金建明,李有海,張海倫,梁朝寧,唐雙焱
1北京工商大學(xué)植物資源研究開發(fā)北京市重點實驗室,北京 100048;2云南民族大學(xué)民族藥資源化學(xué)國家民委-教育部重點實驗室,昆明 650500;3中國科學(xué)院微生物研究所 中國科學(xué)院微生物生理與代謝工程重點實驗室,北京 100101
西洋參(Panax quinquefolium L.)又名花旗參、西洋人參、洋參、美國人參、廣東人參等,是我國傳統(tǒng)的名貴中藥材,為五加科人參屬植物,原產(chǎn)北美洲的加拿大南部和美國北部,現(xiàn)我國的許多地區(qū)都有引種栽培。其味甘、微苦、性寒,可滋陰降火,益氣生津,其藥用價值有鎮(zhèn)靜鎮(zhèn)驚、安神促智、增強機體免疫、抗心律失常、防血管硬塞、保肝、降血脂等功效。對西洋參的化學(xué)成分已經(jīng)有許多研究報道,其主要活性成分是人參皂苷。西洋參總皂苷中絕大多數(shù)屬于達瑪烷四環(huán)三萜類人參皂苷[1,2]。達瑪烷四環(huán)三萜類人參皂苷根據(jù)苷元6 位碳上是否有羥基分為原人參二醇型皂苷如人參皂苷Rb1、Rb2、Rc 和Rd 等(圖1)和原人參三醇型皂苷如人參皂苷Rg1、Rg2、Re 和F1等。
稀有人參皂苷compound K 也稱人參皂苷CK或人參皂苷C-K,其結(jié)構(gòu)為20(S)-原人參二醇-20-O-β-D-吡喃葡萄糖苷(圖1),屬原人參二醇型皂苷。人參皂苷compound K 在人參屬植物中幾乎不存在,首次由土壤細菌降解人參皂苷Rbl、Rb2和Rc 后被發(fā)現(xiàn)并鑒定[3]。研究發(fā)現(xiàn)人參皂苷compound K 是人參等藥材中原人參二醇型皂苷經(jīng)腸道菌降解后產(chǎn)生的在人體內(nèi)發(fā)揮藥效作用的真正代謝活性物[4]。人參皂苷compound K 的生物活性具有多靶點、高活性和低毒性的特點,具有極高的生產(chǎn)價值和應(yīng)用前景,醫(yī)藥價值很高[5,6]。目前制備人參皂苷compound K 的方法多采用微生物發(fā)酵法和酶解法。利用酶解法制備人參皂苷compound K 具有反應(yīng)條件溫和、操作簡便、成本低、產(chǎn)物便于純化等優(yōu)點[5,7]。
圖1 原人參二醇型皂苷結(jié)構(gòu)Fig.1 Chemical structures of protopanaxdiol-type ginsenosides
項目組前期得到了高效催化人參皂苷Rb1產(chǎn)生人參皂苷compound K 的β-糖苷酶。為了研究該β-糖苷酶是否也催化其他原人參二醇型皂苷產(chǎn)生人參皂苷compound K,實驗采用原人參二醇型皂苷含量高的西洋參總皂苷,詳細分析了西洋參總皂苷經(jīng)該β-糖苷酶完全催化水解后的成分組成,為將來進一步采用西洋參總皂苷等高效制備人參皂苷compound K 奠定基礎(chǔ)。
Bruker Avance 400 MHz 型核磁共振波譜儀;Agilent 6520 LC/MS 質(zhì)譜儀;島津Lc-20A 高效液相色譜儀。柱層析硅膠(60~100 目)和薄層層析硅膠板H(青島海洋化工有限公司);RP-C18 反相層析材料和RP-C18 高效薄層板(Merck 公司);乙腈為色譜純,其余試劑均為分析純;西洋參總皂苷購自南京澤朗醫(yī)藥生物科技有限公司。標準品人參皂苷Rb1(批號20110526)、Rb2(批號20120311)、Rc(批號20120617)和Rd(批號20120621)購自寶雞辰光生物科技有限公司。
將β-糖苷酶LacS 突變株接種到LB 培養(yǎng)基中(含100 mmol/L 卡那霉素),接種量1%,37 ℃、280 rpm 搖床培養(yǎng)12 h 后將種子液轉(zhuǎn)接到600 mL 誘導(dǎo)培養(yǎng)基中(含100 mmol/L 卡那霉素),接種量1%,30 ℃、280 rpm 搖床培養(yǎng)21 h。培養(yǎng)液10000 rpm 離心10 min,沉淀加入60 mL Mc 緩沖液重懸(0.2 mol/L 磷酸氫二鈉和0.1 mol/L 檸檬酸,pH=5.5),超聲破細胞后12000 rpm 離心10 min,取上清液于60 ℃水浴中靜置10 min 后再次12000 rpm 離心10 min,上清液即β-糖苷酶液[8]。
約60 mL 酶液加入到75 mL 含7.5 g 西洋參總皂苷的Mc 緩沖液中,水浴恒溫70 ℃反應(yīng)過夜使酶催化水解反應(yīng)完全(19 h)[8]。
135 mL 水解反應(yīng)液用等體積正丁醇萃取三次,合并萃取液,減壓濃縮干燥后進行硅膠柱層析,用二氯甲烷甲醇進行梯度洗脫,分為五個部分(Fr I~Fr V)。各部分再經(jīng)RP-C18 柱層析,采用甲醇水進行梯度洗脫純化各化合物。Fr I 部分1.3 g 經(jīng)50%~80%甲醇水梯度洗脫純化得到化合物1(0.807 g);Fr II 部分0.2 g 用50%~60%甲醇水梯度洗脫得到化合物6(0.049 g);Fr III 部分0.6 g 經(jīng)60%~75%甲醇水梯度洗脫得到化合物2(0.311 g);Fr IV 部分0.58 g 用20%~75%甲醇水梯度洗脫得到化合物3(0.152 g)、7(0.070 g)和4(0.046 g)。Fr V 部分1.036 g 用10%~50%甲醇水梯度洗脫得到化合物5(0.481 g)。
西洋參總皂苷及其水解產(chǎn)物的HPLC 方法在參考文獻的基礎(chǔ)上略有修改[9]。西洋參總皂苷及其水解產(chǎn)物配制成3 mg/mL。HPLC 條件如下:色譜柱:Waters symmetry C18色譜柱(4.6 × 250 mm,5 μm);流動相:乙腈(A)和水(B);二元高壓梯度洗脫:0~2.5 min,20% A;2.5~5 min,20%~25% A;5~10 min,25% A;10~30 min,25%~40% A;30~45 min,40%~70% A;45~60 min,70% A;流速:0.7 mL/min;檢測波長:203 nm;柱溫:35 ℃;進樣量:10 μL。
為了研究β-糖苷酶催化總皂苷后水解產(chǎn)物的成分組成及原人參二醇型皂苷生成人參皂苷compound K 的效率,將西洋參總皂苷經(jīng)β-糖苷酶催化水解過夜(19 h),水解產(chǎn)物經(jīng)TLC 檢測初步證明原人參二醇型皂苷Rb1已經(jīng)完全被水解。HPLC 檢測分析再次確定西洋參總皂苷中的原人參二醇型皂苷人參皂苷Rb1(8)、Rc(9)、Rb2(10)和Rd(11)已經(jīng)完全水解(圖2B)。進一步延長水解反應(yīng)時間后的水解產(chǎn)物經(jīng)HPLC 檢測分析皂苷的成分和含量沒有變化,表明19 h 水解反應(yīng)已經(jīng)完全。水解產(chǎn)物中主要有二個原人參二醇型皂苷:人參皂苷compound K(1)和人參皂苷Mc(2);四個原人參三醇型皂苷:人參皂苷Rg1(3)、人參皂苷Rg2(4)、人參皂苷Re(5)和人參皂苷F1(6);以及擬人參皂苷F11(7)。其中人參皂苷compound K 是西洋參總皂苷水解產(chǎn)物中含量最高的成分。
圖2 西洋參總皂苷水解前(A)和水解后(B)以及人參皂苷compound K(C)的HPLC 色譜圖Fig.2 HPLC chromatograms of total ginsenoside extract of P.quinquefolium before (A)and after (B)hydrolysis as well as ginsenoside compound K standard (C)
化合物1 白色粉末,易溶于甲醇。HR-ESI-MS m/z 645.4342 [M+Na]+(calcd for C36H62NaO8,645.4337);1H NMR (400 MHz,pyridine-d5)δH:5.19 (1H,d,J=7.6 Hz,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 NMR δC: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-20),22.4 (C-21),36.2 (C-22),23.3 (C-23),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)。以上數(shù)據(jù)和文獻報道的基本一致[10],故化合物1 鑒定為人參皂苷compound K,即20(S)-原人參二醇-20-O-β-D-吡喃葡萄糖苷。
化合物2 白色粉末,易溶于甲醇。1H NMR(400 MHz,pyridine-d5)δH:5.67 (1H,s,Araf-H-1),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),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 NMR δC: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)。以上數(shù)據(jù)和文獻報道的基本一致[11],故化合物2 鑒定為人參皂苷Mc,即20(S)-原人參二醇-20-O-α-L-呋喃阿拉伯糖基-(1→6)-β-D-吡喃葡萄糖苷(圖1)。
化合物3 白色粉末,易溶于甲醇。1H NMR(400 MHz,pyridine-d5)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 NMR δC:39.7 (C-1),27.9 (C-2),78.8 (C-3),40.4(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-15),26.6 (C-16),51.4 (C-17),17.6 (C-18),17.8(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)。以上數(shù)據(jù)和文獻報道的基本一致[12],故化合物3 鑒定為人參皂苷Rg1,即6-O-β-D-吡喃葡萄糖基-20(S)-原人參三醇-20-O-β-D-吡喃葡萄糖苷。
化合物4 白色粉末,易溶于甲醇。1H NMR(400 MHz,pyridine-d5)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 NMR δC: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-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),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-6-Glc-1),79.4 (C-6-Glc-2),78.5 (C-6-Glc-3),72.5(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)。以上數(shù)據(jù)和文獻報道的基本一致[13],故化合物4 鑒定為人參皂苷Rg2,即20(S)-原人參三醇-6-O-α-L-吡喃鼠李糖基-(1→2)-β-D-吡喃葡萄糖苷。
化合物5 白色粉末,易溶于甲醇。1H NMR(400 MHz,pyridine-d5)δH:6.47 (1H,s,Rha-H-1),5.24 (1H,d,J=6.4Hz,6-Glc-H-1),5.15 (1H,d,J=8.0 Hz,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(3H,s,H-18),0.96 (3H,s,H-19),0.94 (3H,s,H-30);13C NMR δC: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)。以上數(shù)據(jù)和文獻報道的基本一致[14],故化合物5 鑒定為人參皂苷Re,即20-O-β-D-吡喃葡萄糖基-20(S)-原人參三醇-6-O-α-L-吡喃鼠李糖基-(1→2)-β-D-吡喃葡萄糖苷。
化合物6 白色粉末,易溶于甲醇。1H NMR(400 MHz,pyridine-d5)δH:5.21 (1H,d,J=6.8 Hz,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 NMR δC: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)。以上數(shù)據(jù)和文獻報道的基本一致[15],故化合物6 鑒定為人參皂苷F1,即20(S)-原人參三醇-20-O-β-D-吡喃葡萄糖苷。
化合物7 白色粉末,易溶于甲醇。1H NMR(400 MHz,pyridine-d5)δH:6.48 (1H,s,Rha-H-1),5.25 (1H,d,J=6.8 Hz,Glc-H-1),2.10 (3H,s,H-28),1.78 (3H,d,J=6.4 Hz,Rha-H-6),1.46 (3H,s,H-27),1.33 (3H,s,H-29),1.26 (3H,s,H-21),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 NMR δC: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-6-Glc-1),79.4 (C-6-Glc-2),78.6 (C-6-Glc-3),72.6(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)。以上數(shù)據(jù)和文獻報道的基本一致[16],故化合物7 鑒定為擬人參皂苷F11,即達瑪-3β,6α,12β,25-四羥基-(20S,24R)-環(huán)氧-6-O-α-L-吡喃鼠李糖基-(1→2)-β-D-吡喃葡萄糖苷。
HPLC 檢測分析結(jié)果表明實驗用的西洋參總皂苷中人參皂苷Rb1是含量最高的原人參二醇型皂苷成分,其次是人參皂苷Rd、Rc 和Rb2(圖2A)。文獻也報道在西洋參總皂苷中,人參皂苷Rb1、Rd、Rc和Rb2是其主要原人參二醇型皂苷成分,且人參皂苷Rb1是其中含量最高的原人參二醇型皂苷[1,2,9,17]。研究已表明人參皂苷compound K 可由原人參二醇型皂苷Rb1、Rb2、Rc 和Rd 等通過酶水解而產(chǎn)生[5,7]。從西洋參總皂苷的水解產(chǎn)物中分離得到的原人參二醇型皂苷除人參皂苷compound K 外,另外得到人參皂苷Mc,但沒有分離到原人參二醇型皂苷Rb1、Rb2、Rc 或Rd。HPLC 檢測分析也證明水解產(chǎn)物中只存在原人參二醇型皂苷compound K 和Mc,而不存在主要原人參二醇型皂苷Rb1、Rb2、Rc和Rd(圖2B)。HPLC 檢測分析證明西洋參總皂苷中并不存在人參皂苷compound K 和Mc(圖2A),這表明人參皂苷Rb1、Rb2、Rc 和Rd 已經(jīng)完全被該β-糖苷酶催化水解,而人參皂苷Mc 不能進一步被催化水解產(chǎn)生人參皂苷compound K 或這種催化活性很低。
原人參二醇型皂苷的糖基連接在C-3 或C-20的羥基上。原人參二醇型皂苷Rb1、Rb2、Rc 和Rd在C-3 的羥基上連接有糖基,而水解產(chǎn)物人參皂苷compound K 和Mc 在C-3 的羥基上都沒有糖基,因此推斷該β-糖苷酶能將西洋參中原人參二醇型皂苷的C-3 的羥基的糖基全部水解。
人參皂苷Rc 和Rb2的分子量相同而且結(jié)構(gòu)也很相似,區(qū)別在于連接在苷元C-20 的糖基不同:人參皂苷Rc 是一個呋喃阿拉伯糖基連接在C-20 的葡萄糖基的6 位羥基上,而人參皂苷Rb2是一個吡喃阿拉伯糖基連接在C-20 的葡萄糖基的6 位羥基上(圖1)。人參皂苷Rc 經(jīng)該β-糖苷酶水解可產(chǎn)生人參皂苷Mc,如進一步水解可產(chǎn)生人參皂苷compound K;人參皂苷Rb2經(jīng)該β-糖苷酶水解可產(chǎn)生人參皂苷compound K,但不可能產(chǎn)生人參皂苷Mc。由于從水解產(chǎn)物中只分離到人參皂苷compound K和Mc,表明該β-糖苷酶能水解C-20 的葡萄糖基上的吡喃阿拉伯糖基,但不能水解C-20 的葡萄糖基上的呋喃阿拉伯糖基或水解呋喃阿拉伯糖基的活性很低。
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