恰玛古异硫氰酸酯的酶解制备及抗宫颈癌活性

Enzymatic preparation and antitumor activity of Brassica rapa L.isothiocyanate

优化了恰玛古异硫氰酸酯(Brassica rapa L.isothiocyanate,BRIe)的外源酶解制备工艺,分析、评价了酶解产物的化学成分和抗宫颈癌活性。采用外源黑芥子酶对恰玛古硫代葡萄糖苷进行降解,通过单因素和正交优化实验确定了酶解法的最佳制备工艺:酶量为20μL、酶解温度70℃、pH 6.5、酶解时间2 h、抗坏血酸浓度为0.01 mg/g,在此条件下,异硫氰酸酯的提取率为11.13%;GC-MS鉴定分析所得酶解产物的化学成分有7种,分别为3-氨基-2-恶唑烷酮、2-恶唑烷硫酮、3-丁烯基异硫氰酸酯、2-苯乙基异硫氰酸酯、异硫氰酸异丙酯、异硫氰酸-2-苯乙酯。采用MTT法检测BRIe对宫颈癌Hela、Siha、U14细胞的增殖抑制活性,其中BRIe对Hela细胞的抑制作用最强,72 h IC_(50)为97.48μg/mL;细胞划痕实验显示BRIe抑制了Hela细胞的迁移,PI流式细胞染色显示BRIe将细胞周期阻滞在S期和G2/M期,Hochst 33258、Annexin V-FITC和PI染色表明BRIe诱导了Hela细胞凋亡。研究结果为开发利用恰玛古用于功能食品和抗宫颈癌药物提供了科学依据。

The exogenous enzymatic preparation process of Brassica rapa L.isothiocyanate(BRIe)was optimized,and anti-cervical cancer activity of the enzymatic products were analyzed and evaluated.The exogenous myrosinase was used for the degradation of B.rapa glucosinolate and the optimal preparation process for the enzymatic method was determined by one-way and orthogonal optimization experiments:the amount of myrosinase addition was 20μL,enzymatic temperature was 70℃,pH 6.5,enzyme time was 2 h,and the ascorbic acid concentration was 0.01 mg/g.Under these conditions,the extraction rate of isothiocyanate was 11.13%.There are seven chemical components of the enzymatic hydrolysis products obtained by GC-MS identification and analysis,namely 3-amino-2-oxazolidinone,2-oxazolidinethione,2-oxazolidinethione,3-butenyl isothiocyanate,2-Phenethyl isothiocyanate,isopropyl isothiocyanate,2-phenylethyl isothiocyanate.The proliferation inhibitory activity of BRIe on cervical cancer Hela,Siha and U14 cells was detected by MTT assay,in which BRIe had the strongest inhibitory effect on Hela cells,with a 72 h IC_(50)of 97.48μg/mL;Cell scratch experiments showed that BRIe inhibited the migration of Hela cells,PI flow cytometry staining exhibited that BRIe arrested the cell cycle in the S phase and G2/M phase,and Hoechst 33258,Annexin V-FITC and PI staining demonstrated that BRIe induced apoptosis in Hela cells.The findings provide a scientific basis for the development and utilization of B.rapa for functional foods and anti-cervical cancer drugs.