稀有人参皂苷IH901酶法转化与制备研究

本研究利用酶制剂蜗牛酶,酶法转化三七二醇组皂苷制备稀有人参皂苷IH901,正交实验优化酶解条件,建立酶法转化工艺.结果表明:超声法提取三七总皂苷正交实验优化条件为用75%乙醇溶液,15倍溶剂用量,超声波提取210 min作为最佳条件,三七总皂苷得率为12.21%;酶法转化二醇组人参皂苷制备稀有人参皂苷IH901,正交实验优化的条件为物料比为6/1、反应时间9 h、反应温度为45℃、pH值为3.0,酶解得率为54.24%;经硅胶柱分离获得IH901单体化合物,HPLC测定纯度达98%.酶法转化制备皂苷IH901的工艺方法简便,切实可行,可为中试生产提供参考.     

人参植物高质量RNA的分离及其皂苷生物合成相关新基因GBR6的表达分析

为从富含多糖的人参植物组织中分离出高质量的RNA,使用改进的两种异硫氰酸胍法,可从人参植物根组织中提取到较高产量和质量的总RNA、每克新鲜组织的RNA产量在80～110μg之间;经琼脂糖凝胶电泳分析,可见到28S和18S rRNA两条完整、清晰的条带;紫外分光光度法测得的A260／A280比值位于1．8～2.0．而且,使用该改良法分离的RNA,可广泛用于检测基因表达的RT-PCR与Northern印迹杂交分析以及cDNA文库构建等植物分子生物学研究。     

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。     

西洋参发根的诱导及不同外源物质对发根生长和皂苷含量的影响

利用发根农杆菌A4菌株在西洋参根外植体上直接诱导产生发根,并分析不同浓度的植物激素6-BA、NAA,前体物醋酸镁(Mg(Ac)2)、L-亮氨酸和诱导子茉莉酸甲酯(MJA)、水杨酸(SA)对西洋参(Panax quinquefolium L.)发根生长和皂苷含量的影响。同时,研究MJA与SA组合作用对皂苷含量的影响。采用高效液相色谱法测定发根中单体皂苷Rb1的含量。结果显示,适宜浓度的外源物质均可促进皂苷含量的增加,MJA尤其明显。     

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.     

Ginsenoside Rg1 protects against 6-OHDA-induced neurotoxicity in neuroblastoma SK-N-SH cells via IGF-I receptor and estrogen receptor pathways

Our previous studies have demonstrated that ginsenoside Rg1 is a novel class of potent phytoestrogen and can mimic the action of estradiol in stimulation of MCF-7 cell growth by the crosstalk between insulin-like growth factor-I receptor (IGF-IR)-dependent pathway and estrogen receptor (ER)-dependent pathway. The present study was designed to investigate the neuroprotective effects of ginsenoside Rg1 against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in human neuroblastoma SK-N-SH cells and the possible mechanisms. Pre-treatment with ginsenoside Rg1 resulted in an enhancement of survival, and significant rescue occurred at the concentration of 0.01 μM on cell viability against 6-OHDA-induced neurotoxicity. These effects could be completely blocked by IGF-IR antagonist JB-1 or ER antagonist ICI 182780. 6-OHDA arrested the cells at G₀G₁ phase and prevented S phase entry. Rg1 pre-treatment could reverse the cytostatic effect of 6-OHDA. Ginsenoside Rg1 also could attenuate 6-OHDA-induced decrease in mitochondrial membrane potential. These effects could also be completely blocked by JB-1 or ICI 182780. Furthermore, 6-OHDA-induced up-regulation of Bax and down-regulation of Bcl-2 mRNA and protein expression could be restored by Rg1 pre-treatment. Rg1 pre-treatment could reverse the down-regulation of ERα protein expression induced by 6-OHDA treatment. Cells transfected with the estrogen responsive element (ERE)-luciferase reporter construct exhibited significantly increased ERE-luciferase activity in the Rg1 presence, suggesting that the estrogenic effects of Rg1 were mediated through the endogenous ERs. These results suggest that ginsenoside Rg1 may attenuate 6-OHDA-induced apoptosis and its action might involve the activation of IGF-IR signaling pathway and ER signaling pathway.     

Microbial transformation of ginsenosides Rb1, Rb3 and Rc by Fusarium sacchari

Aims: This study examined the transformation pathways of ginsenosides G‐Rb₁, G‐Rb₃, and G‐Rc by the fungus Fusarium sacchari. Methods and Results: Ginsenosides G‐Rb₁, G‐Rb₃ and G‐Rc were isolated from leaves of Radix notoginseng, and their structural identification was confirmed using NMR. Transformation of G‐Rb₁, G‐Rb₃ and G‐Rc by Fusarium sacchari was respectively experimented. Kinetic evolutions of G‐Rb₁, G‐Rb₃ and G‐Rc and their metabolites during the cell incubation were monitored by HPLC analysis. High‐performance liquid chromatography (HPLC) was used for monitoring the transformation kinetics of bioactive compounds during F. sacchari metabolism. Conclusions: Ginsenoside C‐K was transformed by F. sacchari from G‐Rb₁ via G‐Rd or via G‐F₂, or from G‐Rb₁ via firstly Rd and then G‐F₂, and C‐Mx was transformed by F. sacchari or directly from Rb₃, or from Rb₃ via Gy‐IX, while G‐Mc was transformed by F. sacchari directly from G‐Rc. Furthermore, C‐K could be also formed from G‐Rc via notoginsenoside Fe (N‐Fe). Significance and Impact of the Study: The results showed an important practical application in the preparation of ginsenoside C‐K. As our precious research indicated C‐K possessed much more antitumor activities than C‐Mx and G‐Mc, so according to the transformation pathways proposed by this work, the production of antitumor compound C‐K may be performed by biotransformation of G‐Rb₁ previously isolated from PNLS.     

微波处理对人参皂苷生物转化影响的研究

利用微波技术分别对人参皂苷粗品及人参皂苷转化发酵液进行处理,探讨微波处理对人参皂苷生物转化效果的影响。实验结果通过高效液相色谱分析显示,经微波处理后,人参皂苷峰几乎消失,苷元峰突出,表明微波处理对人参皂苷的转化效果显著。并确定微波的最佳处理条件为微波功率30W,辐射60s。