人参二醇类皂苷的生物转化动态及人参稀有皂苷C-K或F2的制备

doi:10.7503/cjcu20180709

高等学校化学学报 ›› 2019, Vol. 40 ›› Issue (6): 1184.doi: 10.7503/cjcu20180709

人参二醇类皂苷的生物转化动态及人参稀有皂苷C-K或F₂的制备

肖永坤^1,^2,³, 刘春莹⁴, 鱼红闪¹(), 李泰厚²(), 徐龙权¹, 宋建国¹, 林完泽⁵, 孙长凯⁶, 金凤燮¹

1. 大连工业大学生物工程学院, 大连 116034
2. 韩国庆熙大学校韩方药生物技术学科, 韩国京畿道 446-701
3. 葵花药业集团(天津)药物研究院, 天津 300457
4. 大连大学生命科学与技术学院, 大连 116622
5. 韩国韩京国立大学校生物技术学科, 韩国京畿道 456-749
6. 大连理工大学生物医药工程学院, 大连 116024

收稿日期:2018-10-19 出版日期:2019-06-10 发布日期:2019-04-24
作者简介:
联系人简介: 鱼红闪, 男, 博士, 教授, 博士生导师, 主要从事天然产物生物转化研究. E-mail: hongshan@dlpu.edu.cn;李泰厚, 男, 博士, 教授, 博士生导师, 主要从事韩方药成分药理研究. E-mail: drhoo@khu.ac.kr
基金资助:
国家科技重大专项重大新药创制项目(批准号: 2012ZX09503001-003)和国家高端外国专家项目(批准号: GDT20152100019)资助.

Dynamic Biotransformation of Protopanaxadiol-ginsenosides and Preparation of Minor Ginsenosides C-K or $F 2 †$

XIAO Yongkun^1,^2,³, LIU Chunying⁴, YU Hongshan¹(), YI Tea-Hoo²(), XU Longquan¹, SONG Jianguo¹, IM Wan-Teak⁵, SUN Changkai⁶, JIN Fengxie¹

1. College of Biotechnology, Dalian Polytechnic University, Dalian 116034, China
2. Department of Oriental Medicinal Biotechnology, Kyung Hee University, Gyeonggi-do 446-701, Korea
3. Sunflower Pharmaceutical Group(Tianjin) Drug Research Institute, Tianjin 300457, China
4. College of Life Science and Technology, Dalian University, Dalian 116622, China
5. Department of Biotechnology, Hankyoung National University, Kyonggi-do 456-749, Korea
6. Department of Biomedical Engineering, Dalian University of Technology, Dalian 116024, China

Received:2018-10-19 Online:2019-06-10 Published:2019-04-24
Supported by:
†Supported by the Major Projects of China National Science and Technology “Significant New Drugs Creation” Project of China(No.2012ZX09503001?003) and the National High?end Foreign Expert Project of China(No.GDT20152100019).

摘要/Abstract

摘要：

为了低成本有效制备人参稀有皂苷C-K或F₂, 将A. niger g.848菌酶用于转化含有人参皂苷(质量分数)分别为49.6% Rb₁, 25.9% Rd, 19.3% Rc和5.23% Rb₂的西洋参二醇混合皂苷. 霉菌发酵时, 采用人参二醇皂苷诱导物比人参提取液诱导物的产酶总活力提高10%~15%. 所产的2种诱导酶均能水解人参二醇皂苷的3-O-和20-O-多种糖基, 均为人参皂苷酶Ⅰ型; 但是人参二醇皂苷诱导物所产酶几乎全部转化人参二醇皂苷为C-K, 而人参提取液诱导物所产酶则残留中间产物. 使用黑曲霉人参二醇皂苷诱导所产酶, 在转化西洋参二醇皂苷的动态研究中发现, 酶反应1.5~2.5 h, 主要为产物F₂; 酶反应12 h后, 主要产物为C-K皂苷. 基于此, 40 g人参二醇类皂苷在45 ℃粗酶反应24 h, 经处理得到含C-K质量分数为87%的23 g酶反应产物, C-K转化率达85%(摩尔分数). 用40 g西洋参二醇皂苷在45 ℃粗酶反应2.5 h, 经处理得到含有质量分数为58%的F₂和27%的C-K的26 g酶反应产物, F₂转化率为50.4%, C-K转化率为29.5%. 通过人参二醇皂苷诱导的黑曲霉粗酶转化人参二醇类皂苷动态研究, 建立了C-K转化率为85%, F₂转化率为50%的制备方法, 为大批量制备提供了基础依据.

关键词: 人参二醇类皂苷, 人参皂苷生物转化, 人参皂苷F₂, 人参皂苷C-K, 黑曲霉

Abstract:

In order to efficiently produce high-bioactive minor ginsensoide C-K or F₂, the protopanaxadiol(PPD) ginsenosides from American ginseng were converted by the enzyme from Aspergillus niger g.848 strain. The mass fractions of PPD ginsenosides were 49.6% Rb₁, 25.9% Rd, 19.3% Rc and 5.23% Rb₂, respectively. In fermentation, when the PPD-ginsenosides as a ginsenosidase-inducer, the 10%—15% ginsenosidase activity was increased than that of ginseng-extract ginsenosidase-inducer. The two enzymes were all the Ginsenosidase typ1-I. However, the enzyme by the ginsenosidase-inducer could hydrolyze almost PPD-ginsenosides into C-K, but the enzyme by ginseng extract-inducer hydrolyzed PPD-ginsenosides into F₂, C-K, C-Mc and C-Y. When reacted for 1.5 h to 3 h, the main product was F₂. When reacted for over 12 h, the main product was C-K. In the C-K preparation, 40 g of PPD-ginsenosides was reacted at 45 ℃ and pH=5.0 for 24 h to get 23 g product containing over 87%(mass fraction) C-K, the C-K molar yield was 85%. In the F₂ preparation, 40 g PPD-ginsenosides was reacted at 45 ℃ and pH=5.0 for 2.5 h to get 26 g product containing 58%(mass fraction) of F₂ and 27% of C-K, the molar yield was 50.4% for F₂, and 29.5% for C-K. Therefore, according to biotransformation of the enzyme by PPD-ginsenosides-inducer, the 85% of C-K, or 50% of F₂ was successfully prepared from PPD ginsenosides of American ginseng. The results are very usable for ginseng development.

Key words: Protopanaxadiol ginsenoside, Ginsenoside biotransformation, Ginsenoside F₂, Ginsenoside C-K, Aspegillus niger

中图分类号:

O629
O656

TrendMD:

肖永坤, 刘春莹, 鱼红闪, 李泰厚, 徐龙权, 宋建国, 林完泽, 孙长凯, 金凤燮. 人参二醇类皂苷的生物转化动态及人参稀有皂苷C-K或F₂的制备. 高等学校化学学报, 2019, 40(6): 1184.

XIAO Yongkun,LIU Chunying,YU Hongshan,YI Tea-Hoo,XU Longquan,SONG Jianguo,IM Wan-Teak,SUN Changkai,JIN Fengxie. Dynamic Biotransformation of Protopanaxadiol-ginsenosides and Preparation of Minor Ginsenosides C-K or $F 2 †$ . Chem. J. Chinese Universities, 2019, 40(6): 1184.

图/表 7

参考文献 27

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Ginsenoside	Sugar moiety		t_R/min	Area	Peak area ratio(%)
Ginsenoside	3-O-R₁	20-O-R₂	t_R/min	Area	Peak area ratio(%)
Rb₁	β-D-Glc-(1→2)-β-D-Glc-	20(S)-β-D-Glc-(1→6)-β-D-Glc-	38.910	1366983	49.6
Rb₂	β-D-Glc-(1→2)-β-D-Glc-	20(S)-α-L-Arap-(1→6)-β-D-Glc-	39.742	530283	19.3
Rc	β-D-Glc-(1→2)-β-D-Glc-	20(S)-α-L-Araf-(1→6)-β-D-Glc-	40.755	143909	5.2
Rd	β-D-Glc-(1→2)-β-D-Glc-	20(S)-β-D-Glc-	42.555	712247	25.9

Ginsenoside	Sugar moiety		t_R/min	Area	Peak area ratio(%)
Ginsenoside	3-O-R₁	20-O-R₂	t_R/min	Area	Peak area ratio(%)
Rb₁	β-D-Glc-(1→2)-β-D-Glc-	20(S)-β-D-Glc-(1→6)-β-D-Glc-	38.910	1366983	49.6
Rb₂	β-D-Glc-(1→2)-β-D-Glc-	20(S)-α-L-Arap-(1→6)-β-D-Glc-	39.742	530283	19.3
Rc	β-D-Glc-(1→2)-β-D-Glc-	20(S)-α-L-Araf-(1→6)-β-D-Glc-	40.755	143909	5.2
Rd	β-D-Glc-(1→2)-β-D-Glc-	20(S)-β-D-Glc-	42.555	712247	25.9

Carbon site	F₂	C-K	Carbon site	F₂	C-K
Aglycone moiety			C23	23.37	23.37
C1	23.38	39.72	C24	126.10	126.11
C2	26.91	28.41	C25	131.08	131.07
C3	88.98	79.45	C26	22.56	22.52
C4	39.36	39.58	C27	17.94	17.95
C5	56.55	56.53	C28	28.33	28.86
C6	18.65	18.94	C29	16.16	16.19
C7	35.30	35.34	C30	17.54	17.56
C8	40.22	40.25	3-O-Glc
C9	50.35	50.47	C1'	107.12	—
C10	37.14	37.53	C2'	75.93	—
C11	31.06	31.10	C3'	79.45	—
C12	70.34	70.35	C4'	71.80	—
C13	49.63	49.67	C5'	78.50	—
C14	51.58	51.59	C6'	63.25	—
C15	30.93	30.95	20-O-Glc
C16	26.80	26.80	C1'	98.42	98.41
C17	51.81	51.79	C2'	75.28	75.27
C18	16.97	16.53	C3'	78.90	78.40
C19	16.47	16.49	C4'	72.04	71.80
C20	83.46	83.46	C5	78.43	78.22
C21	25.95	25.94	C6'	63.04	63.04
C22	36.32	36.32

Carbon site	F₂	C-K	Carbon site	F₂	C-K
Aglycone moiety			C23	23.37	23.37
C1	23.38	39.72	C24	126.10	126.11
C2	26.91	28.41	C25	131.08	131.07
C3	88.98	79.45	C26	22.56	22.52
C4	39.36	39.58	C27	17.94	17.95
C5	56.55	56.53	C28	28.33	28.86
C6	18.65	18.94	C29	16.16	16.19
C7	35.30	35.34	C30	17.54	17.56
C8	40.22	40.25	3-O-Glc
C9	50.35	50.47	C1'	107.12	—
C10	37.14	37.53	C2'	75.93	—
C11	31.06	31.10	C3'	79.45	—
C12	70.34	70.35	C4'	71.80	—
C13	49.63	49.67	C5'	78.50	—
C14	51.58	51.59	C6'	63.25	—
C15	30.93	30.95	20-O-Glc
C16	26.80	26.80	C1'	98.42	98.41
C17	51.81	51.79	C2'	75.28	75.27
C18	16.97	16.53	C3'	78.90	78.40
C19	16.47	16.49	C4'	72.04	71.80
C20	83.46	83.46	C5	78.43	78.22
C21	25.95	25.94	C6'	63.04	63.04
C22	36.32	36.32

人参二醇类皂苷的生物转化动态及人参稀有皂苷C-K或F₂的制备

Dynamic Biotransformation of Protopanaxadiol-ginsenosides and Preparation of Minor Ginsenosides C-K or $F 2 †$

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t_R/h	Substrate ginsenosides(%)				Produced ginsenosides(%)
t_R/h	Rb₁	Rc	Rb₂	Rd	C-Mc₁	C-O	F₂	C-Mc	C-Y	C-K
0	49.7	19.3	5.2	25.9	—	—	—	—	—	—
0.5	49.6	19.3	5.2	25.9	—	—	—	—	—	—
1.0	49.6	19.3	5.2	25.9	—	—	—	—	—	—
1.5	—	2.0	1.5	—	13.0	4.5	58.1	4.9	—	16.0
2.0	—	2.0	1.7	—	12.3	4.4	55.5	3.5	—	20.0
2.5	—	1.1	0.6	—	7.0	3.9	55.4	4.6	—	27.0
3.0	—	0.8	—	—	5.7	3.5	49.9	5.6	2.1	32.2
6.0	—	—	—	—	4.2	1.9	21.2	10.6	3.7	57.7
12	—	—	—	—	—	—	5.4	13.4	5.3	76.0
24	—	—	—	—	—	—	2.4	6.3	3.8	87.0
30	—	—	—	—	—	—	3.2	5.8	2.7	88.5
36	—	—	—	—	—	—	5.6	3.1	1.4	89.1
M_W	1108.6	10778.6	1078.6	946.6	916.6	916.6	784.5	754.6	754.6	622.4