26:1552-1556,1567天然产物研究与开发Nat Prod ResDev 2014,
6880(2014)10-1552-06文章编号:1001-
西洋参总皂苷酶水解产物成分研究
金建明
12
3
1,2*
2133
,李有海,张海伦,梁朝宁,唐双焱
北京工商大学植物资源研究开发北京市重点实验室,北京100048;
云南民族大学民族药资源化学国家民委-教育部重点实验室,昆明650500;
中国科学院微生物研究所中国科学院微生物生理与代谢工程重点实验室,北京100101
摘要:西洋参总皂苷经β-糖苷酶催化水解,采用HPLC 检测分析确定西洋参总皂苷中的主要原人参二醇型皂
Rd、Rc和Rb2已经完全被水解。水解产物通过反复硅胶柱层析和反向硅胶柱层析分离纯化得到7个皂苷Rb1、
苷,通过NMR谱图分析分别鉴定为人参皂苷compound K (1)、人参皂苷Mc (2)、人参皂苷Rg1(3)、人参皂苷Rg2(4)、人参皂苷Re(5)、人参皂苷F 1(6)和拟人参皂苷F 11(7)。β-糖苷酶催化西洋参总皂苷水解实验表明,西洋参中原人参二醇型皂苷的水解产物是人参皂苷compound K 和人参皂苷Mc 。关键词:西洋参;原人参二醇型皂苷;人参皂苷compound K ;β-糖苷酶中图分类号:R939.9
文献标识码:A
Chemical Constituents of Enzymatic Hydrolysate of Total
Ginsenoside Extract of Panax quinquefolium
2*
JIN Jian-ming 1,,LI You-hai 2,ZHANG Hai-lun 1,LIANG Chao-ning 3,TANG Shuang-yan 3
1
Beijing Key Laboratory of Plant ResourcesResearchand Development ,Beijing Technology and Business University ,
Beijing 100048,China ;2Key Laboratory of Ethnic Medicine ResourceChemistry ,State Ethnic Affairs Commission &Ministry of Education ,Yunnan University of Nationalities ,Yunnan Kunming 650500,China ;3CAS Key Laboratory of
Microbial Physiological and Metabolic Engineering ,Institute of Microbiology ,Chinese
Beijing 100101,China Academy of Sciences ,
Abstract :The total ginsenoside extract of Panax quinquefolium was hydrolyzed by β-glycosidase.HPLC analysis indica-ted that ginsenosides Rb1,Rd,Rcand Rb2were completely hydrolyzed.Seven ginsenosides were isolated from the hydrol-ysate ,and identified as ginsenoside compound K (1),ginsenoside Mc (2),ginsenoside Rg1(3),ginsenoside Rg2(4),ginsenoside Re(5),ginsenoside F 1(6)and pseudo-ginsenoside F 11(7),respectively.The protopanaxdiol-type ginsen-osides in the total ginsenoside extract of P.quinquefolium were converted into ginsenoside compound K and ginsenoside glycosidase.Mc by the β-Key words :Panax quinquefolium ;protopanaxdiol-type ginsenosides ;ginsenoside compound K ;β-glycosidase
西洋参(Panax quinquefolium L.)又名花旗参、
西洋人参、洋参、美国人参、广东人参等,是我国传统为五加科人参属植物,原产北美洲的的名贵中药材,
加拿大南部和美国北部,现我国的许多地区都有引种栽培。其味甘、微苦、性寒,可滋阴降火,益气生津,其药用价值有镇静镇惊、安神促智、增强机体免疫、抗心律失常、防血管硬塞、保肝、降血脂等功效。对西洋参的化学成分已经有许多研究报道,其主要
02-1405-16收稿日期:2014-接受日期:2014-基金项目:国家自然科学基金项目(31160017;21172095);北京市
自然科学基金项目(2142012);云南省教育厅科学研究基金项目(2011Z120)
*通讯作者Tel :86-10-68984937;E-mail :jinjm8@qq.com
活性成分是人参皂苷。西洋参总皂苷中绝大多数属
[1,2]
。达玛烷四环三于达玛烷四环三萜类人参皂苷
萜类人参皂苷根据苷元6位碳上是否有羟基分为原Rb2、Rc和Rd等人参二醇型皂苷如人参皂苷Rb1、
(图1)和原人参三醇型皂苷如人参皂苷Rg1、Rg2、Re和F 1等。
稀有人参皂苷compound K 也称人参皂苷CK
K ,20-或人参皂苷C-其结构为20(S )-原人参二醇-O-D-β-吡喃葡萄糖苷(图1),属原人参二醇型皂苷。
人参皂苷compound K 在人参属植物中几乎不存在,Rb2和Rc后被首次由土壤细菌降解人参皂苷Rbl 、发现并鉴定
[3]
。研究发现人参皂苷compound K 是
Vol. 26金建明等:西洋参总皂苷酶水解产物成分研究1553
人参等药材中原人参二醇型皂苷经肠道菌降解后产
[4]
生的在人体内发挥药效作用的真正代谢活性物。人参皂苷compound K 的生物活性具有多靶点、高活性和低毒性的特点,具有极高的生产价值和应用前
[5,6]
。目前制备人参皂苷com-景,医药价值很高
pound K 的方法多采用微生物发酵法和酶解法。利
rpm 摇床培养12h 后将种子液转接到600mL 诱导培养基中(含100mmol /L卡那霉素),接种量1%,30ħ 、280rpm 摇床培养21h 。培养液10000rpm 离心10min ,沉淀加入60mL Mc 缓冲液重悬(0.2mol /L磷酸氢二钠和0.1mol /L柠檬酸,pH =5.5),超声破细胞后12000rpm 离心10min ,取上清液于60ħ 水浴中静置10min 后再次12000rpm 离心10
[8]
min ,上清液即β-糖苷酶液。1.3
酶水解反应
约60mL 酶液加入到75mL 含7.5g 西洋参总
用酶解法制备人参皂苷compound K 具有反应条件
[5,7]
。温和、操作简便
、成本低、产物便于纯化等优点
皂苷的Mc 缓冲液中,水浴恒温70ħ 反应过夜使酶
[8]
催化水解反应完全(19h )。1.4
分离提取
135mL 水解反应液用等体积正丁醇萃取三次,
图1
Fig. 1
原人参二醇型皂苷结构
Chemical structures of protopanaxdiol-type ginsenosides
合并萃取液,减压浓缩干燥后进行硅胶柱层析,用二氯甲烷甲醇进行梯度洗脱,分为五个部分(Fr I Fr V )。各部分再经RP-C18柱层析,采用甲醇水进行梯度洗脱纯化各化合物。Fr I 部分1.3g 经50% 80%甲醇水梯度洗脱纯化得到化合物1(0.807g );Fr II 部分0.2g 用50% 60%甲醇水梯度洗脱得到化合物6(0.049g );Fr III 部分0.6g 经60% 75%甲醇水梯度洗脱得到化合物2(0.311g );Fr IV 部分0.58g 用20% 75%甲醇水梯度洗脱得到化合物3(0.152g )、7(0.070g )和4(0.046g )。Fr V 部分1.036g 用10% 50%甲醇水梯度洗脱得到化合物5(0.481g )。1.5
HPLC 检测
西洋参总皂苷及其水解产物的HPLC 方法在参
[9]
考文献的基础上略有修改。西洋参总皂苷及其水解产物配制成3mg /mL。HPLC 条件如下:色谱
5柱:Waters symmetry C 18色谱柱(4.6ˑ 250mm ,μm );流动相:乙腈(A )和水(B );二元高压梯度洗
20%A ;2.5 5min ,20% 25%A ;脱:0 2.5min ,
5 10min ,25%A ;10 30min ,25% 40%A ;30 45min ,40% 70%A ;45 60min ,70%A ;流速:
0. 7mL /min;检测波长:203nm ;柱温:35ħ ;进样量:10μL 。
Glc :β-D-glucopyranosyl ;Ara (pyr ):α-L-arabinopyranosyl ;Ara (fur ):L-arabinofuranosyl α-
项目组前期得到了高效催化人参皂苷Rb1产生人参皂苷compound K 的β-糖苷酶。为了研究该β-糖苷酶是否也催化其他原人参二醇型皂苷产生人参
实验采用原人参二醇型皂苷含量皂苷compound K ,
高的西洋参总皂苷,详细分析了西洋参总皂苷经该
β-糖苷酶完全催化水解后的成分组成,为将来进一步采用西洋参总皂苷等高效制备人参皂苷com-pound K 奠定基础。
1
1.1
材料与方法
仪器与试剂
Bruker Avance 400MHz 型核磁共振波谱仪;Ag-
ilent 6520LC /MS质谱仪;岛津Lc-20A 高效液相色
谱仪。柱层析硅胶(60 100目)和薄层层析硅胶板H (青岛海洋化工有限公司);RP-C18反相层析材料
C18高效薄层板(Merck 公司);乙腈为色谱和RP-纯,其余试剂均为分析纯;西洋参总皂苷购自南京泽朗医药生物科技有限公司。标准品人参皂苷Rb1(批号20110526)、Rb2(批号20120311)、Rc(批号20120617)和Rd(批号20120621)购自宝鸡辰光生物科技有限公司。1.2酶液制备
将β-糖苷酶LacS 突变株接种到LB 培养基中
(含100mmol /L卡那霉素),37ħ 、280接种量1%,
2
2.1
结果与分析
西洋参水解产物的组分分析
为了研究β-糖苷酶催化总皂苷后水解产物的
成分组成及原人参二醇型皂苷生成人参皂苷com-pound K 的效率,将西洋参总皂苷经β-糖苷酶催化
1554
天然产物研究与开发Vol. 26
水解过夜(19h ),水解产物经TLC 检测初步证明原人参二醇型皂苷Rb1已经完全被水解。HPLC 检测分析再次确定西洋参总皂苷中的原人参二醇型皂苷
Rc(9)、Rb2(10)和Rd(11)已经人参皂苷Rb1(8)、
完全水解(图2B )。进一步延长水解反应时间后的
水解产物经HPLC 检测分析皂苷的成分和含量没有变化,表明19h 水解反应已经完全。水解产物中主
mAU
[**************]00
mAU [**************]0
要有二个原人参二醇型皂苷:人参皂苷compound K (1)和人参皂苷Mc (2);四个原人参三醇型皂苷:人人参皂苷Rg2(4)、人参皂苷Re(5)参皂苷Rg1(3)、
和人参皂苷F 1(6);以及拟人参皂苷F 11(7)。其中人参皂苷compound K 是西洋参总皂苷水解产物中含量最高的成分。
mAU [**************]0
10%20%30%40%50min 010%20%30%40%50min 010%20%30%40%50min
图2
Fig. 2
西洋参总皂苷水解前(A )和水解后(B )以及人参皂苷compound K (C )的HPLC 色谱图
HPLC chromatograms of total ginsenoside extract of P.quinquefolium before (A )and after (B )hydrolysis as well as ginsen-oside compound K standard (C )
1:人参皂苷compound K (ginsenoside compound K );2:人参皂苷Mc (ginsenoside Mc );3:人参皂苷Rg1(ginsenoside Rg1);4:人参皂苷Rg2(gin-senoside Rg2);5:人参皂苷Re(ginsenoside Re);6:人参皂苷F 1(ginsenoside F 1);8:人参皂苷Rb1(ginsenoside Rb1);9:人参皂苷Rc(ginsen-oside Rc);10:人参皂苷Rb2(ginsenoside Rb2);11:人参皂苷Rd(ginsenoside Rd)
2.2
水解产物结构鉴定
ESI-MS 化合物1白色粉末,易溶于甲醇。HR-
1. 04(3H ,s ,H-29),1. 00(3H ,s ,H-30),0. 94(3H ,s ,H-18),0. 89(3H ,s ,H-19);13C NMRδC :39. 6(C-1),28. 3(C-2),78. 1(C-3),39. 6(C-4),56. 4(C-5),18. 8(C-6),35. 2(C-7),40. 1(C-8),50. 3(C-9),37. 4(C-10),30. 8(C-11),70. 3(C-12),49. 5(C-13),51. 2(C-14),30. 9(C-15),26. 7(C-16),51. 7(C-17),16. 4(C-18),16. 1(C-19),83. 4(C-20),22. 4(C-21),36. 2(C-22),23. 2(C-23),126. 1(C-24),131. 1(C-25),25. 8(C-26),17. 9(C-27),28. 7(C-28),16. 4(C-29),17. 4(C-30),98. 1(C-20-Glc-1),75. 1(C-20-Glc-2),79. 3(C-20-Glc-3),72. 2(C-20-Glc-4),76. 6(C-20-Glc-5),68. 5(C-20-Glc-6),110. 2(C-Ara-1),83. 4(C-Ara-2),78. 9(C-Ara-3),86. 1(C-Ara-4),62. 7(C-Ara-5)。以上数据和文献报道的基本一致
,故化合
20-物2鉴定为人参皂苷Mc ,即20(S )-原人参二醇-[11]
m /z 645.4342[M +Na ]+(calcd for C 36H 62NaO 8,645.4337);1H NMR(400MHz ,pyridine-d 5)δH :
5. 19(1H ,d ,J =7. 6Hz ,Glc-H-1),1. 63(3H ,s ,H-21),1. 60(6H ,s ,H-26,27),1. 23(3H ,s ,H-28),1. 00(3H ,s ,H-29),1. 00(3H ,s ,H-30),0. 95(3H ,s ,H-18),0. 89(3H ,s ,H-19);13C NMRδC :39. 4(C-1),28. 3(C-2),78. 1(C-3),39. 6(C-4),56. 4(C-5),18. 8(C-6),35. 2(C-7),40. 1(C-8),50. 3(C-9),37. 4(C-10),30. 8(C-11),70. 2(C-12),49. 7(C-13),51. 5(C-14),31. 0(C-15),26. 7(C-16),51. 7(C-17),16. 4(C-18),16. 1(C-19),83. 3(C-22. 4(C-21),36. 2(C-22),23. 3(C-23),20),
126. 0(C-24),131. 0(C-25),25. 8(C-26),17. 8(C-27),28. 7(C-28),16. 4(C-29),17. 4(C-30),98. 3(C-20-Glc-1),75. 2(C-20-Glc-2),79. 3(C-20-Glc-3),71. 6(C-20-Glc-4),78. 3(C-20-Glc-5),62. 9(C-20-Glc-6)。以上数据和文献报道的基本一
[10]
故化合物1鉴定为人参皂苷compound K ,即致,20(S )-20-O -D-原人参二醇-β-吡喃葡萄糖苷。1
化合物2白色粉末,易溶于甲醇。H NMR(400MHz ,pyridine-d 5)δH :5. 67(1H ,s ,Araf-H-1),
O -L-(1→6)-D-α-呋喃阿拉伯糖基-β-吡喃葡萄糖苷
(图1)。
1
化合物3白色粉末,易溶于甲醇。H NMR(400MHz ,pyridine-d 5)δH :5. 17(1H ,d ,J =8. 0Hz ,
20-Glc-H-1),5. 02(1H ,d ,J =8. 0Hz ,6-Glc-H-1),2. 07(3H ,s ,H-21),1. 61(3H ,s ,H-26),1. 60(6H ,s ,H-27,29),1. 52(3H ,s ,H-28),1. 16(3H ,s ,H-18),1. 04(3H ,s ,H-19),0. 81(3H ,s ,H-30);13C NMRδC :39. 7(C-1),27. 9(C-2),78. 8(C-3),40. 4
5. 14(1H ,d ,J =6. 0Hz ,Glc-H-1),1. 67(3H ,s ,H-27),1. 63(6H ,s ,H-21,26),1. 23(3H ,s ,H-28),
Vol. 26金建明等:西洋参总皂苷酶水解产物成分研究1555
(C-4),61. 4(C-5),75. 5(C-6),45. 1(C-7),41. 1(C-8),50. 0(C-9),39. 4(C-10),30. 7(C-11),70. 2(C-12),49. 2(C-13),51. 6(C-14),31. 0(C-26. 6(C-16),51. 4(C-17),17. 6(C-18),17. 815),
(C-19),83. 3(C-20),22. 2(C-21),36. 1(C-22),23. 2(C-23),126. 0(C-24),131. 0(C-25),25. 8(C-26),17. 6(C-27),31. 8(C-28),16. 4(C-29),17. 2(C-30),98. 3(C-20-Glc-1),75. 6(C-20-Glc-2),79. 3(C-20-Glc-3),71. 6(C-20-Glc-4),78. 1(C-20-Glc-5),62. 9(C-20-Glc-6),106. 0(C-6-Glc-1),75. 5(C-6-Glc-2),80. 2(C-6-Glc-3),71. 9(C-6-Glc-4),79. 7(C-6-Glc-5),63. 1(C-6-Glc-6)。以
[12]
,上数据和文献报道的基本一致故化合物3鉴
O -D-20定为人参皂苷Rg1,即6-β-吡喃葡萄糖基-
(3H ,s ,H-18),0. 96(3H ,s ,H-19),0. 94(3H ,s ,H-30);13C NMRδC :39. 6(C-1),27. 7(C-2),78. 6
(C-3),39. 4(C-4),60. 8(C-5),74. 6(C-6),46. 0(C-7),41. 2(C-8),49. 7(C-9),40. 0(C-10),30. 7(C-11),70. 2(C-12),49. 0(C-13),51. 4(C-14),30. 9(C-15),26. 6(C-16),51. 7(C-17),17. 3(C-18),17. 2(C-19),83. 3(C-20),22. 3(C-21),36. 0(C-22),23. 3(C-23),126. 0(C-24),130. 9(C-25),25. 8(C-26),17. 6(C-27),32. 2(C-28),17. 5(C-29),17. 8(C-30),98. 2(C-20-Glc-1),75. 1(C-20-Glc-2),79. 1(C-20-Glc-3),71. 5(C-20-Glc-4),78. 2(C-20-Glc-5),62. 9(C-20-Glc-6),101. 9(C-6-Glc-1),79. 4(C-6-Glc-2),78. 4(C-6-Glc-3),72. 5(C-6-Glc-4),78. 3(C-6-Glc-5),63. 1(C-6-Glc-6),101. 8(C-Rha-1),72. 3(C-Rha-2),72. 4(C-Rha-3),74. 1(C-Rha-4),69. 4(C-Rha-5),18. 7(C-Rha-6)。以上数据和文献报道的基本一致
[14]
(S )-20-O -D-原人参三醇-β-吡喃葡萄糖苷。
1
化合物4白色粉末,易溶于甲醇。H NMR(400MHz ,pyridine-d 5)δH :6. 49(1H ,s ,Rha-H-1),5. 26(1H ,d ,J =6. 4Hz ,Glc-H-1),2. 12(3H ,s ,H-28),1. 79(3H ,d ,J =6. 0,Rha-H-6),1. 68(3H ,s ,H-26),1. 63(3H ,s ,H-27),1. 39(3H ,s ,H-30),1. 35(3H ,s ,H-21),1. 19(3H ,s ,H-19),0. 96(3H ,s ,H-18),0. 93(3H ,s ,H-29);13C NMRδC :39. 6(C-1),27. 7(C-2),78. 3(C-3),40. 0(C-4),60. 8(C-5),74. 3(C-6),46. 0(C-7),41. 2(C-8),49. 6(C-9),41. 1(C-10),32. 0(C-11),71. 0(C-12),48. 1(C-51. 6(C-14),31. 2(C-15),26. 8(C-16),54. 613),
(C-17),17. 6(C-18),17. 6(C-19),72. 9(C-20),27. 0(C-21),35. 7(C-22),22. 9(C-23),126. 3(C-24),130. 7(C-25),25. 8(C-26),16. 9(C-27),32. 1(C-28),17. 1(C-29),17. 1(C-30),101. 8(C-79. 4(C-6-Glc-2),78. 5(C-6-Glc-3),72. 56-Glc-1),(C-6-Glc-4),78. 3(C-6-Glc-5),63. 0(C-6-Glc-6),101. 9(C-Rha-1),72. 2(C-Rha-2),72. 4(C-Rha-3),74. 1(C-Rha-4),69. 4(C-Rha-5),18. 7(C-Rha-6)。以上数据和文献报道的基本一致[13],故化合
6-物4鉴定为人参皂苷Rg2,即20(S )-原人参三醇-O -L-(1→2)-D-α-吡喃鼠李糖基-β-吡喃葡萄糖苷。
1
化合物5白色粉末,易溶于甲醇。H NMR(400MHz ,pyridine-d 5)δH :6. 47(1H ,s ,Rha-H-1),5. 24(1H ,d ,J =6. 4Hz ,6-Glc-H-1),5. 15(1H ,d ,J =8. 0Hz ,20-Glc-H-1),2. 09(3H ,s ,H-21),1. 76(3H ,d ,J =6. 0Hz ,Rha-H-6),1. 60(6H ,s ,H-26,27),1. 58(3H ,s ,H-29),1. 35(3H ,s ,H-28),1. 16
,故化合
O-D-物5鉴定为人参皂苷Re,即20-β-吡喃葡萄糖
20(S )-6-O -L-(1→原人参三醇-α-吡喃鼠李糖基-基-2)-D-β-吡喃葡萄糖苷。
1
化合物6白色粉末,易溶于甲醇。H NMR(400MHz ,pyridine-d 5)δH :5. 21(1H ,d ,J =6. 8Hz ,Glc-H-1),2. 00(3H ,s ,H-21),1. 60(9H ,s ,H-26,27,29),1. 47(3H ,s ,H-28),1. 10(3H ,s ,H-18),1. 02(3H ,s ,H-19),0. 98(3H ,S ,H-30);13C NMRδC :39. 6(C-1),28. 1(C-2),78. 3(C-3),40. 3(C-4),61. 7(C-5),67. 7(C-6),47. 5(C-7),41. 2(C-8),49. 9(C-9),39. 4(C-10),30. 9(C-11),70. 2(C-12),49. 1(C-13),51. 4(C-14),30. 8(C-15),26. 6(C-16),51. 6(C-17),17. 6(C-18),17. 5(C-19),83. 3(C-20),22. 3(C-21),36. 1(C-22),23. 2(C-23),126. 0(C-24),131. 0(C-25),25. 8(C-26),17. 8(C-27),32. 0(C-28),16. 5(C-29),17. 4(C-30),98. 3(C-20-Glc-1),75. 1(C-20-Glc-2),78. 5(C-20-Glc-3),71. 5(C-20-Glc-4),79. 3(C-20-Glc-5),62. 9(C-20-Glc-6)。以上数据和文
[15]
,献报道的基本一致故化合物6鉴定为人参皂苷
F 1,20-O -D-即20(S )-原人参三醇-β-吡喃葡萄糖苷。1
化合物7白色粉末,易溶于甲醇。H NMR(400MHz ,pyridine-d 5)δH :6. 48(1H ,s ,Rha-H-1),
5. 25(1H ,d ,J =6. 8Hz ,Glc-H-1),2. 10(3H ,s ,H-28),1. 78(3H ,d ,J =6. 4Hz ,Rha-H-6),1. 46(3H ,s ,H-27),1. 33(3H ,s ,H-29),1. 26(3H ,s ,H-21),
1556
天然产物研究与开发Vol. 26
1. 25(3H ,s ,H-26),1. 21(3H ,s ,H-18),0. 95(3H ,s ,H-19),0. 90(3H ,s ,H-30);13C NMRδC :39. 5(C-1),27. 7(C-2),78. 3(C-3),40. 0(C-4),60. 8(C-5),74. 3(C-6),46. 0(C-7),41. 2(C-8),49. 6(C-9),41. 1(C-10),32. 0(C-11),71. 0(C-12),48. 1(C-13),51. 6(C-14),31. 2(C-15),26. 8(C-16),54. 6(C-17),17. 6(C-18),17. 6(C-19),86. 7(C-20),27. 0(C-21),32. 8(C-22),28. 8(C-23),85. 6(C-24),70. 3(C-25),27. 2(C-26),27. 6(C-27),32. 4(C-28),18. 2(C-29),16. 9(C-30),101. 8(C-79. 4(C-6-Glc-2),78. 6(C-6-Glc-3),72. 66-Glc-1),(C-6-Glc-4),78. 4(C-6-Glc-5),63. 1(C-6-Glc-6),101. 9(C-Rha-1),72. 3(C-Rha-2),72. 4(C-Rha-3),74. 2(C-Rha-4),69. 4(C-Rha-5),18. 7(C-Rha-6)。以上数据和文献报道的基本一致,故化合
3β,6α,12β,25-物7鉴定为拟人参皂苷F 11,即达玛-(20S ,24R)-6-O -L-(1四羟基-环氧-α-吡喃鼠李糖基-D-β-吡喃葡萄糖苷。→2)-[16]
此推断该β-糖苷酶能将西洋参中原人参二醇型皂3的羟基的糖基全部水解。苷的C-人参皂苷Rc和Rb2的分子量相同而且结构也20的糖基不同:人很相似,区别在于连接在苷元C-20的葡参皂苷Rc是一个呋喃阿拉伯糖基连接在C-萄糖基的6位羟基上,而人参皂苷Rb2是一个吡喃
20的葡萄糖基的6位羟基上阿拉伯糖基连接在C-(图1)。人参皂苷Rc经该β-糖苷酶水解可产生人
如进一步水解可产生人参皂苷com-参皂苷Mc ,
pound K ;人参皂苷Rb2经该β-糖苷酶水解可产生人
参皂苷compound K ,但不可能产生人参皂苷Mc 。由于从水解产物中只分离到人参皂苷compound K
20的葡萄糖基上和Mc ,表明该β-糖苷酶能水解C-20的葡萄糖基上的但不能水解C-的吡喃阿拉伯糖基,
呋喃阿拉伯糖基或水解呋喃阿拉伯糖基的活性很低。
参考文献1
Su J (苏健),Li YH (李海舟),Yang CR(杨崇仁). Studies on saponin constituents in roots of Panax quinquefoli-um . China J Chin Mater Med (中国中药杂志),2003,28:830-833. 2
Wang L (王蕾),Wang YP (王英平),Xu SQ (许世泉),et al . A review on studies of the components and pharmacologi-cal activity of Panax quinquefolium L. Spec Wild Econ Anim Plant Res(特产研究),2007,3:73-77. 3
Yoshika I ,Sugawara T ,Imai K ,et al . Soil bacterial hydroly-Rb1,Rb2sis :leading to genuine aglycone. V. on ginsenosides-and Reof the ginseng root saponins. Chem Pharm Bull ,1972,20:2418-2421. 4
Hasegawa H. Proof of the mysterious efficacy of ginseng basic and clinical trials metabolic activation of ginsenoside degly-cosylation by in testinal bacteria and esterification with fatty acid. J Pharmacol Sci ,2004,95:153-157. 5
Zhou W (周伟),Zhou P (周珮). Advances in the study of ginsenoside compound K. Acta Pharm Sin (药学学报),2007,42:917-923. 6
Li XP (李相鹏),Wang P (王鹏),Li YX (李英霞). Progress an in pharmacological actions of ginsenoside compound K ,activemetabolite of protopanaxadiol type saponins. Chin J Pharmacol Toxicol ,2011,25:97-101. 7
Zang P (臧埔),Zhang LX (张连学),et Li X (李学),
al . Researchprogress on ginsenoside CK production by mi-crobial transformation. Food Sci (食品科学),2012,33:323-327.
3讨论
HPLC 检测分析结果表明实验用的西洋参总皂
苷中人参皂苷Rb1是含量最高的原人参二醇型皂苷
Rc和Rb2(图2A )。文成分,其次是人参皂苷Rd、Rd、Rc人参皂苷Rb1、献也报道在西洋参总皂苷中,
和Rb2是其主要原人参二醇型皂苷成分,且人参皂苷Rb1是其中含量最高的原人参二醇型皂苷
[1,2,9,17]
。研究已表明人参皂苷compound K 可由原
Rb2、Rc和Rd等通过酶水解人参二醇型皂苷Rb1、而产生
[5,7]
。从西洋参总皂苷的水解产物中分离得
到的原人参二醇型皂苷除人参皂苷compound K 外,
另外得到人参皂苷Mc ,但没有分离到原人参二醇型Rb2、Rc或Rd。HPLC 检测分析也证明水皂苷Rb1、
解产物中只存在原人参二醇型皂苷compound K 和Mc ,Rb2、Rc而不存在主要原人参二醇型皂苷Rb1、和Rd(图2B )。HPLC 检测分析证明西洋参总皂苷中并不存在人参皂苷compound K 和Mc (图2A ),这Rb2、Rc和Rd已经完全被该β-糖表明人参皂苷Rb1、苷酶催化水解,而人参皂苷Mc 不能进一步被催化水解产生人参皂苷compound K 或这种催化活性很低。
3或C-20原人参二醇型皂苷的糖基连接在C-Rb2、Rc和Rd的羟基上。原人参二醇型皂苷Rb1、
3的羟基上连接有糖基,在C-而水解产物人参皂苷compound K 和Mc 在C-3的羟基上都没有糖基,因
(下转第1567页)
Vol. 26刘韶等:大孔树脂分离纯化陈皮中多甲氧基黄酮类化合物1567
5Li SM ,Pan MH ,Lo CY ,et al .Chemistry and health effects of polymethoxyfiavones and hydrxylated polymethoxyflavones.J Functional Foods ,2009,1:2-12.
et al .HPLC simultaneous determination of contents of five flavonoids in Citrus reticulata ‘Chachi ’from various habi-tats.Chin Tradit Herb Drugs (中草药),2010,41:652-655.9
Jiang L (蒋林),Zheng GD (郑国栋).Li LW (林乐维),
Study on the contents of flavonoids in Citrus reticulata ‘Cha-chi ’from various habitats and different collecting periods .J Chin Med Mater (中药材),2010,33:173-176.
10Wu ZC (吴志成),Liu SZ (刘淑芝),Li ML (李曼玲),et
al .Study on refine techincs of fufang jingzhigguanxin with macroporous resin.Chin J Exp Tradit Med Form (中国实验2011,17(8):11-14.方剂学杂志),
6Wang HX (王辉宪),Cao YB (曹永兵),Liu BF (刘邦夫),
et al .Study on separation and purification of flavonoids from citrus .Che ResApp (化学研究与应用),2008,20:485-489.
7Chen FS (陈复生),Zuo JJ (左锦静),YaoYZ (姚永志).Study on the performance of absorption and separation of macroporous res ins for the flavonoids from citrus .Food Sci Tech (食品科技),2006,7:121-124.
8Zheng GD (郑国栋),Jiang L (蒋林),Yang DB (杨得坡),
櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵
ginsenoside Rg2. Chin J Magn Reson(波谱学杂志),2000,(上接第1556页)
8
Liang CN (梁朝宁),Xiong DD (熊丹丹),Tang SY (唐双焱). A screening method for mutations produce high yield of rare ginsenosides from major ginsenosides. CN[1**********]7. 9,2013-6-20. 9
Meng Q (孟琼),Qian ZM (钱正明),Chen ZY (陈治宇),et al . HPLC characteristics of Panax quinquefolium . Chin J Pharm Anal (药物分析杂志),2010,30:791-795.
10Haruyo K ,Shuichi S ,Yoshiteru I ,et al . Studies on the sapo-nins of ginseng IV. on the structure and enzymatic hydrolysis of ginsenoside-Ra1. Chem Pharm Bull ,1982,30:2393-2398. 11Bae EA ,Choo MK ,Park EK ,et al . Metabolism of ginsenoside
Rcby human intestinal bacteria and its related antiallergic activity. Biol Pharm Bull ,2002,25:743-747.
12Shoji Y ,Kiyoko K ,Osamu T. Study on dammarane-type sapo-nins of roots ,leaves ,flower-buds and fruits of Panax ginseng C. A. Meyer. Chem Pharm Bull ,1979,27:88-92. 13Yang XW (杨秀伟). Complete assignment of 1H and
13
17:9-15.
14Zeng J (曾江),Cui XM (崔秀明),Zhou JM (周家明),et
al . Studies on chemical constituents from rhizomes of Panax notogingseng . J Chin Medi Mater (中药材),2007,30:1388-1391.
15Song JP (宋建平),Zeng J (曾江),Cui XM (催秀明),et
al . Studies on chemical constituents from rhizomes of Panax notoginseng (Ⅱ). J Yunnan Univ (云南大学学报),2007,29:287-290.
16Tanaka O ,Yahara S. Dammarane saponins of leaves of Panax
pseudo-ginseng subsp. Himalaicus . Phytochem ,1978,17:1353-1358.
17Zhai WM (翟为民),Yuan YS (袁永生),Zhou YX (周玉
et al . HPLC fingerprints identification of Panax ginseng 新),
C. A. Mey. ,P . quinquefolium 2001,26:481-482. 志),
L. and
P . notoginseng
(Burk. F. H. Chen ). China J Chin Mater Med (中国中药杂
C
ginsenoside Rg2and 20(S )-NMRchemical shifts of 20(R)-
26:1552-1556,1567天然产物研究与开发Nat Prod ResDev 2014,
6880(2014)10-1552-06文章编号:1001-
西洋参总皂苷酶水解产物成分研究
金建明
12
3
1,2*
2133
,李有海,张海伦,梁朝宁,唐双焱
北京工商大学植物资源研究开发北京市重点实验室,北京100048;
云南民族大学民族药资源化学国家民委-教育部重点实验室,昆明650500;
中国科学院微生物研究所中国科学院微生物生理与代谢工程重点实验室,北京100101
摘要:西洋参总皂苷经β-糖苷酶催化水解,采用HPLC 检测分析确定西洋参总皂苷中的主要原人参二醇型皂
Rd、Rc和Rb2已经完全被水解。水解产物通过反复硅胶柱层析和反向硅胶柱层析分离纯化得到7个皂苷Rb1、
苷,通过NMR谱图分析分别鉴定为人参皂苷compound K (1)、人参皂苷Mc (2)、人参皂苷Rg1(3)、人参皂苷Rg2(4)、人参皂苷Re(5)、人参皂苷F 1(6)和拟人参皂苷F 11(7)。β-糖苷酶催化西洋参总皂苷水解实验表明,西洋参中原人参二醇型皂苷的水解产物是人参皂苷compound K 和人参皂苷Mc 。关键词:西洋参;原人参二醇型皂苷;人参皂苷compound K ;β-糖苷酶中图分类号:R939.9
文献标识码:A
Chemical Constituents of Enzymatic Hydrolysate of Total
Ginsenoside Extract of Panax quinquefolium
2*
JIN Jian-ming 1,,LI You-hai 2,ZHANG Hai-lun 1,LIANG Chao-ning 3,TANG Shuang-yan 3
1
Beijing Key Laboratory of Plant ResourcesResearchand Development ,Beijing Technology and Business University ,
Beijing 100048,China ;2Key Laboratory of Ethnic Medicine ResourceChemistry ,State Ethnic Affairs Commission &Ministry of Education ,Yunnan University of Nationalities ,Yunnan Kunming 650500,China ;3CAS Key Laboratory of
Microbial Physiological and Metabolic Engineering ,Institute of Microbiology ,Chinese
Beijing 100101,China Academy of Sciences ,
Abstract :The total ginsenoside extract of Panax quinquefolium was hydrolyzed by β-glycosidase.HPLC analysis indica-ted that ginsenosides Rb1,Rd,Rcand Rb2were completely hydrolyzed.Seven ginsenosides were isolated from the hydrol-ysate ,and identified as ginsenoside compound K (1),ginsenoside Mc (2),ginsenoside Rg1(3),ginsenoside Rg2(4),ginsenoside Re(5),ginsenoside F 1(6)and pseudo-ginsenoside F 11(7),respectively.The protopanaxdiol-type ginsen-osides in the total ginsenoside extract of P.quinquefolium were converted into ginsenoside compound K and ginsenoside glycosidase.Mc by the β-Key words :Panax quinquefolium ;protopanaxdiol-type ginsenosides ;ginsenoside compound K ;β-glycosidase
西洋参(Panax quinquefolium L.)又名花旗参、
西洋人参、洋参、美国人参、广东人参等,是我国传统为五加科人参属植物,原产北美洲的的名贵中药材,
加拿大南部和美国北部,现我国的许多地区都有引种栽培。其味甘、微苦、性寒,可滋阴降火,益气生津,其药用价值有镇静镇惊、安神促智、增强机体免疫、抗心律失常、防血管硬塞、保肝、降血脂等功效。对西洋参的化学成分已经有许多研究报道,其主要
02-1405-16收稿日期:2014-接受日期:2014-基金项目:国家自然科学基金项目(31160017;21172095);北京市
自然科学基金项目(2142012);云南省教育厅科学研究基金项目(2011Z120)
*通讯作者Tel :86-10-68984937;E-mail :jinjm8@qq.com
活性成分是人参皂苷。西洋参总皂苷中绝大多数属
[1,2]
。达玛烷四环三于达玛烷四环三萜类人参皂苷
萜类人参皂苷根据苷元6位碳上是否有羟基分为原Rb2、Rc和Rd等人参二醇型皂苷如人参皂苷Rb1、
(图1)和原人参三醇型皂苷如人参皂苷Rg1、Rg2、Re和F 1等。
稀有人参皂苷compound K 也称人参皂苷CK
K ,20-或人参皂苷C-其结构为20(S )-原人参二醇-O-D-β-吡喃葡萄糖苷(图1),属原人参二醇型皂苷。
人参皂苷compound K 在人参属植物中几乎不存在,Rb2和Rc后被首次由土壤细菌降解人参皂苷Rbl 、发现并鉴定
[3]
。研究发现人参皂苷compound K 是
Vol. 26金建明等:西洋参总皂苷酶水解产物成分研究1553
人参等药材中原人参二醇型皂苷经肠道菌降解后产
[4]
生的在人体内发挥药效作用的真正代谢活性物。人参皂苷compound K 的生物活性具有多靶点、高活性和低毒性的特点,具有极高的生产价值和应用前
[5,6]
。目前制备人参皂苷com-景,医药价值很高
pound K 的方法多采用微生物发酵法和酶解法。利
rpm 摇床培养12h 后将种子液转接到600mL 诱导培养基中(含100mmol /L卡那霉素),接种量1%,30ħ 、280rpm 摇床培养21h 。培养液10000rpm 离心10min ,沉淀加入60mL Mc 缓冲液重悬(0.2mol /L磷酸氢二钠和0.1mol /L柠檬酸,pH =5.5),超声破细胞后12000rpm 离心10min ,取上清液于60ħ 水浴中静置10min 后再次12000rpm 离心10
[8]
min ,上清液即β-糖苷酶液。1.3
酶水解反应
约60mL 酶液加入到75mL 含7.5g 西洋参总
用酶解法制备人参皂苷compound K 具有反应条件
[5,7]
。温和、操作简便
、成本低、产物便于纯化等优点
皂苷的Mc 缓冲液中,水浴恒温70ħ 反应过夜使酶
[8]
催化水解反应完全(19h )。1.4
分离提取
135mL 水解反应液用等体积正丁醇萃取三次,
图1
Fig. 1
原人参二醇型皂苷结构
Chemical structures of protopanaxdiol-type ginsenosides
合并萃取液,减压浓缩干燥后进行硅胶柱层析,用二氯甲烷甲醇进行梯度洗脱,分为五个部分(Fr I Fr V )。各部分再经RP-C18柱层析,采用甲醇水进行梯度洗脱纯化各化合物。Fr I 部分1.3g 经50% 80%甲醇水梯度洗脱纯化得到化合物1(0.807g );Fr II 部分0.2g 用50% 60%甲醇水梯度洗脱得到化合物6(0.049g );Fr III 部分0.6g 经60% 75%甲醇水梯度洗脱得到化合物2(0.311g );Fr IV 部分0.58g 用20% 75%甲醇水梯度洗脱得到化合物3(0.152g )、7(0.070g )和4(0.046g )。Fr V 部分1.036g 用10% 50%甲醇水梯度洗脱得到化合物5(0.481g )。1.5
HPLC 检测
西洋参总皂苷及其水解产物的HPLC 方法在参
[9]
考文献的基础上略有修改。西洋参总皂苷及其水解产物配制成3mg /mL。HPLC 条件如下:色谱
5柱:Waters symmetry C 18色谱柱(4.6ˑ 250mm ,μm );流动相:乙腈(A )和水(B );二元高压梯度洗
20%A ;2.5 5min ,20% 25%A ;脱:0 2.5min ,
5 10min ,25%A ;10 30min ,25% 40%A ;30 45min ,40% 70%A ;45 60min ,70%A ;流速:
0. 7mL /min;检测波长:203nm ;柱温:35ħ ;进样量:10μL 。
Glc :β-D-glucopyranosyl ;Ara (pyr ):α-L-arabinopyranosyl ;Ara (fur ):L-arabinofuranosyl α-
项目组前期得到了高效催化人参皂苷Rb1产生人参皂苷compound K 的β-糖苷酶。为了研究该β-糖苷酶是否也催化其他原人参二醇型皂苷产生人参
实验采用原人参二醇型皂苷含量皂苷compound K ,
高的西洋参总皂苷,详细分析了西洋参总皂苷经该
β-糖苷酶完全催化水解后的成分组成,为将来进一步采用西洋参总皂苷等高效制备人参皂苷com-pound K 奠定基础。
1
1.1
材料与方法
仪器与试剂
Bruker Avance 400MHz 型核磁共振波谱仪;Ag-
ilent 6520LC /MS质谱仪;岛津Lc-20A 高效液相色
谱仪。柱层析硅胶(60 100目)和薄层层析硅胶板H (青岛海洋化工有限公司);RP-C18反相层析材料
C18高效薄层板(Merck 公司);乙腈为色谱和RP-纯,其余试剂均为分析纯;西洋参总皂苷购自南京泽朗医药生物科技有限公司。标准品人参皂苷Rb1(批号20110526)、Rb2(批号20120311)、Rc(批号20120617)和Rd(批号20120621)购自宝鸡辰光生物科技有限公司。1.2酶液制备
将β-糖苷酶LacS 突变株接种到LB 培养基中
(含100mmol /L卡那霉素),37ħ 、280接种量1%,
2
2.1
结果与分析
西洋参水解产物的组分分析
为了研究β-糖苷酶催化总皂苷后水解产物的
成分组成及原人参二醇型皂苷生成人参皂苷com-pound K 的效率,将西洋参总皂苷经β-糖苷酶催化
1554
天然产物研究与开发Vol. 26
水解过夜(19h ),水解产物经TLC 检测初步证明原人参二醇型皂苷Rb1已经完全被水解。HPLC 检测分析再次确定西洋参总皂苷中的原人参二醇型皂苷
Rc(9)、Rb2(10)和Rd(11)已经人参皂苷Rb1(8)、
完全水解(图2B )。进一步延长水解反应时间后的
水解产物经HPLC 检测分析皂苷的成分和含量没有变化,表明19h 水解反应已经完全。水解产物中主
mAU
[**************]00
mAU [**************]0
要有二个原人参二醇型皂苷:人参皂苷compound K (1)和人参皂苷Mc (2);四个原人参三醇型皂苷:人人参皂苷Rg2(4)、人参皂苷Re(5)参皂苷Rg1(3)、
和人参皂苷F 1(6);以及拟人参皂苷F 11(7)。其中人参皂苷compound K 是西洋参总皂苷水解产物中含量最高的成分。
mAU [**************]0
10%20%30%40%50min 010%20%30%40%50min 010%20%30%40%50min
图2
Fig. 2
西洋参总皂苷水解前(A )和水解后(B )以及人参皂苷compound K (C )的HPLC 色谱图
HPLC chromatograms of total ginsenoside extract of P.quinquefolium before (A )and after (B )hydrolysis as well as ginsen-oside compound K standard (C )
1:人参皂苷compound K (ginsenoside compound K );2:人参皂苷Mc (ginsenoside Mc );3:人参皂苷Rg1(ginsenoside Rg1);4:人参皂苷Rg2(gin-senoside Rg2);5:人参皂苷Re(ginsenoside Re);6:人参皂苷F 1(ginsenoside F 1);8:人参皂苷Rb1(ginsenoside Rb1);9:人参皂苷Rc(ginsen-oside Rc);10:人参皂苷Rb2(ginsenoside Rb2);11:人参皂苷Rd(ginsenoside Rd)
2.2
水解产物结构鉴定
ESI-MS 化合物1白色粉末,易溶于甲醇。HR-
1. 04(3H ,s ,H-29),1. 00(3H ,s ,H-30),0. 94(3H ,s ,H-18),0. 89(3H ,s ,H-19);13C NMRδC :39. 6(C-1),28. 3(C-2),78. 1(C-3),39. 6(C-4),56. 4(C-5),18. 8(C-6),35. 2(C-7),40. 1(C-8),50. 3(C-9),37. 4(C-10),30. 8(C-11),70. 3(C-12),49. 5(C-13),51. 2(C-14),30. 9(C-15),26. 7(C-16),51. 7(C-17),16. 4(C-18),16. 1(C-19),83. 4(C-20),22. 4(C-21),36. 2(C-22),23. 2(C-23),126. 1(C-24),131. 1(C-25),25. 8(C-26),17. 9(C-27),28. 7(C-28),16. 4(C-29),17. 4(C-30),98. 1(C-20-Glc-1),75. 1(C-20-Glc-2),79. 3(C-20-Glc-3),72. 2(C-20-Glc-4),76. 6(C-20-Glc-5),68. 5(C-20-Glc-6),110. 2(C-Ara-1),83. 4(C-Ara-2),78. 9(C-Ara-3),86. 1(C-Ara-4),62. 7(C-Ara-5)。以上数据和文献报道的基本一致
,故化合
20-物2鉴定为人参皂苷Mc ,即20(S )-原人参二醇-[11]
m /z 645.4342[M +Na ]+(calcd for C 36H 62NaO 8,645.4337);1H NMR(400MHz ,pyridine-d 5)δH :
5. 19(1H ,d ,J =7. 6Hz ,Glc-H-1),1. 63(3H ,s ,H-21),1. 60(6H ,s ,H-26,27),1. 23(3H ,s ,H-28),1. 00(3H ,s ,H-29),1. 00(3H ,s ,H-30),0. 95(3H ,s ,H-18),0. 89(3H ,s ,H-19);13C NMRδC :39. 4(C-1),28. 3(C-2),78. 1(C-3),39. 6(C-4),56. 4(C-5),18. 8(C-6),35. 2(C-7),40. 1(C-8),50. 3(C-9),37. 4(C-10),30. 8(C-11),70. 2(C-12),49. 7(C-13),51. 5(C-14),31. 0(C-15),26. 7(C-16),51. 7(C-17),16. 4(C-18),16. 1(C-19),83. 3(C-22. 4(C-21),36. 2(C-22),23. 3(C-23),20),
126. 0(C-24),131. 0(C-25),25. 8(C-26),17. 8(C-27),28. 7(C-28),16. 4(C-29),17. 4(C-30),98. 3(C-20-Glc-1),75. 2(C-20-Glc-2),79. 3(C-20-Glc-3),71. 6(C-20-Glc-4),78. 3(C-20-Glc-5),62. 9(C-20-Glc-6)。以上数据和文献报道的基本一
[10]
故化合物1鉴定为人参皂苷compound K ,即致,20(S )-20-O -D-原人参二醇-β-吡喃葡萄糖苷。1
化合物2白色粉末,易溶于甲醇。H NMR(400MHz ,pyridine-d 5)δH :5. 67(1H ,s ,Araf-H-1),
O -L-(1→6)-D-α-呋喃阿拉伯糖基-β-吡喃葡萄糖苷
(图1)。
1
化合物3白色粉末,易溶于甲醇。H NMR(400MHz ,pyridine-d 5)δH :5. 17(1H ,d ,J =8. 0Hz ,
20-Glc-H-1),5. 02(1H ,d ,J =8. 0Hz ,6-Glc-H-1),2. 07(3H ,s ,H-21),1. 61(3H ,s ,H-26),1. 60(6H ,s ,H-27,29),1. 52(3H ,s ,H-28),1. 16(3H ,s ,H-18),1. 04(3H ,s ,H-19),0. 81(3H ,s ,H-30);13C NMRδC :39. 7(C-1),27. 9(C-2),78. 8(C-3),40. 4
5. 14(1H ,d ,J =6. 0Hz ,Glc-H-1),1. 67(3H ,s ,H-27),1. 63(6H ,s ,H-21,26),1. 23(3H ,s ,H-28),
Vol. 26金建明等:西洋参总皂苷酶水解产物成分研究1555
(C-4),61. 4(C-5),75. 5(C-6),45. 1(C-7),41. 1(C-8),50. 0(C-9),39. 4(C-10),30. 7(C-11),70. 2(C-12),49. 2(C-13),51. 6(C-14),31. 0(C-26. 6(C-16),51. 4(C-17),17. 6(C-18),17. 815),
(C-19),83. 3(C-20),22. 2(C-21),36. 1(C-22),23. 2(C-23),126. 0(C-24),131. 0(C-25),25. 8(C-26),17. 6(C-27),31. 8(C-28),16. 4(C-29),17. 2(C-30),98. 3(C-20-Glc-1),75. 6(C-20-Glc-2),79. 3(C-20-Glc-3),71. 6(C-20-Glc-4),78. 1(C-20-Glc-5),62. 9(C-20-Glc-6),106. 0(C-6-Glc-1),75. 5(C-6-Glc-2),80. 2(C-6-Glc-3),71. 9(C-6-Glc-4),79. 7(C-6-Glc-5),63. 1(C-6-Glc-6)。以
[12]
,上数据和文献报道的基本一致故化合物3鉴
O -D-20定为人参皂苷Rg1,即6-β-吡喃葡萄糖基-
(3H ,s ,H-18),0. 96(3H ,s ,H-19),0. 94(3H ,s ,H-30);13C NMRδC :39. 6(C-1),27. 7(C-2),78. 6
(C-3),39. 4(C-4),60. 8(C-5),74. 6(C-6),46. 0(C-7),41. 2(C-8),49. 7(C-9),40. 0(C-10),30. 7(C-11),70. 2(C-12),49. 0(C-13),51. 4(C-14),30. 9(C-15),26. 6(C-16),51. 7(C-17),17. 3(C-18),17. 2(C-19),83. 3(C-20),22. 3(C-21),36. 0(C-22),23. 3(C-23),126. 0(C-24),130. 9(C-25),25. 8(C-26),17. 6(C-27),32. 2(C-28),17. 5(C-29),17. 8(C-30),98. 2(C-20-Glc-1),75. 1(C-20-Glc-2),79. 1(C-20-Glc-3),71. 5(C-20-Glc-4),78. 2(C-20-Glc-5),62. 9(C-20-Glc-6),101. 9(C-6-Glc-1),79. 4(C-6-Glc-2),78. 4(C-6-Glc-3),72. 5(C-6-Glc-4),78. 3(C-6-Glc-5),63. 1(C-6-Glc-6),101. 8(C-Rha-1),72. 3(C-Rha-2),72. 4(C-Rha-3),74. 1(C-Rha-4),69. 4(C-Rha-5),18. 7(C-Rha-6)。以上数据和文献报道的基本一致
[14]
(S )-20-O -D-原人参三醇-β-吡喃葡萄糖苷。
1
化合物4白色粉末,易溶于甲醇。H NMR(400MHz ,pyridine-d 5)δH :6. 49(1H ,s ,Rha-H-1),5. 26(1H ,d ,J =6. 4Hz ,Glc-H-1),2. 12(3H ,s ,H-28),1. 79(3H ,d ,J =6. 0,Rha-H-6),1. 68(3H ,s ,H-26),1. 63(3H ,s ,H-27),1. 39(3H ,s ,H-30),1. 35(3H ,s ,H-21),1. 19(3H ,s ,H-19),0. 96(3H ,s ,H-18),0. 93(3H ,s ,H-29);13C NMRδC :39. 6(C-1),27. 7(C-2),78. 3(C-3),40. 0(C-4),60. 8(C-5),74. 3(C-6),46. 0(C-7),41. 2(C-8),49. 6(C-9),41. 1(C-10),32. 0(C-11),71. 0(C-12),48. 1(C-51. 6(C-14),31. 2(C-15),26. 8(C-16),54. 613),
(C-17),17. 6(C-18),17. 6(C-19),72. 9(C-20),27. 0(C-21),35. 7(C-22),22. 9(C-23),126. 3(C-24),130. 7(C-25),25. 8(C-26),16. 9(C-27),32. 1(C-28),17. 1(C-29),17. 1(C-30),101. 8(C-79. 4(C-6-Glc-2),78. 5(C-6-Glc-3),72. 56-Glc-1),(C-6-Glc-4),78. 3(C-6-Glc-5),63. 0(C-6-Glc-6),101. 9(C-Rha-1),72. 2(C-Rha-2),72. 4(C-Rha-3),74. 1(C-Rha-4),69. 4(C-Rha-5),18. 7(C-Rha-6)。以上数据和文献报道的基本一致[13],故化合
6-物4鉴定为人参皂苷Rg2,即20(S )-原人参三醇-O -L-(1→2)-D-α-吡喃鼠李糖基-β-吡喃葡萄糖苷。
1
化合物5白色粉末,易溶于甲醇。H NMR(400MHz ,pyridine-d 5)δH :6. 47(1H ,s ,Rha-H-1),5. 24(1H ,d ,J =6. 4Hz ,6-Glc-H-1),5. 15(1H ,d ,J =8. 0Hz ,20-Glc-H-1),2. 09(3H ,s ,H-21),1. 76(3H ,d ,J =6. 0Hz ,Rha-H-6),1. 60(6H ,s ,H-26,27),1. 58(3H ,s ,H-29),1. 35(3H ,s ,H-28),1. 16
,故化合
O-D-物5鉴定为人参皂苷Re,即20-β-吡喃葡萄糖
20(S )-6-O -L-(1→原人参三醇-α-吡喃鼠李糖基-基-2)-D-β-吡喃葡萄糖苷。
1
化合物6白色粉末,易溶于甲醇。H NMR(400MHz ,pyridine-d 5)δH :5. 21(1H ,d ,J =6. 8Hz ,Glc-H-1),2. 00(3H ,s ,H-21),1. 60(9H ,s ,H-26,27,29),1. 47(3H ,s ,H-28),1. 10(3H ,s ,H-18),1. 02(3H ,s ,H-19),0. 98(3H ,S ,H-30);13C NMRδC :39. 6(C-1),28. 1(C-2),78. 3(C-3),40. 3(C-4),61. 7(C-5),67. 7(C-6),47. 5(C-7),41. 2(C-8),49. 9(C-9),39. 4(C-10),30. 9(C-11),70. 2(C-12),49. 1(C-13),51. 4(C-14),30. 8(C-15),26. 6(C-16),51. 6(C-17),17. 6(C-18),17. 5(C-19),83. 3(C-20),22. 3(C-21),36. 1(C-22),23. 2(C-23),126. 0(C-24),131. 0(C-25),25. 8(C-26),17. 8(C-27),32. 0(C-28),16. 5(C-29),17. 4(C-30),98. 3(C-20-Glc-1),75. 1(C-20-Glc-2),78. 5(C-20-Glc-3),71. 5(C-20-Glc-4),79. 3(C-20-Glc-5),62. 9(C-20-Glc-6)。以上数据和文
[15]
,献报道的基本一致故化合物6鉴定为人参皂苷
F 1,20-O -D-即20(S )-原人参三醇-β-吡喃葡萄糖苷。1
化合物7白色粉末,易溶于甲醇。H NMR(400MHz ,pyridine-d 5)δH :6. 48(1H ,s ,Rha-H-1),
5. 25(1H ,d ,J =6. 8Hz ,Glc-H-1),2. 10(3H ,s ,H-28),1. 78(3H ,d ,J =6. 4Hz ,Rha-H-6),1. 46(3H ,s ,H-27),1. 33(3H ,s ,H-29),1. 26(3H ,s ,H-21),
1556
天然产物研究与开发Vol. 26
1. 25(3H ,s ,H-26),1. 21(3H ,s ,H-18),0. 95(3H ,s ,H-19),0. 90(3H ,s ,H-30);13C NMRδC :39. 5(C-1),27. 7(C-2),78. 3(C-3),40. 0(C-4),60. 8(C-5),74. 3(C-6),46. 0(C-7),41. 2(C-8),49. 6(C-9),41. 1(C-10),32. 0(C-11),71. 0(C-12),48. 1(C-13),51. 6(C-14),31. 2(C-15),26. 8(C-16),54. 6(C-17),17. 6(C-18),17. 6(C-19),86. 7(C-20),27. 0(C-21),32. 8(C-22),28. 8(C-23),85. 6(C-24),70. 3(C-25),27. 2(C-26),27. 6(C-27),32. 4(C-28),18. 2(C-29),16. 9(C-30),101. 8(C-79. 4(C-6-Glc-2),78. 6(C-6-Glc-3),72. 66-Glc-1),(C-6-Glc-4),78. 4(C-6-Glc-5),63. 1(C-6-Glc-6),101. 9(C-Rha-1),72. 3(C-Rha-2),72. 4(C-Rha-3),74. 2(C-Rha-4),69. 4(C-Rha-5),18. 7(C-Rha-6)。以上数据和文献报道的基本一致,故化合
3β,6α,12β,25-物7鉴定为拟人参皂苷F 11,即达玛-(20S ,24R)-6-O -L-(1四羟基-环氧-α-吡喃鼠李糖基-D-β-吡喃葡萄糖苷。→2)-[16]
此推断该β-糖苷酶能将西洋参中原人参二醇型皂3的羟基的糖基全部水解。苷的C-人参皂苷Rc和Rb2的分子量相同而且结构也20的糖基不同:人很相似,区别在于连接在苷元C-20的葡参皂苷Rc是一个呋喃阿拉伯糖基连接在C-萄糖基的6位羟基上,而人参皂苷Rb2是一个吡喃
20的葡萄糖基的6位羟基上阿拉伯糖基连接在C-(图1)。人参皂苷Rc经该β-糖苷酶水解可产生人
如进一步水解可产生人参皂苷com-参皂苷Mc ,
pound K ;人参皂苷Rb2经该β-糖苷酶水解可产生人
参皂苷compound K ,但不可能产生人参皂苷Mc 。由于从水解产物中只分离到人参皂苷compound K
20的葡萄糖基上和Mc ,表明该β-糖苷酶能水解C-20的葡萄糖基上的但不能水解C-的吡喃阿拉伯糖基,
呋喃阿拉伯糖基或水解呋喃阿拉伯糖基的活性很低。
参考文献1
Su J (苏健),Li YH (李海舟),Yang CR(杨崇仁). Studies on saponin constituents in roots of Panax quinquefoli-um . China J Chin Mater Med (中国中药杂志),2003,28:830-833. 2
Wang L (王蕾),Wang YP (王英平),Xu SQ (许世泉),et al . A review on studies of the components and pharmacologi-cal activity of Panax quinquefolium L. Spec Wild Econ Anim Plant Res(特产研究),2007,3:73-77. 3
Yoshika I ,Sugawara T ,Imai K ,et al . Soil bacterial hydroly-Rb1,Rb2sis :leading to genuine aglycone. V. on ginsenosides-and Reof the ginseng root saponins. Chem Pharm Bull ,1972,20:2418-2421. 4
Hasegawa H. Proof of the mysterious efficacy of ginseng basic and clinical trials metabolic activation of ginsenoside degly-cosylation by in testinal bacteria and esterification with fatty acid. J Pharmacol Sci ,2004,95:153-157. 5
Zhou W (周伟),Zhou P (周珮). Advances in the study of ginsenoside compound K. Acta Pharm Sin (药学学报),2007,42:917-923. 6
Li XP (李相鹏),Wang P (王鹏),Li YX (李英霞). Progress an in pharmacological actions of ginsenoside compound K ,activemetabolite of protopanaxadiol type saponins. Chin J Pharmacol Toxicol ,2011,25:97-101. 7
Zang P (臧埔),Zhang LX (张连学),et Li X (李学),
al . Researchprogress on ginsenoside CK production by mi-crobial transformation. Food Sci (食品科学),2012,33:323-327.
3讨论
HPLC 检测分析结果表明实验用的西洋参总皂
苷中人参皂苷Rb1是含量最高的原人参二醇型皂苷
Rc和Rb2(图2A )。文成分,其次是人参皂苷Rd、Rd、Rc人参皂苷Rb1、献也报道在西洋参总皂苷中,
和Rb2是其主要原人参二醇型皂苷成分,且人参皂苷Rb1是其中含量最高的原人参二醇型皂苷
[1,2,9,17]
。研究已表明人参皂苷compound K 可由原
Rb2、Rc和Rd等通过酶水解人参二醇型皂苷Rb1、而产生
[5,7]
。从西洋参总皂苷的水解产物中分离得
到的原人参二醇型皂苷除人参皂苷compound K 外,
另外得到人参皂苷Mc ,但没有分离到原人参二醇型Rb2、Rc或Rd。HPLC 检测分析也证明水皂苷Rb1、
解产物中只存在原人参二醇型皂苷compound K 和Mc ,Rb2、Rc而不存在主要原人参二醇型皂苷Rb1、和Rd(图2B )。HPLC 检测分析证明西洋参总皂苷中并不存在人参皂苷compound K 和Mc (图2A ),这Rb2、Rc和Rd已经完全被该β-糖表明人参皂苷Rb1、苷酶催化水解,而人参皂苷Mc 不能进一步被催化水解产生人参皂苷compound K 或这种催化活性很低。
3或C-20原人参二醇型皂苷的糖基连接在C-Rb2、Rc和Rd的羟基上。原人参二醇型皂苷Rb1、
3的羟基上连接有糖基,在C-而水解产物人参皂苷compound K 和Mc 在C-3的羟基上都没有糖基,因
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Vol. 26刘韶等:大孔树脂分离纯化陈皮中多甲氧基黄酮类化合物1567
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