冬凌草毛状根的优化培养及其提取物的肿瘤细胞增殖抑制活性检测

doi:10.13560/j.cnki.biotech.bull.1985.2018-0207

生物技术通报 ›› 2018, Vol. 34 ›› Issue (10): 122-128.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0207

冬凌草毛状根的优化培养及其提取物的肿瘤细胞增殖抑制活性检测

陈婉琪^{1, 2}, 张鸿^{1, 2}, 王进峰^{1, 2}, 陈俊杰^{1, 2}, 林晓文^{1, 2}, 罗结华^{1, 2}, 许源³, 陈艳芳^{1, 2}, 陆幸妍^{1, 2}

1. 广东药科大学生命科学与生物制药学院,广州 510006;
2. 广东省生物技术候选药物研究重点实验室,广州 510006;
3. 广东省药物新剂型重点实验室,广州 510006

收稿日期:2018-03-13 出版日期:2018-10-26 发布日期:2018-11-07
作者简介:陈婉琪,女,研究方向：生物制药;E-mail：kiki.chenwanqi@gmail.com;张鸿同为本文第一作者
基金资助:
2015年大学生创新创业训练计划项目（201510573011）,2014年大学生创新创业训练计划项目（201410573041）

Optimal Cultivation of Rabdosia rubescens Hairy Roots and the Inhibition Test of Its Extract on Tumor Cell Growth

CHEN Wan-qi^{1, 2}, ZHANG Hong^{1, 2}, WANG Jin-feng^{1, 2}, CHEN Jun-jie^{1, 2}, LIN Xiao-wen^{1, 2}, LUO Jie-hua^{1, 2}, XU Yuan³, CHEN Yan-fang^{1, 2}, LU Xing-yan^{1, 2}

1. School of Life Science and Biopharmaceutics,Guangdong Pharmaceutical University,Guangzhou 510006;
2. Guangdong Province Key Laboratory of Biotechnology Drug Candidate,Guangzhou 510006;
3. Guangdong Province Key Laboratory of Advanced Drug Delivery,Guangzhou 510006

Received:2018-03-13 Published:2018-10-26 Online:2018-11-07

摘要/Abstract

摘要： 探究并建立冬凌草毛状根的优化培养方案并检测其提取物的抗肿瘤活性。用发根农杆菌ATCC11325及ATCC15834诱导冬凌草外植体生成毛状根,并探究培养基种类、蔗糖浓度等条件对冬凌草毛状根生长的影响,测定其生长曲线;超声法制备毛状根粗提物,大孔吸附树脂进一步纯化粗提物获得提取物,用高效液相色谱法检测冬凌草甲素含量;提取物配制成不同浓度试液作用于癌细胞株BEL-7402、A-549、SGC-7901和HGC,用CCK-8法测定细胞存活率。仅ATCC15834可诱导冬凌草从叶柄和茎部切口处长出不定根,叶片外植体未见不定根萌发,不定根遗传鉴定符合毛状根特征。毛状根在含3%蔗糖B5培养基中增殖率最高,其生长曲线近似“S”型,25 d达到对数中期,70 d达到平台期,其冬凌草甲素含量分别为0.0171%、0.0022%。当毛状根提取物含量为1 mg/mL时,对四种癌细胞生长的抑制率均达95%以上。以ATCC15834侵染冬凌草叶柄和茎部外植体诱导出的冬凌草毛状根提取物在含3%蔗糖的B5培养基中摇瓶培养25 d再继代培养70 d得率最高;冬凌草毛状根提取物中含有冬凌草甲素,且能有效抑制体外培养肿瘤细胞生长。

关键词: 冬凌草毛状根诱导, 冬凌草甲素, 优化培养, 毛状根提取物, 抗肿瘤活性

Abstract: The objective of this study is to explore and establish the optimal method for cultivating Rabdosia rubescens hairy roots and to test the anti-tumor activity of its extracts. Agrobacterium rhizogenes ATCC11325 and ATCC15834 were used to induce the growth of hairy roots in the R. rubescens explants,the effects of different types of medium and different sucrose concentrations on the growth of R. rubescens hairy roots were investigated,and thereafter,their growth curves were measured. Ultrasound was employed to prepare the crude extract from R. rubescens hairy roots,then the crude extracts were purified via macroporous adsorption resin,and further,the content of oridonin in the extract was determined by high performance liquid chromatography（HPLC）. The purified extracts were formulated into test solutions with different concentrations to be applied on the cancer cell line BEL-7402,A-549,SGC-7901,and HGC,and the cell viability were determined by the CCK-8 method. As results,only ATCC15834 induced adventitious roots from the petiole and the incision on stems,however,no adventitious roots grew in the leaf explants. The genetic identification of adventitious roots was consistent with the characteristics of hairy roots. Hairy roots had the highest proliferation rate in 3% sucrose B5 medium,and the growth curve was similar to a “S” type,the mid-logarithmic phase reached at the 25th day and the plateau phase was at the 70th day. The rude extract from hairy roots by ultrasound and macroporous resin extract were analyzed to have the content of oridonin of 0.0171% and 0.0022%,respectively. When the content of extract from hairy roots was 1 mg/mL,the inhibition rate to 4 cancer cells reached 95% or more. Conclusively,the extract rate from the hairy roots of R. rube is the highest when the hairy roots induced by ATCC15834 to infect the petiole and stem explant of R. rubescens are cultured in 3% sucrose B5 medium with shake flasks for 25 days and then subculturing them for 70 days. The extract from R. rubescens hairy roots contains oridonin that can effectively inhibit the growth of in vitro tumor cells.

Key words: Rabdosia rubescens hairy roots induction, oridonin, optimal method in cultivation, hairy roots extract, anti-tumor activity

陈婉琪, 张鸿, 王进峰, 陈俊杰, 林晓文, 罗结华, 许源, 陈艳芳, 陆幸妍. 冬凌草毛状根的优化培养及其提取物的肿瘤细胞增殖抑制活性检测[J]. 生物技术通报, 2018, 34(10): 122-128.

CHEN Wan-qi, ZHANG Hong, WANG Jin-feng, CHEN Jun-jie, LIN Xiao-wen, LUO Jie-hua, XU Yuan, CHEN Yan-fang, LU Xing-yan. Optimal Cultivation of Rabdosia rubescens Hairy Roots and the Inhibition Test of Its Extract on Tumor Cell Growth[J]. Biotechnology Bulletin, 2018, 34(10): 122-128.

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冬凌草毛状根的优化培养及其提取物的肿瘤细胞增殖抑制活性检测

Optimal Cultivation of Rabdosia rubescens Hairy Roots and the Inhibition Test of Its Extract on Tumor Cell Growth

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