美国豆芋花总皂苷制备及其α-葡萄糖苷酶抑制活性
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摘要
为高效利用美国豆芋花皂苷类化合物,以皂苷得率和纯度为指标,研究美国豆芋花总皂苷的提取工艺及大孔树脂纯化工艺,并对制备的美国豆芋花总皂苷的抗氧化及α-葡萄糖苷酶活性抑制能力进行研究。结果表明,美国豆芋花总皂苷的提取工艺为提取溶剂90%乙醇,料液比1∶20,温度70℃,提取时间2 h。得到的提取物石油醚脱脂后用水饱和正丁醇萃取,D-101大孔树脂吸附柱纯化的工艺为上样皂苷质量浓度1 mg·m L~(-1),上样量40 m L·g~(-1)树脂,洗脱剂为80%乙醇(pH值6),洗脱剂用量4 BV(树脂柱体积)。制备的美国豆芋花总皂苷样得率4.24%,纯度达65.35%,比粗提物(纯度15.27%)提高了约3倍;抗氧化性能(清除DPPH和ABTS自由基IC50值分别为51.08、29.24μg·m L~(-1))和α-葡萄糖苷酶活性抑制能力(IC50值为83.62μg·m L~(-1))也均优于粗提物(清除DPPH、ABTS自由基IC50值分别为286.51、69.92μg·m L~(-1),α-葡萄糖苷酶的抑制浓度(IC50)值为1 043.57μg·m L~(-1)),表明该工艺可用于高活性美国豆芋花总皂苷的富集和纯化。此外,美国豆芋花总皂苷抑制α-葡萄糖苷酶活性的抑制类型为竞争型抑制,抑制常数为49.68μg·m L~(-1)。本研究结果为开发具有降血糖功能的医药中间体或功能性食品提供了新资源和实践基础。
To efficiently use the saponin from the Apios Americana Medikus flower,we taking saponin yield and purity as response indicators,the reflux extraction and macroporous resin purification were used in the present study to explore an appropriate extraction technology. The antioxidant and α-glucosidase inhibition activity were also studied for the refined saponin. The results showed that the optimal reflux conditions were ethanol concentration 90%,material/liquid ration1∶ 20,extraction temperature 70 ℃,and extraction duration 2 h.,The crude saponin was then partitioned in a separatory funnel with n-butanol after defatted and before washed through the D-101 macroporous resin. The optimum purification condition are 1 mg·m L~(-1)of saponons eluted by 80% ethanol at pH value 6 with a volume of 4 BV( resin bed volume),and the loaded amount was 40 m L·g-1resin. The yield of the refined saponin was 4. 24% while the purity reached65. 35%,which was 3 times higher than that of crude extract. The refined saponin showed strong DPPH and ABTS radical scavenging capacity,IC50 index for each assay was 51. 08 μg·m L~(-1)and 29. 24 μg·m L~(-1),respectively.Furthermore,it possessed stronger α-glucosidase inhibitory ability than the positive control of acarbose,IC50 index was83. 62 μg·m L~(-1). All the active indices were better than that of crude extract( IC50 index for DPPH,ABTS radical scavenging capacity and α-glucosidaseinhibitory ability were 286. 51 μg·m L~(-1),69. 92 μg·m L~(-1),and 1 043. 57μg·m L~(-1)respectively.). The result showed that the above reflux extraction and macroporous resin purification method was feasible in thepreparation of saponin from the Apios americana Medikus flower. Kinetic analysis showed that the inhibitory mechanism of the saponin from the flower of Apios Americana Medikus on α-glucosidase was a typical competitive inhibition. The inhibitory constant was 49. 68 μg·m L~(-1). The result would provide new resources and practical basis for the further development of the hypoglycemic pharmaceutical intermediates or functional foods.
To efficiently use the saponin from the Apios Americana Medikus flower,we taking saponin yield and purity as response indicators,the reflux extraction and macroporous resin purification were used in the present study to explore an appropriate extraction technology. The antioxidant and α-glucosidase inhibition activity were also studied for the refined saponin. The results showed that the optimal reflux conditions were ethanol concentration 90%,material/liquid ration1∶ 20,extraction temperature 70 ℃,and extraction duration 2 h.,The crude saponin was then partitioned in a separatory funnel with n-butanol after defatted and before washed through the D-101 macroporous resin. The optimum purification condition are 1 mg·m L~(-1)of saponons eluted by 80% ethanol at pH value 6 with a volume of 4 BV( resin bed volume),and the loaded amount was 40 m L·g-1resin. The yield of the refined saponin was 4. 24% while the purity reached65. 35%,which was 3 times higher than that of crude extract. The refined saponin showed strong DPPH and ABTS radical scavenging capacity,IC50 index for each assay was 51. 08 μg·m L~(-1)and 29. 24 μg·m L~(-1),respectively.Furthermore,it possessed stronger α-glucosidase inhibitory ability than the positive control of acarbose,IC50 index was83. 62 μg·m L~(-1). All the active indices were better than that of crude extract( IC50 index for DPPH,ABTS radical scavenging capacity and α-glucosidaseinhibitory ability were 286. 51 μg·m L~(-1),69. 92 μg·m L~(-1),and 1 043. 57μg·m L~(-1)respectively.). The result showed that the above reflux extraction and macroporous resin purification method was feasible in thepreparation of saponin from the Apios americana Medikus flower. Kinetic analysis showed that the inhibitory mechanism of the saponin from the flower of Apios Americana Medikus on α-glucosidase was a typical competitive inhibition. The inhibitory constant was 49. 68 μg·m L~(-1). The result would provide new resources and practical basis for the further development of the hypoglycemic pharmaceutical intermediates or functional foods.
引文
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[20]Sungeun A,Muhammad H S.Anti-inflammatory activity of ginsenosides in LPS-stimulated RAW 264.7 cells[J].Science Bulletin,2015,60(8):773-784
[21]李鹏飞,于婷,李雪,高美须,王志东,崔承弼.电子束辐照对人参抗肿瘤作用的影响[J].核农学报,2016,30(8):1524-1533
[22]吕梦.苦瓜籽皂苷的提取工艺及抑菌活性[J].天津农业科学,2016,22(7):33-36
[23]刘星,余江丽,刘敏,舒积成,黄慧.近10年甾体皂苷的生物活性研究进展[J].中国中药杂志,2015,40(13):2518-2523
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[31]张家瑞.大孔树脂纯化覆盆子黄酮的研究[J].现代食品科技,2013,29(4):817-821
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[35]彭拓华,陈洁炜,黄湘兰,张少俊,钟红兰.大孔树脂富集纯化人参总皂苷工艺条件优选[J].中医药信息,2015,32(1):3-6
[36]王晓林,杜晓宇,钟方丽.徐欣.刺玫叶总皂苷富集工艺条件优化[J].广东农业科学,2015,28(8):77-82
[37]Zhang L,Tu Z C,Yuan T,Wang H,Xie X,Fu Z F.Antioxidants andα-glucosidase inhibitors form Ipomoea batatas leaves identified by bioassay-guided approach and structure-activity relationships[J].Food Chemistry,2016,208(1):61-67
[38]朱运平,李秀婷,李里特.天然α-葡萄糖苷酶抑制剂来源及应用研究现状[J].中国食品学报,2011,11(4):154-160
[39]彭晓赟,陈紫东,尹志芳,钟桐,杨小琴,赵运林.苦瓜根总皂苷的提取及其对α-葡萄糖苷酶活性抑制作用的研究[J].中成药,2015,37(10):2312-2314
[40]陈丽华,潘自红,曹云丽,马庆一.知母中活性成分对α-萄糖苷酶的抑制研究[J].食品与机械,2013,29(5):147-150,157
[41]陆建美,闫鸿丽,王艳芳,胡雪岩,俞捷,尚云青.滇黄精及其活性成分群对α-糖苷酶活性抑制作用研究[J].中国现代中药,2015,17(3):200-203
[2]Zhang Y Z,Kouzuma Y M T,Miyaji T,Yonekura M.Purification,characterization,and c DNA cloning of a Bowman-Birk type trypsin inhibitor from Apios americana Medikus tubers[J].Bioscience Biotechnology and Biochemistry,2008,72(1):171-178
[3]Iwai K,Matsue H.Ingestion of Apios americana Medikus tuber suppresses blood pressure and improves plasma lipids in spontaneously hypertensive rats[J].Nutrition Research,2007,27(4):218-224
[4]Zhang Y Z,Zhou C S,Tang S M,Yu X J,Kouzuma Y,Yonekura M.Effect of AATI,a Bowman-Birk type inhibitor from Apios americana,on proliferation of cancer cell lines[J].Food Chemistry,2011,128(4):909-915
[5]Nara K,Nihei K I,Ogasawara Y,Koga H,Kato Y.Novel isoflavone diglycoside in groundnut(Apios americana Medik)[J].Food Chemistry,2011,124(3):703-710
[6]Takashima M,Nara K,Niki E,Yoshida Y,Hagihara Y,Stowe M,Horie M.Evaluation of biological activities of a groundnut(Apios americana Medik)extract containing a novel isoflavone[J].Food Chemistry,2013,138(1):298-305
[7]Juliarni,Hoshikawa K.Characteristics of dry matter production and yield of Apios(Apios americana Medikus)clones[J].Japanese Journal of Crop Science,1996,65(1):98-102
[8]Juliarni,Hoshikawa K.The growth of Apios(Apios americana Medikus),a new crop,under field conditions[J].Japanese Journal of Crop Science,1995,64(2):323-327
[9]安家炜,张有做,高前欣,舒恩成,裘耀东,倪勤学.美国豆芋地上部位有效成分及α-葡萄糖苷酶抑制活性研究[J].核农学报,2014,28(12):2275-2282
[10]孙晓博,张凯,李兰林,向大雄.大孔吸附树脂分离纯化单叶铁线莲总皂苷的研究[J].中草药,2011,42(10):1977-1981
[11]姬丽婷,刘晔,谢洪,倪勤学.正交设计优化鸡毛竹叶子抗氧化物的提取工艺[J].竹子研究汇刊,2011,30(3):53-57
[12]张若洁,徐永霞,王鲁峰,陈清婵,徐晓云,潘思轶,王可兴.大孔树脂纯化芦笋总皂苷的工艺研究[J].中草药,2012,43(6):1097-1100
[13]Ni Q X,Zhang Y Z,Xu G Z,Gao Q X,Gong L X,Zhang Y.Influence of harvest season and drying method on the antioxidant activity and active compounds of two bamboo grass leaves[J].Journal of Food Processing and Preservation,2014,38(4):1565-1576
[14]袁柱,孙彦,陆继肖,冯子蓉.不同品种苜蓿青贮总皂苷含量变化的研究[J].草地学报,2015,23(1):194-199
[15]Brand-william W,Curyelier M E,Benset C.Use of a free radical method to evaluate antioxidant activity[J].Lebensm-Wiss Technol,1995,28(1):25-30
[16]Re R,Pellegrini N,Proteggente A,Pannala A,Yang M,RiceEvans C.Antioxidant activity applying and improved ABTS radical cation decolorization assay[J].Free Radical Biology&Medicine,1999,26(9/10):1231-1237
[17]王姝,倪勤学,许光治,杨冬冬,高前欣,张有做.地被竹与淡竹叶提取物抗氧化活性的比较研究[J].食品工业科技,2014,35(22):135-144
[18]Dong H Q,Li M,Zhu F,Liu F L,Huang J B.Inhibitory potential of trilobatin from Lithocarpus polystachyus Rehd againstα-glucosidase andα-amylase linked to type 2 diabetes[J].Food Chemistry,2012,130(2):261-266
[19]张蕾,王姝,倪勤学,许光治,高前欣,张有做.鹅毛竹叶提取物抗氧化及酪氨酸酶抑制活性的研究[J].浙江林业科技,2014,34(5):8-15
[20]Sungeun A,Muhammad H S.Anti-inflammatory activity of ginsenosides in LPS-stimulated RAW 264.7 cells[J].Science Bulletin,2015,60(8):773-784
[21]李鹏飞,于婷,李雪,高美须,王志东,崔承弼.电子束辐照对人参抗肿瘤作用的影响[J].核农学报,2016,30(8):1524-1533
[22]吕梦.苦瓜籽皂苷的提取工艺及抑菌活性[J].天津农业科学,2016,22(7):33-36
[23]刘星,余江丽,刘敏,舒积成,黄慧.近10年甾体皂苷的生物活性研究进展[J].中国中药杂志,2015,40(13):2518-2523
[24]王曙东,乔立业,曹燕丽,苏华,任海祥.三七总皂苷滴鼻液的制备与质量控制[J].医药导报,2013,32(5):653-655
[25]梁晓芳,王步军.大豆中异黄酮和皂苷的提取、制备研究进展[J].大豆科学,2014,33(1):128-134
[26]汪晶晶,刘奕训,文迪,余河水,康利平,庞旭,赵阳,马百平,陈韵岱.盾叶薯蓣中甾体皂苷及其体外血小板活性研究[J].2014,39(19):3782-3787
[27]赵启铎,舒乐新,蔡广知.酸枣仁总黄酮和总皂苷部位制备工艺的稳定性考察[J].中国实验方剂学杂志,2014,20(24):27-29
[28]刘建林,武秋爽,王一涛,张庆文,陆金健.几种人参属中药提取物的制备及体外抗卵巢癌活性比较[J].2016,22(8):105-110
[29]刘玥.大孔吸附树脂在中药提取纯化中的应用进展[J].世界中医药,2013,8(2):232-234
[30]赖谱富,赖锋连,陈君琛,沈恒胜.多年生藤本豆总黄酮纯化及抑菌活性研究[J].核农学报,2015,29(8):1539-1546
[31]张家瑞.大孔树脂纯化覆盆子黄酮的研究[J].现代食品科技,2013,29(4):817-821
[32]罗仓学,杨转萍,李祥.大孔树脂分离纯化黄姜中薯蓣皂苷工艺研究[J].食品科技,2011,36(5):229-236
[33]刘建萍,蔡淑娴,刘仲华,陈暄,郭巧生,由宝昌,黎星辉.大孔树脂纯化地肤子总皂苷的研究[J].江苏农业科学,2008,79(2):178-180,231
[34]顾维,楼凤昌,沈晓兰.大孔树脂纯化薤白总皂苷的研究[J].药学与临床研究,2009,17(6):455-458
[35]彭拓华,陈洁炜,黄湘兰,张少俊,钟红兰.大孔树脂富集纯化人参总皂苷工艺条件优选[J].中医药信息,2015,32(1):3-6
[36]王晓林,杜晓宇,钟方丽.徐欣.刺玫叶总皂苷富集工艺条件优化[J].广东农业科学,2015,28(8):77-82
[37]Zhang L,Tu Z C,Yuan T,Wang H,Xie X,Fu Z F.Antioxidants andα-glucosidase inhibitors form Ipomoea batatas leaves identified by bioassay-guided approach and structure-activity relationships[J].Food Chemistry,2016,208(1):61-67
[38]朱运平,李秀婷,李里特.天然α-葡萄糖苷酶抑制剂来源及应用研究现状[J].中国食品学报,2011,11(4):154-160
[39]彭晓赟,陈紫东,尹志芳,钟桐,杨小琴,赵运林.苦瓜根总皂苷的提取及其对α-葡萄糖苷酶活性抑制作用的研究[J].中成药,2015,37(10):2312-2314
[40]陈丽华,潘自红,曹云丽,马庆一.知母中活性成分对α-萄糖苷酶的抑制研究[J].食品与机械,2013,29(5):147-150,157
[41]陆建美,闫鸿丽,王艳芳,胡雪岩,俞捷,尚云青.滇黄精及其活性成分群对α-糖苷酶活性抑制作用研究[J].中国现代中药,2015,17(3):200-203