was kept at 2.9 minute and wasdetected at 254 nm by UV absorption.This analysis was performed on mother liquorto set molar ratio of genistein and PLLA. Liquorwas taken from the reaction mixture at each 18