银杏叶单组分双黄酮分离制备及抗氧化活性研究
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摘要
从银杏叶中分离制备高纯度双黄酮对照品,探讨其抗氧化活性强弱。以60%乙醇-水提取的银杏叶浸膏为原料,反溶剂沉淀得到含量80.40%的双黄酮粗品,再以甲醇-水为流动相,半制备色谱梯度洗脱。结果得到了4种符合中药化学对照品要求的高纯度双黄酮,在最佳制备条件下,4种双黄酮纯度:阿曼托黄素(98.45%)、白果素(98.66%)、银杏黄素异构体(98.87%)、金松双黄酮(99.29%),其产率(mg/kg)分别为:22.5、23.8、192.5、71.8,考察了它们的抗氧化活性,并采用60%乙腈和有机酸水溶液对银杏黄素异构体进行了完全分离。建立的制备方法快速简便,所得单组分双黄酮纯度、产量高。本工作为单组分银杏双黄酮新药的研究与开发提供重要的技术支持。
To isolate and prepare the biflavonoids reference substance with high-purity from Ginkgo biloba L.,then investigate their antioxidant activity.High-purity biflavonoids were prepared by 60% ethanol-water extract of Ginkgo biloba L,through antisolvent precipitation and semi-preparative HPLC.Crude biflavonoids(purity:80.40%) was obtained by antisolvent precipitation process,then purified by semi-preparative HPLC with the mobile phase comprising of methanol-water in a gradient elution manner.Under the optimal preparation conditions,four biflavonoids were obtained and identified as amentoflavone(98.45%),bilobetin(98.66%),ginkgetin isomers(98.87%) and sciadopitysin(99.29%) with the yield of 22.5,23.8,192.5,71.8(mg/kg) respectively,which met the requirements of TCM reference standards,and examined their antioxidant activity,then completely separate the ginkgetin isomers by the use of 60% acetonitrile and organic acid aqueous solution.The preparation method is rapid and simple,and the obtained biflavonoids have high purity and high yield.This study provides important technical support for the research and development of one-component ginkgo biflavonoids.
To isolate and prepare the biflavonoids reference substance with high-purity from Ginkgo biloba L.,then investigate their antioxidant activity.High-purity biflavonoids were prepared by 60% ethanol-water extract of Ginkgo biloba L,through antisolvent precipitation and semi-preparative HPLC.Crude biflavonoids(purity:80.40%) was obtained by antisolvent precipitation process,then purified by semi-preparative HPLC with the mobile phase comprising of methanol-water in a gradient elution manner.Under the optimal preparation conditions,four biflavonoids were obtained and identified as amentoflavone(98.45%),bilobetin(98.66%),ginkgetin isomers(98.87%) and sciadopitysin(99.29%) with the yield of 22.5,23.8,192.5,71.8(mg/kg) respectively,which met the requirements of TCM reference standards,and examined their antioxidant activity,then completely separate the ginkgetin isomers by the use of 60% acetonitrile and organic acid aqueous solution.The preparation method is rapid and simple,and the obtained biflavonoids have high purity and high yield.This study provides important technical support for the research and development of one-component ginkgo biflavonoids.
引文
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4 Kang SS,Lee JY,Choi YK,et al.Neuroprotective effects of naturally occurring bioflavonoids[J].Bioorg Med Chem Lett,2005,15:3588-3591.
5 Lian N,Tong J,Li W,et al.Ginkgetin ameliorates experimental atherosclerosis in rats[J].Biomed Pharmacother,2018,102:510-516.
6 Kong FS,Yan CY,Pang XX,et al.Study on preparation of sciadopitysin from Ginkgo biloba leaves[J].Chin Tradit Pat Med(中成药),2010,32:878-879.
7 Li B,Hu GS,Hu LL,et al.Isolation and determination of bioflavonoids in Ginkgo biloba leaves[J].Chin Tradit Herb Drugs(中草药),2014,45:2552-2555.
8 Zhang T,Si JG,Chen JH,et al.Antioxidant constituents from Periplaneta americana[J].Nat Prod Res Dev(天然产物研究与开发),2017,29:1867-1872.
9 Yu S,Yan H,Zhang L,et al.A review on the phytochemistry,pharmacology,and pharmacokinetics of amentoflavone,a naturally-occurring biflavonoid[J].Molecules,2017,22:299.
10 Lobstein-Guth A,Briancon-Scheid F,Victoire C,et al.Isolation of amentoflavone from Ginkgo biloba[J].Planta Med,1988,54:555-556.
11 Joly M,Haag-Berrurier M,Anton R.La 5′-méthoxybilobétine,une biflavone xtradite du Ginkgo biloba[J].Phytochemistry,1980,19:1999-2002.
12 Krauze-Baranowska M,Baczek T,Gfód D,et al.HPLC separation of O-acylated F-lavonoids and biflavones from some species of Gymnospermae[J].Chromatographia,2004,60(1-2):9-15.
13 Krauze-Baranowska M,Sowiński P.2,3-Dihydrobiflavone from Ginkgo biloba[J].Planta Med,1999,65:482-484.
14 Editorial department of Chinese new drug journal.Technical requirements for research on new Chinese medicines(ⅱ)[J].Chin J New Drug(中国新药杂志),2000,9:276-281.
2 Miki K,Nagai T,Suzuki K,et al.Anti-influenza virus activity of biflavonoids.Bioorg[J].Med Chem Lett,2007,17(:772-775.
3 Jeong EJ,Hwang L,Lee M,et al.Neur-oprotective biflavonoids of chamaecyparis obtusa leaves against glutamate-induced oxidative stress in HT22 hippocampal cells[J].Food Chem Toxicol,2014,64:397-402.
4 Kang SS,Lee JY,Choi YK,et al.Neuroprotective effects of naturally occurring bioflavonoids[J].Bioorg Med Chem Lett,2005,15:3588-3591.
5 Lian N,Tong J,Li W,et al.Ginkgetin ameliorates experimental atherosclerosis in rats[J].Biomed Pharmacother,2018,102:510-516.
6 Kong FS,Yan CY,Pang XX,et al.Study on preparation of sciadopitysin from Ginkgo biloba leaves[J].Chin Tradit Pat Med(中成药),2010,32:878-879.
7 Li B,Hu GS,Hu LL,et al.Isolation and determination of bioflavonoids in Ginkgo biloba leaves[J].Chin Tradit Herb Drugs(中草药),2014,45:2552-2555.
8 Zhang T,Si JG,Chen JH,et al.Antioxidant constituents from Periplaneta americana[J].Nat Prod Res Dev(天然产物研究与开发),2017,29:1867-1872.
9 Yu S,Yan H,Zhang L,et al.A review on the phytochemistry,pharmacology,and pharmacokinetics of amentoflavone,a naturally-occurring biflavonoid[J].Molecules,2017,22:299.
10 Lobstein-Guth A,Briancon-Scheid F,Victoire C,et al.Isolation of amentoflavone from Ginkgo biloba[J].Planta Med,1988,54:555-556.
11 Joly M,Haag-Berrurier M,Anton R.La 5′-méthoxybilobétine,une biflavone xtradite du Ginkgo biloba[J].Phytochemistry,1980,19:1999-2002.
12 Krauze-Baranowska M,Baczek T,Gfód D,et al.HPLC separation of O-acylated F-lavonoids and biflavones from some species of Gymnospermae[J].Chromatographia,2004,60(1-2):9-15.
13 Krauze-Baranowska M,Sowiński P.2,3-Dihydrobiflavone from Ginkgo biloba[J].Planta Med,1999,65:482-484.
14 Editorial department of Chinese new drug journal.Technical requirements for research on new Chinese medicines(ⅱ)[J].Chin J New Drug(中国新药杂志),2000,9:276-281.