黑曲霉Aspergillus niger對(duì)人參皂苷Re的微生物轉(zhuǎn)化

1、黑曲霉Aspergillus niger對(duì)人參皂苷Re的微生物轉(zhuǎn)化    摘要 目的：篩選長(zhǎng)白山人參土壤中的活性微生物，轉(zhuǎn)化單體人參皂苷產(chǎn)生稀有人參皂苷成份。方法：從長(zhǎng)白山人參根際土壤中分離各類菌株，對(duì)單體人參皂苷Re進(jìn)行微生物轉(zhuǎn)化，通過硅膠柱層析等方法對(duì)轉(zhuǎn)化產(chǎn)物進(jìn)行分離純化，采用波譜解析及理化常數(shù)對(duì)其進(jìn)行結(jié)構(gòu)鑒定。結(jié)果：從長(zhǎng)白山人參根際土壤中分離各類真菌菌株68株，3株真菌對(duì)三醇組人參皂苷Re具有轉(zhuǎn)化作用，其中黑曲霉Aspergillus niger的轉(zhuǎn)化活性較強(qiáng)，轉(zhuǎn)化產(chǎn)物為人參皂苷Rg1、Rg2和Rh1。結(jié)論：首次報(bào)道黑曲霉能將人參皂苷Re轉(zhuǎn)化為人參

2、皂苷Rg1、Rg2和Rh1這一轉(zhuǎn)化過程。關(guān)鍵詞 人參皂苷Re；人參皂苷Rg1；人參皂苷Rg2；人參皂苷Rh1；黑曲霉；生物轉(zhuǎn)化中圖分類號(hào) R285 文獻(xiàn)標(biāo)識(shí)碼 A 文章編號(hào) 1674-4721（2011）11（c）-073Microbial transformation of Ginsenoside Re by Aspergillus nigerWU Xiuli1, LIU Cheng2, CHEN Jing1*1.School of Pharmacy, Ningxia Medical University, Ningxia Research Center of Modern Hui Medi

3、cine Engineering and Technology, Ningxia Hui Autonomous Region, Yinchuan 750004, China; 2.Science and Technology Center, Ningxia Medical University, Ningxia Hui Autonomous Region, Yinchuan 750004, ChinaAbstract Objective: Samples of Ginseng root soils, collect from Changbai Mountain, are used to scr

4、een active microorganism which can transform Ginsenoside Re, so as to obtain rare anti-tumor components. Methods: The strains were isolated and screened on liquid transfer medium and yield transfer process for active strains. Then the active strains were tested for their biotransformation properties

5、 by using Ginsenoside Re. The biotransformation products were separated and purified through different column chromatographies and identified by spectral analysis and physical constants. Results: Total 68 fungal strains were isolated and 3 active strains showed positive activity on Ginsenoside Re. O

6、ne strain, Aspergillus niger, was found to have the strong activity on Re. Conclusion: This is the first report on the transformation G-Re to G-Rg1, G-Rg2, and G-Rh1 by microorganism Aspergillus niger.Key words Ginsenoside Re; Ginsenoside Rg1; Ginsenoside Rg2; Ginsenoside Rh1; Aspergillus niger; Bio

7、transformation人參及其制品中的主要活性成分是人參皂苷，由于人參皂苷分子結(jié)構(gòu)中糖基側(cè)鏈的不同而顯示出不同的性質(zhì)和藥理活性1-2。例如人參皂苷Rb1有促進(jìn)神經(jīng)細(xì)胞生長(zhǎng)的作用、降低細(xì)胞內(nèi)鈣、抗氧化、清除自由基和改善心肌缺血再灌注損傷等作用3；人參皂苷Rd能促進(jìn)T細(xì)胞增殖，提高天然殺傷細(xì)胞（NK）的活性4；20（S）-原人參二醇苷元（PPD）具有抗癌活性5等。人參皂苷Re是抗心律失常有效成分，可抑制嗎啡誘發(fā)小鼠產(chǎn)生的耐藥性等作用；人參皂苷Rg1具有使中樞神經(jīng)興奮、抗疲勞、改善記憶、學(xué)習(xí)功能等作用6；人參皂苷Rg2可抑制兔血小板釋放反應(yīng)等作用。為了獲得具有極高藥用價(jià)值的稀有人參皂苷，從20

8、世紀(jì)80年代始，國(guó)內(nèi)外化學(xué)及生物技術(shù)工程研究人員便開始了人參皂苷的結(jié)構(gòu)改造工程，并取得了很好的結(jié)果7-13。因此，從高含量人參皂苷成分獲得稀有人參皂苷成分的工作備受關(guān)注。目前，用于人參皂苷糖基改造的主要方法有化學(xué)法、酶法和真菌代謝法14-15。真菌代謝法具有條件溫和、不破壞皂苷結(jié)構(gòu)、專屬性、得率高、無(wú)污染等特點(diǎn)，被廣泛應(yīng)用。1 材料與方法1.1 材料1.2 人參根際真菌的分離取長(zhǎng)白山2年和5年人參根際土壤樣品于4050烘23 h，取1 g烘干的土樣溶于10 ml無(wú)菌蒸餾水中，將土樣從10-1稀釋至10-6，選擇10-3、10-4、10-5、10-6 4個(gè)濃度，分別取0.1 ml用無(wú)菌三角爬把溶

9、液均勻涂布在含有慶大霉素（50 g/ml）的PDA平板培養(yǎng)基表面，25倒置培養(yǎng)7 d。挑取單一菌落經(jīng)反復(fù)劃線分離得純培養(yǎng)，傳至PDA斜面培養(yǎng)7 d，4保藏備用。1.3 活性菌株的篩選液體篩選方法參見文獻(xiàn)17，取上清進(jìn)行TLC分析。在氯仿甲醇（3:1）系統(tǒng)中展開后，揮干溶劑，噴10%（V/V）H2SO4-EtOH于105下顯色，與人參皂苷Re所顯示的斑點(diǎn)比較，獲得液體篩選的陽(yáng)性菌株，進(jìn)行放大實(shí)驗(yàn)。1.4 人參皂苷Re轉(zhuǎn)化產(chǎn)物的粗處理將具有轉(zhuǎn)化作用的真菌，分別進(jìn)行液體發(fā)酵轉(zhuǎn)化人參皂苷Re，轉(zhuǎn)化液分別進(jìn)行如下純化過程。離心，上清液用水飽和的正丁醇分別萃取3次，合并正丁醇萃取部分，濃縮得提取物A。菌絲

10、體用甲醇提取1次，回收甲醇得提取物B，合并A、B兩部分提取物。水溶解，經(jīng)HPD100型大孔吸附樹脂吸附，蒸餾水洗滌去除雜質(zhì)，再用95%乙醇洗脫，收集95%乙醇洗脫液，濃縮。2 結(jié)果2.1 活性菌株的篩選從長(zhǎng)白山的2年和5年人參根際土壤樣品中共分離獲得各種類型真菌68株，通過液體篩選方法，發(fā)現(xiàn)具有轉(zhuǎn)化人參皂苷Re的活性菌株3株，分別記為SRS-67、SRS-68和SRS-69（圖1）。其中SRS-69（黑曲霉）對(duì)人參皂苷Re有較強(qiáng)的轉(zhuǎn)化活性，另外兩株真菌菌株SRS-67和SRS-68正在鑒定中。2.2 人參皂苷Re的轉(zhuǎn)化產(chǎn)物的分離純化真菌SRS-67和SRS-68的轉(zhuǎn)化濃縮液，經(jīng)PTLC，重結(jié)晶

11、純化，分別得到單體化合物1及2。SRS-69部分的濃縮液0.19 g，甲醇溶解，硅膠（0.5 g）拌樣，進(jìn)行硅膠柱色譜進(jìn)行分離（15 g），洗脫系統(tǒng)為CHCl3-MeOH （1001, 1002, 1003, 1004, 100:5, 1007, 1009, 10011， 10013, 0.8 L each solvent）得到3個(gè)部分（F1-F3）。F3 組分經(jīng)C18硅膠柱色譜，用不同比例甲醇-水洗脫，得到兩個(gè)部分F3-1和F3-2。F3-1組分繼續(xù)經(jīng)反向HPLC分離得到化合物3（18.2 mg）；F3-2部分反復(fù)重結(jié)晶（甲醇）得到化合物4（24.5 mg）；F2組分通過制備TLC色譜，氯仿

12、甲醇10025作為展開劑，分離得到化合物5（12.9 mg）。2.3 人參皂苷Re的轉(zhuǎn)化產(chǎn)物的結(jié)構(gòu)鑒定SRS-67、SRS-68和黑曲霉的轉(zhuǎn)化產(chǎn)物1-3經(jīng)TLC和HNM數(shù)據(jù)分析，鑒定其為同一化合物。3 討論根據(jù)轉(zhuǎn)化底物和產(chǎn)物的結(jié)構(gòu)特征推測(cè)其轉(zhuǎn)化途徑：真菌SRS-67和SRS-68的轉(zhuǎn)化機(jī)制為產(chǎn)生-鼠李糖苷水解酶，水解掉人參皂苷Re的-鼠李糖而生成人參皂苷Rg1，如圖2所示；黑曲霉將人參皂苷Re轉(zhuǎn)化為 G-Rg1, G-Rg2和G-Rh1，說(shuō)明黑曲霉不但產(chǎn)生-鼠李糖苷酶活性，水解G-Re和G-Rg2的C-6 位O-葡萄糖（21）-鼠李糖苷鍵連接，而且產(chǎn)生-葡萄糖苷水解酶，該酶立體選擇性水解C-2

13、0位葡萄糖，而對(duì)于C-6位葡萄糖并不起作用，因此僅產(chǎn)生人參皂苷Rh1而沒有得到人參皂苷F1，轉(zhuǎn)化過程如圖2所示。這一轉(zhuǎn)化途徑與其他生物體系如小鼠的胃、大腸或者內(nèi)生菌的轉(zhuǎn)化過程不同，為首次報(bào)道。圖2 人參皂苷Re 可能的轉(zhuǎn)化途徑參考文獻(xiàn)1 Chen X, Zhou QL, Wang BX.The metabolism of ginsenoside Rbl by intestinal Bacteda J. Acta Pharm. Sin,1999,34(6):410-414.2 莊毅.菌質(zhì)中藥的一個(gè)新領(lǐng)域J.中藥新藥與臨床藥理(Traditional Chinese Drug Research C

14、linical Pharmacology),1992,3(2):49-51.3 Rudakewich M, Ba F, Benishin CG. Neurotrophic and neuroprotective actions of ginsenoside Rb1 and Rg1J. Planta Medica,2001,67(6):533-537.4 李泰平.人參皂苷藥理活性的研究進(jìn)展M.生物學(xué)教學(xué)(Biology Teaching),2003,28(4):34-37.5 Takahide O, Masayo M, Shizuo O. Mechanism of action of ginse

15、noside Rh2: uptake and metabolism of ginsenoside Rh2 by cultured B16 melanoma cells J. J Pharm Science,1991,80(12):1141-1146.7 Zhang C,Yu H,Bao Y,et al. Purification and characterization of ginsenoside-, 3-ginsenosidase from ginsengJ.Chem Pharm Bull,2001,49(7):795-798.8 Ko SR, Choi KJ, Suzuki K, et

16、al. Enzymatic preparation of ginsenosides Rg2, Rh1, and F1J.Chem Pharm Bull,2003,51(4):404-408.9 Dong AL, Ye M, Guo HZ, et al. Microbial transformation of ginsenosides Rb1 by Rhizopus stolonifer and Curvularia lunataJ. Biotechnol Lett,2003,25(4):339-344.10 Hyun C, Geun-Eog.Transformation of ginsenos

17、ides Rg1 and Re from Panax ginseng by food microorganismsJ.Biotechnol Lett,2005,27(11):765-771.11 Cheng LQ, Kim MK, Lee JW, et al. Conversion major ginsenoside Rb1 to ginsenoside F2 by Caulobacter leidyiaJ.Biotechnol Lett,2006,28 (14): 1121-1127.12 Chen GT, Yang M, Lu ZQ, et al. Microbial transforma

18、tion of 20(S)-protopanaxatriol-type saponins by Absidia coeruleaJ.J Nat Prod,2007,70(70):1203-1206.13 Cheng LQ, Na JR, Bang MH,et al.Conversion of major ginsenosides Rb1 to 20(S)-ginsenoside Rg3 by Microbacterium sp. GS514J.Phytochemistry,2008,69(1):218-224.14 Akao T, Kanaoka M. Appearance of compou

19、nd K, a major metabolite of ginseng Rb1 by intestinal bacteria, in rat plasma after oral administration-measurement of compound K by enzyme immunoassayJ.Biol Pharm Bull,1998,21(3):245-249.15 Chen YG, Lü YP, Gui SH, et al. Preparation of protopanaxadiol and its epimer 20(R)-protopanaxadiol from leaf saponins of Panax notoginsengJ.Fine Chem,2003,20(7):425-430.16 Pitt JI.A Laboratory Guide to Common Penicillium SpeciesM.Australia: CSIRO Division of Food Research,1985:64-65.17 Dong AL,Cui YJ, Guo HZ,et al. Microbiological Transfor