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。     

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。     

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.     

人参皂苷IH901抗肝癌生长及其侵袭转移的作用机理

人参皂苷IH901是一种天然二醇组人参皂苷肠道细菌最终代谢产物.目前研究表明,IH901具有抗肝癌活性,然而其作用机理尚不清楚.采用人肝癌细胞株SMMC-7721,HepG2,MHCC97-H来建立体内外的抗肿瘤模型,以探讨人参皂苷IH901的抗肝癌活性及其作用机理.结果发现,人参皂苷IH901能够通过细胞周期中亚二倍体峰的增加、线粒体跨膜电位（ΔΨm）崩溃、DNA梯形条带的形成和提高Caspase-9/Caspase-3的表达量、促进凋亡来抑制肝癌细胞的生长.通过抑制细胞的迁移和降低VEGF/bFGF的表达来抑制肝癌细胞的侵袭转移.裸鼠体内实验均表明了人参皂苷在体内外的抗肝癌活性,并且在分子、细胞、动物水平初步揭示了IH901抗肝癌生长及其侵袭转移的作用机理,为其作为抗肝癌药物的开发提供理论支持.     

三七叶、人参叶和西洋参叶皂苷类成分的研究(英文)

三七叶、人参叶和西洋参叶其皂苷类成分相近,但专属性成分各异,皂苷类成分的分布比例也各不相同。本文建立了HPLC-UV法测定上述皂苷成分的方法,经过方法学考察,各种皂苷成分精密度好、加样回收率高,方法可靠。11种皂苷成分总含量顺序为:西洋参叶>人参叶>三七叶;二醇组皂苷成分含量:西洋参叶>三七叶>人参叶;三醇组皂苷成分含量:人参叶>西洋参叶>三七叶。西洋参叶中二醇组皂苷和人参叶中三醇组皂苷含量明显高于其他。西洋参叶中人参皂苷Rb3和Rd的含量之和占11种皂苷成分的60%以上。鉴于其中人参皂苷的高含量,三七叶、人参叶和西洋参叶应该作为皂苷来源得到充分利用;不同的皂苷成分有不同的药理活性,应基于它们的皂苷组成和比例选择性进行研究和开发。     

复合酶解法提取三七皂苷的实验研究

以三七提取液中总皂苷的含量和提取物得率为指标,考察了乙醇回流法、渗漉法、纤维素酶解法、果胶酶解法、复合酶解法的优劣,并采用单因素法和四因素（纤维素酶用量、果胶酶用量、酶解温度、乙醇浓度）三水平正交设计法对复合酶解法提取工艺条件进行优选,得到如下较理想的提取工艺条件：纤维素酶用量为15U／g（生药）、果胶酶用量为140U／g（生药）,酶解pH值为4．5,酶解温度为50℃,乙醇浓度为80％,提取时间为2．5h。所得三七提取液中总皂苷的含量为12．01％,提取物得率为35．82％。     

基于UPLC法测定离体大鼠及人源肠道菌对三七中人参皂苷代谢的差异

为探究人与大鼠肠道菌群对三七水煎液中三醇型人参皂苷Rg1、Re及二醇型人参皂苷Rb1、Rd体外代谢的差异性及发现其代谢产物原人参二醇PPD与原人参三醇PPT,实验利用UPLC方法测定三七水煎液分别与人、大鼠肠道菌群在厌氧条件下共培养24h后的孵育液中4种皂苷的含量及代谢产物PPD与PPT的含量。结果表明三七中含有三醇型人参皂苷Rg19.4500mg/g、Re1.8872mg/g,二醇型人参皂苷Rb18.5816mg/g、Rd1.9456mg/g。与人源肠道菌共培养后,三七中含有的二醇型、三醇型人参皂苷含量显著降低,重要的是,在培养液中检测到代谢产物PPD和PPT的存在,含量分别为0.2136mg/g及0.0344mg/g,与大鼠肠道菌共培养后,三七中含有的二醇型皂苷含量有轻微降低,而三醇型皂苷含量未见明显变化,但有少量PPT(0.0184mg/g)的生成。由此可见:在体外条件下,三七水煎液中人参皂苷会被人肠道菌群降解生成代谢产物PPD和PPT,而大鼠肠道菌群的降解产物却仅有PPT生成,二者存在种属差异。     

密闭微波辅助间歇提取RP-HPLC测定三七中三种皂苷类化合物

本文建立了采用微波辅助间歇提取三七样品中的皂苷类化合物,并以反相高效液相色谱（RP—HPLC）测定其中三七皂苷R1、人参皂苷Rg1和人参皂苷Rb1含量的实验方法。分别通过单因素实验和正交实验设计,优化了萃取溶剂浓度、溶剂用量、微波功率和微波辐射时间等提取条件。与传统的索氏提取法、冷浸法和超声波提取法比较,微波辅助萃取法具有快速、提取率高、溶剂消耗少等优点,间歇萃取较连续萃取更是大大节省了微波能耗。     

虎杖中白藜芦醇的酶法制备

以白藜芦醇得率为指标,通过对酶制剂的筛选和酶解条件的考察,分别选出酶法提取和酶法转化制备虎杖中白藜芦醇的最佳条件,并对两种酶法进行比较.结果显示:(1)酶法提取最佳酶解条件为:酶解温度45℃,酶解时间60 min,最适pH 5.0,底物浓度17%;酶法转化最佳酶解条件为:酶解温度40℃,酶解时间48 h,最适pH 5.0,底物浓度15%.(2)与空白样品(不加酶)相比,酶法提取白藜芦醇得率增加了3.12 mg/g;酶法转化白藜芦醇得率增加了12.72 mg/g.研究表明,两种方法与不加酶提取相比均提高了白藜芦醇的得率,但酶法转化效果更显著,可用于白藜芦醇的制备.     

黑根霉对人参皂苷Re的生物转化

利用菌种黑根霉Rhizopus sp.对人参皂苷Re进行生物转化,并对人参皂苷Re及其发酵产物进行HPLC系统分析比较,经液相色谱-质谱分析得出人参皂苷Re转化率为92.16%,并制备出人参皂苷Re发酵产物中峰值升高的成分,转化后的人参皂苷发酵产物中化合物1确定为人参皂苷Rg2,化合物2为Rg2的同分异构体,得率为10.13%;化合物3和化合物4确定为人参皂苷Rg5/Rk1,得率为29.23%。从结果初步推测得出人参皂苷Re被黑根霉转化为人参皂苷Rg2的机理,人参皂苷Re转化成人参皂苷Rg5/Rk1的机理还有待于进一步研究。     

人参皂苷F1转化菌株的原生质体诱变育种

菌核青霉2246(Penicillium sclerotiorum)能够将人参皂苷Rg1转化为人参皂苷F1.以此菌为出发菌株,进行原生质体制备和再生的研究,确定原生质体的最佳形成条件:菌丝体培养24 h,用5 mg/mL溶壁酶、5mg/mL纤维素酶和5 mg/mL蜗牛酶的混合酶液进行酶解,以0.8 mol/L的KCI作为渗透压稳定剂,31℃水浴振摇2h.并对形成的原生质体进行亚硝基胍复合紫外线照射诱变,结果得到1株转化率显著提高、遗传性能稳定的诱变株( NU-1),其转化率由16.7％提高到30.5％.     

Simple and efficient preparation of ginsenoside (S)-Rg2 from ginsenoside Re by biotransformation with Cellulosimicrobium sp. 21

A novel ginsenoside-hydrolyzing strain was isolated from ginseng-cultivation soil in Changbai Mountain (China). The strain was identified as Cellulosimicrobium sp. 21 by 16S rDNA sequencing. Using the β-glucosidases secreted from Cellulosimicrobium sp. 21, protopanaxatriol-type ginsenoside Re was converted to the highly active neuroprotective molecule (S)-Rg2 by removal of the C-20-glucopyranosyl residue. The α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranose at the C-6 position of Rg2 was not further attacked by Cellulosimicrobium sp. 21, so the transformation shows high specificity. To simplify the transformation and product-preparation process, a simple and efficient transformation system was developed in a phosphate buffer system instead of organic media. The optimum conditions for transforming ginsenoside Re into Rg2 by Cellulosimicrobium sp. 21 were determined through single-factor experiments and response surface methodology. Under the optimized conditions: transformation buffer, 50 mM phosphate buffer, at pH: 7.00; temperature: 27.6°C; substrate concentration: 0.50 mg/ml; biotransformation period: 12 h; the biotransformation efficiency reached 89.8% (molar ratio) in 2-L reaction system. This simple biotransformation with high specificity and efficiency has potential for use in Rg2 preparation in the pharmaceutical industry.     

Highly selective microbial transformation of major ginsenoside Rb(1) to gypenoside LXXV by Esteya vermicola CNU120806

Aims

This study examined the biotransformation pathway of ginsenoside Rb₁ by the fungus Esteya vermicola CNU 120806.

Methods and Results

Ginsenosides Rb₁ and Rd were extracted from the root of Panax ginseng. Liquid fermentation and purified enzyme hydrolysis were employed to investigate the biotransformation of ginsenoside Rb₁. The metabolites were identified and confirmed using NMR analysis as gypenoside XVII and gypenoside LXXV. A mole yield of 95·4% gypenoside LXXV was obtained by enzymatic conversion (pH 5·0, temperature 50°C). Ginsenoside Rd was used to verify the transformation pathway under the same reaction condition. The product Compound K (mole yield 49·6%) proved a consecutive hydrolyses occurred at the C‐3 position of ginsenoside Rb₁.

Conclusions

Strain CNU 120806 showed a high degree of specific β‐glucosidase activity to convert ginsenosides Rb₁ and Rd to gypenoside LXXV and Compound K, respectively. The maximal activity of the purified glucosidase for ginsenosides transformation occurred at 50°C and pH 5·0. Compared with its activity against pNPG (100%), the β‐glucosidase exhibited quite lower level of activity against other aryl‐glycosides. Enzymatic hydrolysate, gypenoside LXXV and Compound K were produced by consecutive hydrolyses of the terminal and inner glucopyranosyl moieties at the C‐3 carbon of ginsenoside Rb₁ and Rd, giving the pathway: ginsenoside Rb₁→ gypenoside XVII → gypenoside LXXV; ginsenoside Rd→F₂→Compound K, but did not hydrolyse the 20‐C, β‐(1‐6)‐glucoside of ginsenoside Rb₁ and Rd.

Significance and Impact of the Study

The results showed an important practical application on the preparation of gypenoside LXXV. Additionally, this study for the first time provided a high efficient preparation method for gypenoside LXXV without further conversion, which also gives rise to a potential commercial enzyme application.     

Biotransformation of Panax notoginseng saponins into ginsenoside compound K production by Paecilomyces bainier sp. 229

Aims: Development and optimization of an efficient and inexpensive biotransformation process for ginsenoside compound K production by Paecilomyces bainier sp. 229. Methods and Results: We have determined the optimum culture conditions required for the efficient production of ginsenoside compound K by P. bainier sp. 229 via biotransformation of ginseng saponin substrate. The optimal medium constituents were determined to be: 30 g sucrose, 30 g soybean steep powder, 1 g wheat bran powder, 1 g (NH₄)₂SO₄, 2 g MgSO₄·7H₂O and 1 g CaCl₂ in 1 l of distilled water. An inoculum size of 5–7·5% with an optimal pH range of 4·5–5·5 was essential for high yield. Conclusions: The Mol conversion quotient of ginseng saponins increased from 21·2% to 72·7% by optimization of the cultural conditions. Scale‐up in a 10 l fermentor, under conditions of controlled pH and continuous air supply in the optimal medium, resulted in an 82·6% yield of ginsenoside compound K. Significant and Impact of the Study: This is the first report on the optimization of culture conditions for the production of ginsenoside compound K by fungal biotransformation. The degree of conversion is significantly higher than previous reports. Our method describes an inexpensive, rapid and efficient biotransformation system for the production of ginsenoside compound K.     

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。     

酵母菌半连续转化人参皂苷Rb1的条件优化

以单因素实验为基础,通过多因素方差分析实验对人参皂苷半连续转化的条件进行优化,选出最佳条件组合,得到最佳的补料方式,补料浓度为6%,补料体积为24mL,补料周期为12h,在此条件下人参皂苷Rb1生物转化达33.5%左右。在最佳补料条件下进行人参皂苷酵母菌转化,其稳定性好,转化率高,对工业生产有积极的推动作用。     

Protopanaxadiol 6-hydroxylase and its role in regulating the ginsenoside heterogeneity in Panax notoginseng cells

Various structure-similar plant secondary metabolites like ginseng saponins (ginsenosides) possess different or even totally opposite biological activities. Intentional manipulation of the ginsenoside heterogeneity in cellular biosynthesis is of great interest and significance [Zhong and Yue (2005); Adv Biochem Eng Biotechnol 100:53-88]. In this work, CO-binding spectra of microsomes prepared from the suspended cells of Panax notoginseng showed increases in absorption at 450 nm compared with the control without CO sparging, and protopanaxadiol 6-hydroxylase (P6H), a new enzyme catalyzing the conversion of ginsenoside aglycone protopanaxadiol into protopanaxatriol, was found. P6H was dependent on NADPH and molecular oxygen. The enzymatic reaction was inhibited by carbon monoxide and partially reversible upon illumination with blue light, and sensitive to cytochrome P450 inhibitors. The results supported the contention that P6H was a cytochrome P450-dependent hydroxylase, whose catalytic product was confirmed to be protopanaxatriol by HPLC-MS. Induction of P6H activity by phenobarbital, a cytochrome P450 inducer, was observed. A maximal activity of P6H was obtained with addition of 0.5 mM phenobarbital on day 4 of shake-flask cultivation. The maximum content of protopanaxatriol-type ginsenosides (Rg(1) and Re, Rg group) and the maximum ratio of the content of protopanaxatriol: protopanaxadiol reached 6.88 +/- 0.21 mg g(-1) dry weight and 7.0, respectively, which was about 1.4 and 2.0-fold that of respective controls (without addition of phenobarbital). Oxidative burst was also observed in the cell cultures with addition of phenobarbital. P6H was concluded as a key enzyme in regulating Rg-group ginsenoside biosynthesis in P. notoginseng cells.