人参二醇类皂苷的生物转化动态及人参稀有皂苷C-K或F2的制备

为了低成本有效制备人参稀有皂苷C-K或F₂, 将A. niger g.848菌酶用于转化含有人参皂苷(质量分数)分别为49.6% Rb₁, 25.9% Rd, 19.3% Rc和5.23% Rb₂的西洋参二醇混合皂苷. 霉菌发酵时, 采用人参二醇皂苷诱导物比人参提取液诱导物的产酶总活力提高10%~15%. 所产的2种诱导酶均能水解人参二醇皂苷的3-O-和20-O-多种糖基, 均为人参皂苷酶Ⅰ型; 但是人参二醇皂苷诱导物所产酶几乎全部转化人参二醇皂苷为C-K, 而人参提取液诱导物所产酶则残留中间产物. 使用黑曲霉人参二醇皂苷诱导所产酶, 在转化西洋参二醇皂苷的动态研究中发现, 酶反应1.5~2.5 h, 主要为产物F₂; 酶反应12 h后, 主要产物为C-K皂苷. 基于此, 40 g人参二醇类皂苷在45 ℃粗酶反应24 h, 经处理得到含C-K质量分数为87%的23 g酶反应产物, C-K转化率达85%(摩尔分数). 用40 g西洋参二醇皂苷在45 ℃粗酶反应2.5 h, 经处理得到含有质量分数为58%的F₂和27%的C-K的26 g酶反应产物, F₂转化率为50.4%, C-K转化率为29.5%. 通过人参二醇皂苷诱导的黑曲霉粗酶转化人参二醇类皂苷动态研究, 建立了C-K转化率为85%, F₂转化率为50%的制备方法, 为大批量制备提供了基础依据.     

Purification and characterization of new special ginsenosidase hydrolyzing multi-glycisides of protopanaxadiol ginsenosides, ginsenosidase type I

In this paper, the new type ginsenosidase which hydrolyzing multi-glycosides of ginsenoside, named ginsenoside type I from Aspergillus sp.g48p strain was isolated, characterized and generally described. The enzyme molecular weight was about 80 kDa. Ginsenosidase type I can hydrolyze different glycoside of protopanaxadiol type ginsenosides (PPD); i.e., can hydrolyze the 3(carbon)-O-beta-glucoside of Rb1, Rb2, Rb3, Rc, Rd; can hydrolyze 20(carbon)-O-beta-glucoside of Rb1, 20-O-beta-xyloside of Rb3, 20-O-alpha-arabinoside(p) of Rb2 and 20-O-alpha-arabinoside(f) of Rc to produce mainly F2, compound-K (C-K) and small Rh2, but can not hydrolyze the glycosides of protopanaxatriol type ginsenoside (PPT) such as Re, Rf, Rg1. So, when the ginsenosidase type I hydrolyzed ginsenosides, the enzyme selected ginsenoside-aglycone type, can hydrolyze different glycosides of PPD type ginsenoside; however no selected glycoside type, can hydrolyze multi-glycosides of PPD type ginsenosides. These properties were novel properties, and differentiated with the other previously described glycosidases.     

重组嗜热β-葡萄糖苷酶转化稀有人参皂苷Rd和CK

利用高效液相色谱(HPLC)法,对重组嗜热β-葡萄糖苷酶(Fpglu1)转化稀有人参皂苷(Rd和CK)进行研究,并表征了其催化动力学参数.利用同源模建和分子动力学模拟等生物信息学技术,探究了Fpglu1转化人参皂苷的结构基础及其相互作用.结果表明,Fpglu1能够水解人参总皂苷生成稀有皂苷Rd和CK,其催化人参皂苷Rb_1,Rb_2和Rc的K_m值分别为0.318,1.840和5.269 mmol/L;酶的转换数(k_(cat))值分别为144.191,0.572和0.011 s~(-1).当转化时间分别为6和102 h时,Rd和CK的产率达到最大,分别为60%和93%.通过对该酶的结构预测及皂苷分子的对接研究发现,底物位于由疏水性氨基酸构成的底物口袋中,氨基酸残基Glu194和Glu367是参与催化作用的关键,且实验测得的酶促反应动力学参数(K_m)与对接的相互作用能量值存在线性关系.     

Comparative study on chemical components and anti‐inflammatory effects of Panax notoginseng flower extracted by water and methanol

Methanol and water are commonly used solvents for chemical analysis and traditional decoction, respectively. In the present study, a high‐performance liquid chromatography with ultraviolet detection method was developed to quantify 11 saponins in Panax notoginseng flower extracted by aqueous solution and methanol, and chemical components and anti‐inflammatory effects of these two extracts were compared. The separation of 11 saponins, including notoginsenoside Fc and ginsenoside Rc, was well achieved on a Zorbax SB C18 column. This developed method provides an adequate linearity (r ² > 0.999), repeatability (RSD < 4.26%), inter‐ and intraday variations (RSD < 3.20%) with recovery (94.7–104.1%) of 11 saponins concerned. Our data indicated that ginsenoside biotransformation in PNF was found, when water was used as the extraction solvent, but not methanol. Specifically, the major components of Panax notoginseng flower, ginsenosides Rb1, Rc, Rb2, Rb3, and Rd, can be near completely transformed to the minor components, gypenoside XVII, notoginsenoside Fe, ginsenoside Rd2, notoginsenoside Fd, and ginsenoside F2, respectively. Total protein isolated from Panax notoginseng flower is responsible for this ginsenoside biotransformation. Additionally, methanol extract exerted the stronger anti‐inflammatory effects than water extract in lipopolysaccharide‐induced RAW264.7 cells. This difference in anti‐inflammatory action might be attributed to their chemical difference of saponins.     

Major ginsenoside contents in rhizomes of Panax sokpayensis and Panax bipinnatifidus

This study compared eight major ginsenosides (Rg1, Rg2, Rf, Re, Rd, Rc, Rb1 and Rb2) between Panax sokpayensis and Panax bipinnatifidus collected from Sikkim Himalaya, India. High-performance liquid chromatographic analysis revealed that all major ginsenosides were present in the rhizomes of P. sokpayensis except ginsenoside Rc, whereas ginsenoside Rf, Rc and Rb2 were not detected in P. bipinnatifidus.     

高效液相色谱-飞行时间质谱联用法分析人参及人参皂苷与山楂配伍过程中的水解行为

利用高效液相色谱-飞行时间质谱联用的方法,分别对人参配伍山楂前后人参皂苷的变化进行分析,同时对人参皂苷Re、Rg1、Rb1、Rd与山楂配伍的水解规律进行系统研究,并与单独煎煮液、仿山楂配伍pH值煎煮液的水解产物进行比较,结果发现人参与山楂配伍后人参皂苷Rg1、Rb1含量明显减少,而人参皂苷Re、Rd、Rg2、Rg3、F2、Rh1含量明显增加,其中人参皂苷Re与山楂配伍后水解产物为人参皂苷20(R)-Rg2、20(S)-Rg2,仿山楂配伍pH值水解产物为人参皂苷20(R)-Rg2、20(S)-Rg2、Rg4、Rg6;人参皂苷Rg1与山楂配伍后水解产物为20(S)-Rh1、20(R)-Rh1,仿山楂pH值水解产物为20(S)-Rh1、20(R)-Rh1、Rh4、Rk3;人参皂苷Rb1与山楂配伍后水解产物为Rd、20(S)-Rg3,仿山楂pH值水解产物为F2、20(S)-Rg3;人参皂苷Rd与山楂配伍后水解产物为F2、20(S)-Rg3、20(R)-Rg3,仿山楂pH值水解产物为20(S)-Rg3、20(R)-Rg3。研究表明,不同人参皂苷和山楂配伍后与仿山楂pH值的水解产物并不相同,人参与山楂配伍改变了人参皂苷成分的种类及含量。本研究为临床方剂中人参与山楂配伍后成分的变化提供物质基础数据。     

Marked production of ginsenosides Rd, F2, Rg3, and compound K by enzymatic method

The hydrolysis of protopanaxadiol-type saponin mixture by various glycoside hydrolases was examined. Among these enzymes, crude preparations of lactase from Aspergillus oryzae, beta-galactosidase from A. oryzae, and cellulase from Trichoderma viride were found to produce ginsenoside F(2) [3-O-(beta-D-glucopyranosyl)-20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol], compound K [20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol], and ginsenoside Rd {3-O-[beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl]-20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol}, respectively, from protopanaxadiol-type saponin mixture in large quantities. Moreover, the crude preparation of lactase from Penicillium sp. having a high producing activity of ginsenoside Rh(1) (6-O-beta-D-glucopyranosyl-20(S)-protopanaxatriol) from protopanaxatriol-type saponin mixture gave ginsenoside Rd as a main product, ginsenoside Rg(3) {3-O-[beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl]-20(S)-protopanaxadiol}, and compound K from protopanaxadiol-type saponin mixture. The hydrolytic pathways of ginsenosides Rb(1), Rb(2), and Rc to ginsenosides Rd, Rg(3), and F(2), and compound K by crude preparations of four glycoside hydrolases were also studied. This is the first report on the enzymatic preparation of an intestinal bacterial metabolite, ginsenoside F(2), in quantity, and a considerable amount of a minor saponin, ginsenoside Rg(3), from a protopanaxadiol-type saponin mixture.     

动态泡沫浮选法分离富集人参提取液中的二醇型人参皂苷

采用动态泡沫浮选法分离富集人参提取液中的二醇型人参皂苷, 用高效液相色谱法测定6种人参皂苷Rg₁, Re, Rb₁, Rc, Rb₂和Rd的含量. 考察了浮选液pH值、电解质NaCl浓度、载气流量、料液浓度及料液流速对人参皂苷浮选率的影响, 确定了动态泡沫浮选的最佳条件, 并与溶剂提取法、溶剂浮选法以及静态泡沫浮选法进行了比较. 结果表明, 动态泡沫浮选法对二醇型人参皂苷Rb₁, Rc, Rb₂和Rd具有高富集效率, 回收率分别为93.3%, 98.6%, 96.9%和98.3%, 而对三醇型人参皂苷Rg1和Re的富集效率却很低, 回收率分别为4.8%和4.2%. 该法是分离纯化二醇型人参皂苷的一种简便有效的方法.     

Determination of Seven Major Ginsenosides in Different Parts of Panax quinquefolius L.(American Ginseng) with Different Ages

Ginsenosides Rg1,Re,Rb1,Rc,Rb2,Rb3,and Rd in different parts of the American ginseng plant were investigated.The extraction process was a pressurized microwave-assisted extraction(PMAE).The seven ginsenosides were separated and determined by high-performance liquid chromatography(HPLC) with a ultraviolet(UV) detector,at 203 nm.The experiment results showed significant variations in the individual ginsenoside contents of the American ginseng in different parts and ages of the plant.The results demonstrated that the leaves,root hairs,and rhizomes of Panax quinquefolius L.contained higher ginsenoside contents,followed by the main roots and stems.The leaves contained dramatically higher levels of ginsenoside Rg1,Rb3,and Rd than the other four parts.Higher contents of Rb1 and Re were present in the main roots,root hairs,and rhizomes.The amount of ginsenoside content in the stems was the lowest.The total content of the seven ginsenosides in main roots,root hairs and rhizomes increased with the age of the plant.In contrast,the ginsenoside contents in the leaves and stems decreased with a year of growth.     

Biotransformation of the saponins in Panax notoginseng leaves mediated by gut microbiota from insomniac patients

Saponins extracted from Panax notoginseng leaves by methanol or water could be orally administrated for insomnia with very low bioavailability, which might be bio-converted by gut microbiota to generate potential bioactive products. Moreover, gut microbiota profiles from insomniac patients are very different from healthy subjects. We aimed to compare the metabolic characteristics and profiles of the two saponins extract by incubation with gut microbiota from insomniac patients. The ginsenosides, notoginsenosides, and metabolites were identified and relatively quantified by high-performance liquid chromatography-tandem mass spectrometry. Gut microbiota was profiled by 16S ribosomal RNA gene sequencing. The results showed that saponins were very different between methanol or water extract groups, which were metabolized by gut microbiota to generate similar yields. The main metabolites included ginsenoside Rd, ginsenoside F₂, ginsenoside C-Mc or ginsenoside C-Y, ginsenoside C-Mx, ginsenoside compound K, and protopanaxadiol in both groups, while gypenoside XVII, notoginsenoside Fe, ginsenoside Rd₂, and notoginsenoside Fd were the intermediates in the methanol group. Moreover, the microbial, Faecalibacterium prausnitzi, could bio-convert the saponins to obtain the corresponding metabolites. Our study implied that saponins extracted from P. notoginseng leaves by methanol or water could be used for insomniac patients due to gut microbiota biotransformation.     

泡沫浮选-固相提取分离富集三七提取液中的人参皂苷

采用泡沫浮选-固相提取联用法,分离富集三七中的R1,Rg1,Re,Rc,Rb2,Rb3,Rd和Rb1,并用液相色谱法测定其含量,检测灵敏度和选择性都有所提高.对泡沫浮选过程的载气流量、浮选时间、样品溶液pH值和固相提取柱的洗脱条件进行了优化.原人参二醉型皂苷R1,Rc,Rb2,Rb3,Rd和Rb1的回收率在85.0%9...     

重组嗜热β-葡萄糖苷酶转化稀有人参皂苷Rd和CK

利用高效液相色谱(HPLC)法, 对重组嗜热β-葡萄糖苷酶(Fpglu1)转化稀有人参皂苷(Rd和CK)进行研究, 并表征了其催化动力学参数. 利用同源模建和分子动力学模拟等生物信息学技术, 探究了Fpglu1转化人参皂苷的结构基础及其相互作用. 结果表明, Fpglu1能够水解人参总皂苷生成稀有皂苷Rd和CK, 其催化人参皂苷Rb₁, Rb₂和Rc的K_m值分别为0.318, 1.840和5.269 mmol/L; 酶的转换数(k_cat)值分别为144.191, 0.572和0.011 s^-1. 当转化时间分别为6和102 h时, Rd和CK的产率达到最大, 分别为60%和93%. 通过对该酶的结构预测及皂苷分子的对接研究发现, 底物位于由疏水性氨基酸构成的底物口袋中, 氨基酸残基Glu194和Glu367是参与催化作用的关键, 且实验测得的酶促反应动力学参数(K_m)与对接的相互作用能量值存在线性关系.     

Two Key Amino Acids Variant of α-l-arabinofuranosidase from Bacillus subtilis Str. 168 with Altered Activity for Selective Conversion Ginsenoside Rc to Rd

α-l-arabinofuranosidase is a subfamily of glycosidases involved in the hydrolysis of l-arabinofuranosidic bonds, especially in those of the terminal non-reducing arabinofuranosyl residues of glycosides, from which efficient glycoside hydrolases can be screened for the transformation of ginsenosides. In this study, the ginsenoside Rc-hydrolyzing α-l-arabinofuranosidase gene, BsAbfA, was cloned from Bacilus subtilis, and its codons were optimized for efficient expression in E. coli BL21 (DE3). The recombinant protein BsAbfA fused with an N-terminal His-tag was overexpressed and purified, and then subjected to enzymatic characterization. Site-directed mutagenesis of BsAbfA was performed to verify the catalytic site, and the molecular mechanism of BsAbfA catalyzing ginsenoside Rc was analyzed by molecular docking, using the homology model of sequence alignment with other β-glycosidases. The results show that the purified BsAbfA had a specific activity of 32.6 U/mg. Under optimal conditions (pH 5, 40 °C), the kinetic parameters K_m of BsAbfA for pNP-α-Araf and ginsenoside Rc were 0.6 mM and 0.4 mM, while the K_cat/K_m were 181.5 s⁻¹ mM⁻¹ and 197.8 s⁻¹ mM⁻¹, respectively. More than 90% of ginsenoside Rc could be transformed by 12 U/mL purified BsAbfA at 40 °C and pH 5 in 24 h. The results of molecular docking and site-directed mutagenesis suggested that the E173 and E292 variants for BsAbfA are important in recognizing ginsenoside Rc effectively, and to make it enter the active pocket to hydrolyze the outer arabinofuranosyl moieties at C₂₀ position. These remarkable properties and the catalytic mechanism of BsAbfA provide a good alternative for the effective biotransformation of the major ginsenoside Rc into Rd.     

重组内切纤维素酶Fpendo5A转化三七总皂苷

从嗜热细菌基因组中克隆到1个新的纤维素酶基因,并在大肠杆菌中进行了高效可溶性表达,粗酶液经镍柱亲和层析进行分离纯化.利用快速分离液相色谱-四极杆飞行时间质谱联用仪(RRLC/Q-TOF-MS)对重组内切纤维素酶Fpendo5A转化三七总皂苷的产物结构进行了鉴定,并进一步阐明其转化机制.结果表明,该酶的最适反应温度和pH值分别为80℃和5.5.Fpendo5A能够催化三七总皂苷中的主要皂苷成分,即Ra_1,Rb_1,Rc,Rd和Rg_3的侧链糖基的水解反应,但对于不同的皂苷底物,Fpendo5A选择性催化的侧链糖基类型不同.经鉴定,Fpendo5A转化Ra_1,Rb_1,Rc,Rd和Rg_3的转化产物分别为Rb_2,GypⅩⅦ,CMC_1,F_2和Rh_2.由此可见,Fpendo5A通过水解Rb_1,Rc,Rd和Rg3的C3位的β-(1,2)糖苷键分别生成GypⅩⅦ,CMC1,F2和Rh2.在转化Ra_1时,Fpendo5A通过水解Ra_1的C20位的α-(1,4)木糖苷键生成Rb2.     

Analysis of Low-polar Ginsenosides in Steamed Panax Ginseng at High-temperature by HPLC-ESI-MS/MS

A high performance liquid chromatography coupled with electrospray ionization-tandem mass spectrometry( HPLC-ESI-MS/MS) method was developed for the analysis and identification of ginsenosides in the extracts of raw Panax ginseng(RPG) and steamed Panax ginseng at high temperatures(SPGHT). A total of 25 ginsenosides were extracted include of which 10 low-polar ginsenosides, such as ginsenosides F4, Rk3, Rh4, 20S-Rg3, 20R-Rg3 and so on, were identified according to their HPLC retention time and MS/MS data. The results indicated that the low polar ginsenosides were seldom found in RPG. For the exploration of the transformation pattern of the ginsenosides in steam processing, the standards of ginsenosides Re, Rg1, Rb1, Rc, Rb2, Rb3 and Rd were selected and hydrolyzed at a temperature of 120 ℃. The results show that these polar ginsenosides can be converted to low-polar ginsenosides such as Rg2, Rg6, F4, Rk3 and Rg5 by hydrolyzing the sugar chains.     

Studies on absorption, distribution, excretion and metabolism of ginseng saponins. VII. Comparison of the decomposition modes of ginsenoside-Rb1 and -Rb2 in the digestive tract of rats.

In order to clarify some similarities and differences of decomposition modes between 20(S)-protopanaxadiol (20(S)-ppd) saponins, represented by ginsenoside Rb1 (Rb1) and ginsenoside Rb2 (Rb2), the decompositions of Rb1 and Rb2 in the rat gastrointestinal tract, 0.1 N HCl and crude hesperidinase were investigated in detail. As in the case of Rb2 reported previously, Rb1 was hydrolyzed to 20(R,S)-ginsenoside Rg3 in 0.1 N HCl. On the other hand, hydroperoxidation of Rb1 occurred in rat stomach; the major hydroperoxide was separated and identified as the 25-hydroperoxy-23-ene derivative of Rb1 (VIII) by 1H- and 13C-nuclear magnetic resonance and fast atom bombardment mass spectrometry. The decomposition modes of 20(S)-ppd saponins (Rb1 and Rb2) differed from that of 20(S)-protopanaxatriol saponin (Rg1) in rat stomach. In rat large intestine, five decomposition products of Rb1 were observed by thin-layer chromatography, and these were identified as gypenoside XVII (G-XVII), ginsenoside Rd (Rd), ginsenoside F2 (F2), compound K (C-K) and VIII. The decomposition modes of Rb1 and Rb2, both 20(S)-ppd saponins, are considered to be different because of the hydrolysis rate in the terminal sugar moiety at the C-20 hydroxyl group in the rat large intestine. Using crude hesperidinase, Rb1 was decomposed to G-XVII, F2 and C-K, and Rb2 was decomposed to 3-O-beta-D-glucopyranosyl-20-O-[alpha-L-arabinopyranosyl(1----6)-b eta-D- glucopyranosyl]-20-(S)-ppd, F2 and C-K. Consequently, it appears that hydrolysis by beta-glucosidase, which is present in the rat large intestine, is distinct from that by crude hesperidinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on Intestinal Transport of Ginsenoside Compatibility with Veratrum nigrum via Caco-2 Cell Monolayer Model Coupled with UPLC-ESI-MS Method

The Caco-2 cells have been recognized as effective tools to be applied to imitating the drug absorption in human intestine for the transport of drug. Herein, Caco-2 cell monolayer model was used to study the transport of the ginsenoside compatibility with Veratrum nigrum in different proportions. A specific high performance liquid chromatography-electrospray ionization-mass spectrometry(HPLC-ESI-MS) method was developed for the semiquantitative determination of ginsenoside in intestinal transport with Dioscin as an internal standard. For the Caco-2 model constructed, two influencing factors were investigated, including time and concentration. The results suggest that the absorption of ginsenoside Re, Rg1, Rb1, Rc, Rb2 and Rd are time- and concentration-dependent and the excretions of Rb1, Rc, Rb2 and Rd have a relatronship with some transport proteins. The bioavailability of the ginsenosides has reduced compared to the single Panax ginseng extract when compatibility with a certain amount of Veratrum nigrum.     

人参中人参皂苷的直接高压微波辅助降解

采用高效液相色谱-电喷雾质谱联用法测定了人参提取液中的人参皂苷. 考察了天然人参皂苷发生降解的条件, 同时研究了单体人参皂苷Rg1, Re, Rb1, Rc, Rb2和Rd的降解, 并对降解产物进行了分析. 结果表明, 随着提取压力的升高, 提取液中天然人参皂苷的含量逐渐减少, 同时产生多种次级人参皂苷. 当微波提取压力达到600 kPa, 提取时间为10 min时, 提取液中的主要天然人参皂苷达到完全降解, 次级人参皂苷Rg3含量达到最高. 在单体人参皂苷Rb1, Rc, Rb2和Rd的降解产物中均得到人参皂苷Rg3.     

Identification and Evaluation of a Panel of Ginsenosides from Different Red Ginseng Extracts with Nootropic Effect

Red ginseng has been gradually discovered to have pharmacological and physiological effects. It is well known that the most important bioactive components of ginseng are ginsenosides. The nootropic effect of ginsenosides from nine different red ginseng extracts was evaluated here. Nine groups of mice were perfused with different concentrations of nine red ginseng extracts, respectively, and two groups of mice with distilled water. The nootropic effect of ginsenosides on mice was evaluated with behavior tests and a biochemical indicator study. The extracts were identified by rapid resolution liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry(RRLC-Q-TOF-MS). Furthermore, principal component analysis(PCA) was used to analyze the contribution of chemical components from different ginseng groups. The extracts with the most and the weakest effective nootropic were found. It is notable that extract processing is a very important factor to decide pharmacological functions of ginseng extracts. As a conclusion, the most effective extract method for ginsenosides has been found. A panel of 13 ginsenosides has been screened out as chemical markers with nootropic effect, which include high level ginsenosides Ra0, Rb1, Rc, Rb2, Rb3, Re, Rd, and Rg1 and low level ginsenosides mRb1, mRc, mRb2, mRd, and F2. Low level ginsenosides were first time to be discovered as possible nootropic compounds. This method may shed light on fast discovery of bioactive compounds of medicinal plants with low level compounds.     

高效液相色谱质谱质谱法分析人参皂甙

 以反相高效液相色谱法分离了 9种人参皂甙。操作条件为乙腈 水梯度洗脱 ,二极管阵列检测器检测并在2 0 2nm下提取色谱图。利用三级四极杆质谱研究了 9种人参皂甙的一级质谱 (主要给出相对分子质量信息 )和二级质谱 (提供碎片结构信息 )。通过它们质谱图的差异对其进行了鉴别 ,并将方法用于实际样品中的 9种人参皂甙的定性。