稀有人参皂苷的酶转化研究进展

人参皂苷是五加科人参属植物的主要活性成分之一。研究表明,人参皂苷经过体内代谢后生成的稀有皂苷在抗肿瘤、抗炎、抗衰老等方面的药理作用要强于原型皂苷,因此,稀有皂苷的制备成为目前人参研究领域的热点内容。人参皂苷的酶转化是稀有人参皂苷制备的主要途径之一,近些年取得了突破性的进展,以不同种类的糖苷酶为切入点,将相关研究内容进行了综述,分别讨论了不同类型的糖苷酶在稀有皂苷生物转化过程中的应用,以期为稀有皂苷的大规模开发利用奠定基础。     

一种真菌对人参皂苷Rg3的转化

[目的]筛选长白山人参土壤中的活性微生物,转化人参总皂苷及单体人参皂苷产生稀有抗肿瘤成份.[方法]从长白山人参根际土壤中分离各类菌株,对人参总皂苷及单体人参皂苷进行微生物转化,并通过硅胶柱层析等方法对转化产物进行分离纯化,采用波谱解析及理化常数对其进行结构鉴定;结合菌落形态、产孢结构、孢子形态特征以及菌株ITS rDNA核酸序列分析,对活性菌株进行鉴定.[结果]从长白山人参根际土壤中分离各类真菌菌株68株,有12株菌株对人参总皂苷有转化活性,其中菌株SYP2353对二醇组人参皂苷Rg3具有较强的转化活性.[结论]阳性菌株SYP2353被鉴定为疣孢漆斑菌(Myrothecium verrucaria),能将人参皂苷Rg3转化为稀有人参皂苷Rh2及二醇组人参皂苷苷元PPD,为稀有人参皂苷Rh2的制备提供了新的方法.     

真菌β-葡萄糖苷酶在转化人参皂苷单体中的应用进展

人参皂苷是一类具有抗疲劳及提高免疫力等功能的固醇类化合物,其中含量极少的稀有人参皂苷Rg_3、Rh_2等还具有抗癌的功效,是主要活性成分,拥有广阔的应用前景。研究发现真菌可以产生能够水解人参皂苷糖基的β-葡萄糖苷酶,可以有效水解人参皂苷的糖基,将大量的常见皂苷转化为稀有皂苷,是大量获得稀有人参皂苷的新途径。本文对人参皂苷合成途径、糖基分布及数量与抗肿瘤的效果、β-葡萄糖苷酶的性质及其催化人参皂苷单体转换的规律进行了综述。相信随着现代分子生物学技术和酶工程的发展,工业上大规模获得稀有人参皂苷将有望实现。     

人参皂苷单体定向转化的生物催化及应用进展

人参是我国传统中药,药效显著、应用广泛。通过定向修饰与转化人参皂苷糖基可产生高抗癌活性稀有人参皂苷。传统化学法由于制备工艺极其复杂、成本过高,不能应用于临床,微生物及其酶系转化成为解决该瓶颈问题的最可行手段。有关全细胞催化、糖苷酶重组表达、固定化及其催化分子识别机制和溶剂工程的生物转化已有大量综述报道,但尚无在人参皂苷转化应用中的系统研究。文中通过对人参皂苷单体生物转化理论和应用研究最新进展的回顾,结合目前广泛采用的生物催化方法的讨论,系统梳理归纳了能够改善产物专一性、提高催化效率,且具有工业应用前景的人参皂苷单体定向转化方法。基于酶分子设计以及离子液体溶剂工程,对人参皂苷单体抗癌药物和食品、保健品市场的开发、规模化制备进行了展望。     

人参皂苷等萜类化合物生物合成途径及HMGR的研究进展

人参皂苷是人参的主要有效成分之一,属典型的萜类化合物。本文对萜类生物合成途径及HMG-CoA还原酶进行了综述。人参皂苷等萜类生物合成分为甲羟戊酸和丙酮酸两种途径,两者都是以异戊烯基焦磷酸为主要的中间产物。大量研究资料表明HMG-CoA还原酶是甲羟戊酸途径的第一个限速关键酶,这对人参皂苷生物合成途径及其调控的深入研究具有一定的参考价值。     

糖苷酶转化人参皂苷研究进展

人参皂苷为人参主要的药理活性组成部分,通过水解二醇系人参皂苷的糖苷配基是制备稀有人参皂的常用方法。酶法转化因其底物高度专一、条件温和、副产物少等潜在优势而被作为结构修饰和生理研究的主要技术手段。本文主要对糖苷酶转化人参皂苷研究进展进行了综述,为其工业化生产高活性皂苷提供理论依据。     

甾体皂苷生物转化研究进展

甾体皂苷是中药中一类较为复杂的糖苷类化合物,具有多方面的药理活性。生物转化是利用各种酶系或微生物对天然活性化合物进行生物合成与结构修饰。利用生物转化技术可以对甾体皂苷类化合物完成化学法难以进行的结构改造和修饰,从而获得具有更高药用价值的目标化合物。本文对近几年利用微生物和酶法转化对甾体皂苷结构修饰的研究进展进行了综述,并分析了甾体皂苷生物转化研究中存在的问题,展望了其研究的前景。     

人参皂苷生物合成和次生代谢工程

人参皂苷属于植物三萜皂苷类化合物,是传统名贵药材人参和西洋参的主要活性成分,具有抗炎、抗氧化作用,还有广泛的抗肿瘤作用。人参皂苷与植物甾醇共享前期代谢途径,通过2, 3-氧化鲨烯环化步骤进入三萜代谢分支途径,在三萜碳环骨架复杂修饰的基础上形成人参皂苷。综述了近年人参皂苷生物合成途径及关键酶基因研究的最新进展,揭示了人参皂苷生物合成的基本途径,对途径中关键酶的基因进行了综述,并结合次生代谢工程技术, 探讨了该技术在人参皂苷生物合成中的应用前景。     

稀有人参皂苷IH901在体内外的生物转化及其生物活性

人参皂苷IH901是近年人参代谢组学研究中新发现的一种稀有人参皂苷。IH901在天然人参中并不存在,系口服人参后通过系列肠道微生物在体内代谢转化,最终入血的主要代谢产物之一。最新药理学研究表明,IH901在抗肿瘤、抗炎、抗糖尿病和抗衰老等方面均表现出良好的生物活性,是人参在体内发挥活性作用的主要物质。近年来,在体内转化IH901的理论指导下,国内外学者通过体外酶转化和微生物转化等生物工程技术在大规模提取制备IH901等研究方面均取得突破性的进展。以下综述了稀有人参皂苷IH901在体内外的生物转化及其生物活性等研究进展。     

鬼臼毒素及其衍生物生物合成研究进展

鬼臼毒素(Podophyllotoxin,PTOX)是来源于中药八角莲、山荷叶和桃儿七等鬼臼属植物的芳基四氢萘类木脂素。其化学半合成衍生物依托泊苷和替尼泊苷被用于多种癌症的临床治疗。作为天然产物来源新药创制的典型代表,鬼臼毒素目前依赖天然提取,供求矛盾日渐突出。生物合成具有不受资源限制、反应条件友好等优势,是鬼臼毒素及其衍生物生产的新方式。文中总结了鬼臼毒素在植物中的生物合成途径的研究进展,阐述了合成途径中关键酶的功能及其亚细胞定位,进而介绍了以模式植物烟草为底盘的鬼臼毒素合成生物学研究。最后总结了利用微生物对鬼臼毒素进行异源表达及生物转化的研究进展,以期为利用微生物细胞工厂高效合成鬼臼毒素及其衍生物提供参考。     

人参皂苷化合物生物合成进展

人参皂苷是我国许多传统名贵药用植物的主要活性物质,在医药、保健品、营养品和化妆品开发领域有广阔的应用前景,特别是稀有人参皂苷具有显著的抗肿瘤、保护神经系统、保肝护肝等药理活性.但其在植物中含量低且不稳定,严重影响了人参皂苷类物质的开发利用.自20世纪末以来,随着生物技术的不断发展和多种生物基因组的测序和解析,使得人参皂...     

Microbial conversion of ginsenoside Rd from Rb1 by the fungus mutant Aspergillus niger strain TH-10a

Ginsenoside Rd, one of the ginsenosides with significant pharmaceutical activities, is getting more and more attractions on its biotransformation. In this study, a novel fungus mutant, the Aspergillus niger strain TH-10a, which can efficiently convert ginsenoside Rd from Rb1, was obtained through screening survival library of LiCl and ultraviolet (UV) irradiation. The transformation product ginsenoside Rd, generated by removing the outer glucose residue from the position C20 of ginsenoside Rb1, was identified through high-performance liquid chromatography (HPLC) analysis. Factors for the microbial culture and biotransformation were investigated in terms of the carbon sources, the nitrogen sources, pH values, and temperatures. This showed that maximum mycelia growth could be obtained at 28°C and pH 6.0 with cellobiose and tryptone as the carbon source and the nitrogen source, respectively. The highest transformation rate (～86%) has been achieved at 32°C and pH 5.0 with the feeding time of substrate 48 hr. Also, Aspergillus niger strain TH-10a could tolerate even 40 mg/mL ginseng root extract as substrate with 60% bioconversion rate after 72 hr of treatment at the optimal condition. Our results highlight a novel ginsenoside Rd transformation fungus and illuminate its potentially practical application in the pharmaceutical industries.     

Ginsenosides from American ginseng: chemical and pharmacological diversity

Ginseng occupies a prominent position in the list of best-selling natural products in the world. Compared to the long history of use and widespread research on Asian ginseng, the study of American ginseng is relatively limited. In the past decade, some promising advances have been achieved in understanding the chemistry, pharmacology and structure-function relationship of American ginseng. To date, there is no systematic review of American ginseng. In this review, the different structures of the ginsenosides in American ginseng are described, including naturally occurring compounds and those resulting from steaming or biotransformation. Preclinical and clinical studies published in the past decade are also discussed. Highlighted are the chemical and pharmacological diversity and potential structural-activity relationship of ginsenosides. The goal is that this article is a useful reference to chemists and biologists researching American ginseng, and will open the door to agents in drug discovery.     

Mechanistic studies on protopanaxadiol, Rh2, and ginseng (Panax quinquefolius) extract induced cytotoxicity in intestinal Caco-2 cells

Certain ginsenosides, also known as triterpene glycosides, have been recently reported to have a characteristic effect on cultured intestinal and leukemia cell growth. Ginsenoside aglycones 20(S)-protopanaxadiol (PD), 20(S)-protopanaxatriol (PT), and ginsenoside Rh2 have been identified as having a strong effect on reducing cell viability. Furthermore, ginsenoside Rh2 is thought to be a rare ginsenoside not found in all ginseng products. Rather, Rh2 has been recently reported to be a breakdown product of thermal processing of North American ginseng. In this study, pure ginsenosides PD, PT, Rh2 standards and an enriched Rh2 fraction derived from ginseng leaf were tested in cultured Caco-2 cells for relative cytotoxic potency. PD and Rh2 LC50 were similar after 24 to 72 h, whereas a drop in PT LC50 occurred later at 48 and 72 h. Furthermore, PD and Rh2 affected membrane integrity as indicated by LDH secretion earlier than PT and the enriched Rh2 fraction (P < or = 0.05). Ginsenoside Rh2 showed the greatest (P < or = 0.05) build up of necrotic cells (18.3 +/- 0.1%) at the respective LC50 after 24 h and PD (21.3 +/- 0.3%) showed the largest effect after 44 h of exposure. The effect on apoptotic cells at 44 h of treatment were significantly different (P < or = 0.05) for Rh2 (21 +/- 0.4%), PD (14.6 +/- 0.1%), enriched Rh2 leaf fraction (9.9 +/- 0.6%), and PT (2.3 +/- 0.1%) treatments. Caco-2 caspase-3 activity was different between ginsenoside exposure; Rh2 (10.6 +/- 0.3 nM pNA) had the greatest (P < or = 0.05) activity followed by the enriched Rh2 leaf fraction (8.3 +/- 0.2 nM pNA), PT (7.3 +/- 0.3 nM pNA). The PD (4.8 +/- 0.04 nM pNA) treatment was similar to untreated cells (4.3 +/- 0.05 nM pNA) in caspase-3 activity. These results show variable bioactive response in cultured intestinal cell to specific ginsenosides and an enriched Rh2 North American ginseng extract which may be explained on basis of hydrophobic/hydrophilic balance.     

Enhanced Production of β-D-glycosidase and α-L-arabinofuranosidase in Recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> in Fed-batch Culture for the Biotransformation of Ginseng Leaf Extract to Ginsenoside Compound K

Ginsenoside compound K is an essential ingredient in nutritional supplements, cosmetics, and traditional medicines. However, cultivation for the production of enzymes involved in ginsenoside biotransformation has not been attempted in a fermenter. The host strain Escherichia coli ER2566 and the constitutive pHCE vector were selected for the efficient production of β-D-glycosidase, and expression medium composition to produce Sulfolobus solfataricus β-glycosidase expressed in E. coli was optimized in flask and batch cultures. The total activity of β-Dglycosidase in fed-batch culture using a fermenter increased 14-fold before optimization. S. solfataricus β-D-glycosidase and Thermotoga petrophila α-L-arabinofuranosidase were produced in a fed-batch culture. These two enzymes completely converted protopanaxadiol-type ginsenosides in ginseng leaf extract obtained from discarded ginseng leaves as a renewable substrate to compound K. The effective bioprocess for compound K production developed here will contribute to the industrial biological production of compound K.     

人参药材中人参皂苷与生态因子的相关性及人参生态区划

为了扩大人参(Panax ginseng)栽培面积, 解决人参资源日益短缺的问题, 研究了人参皂苷与生态因子之间的相关性。利用超高效液相(UPLC)色谱法, 测定了辽宁、吉林和黑龙江三省不同产区人参样品中3种人参皂苷(Rg1、Re和Rb1)的含量, 并基于“中药材产地适宜性分析地理信息系统”(TCMGIS)平台, 获得采样区域10个生态因子(包括活动积温、年平均气温、海拔、相对湿度、年日照时数、年降水量、7月最高气温、7月平均气温、1月最低气温和1月平均气温等)数据; 利用因子分析法对16个人参基地进行因子得分评价, 得分最高的是吉林和辽宁的人参基地, 故将吉林和辽宁的人参基地作为人参生态适宜性分析的最佳区域; 通过偏最小二乘回归法建立3种人参皂苷成分与上述10个生态因子间的回归方程并获取其相应的权重, 结果发现多个温度因子与人参皂苷含量呈强负相关关系, 说明热量因子对人参皂苷活性成分的累积起主要作用, 而水分因子、地理因子和光照因子与人参皂苷含量呈弱相关关系; 以因子得分最高的吉林和辽宁人参基地为基点区域, 分别对3种人参皂苷进行单成分生态适宜性区划以及综合区划, 得知3种人参皂苷成分积累的最佳区域主要集中在长白山脉, 而燕山山脉和太行山脉只有少量分布区域。     

Biotransformation pathways of ginsenoside Rb1 to compound K by β-glucosidases in fungus Paecilomyces Bainier sp. 229

Ginsenoside Rb1 is the most abundant ginsenoside in Panax (ginseng). The hydrolysis of this ginsenoside produces compound K, the biologically active ginsenoside of ginseng. We previously identified a fungus Paecilomyces Bainier sp. 229 (sp. 229), which can efficiently convert ginsenoside Rb1 to compound K. In this report, the ginsenoside hydrolyzing β-glucosidases were isolated from sp. 229 and the pathway of the biotransformation of ginsenoside Rb1 to compound K by sp. 229 was investigated. Based on reverse-phase HPLC and TLC analysis, we found the main metabolic pathway is as follows: ginsenoside Rb1 → ginsenoside Rd → ginsenoside F2 → compound K. Moreover, the results showed that there were other metabolic pathways: ginsenoside Rb1 → ginsenoside XVII → ginsenoside F2 → compound K and ginsenoside Rb1 → ginsenoside Rg3 → ginsenoside Rh2. These processes would allow the specific bioconversion of ginsenoside Rb1 to various ginsenosides using an appropriate combination of specific microbial enzymes.