Ginsenosides, the main pharmacologically active natural compounds in ginseng （Panax ginseng）, are mostly the glycosylated products of protopanaxadiol （PPD） and protopanaxatriol （PPT）. No uridine diphosphate glyc...Ginsenosides, the main pharmacologically active natural compounds in ginseng （Panax ginseng）, are mostly the glycosylated products of protopanaxadiol （PPD） and protopanaxatriol （PPT）. No uridine diphosphate glycosyltransferase （UGT）, which catalyzes PPT to produce PPT-type ginsenosides, has yet been reported. Here, we show that UGTPgl, which has been demonstrated to regio-specifically glycosylate the C20-OH of PPD, also specifically glycosylates the C20-OH of PPT to produce bioactive ginsenoside FI. We report the characterization of four novel UGT genes isolated from P. ginseng, sharing high deduced amino acid identity （〉84%） with UGTPgl. We demonstrate that UGTPgl00 specifically glycosylates the C6-OH of PPT to produce bioactive ginsenoside Rhl, and UGTPgl01 catalyzes PPT to produce F1, followed by the generation of ginsenoside Rgl from FI. However, UGTPgl02 and UGTPgl03 were found to have no detectable activity on PPT. Through structural modeling and site-directed mutagenesis, we identified several key amino acids of these UGTs that may play important roles in determining their activities and substrate regio-specificities. Moreover, we constructed yeast recombinants to biosynthesize F1 and Rhl by introducing the genetically engineered PPT-producing pathway and UGTPgl or UGTPgl00. Our study reveals the possible biosynthetic pathways of PPT-type ginsenosides in Panax plants, and provides a sound manufacturing approach for bioactive PPT-type ginsenosides in yeast via synthetic biology strategies.展开更多
采用硅胶柱层析法分离人参叶三醇皂苷,以氯仿-甲醇为流动相洗脱,得到了纯度较高的人参皂苷Re。结果表明,利用硅胶柱层析柱从35 g叶三醇皂苷中分离得到人参皂苷Re7.36 g,得率为21.03%。采用重结晶法对经硅胶柱分离制备的5 g Re进行提纯,...采用硅胶柱层析法分离人参叶三醇皂苷,以氯仿-甲醇为流动相洗脱,得到了纯度较高的人参皂苷Re。结果表明,利用硅胶柱层析柱从35 g叶三醇皂苷中分离得到人参皂苷Re7.36 g,得率为21.03%。采用重结晶法对经硅胶柱分离制备的5 g Re进行提纯,将结晶产品分开收集,第2次结晶效果最好,得产品2.43 g,得率为48.68%,纯度为98.89%。展开更多
[Objectives]To detect the protective effects of six protopanaxatriols(PPTs)on hypoxia/reoxygenation(H/R)induced cardiomyocyte injury by different treatments.[Methods]The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetra...[Objectives]To detect the protective effects of six protopanaxatriols(PPTs)on hypoxia/reoxygenation(H/R)induced cardiomyocyte injury by different treatments.[Methods]The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide(MTT)assay was used for detecting the protective effects of six PPTs including ginsenoside Rg1,Re,Rf,Rg2,(R)Rh1 and(S)Rh1 on cell viability reduced by H/R in different treatments.And the adenosine triphosphate(ATP)content and mitochondrial membrane potential(MMP)were used for detecting the mitochondrial function change during PPTs treatment.[Results]Among six PPTs,ginsenoside Rg1,Re,Rf,Rg2 and(R)Rh1 at the concentration of 12.5μM significantly increased the cell survival when treated before and during H/R.These five PPTs also significantly increased the ATP content and MMP reduced by H/R in the same manner.In comparison,only Rg1 significantly increased the cell viability compared with H/R group by pretreating and treating the cells during hypoxia process.[Conclusions]Different treatments affect the protective effects of PPTs.When treated before and during H/R,ginsenoside Rg1,Re,Rf,Rg2 and(R)Rh1 protect the cardiomyocyte against H/R injury mitochondrial function,and only ginsenoside Rg1 has protective effects when treated before and during hypoxia process.展开更多
Five minor compounds isolated from the leaves of Panax ginseng C. A. Meyer were characterized as 20（R）-protopanaxatriol （1）, daucosterin （2）, 3β, 12β-dihydroxy-dammar-20 （22）, 24-diene-3-O-β-D-glucopyranosi...Five minor compounds isolated from the leaves of Panax ginseng C. A. Meyer were characterized as 20（R）-protopanaxatriol （1）, daucosterin （2）, 3β, 12β-dihydroxy-dammar-20 （22）, 24-diene-3-O-β-D-glucopyranoside （3）, 20 （R）-protopanaxadiol-3-O-β-D-glucopyranoside （4） and ginsenoside-Rh2 （5）, respectively, on the basis of spectral analyses and chemical evidence. The two new saponins, 3 and 4, were named as ginsenoside-Rh3 and 20（R）-ginsenoside-Rh2.Nine other major saponins obtained simultaneously were identical with ginsenoside-Rh1（6）,-Rg3 （7）, -Rg2 （8）, -Rg1 （9）,-Re（10）,-Rd （11）, -Rc （12）, -Rb2（13） and Rb1 （14）, respectively.展开更多
文摘Ginsenosides, the main pharmacologically active natural compounds in ginseng （Panax ginseng）, are mostly the glycosylated products of protopanaxadiol （PPD） and protopanaxatriol （PPT）. No uridine diphosphate glycosyltransferase （UGT）, which catalyzes PPT to produce PPT-type ginsenosides, has yet been reported. Here, we show that UGTPgl, which has been demonstrated to regio-specifically glycosylate the C20-OH of PPD, also specifically glycosylates the C20-OH of PPT to produce bioactive ginsenoside FI. We report the characterization of four novel UGT genes isolated from P. ginseng, sharing high deduced amino acid identity （〉84%） with UGTPgl. We demonstrate that UGTPgl00 specifically glycosylates the C6-OH of PPT to produce bioactive ginsenoside Rhl, and UGTPgl01 catalyzes PPT to produce F1, followed by the generation of ginsenoside Rgl from FI. However, UGTPgl02 and UGTPgl03 were found to have no detectable activity on PPT. Through structural modeling and site-directed mutagenesis, we identified several key amino acids of these UGTs that may play important roles in determining their activities and substrate regio-specificities. Moreover, we constructed yeast recombinants to biosynthesize F1 and Rhl by introducing the genetically engineered PPT-producing pathway and UGTPgl or UGTPgl00. Our study reveals the possible biosynthetic pathways of PPT-type ginsenosides in Panax plants, and provides a sound manufacturing approach for bioactive PPT-type ginsenosides in yeast via synthetic biology strategies.
基金Supported by the National Natural Science Foundation of China (No. 81603342)the Guangdong Provincial Key Laboratory of Traditional Chinese Medicine Informatization (No. 2021B1212040007)+5 种基金the Guangdong Basic and Applied Basic Re-search Foundation (No. 2022A1515012641No. 2024A1515012948)the Guangdong Provincial Bureau of Traditional Chinese Medi-cine Research Project (No. 20221107)the Guangzhou Science and Technology Projects (No. 2024A03J0154No. 2023B01J1004)the Foshan “Summit Plan” of Building High-Level Hospitals。
基金Supported by General Colleges and Universities Youth Innovative Talents Project of Guangdong Province(2019GKQNCX134)Guangdong Doctoral Workstation Funds。
文摘[Objectives]To detect the protective effects of six protopanaxatriols(PPTs)on hypoxia/reoxygenation(H/R)induced cardiomyocyte injury by different treatments.[Methods]The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide(MTT)assay was used for detecting the protective effects of six PPTs including ginsenoside Rg1,Re,Rf,Rg2,(R)Rh1 and(S)Rh1 on cell viability reduced by H/R in different treatments.And the adenosine triphosphate(ATP)content and mitochondrial membrane potential(MMP)were used for detecting the mitochondrial function change during PPTs treatment.[Results]Among six PPTs,ginsenoside Rg1,Re,Rf,Rg2 and(R)Rh1 at the concentration of 12.5μM significantly increased the cell survival when treated before and during H/R.These five PPTs also significantly increased the ATP content and MMP reduced by H/R in the same manner.In comparison,only Rg1 significantly increased the cell viability compared with H/R group by pretreating and treating the cells during hypoxia process.[Conclusions]Different treatments affect the protective effects of PPTs.When treated before and during H/R,ginsenoside Rg1,Re,Rf,Rg2 and(R)Rh1 protect the cardiomyocyte against H/R injury mitochondrial function,and only ginsenoside Rg1 has protective effects when treated before and during hypoxia process.
文摘Five minor compounds isolated from the leaves of Panax ginseng C. A. Meyer were characterized as 20（R）-protopanaxatriol （1）, daucosterin （2）, 3β, 12β-dihydroxy-dammar-20 （22）, 24-diene-3-O-β-D-glucopyranoside （3）, 20 （R）-protopanaxadiol-3-O-β-D-glucopyranoside （4） and ginsenoside-Rh2 （5）, respectively, on the basis of spectral analyses and chemical evidence. The two new saponins, 3 and 4, were named as ginsenoside-Rh3 and 20（R）-ginsenoside-Rh2.Nine other major saponins obtained simultaneously were identical with ginsenoside-Rh1（6）,-Rg3 （7）, -Rg2 （8）, -Rg1 （9）,-Re（10）,-Rd （11）, -Rc （12）, -Rb2（13） and Rb1 （14）, respectively.
