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目的:利用转基因何首乌毛状根对瑞香素(I)进行生物转化研究,对其生物转化产物进行分离、鉴定,并建立其产物的时效曲线图。方法:将瑞香素投入何首乌毛状根悬浮培养体系中,共培养36 h后,利用TLC和HPLC进行产物检测,硅胶柱层析法分离纯化,核磁共振技术进行结构鉴定,利用HPLC考察共培养时间对转化产物(Ⅱ)、降解产物含量的影响及分析转化产物降解的原因。结果:瑞香素在何首乌毛状根悬浮培养体系中发生了生物转化反应。分离鉴定出一个产物:瑞香素-8-O-β-D-葡萄糖苷(Ⅱ)。何首乌毛状根悬浮培养体系转化生成Ⅱ的最佳共培养时间为36 h,总摩尔转化率为32.11%。蔗糖-水培养基(仅含有蔗糖和水)可以使何首乌毛状根悬浮培养体系转化产率大幅增加,总摩尔转化率达72.44%。初步推断产物(Ⅱ)的降解与MS培养基中金属离子有关。结论:使用何首乌毛状根悬浮培养体系可大量生物合成天然药材中含量极低的活性化合物——瑞香素-8-O-β-D-葡萄糖苷。
OBJECTIVE: To study the bioconversion of daphnetin (I) with the hairy root of Polygonum multiflorum Thunb., And to isolate and identify its bioconversion product. The time-course curve of its product was established. Methods: Daphnetin was put into the hairy root suspension culture system of Polygonum multiflorum Thunb.For 36 h, the products were detected by TLC and HPLC, separated and purified by silica gel column chromatography, and identified by NMR. The co-culture time On the transformation products (Ⅱ), the impact of degradation products and analysis of the degradation of transformation products. Results: Daphnetin occurred in the Polygonum multiflorum hairy stem suspension culture system. A product was isolated and identified: Daphnetin-8-O-β-D-glucoside (Ⅱ). The optimum co-culture time for transformation Ⅱ of hairy root suspension culture of Polygonum multiflorum was 36 h and the total molar conversion was 32.11%. Sucrose - water medium (containing only sucrose and water) can make Polygonum multiflorum root suspension culture system conversion yield increased significantly, the total molar conversion rate of 72.44%. It is concluded that the degradation of product (Ⅱ) is related to the metal ions in MS medium. Conclusion: The Polygonum multiflorum hairy root suspension culture system can biosynthesize a very small amount of the active compound, daphnetin-8-O-β-D-glucoside, in natural medicine.