Ginsenoside Re impacts on biotransformation products of ginsenoside Rb1 by Cellulosimicrobium cellulans sp. 21 and its mechanisms

Ginsenoside Rg3, a known anti-cancer agent, is usually prepared by enzyme-mediated and acid hydrolysis of ginsenoside Rb1 and Rd. In this study, we used the bacterium Cellulosimicrobium cellulans sp. 21 to transform Rb1 into Rg3. When Rb1 was used as the sole substrate, the transformation products included Rg3, Rh2, C-K and PPD. However, when Rb1 and Re were mixed, the yield of Rg3 was significantly higher, indicating that Re attenuates the activity of β-1,2-glucosidase secreted by C. cellulans sp. 21. β-1,2-glucosidase hydrolyzes the β-1,2-glucose moiety at the C-3 position of Rb1, but Re dose not modify enzymes that produce Rg3 by hydrolyzing glucose at the C-20 position in aglycon. We also tested the inhibitory effects from various ginsenosides on β-1,2-glucosidase, and discovered that sugar chains played key roles in inhibiting β-1,2 glucosidase activity, whereas aglycones of protopanaxadiol and protopanaxatriol had little inhibitory effects. Some sugar chains with different linkages, such as C-20, C-3 and C-6, exhibited different inhibitory effects. Overall, our findings demonstrate that a combination of substrates, in addition to microorganism-secreted enzymes, can be used for selective biotransformation. This approach provides a novel strategy for natural product preparations via microbial transformation.     

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。     

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.     

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.     

Microbial transformation of ginsenoside Rg3 to ginsenoside Rh2 by <Emphasis Type="Italic">Esteya vermicola</Emphasis> CNU 120806

In the present investigation, we successfully employed a cell-free extract of Esteya vermicola CNU 120806 to convert ginsenoside Rg3 to Rh2. Three important factors including pH, temperature and substrate concentration were optimized for the preparation of Rh₂. The optimal condition was obtained as follows: 50°C, pH 5.0 and substrate concentration of 3 mg ml⁻¹. The yield of conversion was up to 90.7%. In order to identify the specificity of the β-glucosidase activity of Esteya vermicola CNU 120806, ginsenoside Re (protopanaxatriol saponins) was treated under the same reaction system. Interestingly, no new metabolite was generated, which elucidated that the enzymatic process only occurred by hydrolysis of the terminal glucopyranosyl moieties at the C-3 carbon of ginsenoside Rg₃. The crude enzyme extract can be used for commercial ginsenoside Rh₂ preparation.     

Biotransformation of ginsenosides Re and Rg1 into ginsenosides Rg2 and Rh1 by recombinant β-glucosidase

Ginsenosides Re and Rg1 were transformed by recombinant β-glucosidase (Bgp1) to ginsenosides Rg2 and Rh1, respectively. The bgp1 gene consists of 2,496?bp encoding 831 amino acids which have homology to the glycosyl hydrolase families 3 protein domain. Using 0.1?mg enzyme ml(-1) in 20?mM sodium phosphate buffer at 37°C and pH 7.0, the glucose moiety attached to the C-20 position of ginsenosides Re and Rg1, was removed: 1?mg ginsenoside Re ml(-1) was transformed into 0.83?mg Rg2?ml(-1) (100% molar conversion) after 2.5?h and 1?mg ginsenoside Rg1?ml(-1) was transformed into 0.6?mg ginsenoside Rh1?ml(-1) (78% molar conversion) in 15?min. Using Bgp1 enzyme, almost all initial ginsenosides Re and Rg1 were converted completely to ginsenosides Rg2 and Rh1. This is the first report of the conversion of ginsenoside Re to ginsenoside Rg2 and ginsenoside Rg1 to ginsenoside Rh1 using the recombinant β-glucosidase.     

Transformation of Ginsenosides Rb1 and Re from <Emphasis Type="Italic">Panax ginseng</Emphasis> by Food Microorganisms

Ginsenosides Rb1 and Re, respectively belonging to the major protopanaxadiol and protopanaxatriol ginsenosides, were transformed using cell-free extracts from food microorganisms. Rb1 was transformed into compound K via Rd and F2 by Bifidobacterium sp. Int57, Bif. sp. SJ32, Aspergillus niger and A.␣usamii. Lactobacillus delbrueckii, and Leuconostoc paramesenteroides transformed Rb1 into Rh2 via Rd and F2. Bifidobacterium sp. SH5 transformed Rb1 into F2 via Rd. Re was transformed into Rh1 via Rg2 by Bif. sp. Int57 and Bif. sp. SJ32. A. niger transformed Re into Rh1 via Rg1. A. usamii transformed Re into Rg2. Transformation of Rb1 proceeded at a higher rate and needed less amount of enzymes than that of Re. Taken together, these processes would allow a specific bioconversion process possible to obtain specific ginsenosides using an appropriate combination of ginsenoside substrates and specific microbial enzymes.     

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.     

Conversion of major ginsenoside Rb1 to 20(S)-ginsenoside Rg3 by Microbacterium sp. GS514

Ginseng saponin, the most important secondary metabolite in ginseng, has various pharmacological activities. Many studies have been directed towards converting major ginsenosides to the more active minor ginsenoside, Rg3. Due to the difficulty in preparing ginsenoside Rg3 enzymatically, the compound has been mainly produced by either acid treatment or heating. A microbial strain GS514 was isolated from soil around ginseng roots in a field and used for enzymatic preparation of the ginsenoside Rg3. Blast results of the 16S rRNA gene sequence of the strain GS514 established that the strain GS514 belonged to the genus Microbacterium. Its 16S rRNA gene sequence showed 98.7%, 98.4% and 96.1% identity with those of M. esteraromaticum, M. arabinogalactanolyticum and M. lacticum. Strain GS514 showed a strong ability to convert ginsenoside Rb1 or Rd into Rg3. Enzymatic production of Rg3 occurred by consecutive hydrolyses of the terminal and inner glucopyranosyl moieties at the C-20 carbon of ginsenoside Rb1 showing the biotransformation pathway: Rb1-->Rd-->Rg3.     

Efficient elicitation of ginsenoside biosynthesis in cell cultures ofPanax notoginseng by using self-chemically-synthesized jasmonates

A series of fluorine and hydroxyl containing jasmonate derivatives, which were chemically synthesized in our institute, were investigated for their effects on the biosynthesis and heterogeneity of ginsenosides in suspension cultures ofPanax notoginseng cells. Compared to the control (without addition of elicitors), 100 μM of each of the jasmonate was added on day 4 to the suspension cultures ofP. notoginseng cells. It was observed that, jasmonates greatly enhanced the ginsenoside content and the ratio of Rb group to Rg group (i.e. (Rb₁+Rd)/(Rg₁+Re)) in theP. notoginseng cells. Some of the synthetic jasmonates, such as pentafluoropropyl jasmonate (PFPJA), 2-hydroxyethyl jasmonate (HEJA) and 2-hydroxyethoxyethyl jasmonate (HEEJA), could promote the ginsenoside content to 2.55±0.11, 3.65±0.13 and 2.94±0.06 mg/100 mg DW, respectively, compared to that of 0.64±0.06 mg/100 mg DW for the control and 2.17±0.04 mg/100 mg DW by the commercially available methyl jasmonate (MJA); and they could change the respective Rb: Rg ratio to 1.60±0.04, 1.87±0.01 and 1.56±0.05, compared to that of 0.47±0.01 for the control and 1.42±0.06 by MJA. The results suggest that suitable esterification of MJA with fluorine or hydroxyl group could increase the elicitation activity to induce plant secondary metabolism. The information obtained from this study is useful for hyper-production of heterogeneous products by plant cell cultures.     

Efficient biotransformation for preparation of pharmaceutically active ginsenoside Compound K by Penicillium oxalicum sp. 68

Pathogenic fungus Penicillium oxalicum sp. 68 was screened from soil and identified by ITS sequencing. The strain was found to be able to transform protopanaxadiol-type ginsenosides to produce a series of bioactive metabolites. Glycosidase from the culture of P. oxalicum sp. 68 was partially purified with a simple two-step procedure consisting of DEAE-cellulose chromatography and ammonium sulfate precipitation. Bioactive ginsenoside Compound K was prepared selectively and efficiently by biotransformation of ginsenosides Rb1, Rb2, Rc and Rd using the partially purified glycosidase. The optimal conditions for transforming Rb1 into Compound K were pH 4.0, 55 °C and 0.5 mg mL^?1 Rb1. The sole product is Compound K and the maximum yield reached 87.7 % (molar ratio). The transformation pathways of Rb1, Rb2, Rc and Rd are Rb1→Rd→F2→Compound K, Rb2→CO→CY→Compound K, Rc→Mb→Mc→Compound K and Rd→F2→Compound K, respectively. This biotransformation method showed great potential for preparing minor bioactive ginsenosides, especially Compound K, in the pharmaceutical industry because of its high specificity and favorable environmental compatibility.     

Enzymatic biotransformation of ginsenoside Rb1 to 20(<Emphasis Type="Italic">S</Emphasis>)-Rg3 by recombinant β-glucosidase from <Emphasis Type="Italic">Microbacterium esteraromaticum</Emphasis>

Microbacterium esteraromaticum was isolated from ginseng field. The β-glucosidase gene (bgp1) from M. esteraromaticum was cloned and expressed in Escherichia coli BL21 (DE3). The bgp1 gene consists of 2,496 bp encoding 831 amino acids which have homology to the glycosyl hydrolase family 3 protein domain. The recombinant β-glucosidase enzyme (Bgp1) was purified and characterized. The molecular mass of purified Bgp1 was 87.5 kDa, as determined by SDS-PAGE. Using 0.1 mg ml⁻¹ enzyme in 20 mM sodium phosphate buffer at 37°C and pH 7.0, 1.0 mg ml⁻¹ ginsenoside Rb1 was transformed into 0.444 mg ml⁻¹ ginsenoside Rg3 within 6 h. The Bgp1 sequentially hydrolyzed the outer and inner glucose attached to the C-20 position of ginsenosides Rb1. Bgp1 hydrolyzed the ginsenoside Rb1 along the following pathway: Rb1 → Rd → 20(S)-Rg3. This is the first report of the biotransformation of ginsenoside Rb1 to ginsenoside 20(S)-Rg3 using the recombinant β-glucosidase.     

HPLC法同时测定人参皂苷Rb1、Rc、Rd、Rg3、CK和Rh2

目的:建立高效液相色谱法同时测定人参皂苷Rb1、Rc、Rd、Rg3、CK和Rh2的方法.方法:采用ODSC18(4.6 mm×150 mm)色谱柱,流动相乙腈-0.05%磷酸水,梯度洗脱,流速1 Ml/min,检测波长203 nm,柱温35 ℃.结果:人参皂苷Rb1、Rc、Rd、Rg3、CK和Rh2分离效果良好,线性关...     

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.     

Improvement of ginsenoside production by jasmonic acid and some other elicitors in hairy root culture of ginseng (Panax ginseng C. A. Meyer)

Summary Hairy root cultures of Panax ginseng, established after the infection of root sections with Agrobacterium rhizogenes KCTC 2703, were cultured in phytohormone-free Murashige and Skoog (MS) liquid medium containing different concentrations of jasmonic acid and some other elicitors, in order to promote ginsenoside accumulation. Jasmonic acid in the range 1.0−5.0 mg l⁻¹ (4.8–23.8 μM) strongly improved total ginsenoside production in ginseng hairy roots. Peptone (300 mg l⁻¹) also showed some effect on ginsenoside improvement; however its effect was much weaker than that of jasmonic acid. Ginsenoside content and productivity were 58.65 and 504.39 mg g⁻¹, respectively. The Rb group of ginsenoside content was increased remarkably by jasmonic acid, while Rg group ginsenoside content changed only slightly compared to controls. However, jasmonic acid also strongly inhibited ginseng hairy root growth.     

Enzymatic synthesis of two ginsenoside Re-beta-xylosides.

Two new beta-xylosyl derivatives of ginsenoside Re, 20(S)-protopanaxatriol 6-O-alpha-L-rhamnopyranosyl-(1 --> 2)-[beta-D-xylopyranosyl-(1 --> 4)]-beta-D-glucopyranosyl-20-O-beta-D-glucopyranoside and 20(S)-protopanaxatriol 6-O-alpha-L-rhamnopyranosyl-(1 --> 2)-[beta-D-xylopyranosyl-(1 --> 6)]-beta-D-glucopyranosyl-20-O-beta-D-glucopyranoside, were respectively synthesized from p-nitrophenyl beta-D-xylopyranoside and phenyl beta-D-xylopyranoside as donors and ginsenoside Re as the acceptor in 25% acetone and acetonitrile by a cellulase preparation from Trichoderma viride and a beta-galactosidase preparation from Aspergillus oryzae. The latter enzyme preparation also catalyzed the hydrolysis of ginsenoside Re to the minor saponin, ginsenoside Rg2.     

人参皂苷Rb1、Rg1、Re对白血病细胞株KG1α增殖的影响

目的:探讨人参皂苷Rb1、Rg1、Re对急性髓系白血病细胞株(KG1α)增殖的影响.方法:取对数生长期KG1α细胞,分设人参皂苷Rb1、Rg1、Re组和常规培养组,以MTT比色法检测作用24h、48h、72h时对KG1α细胞增殖抑制作用,并计算Rb1的IC_(50)值,以此浓度为工作浓度,设常规培养组和处理组,台盼蓝计数法观察对KG1α细胞增殖的影响;流式细胞术测定细胞周期分布的变化.结果:MTT、台盼蓝计数显示人参皂苷单体Rb1、Rg1可抑制KG1α细胞增殖,呈浓度依赖性,以Rb1抑制效应最佳,于作用48h抑制率最高.台盼蓝计数显示人参皂苷单体Rb1-120μmol/L作用48h时抑制率达50.22%;流式细胞术结果提示,与对照组比较,Rb1-120μmol/L组G_2/M期KG1α细胞比例增加(P<0.05).结论:Rb1可抑制KG1α细胞体外增殖,其增殖抑制作用与将KG1α细胞阻滞于G_2/M期有关.     

Isolation and characterization of novel ginsenoside-hydrolyzing glycosidase from Microbacterium esteraromaticum that transforms ginsenoside Rb2 to rare ginsenoside 20(S)-Rg3

Ginsenoside Rb2 was transformed by recombinant glycosidase (Bgp2) into ginsenosides Rd and 20(S)-Rg3. The bgp2 gene consists of 2,430 bp that encode 809 amino acids, and this gene has homology to the glycosyl hydrolase family 2 protein domain. SDS-PAGE was used to determine that the molecular mass of purified Bgp2 was 87 kDa. Using 0.1 mg ml^?1 of enzyme in 20 mM sodium phosphate buffer at 40 °C and pH 7.0, 1.0 mg ml^?1 ginsenoside Rb2 was transformed into 0.47 mg ml^?1 ginsenoside 20(S)-Rg3 within 120 min, with a corresponding molar conversion yield of 65 %. Bgp2 hydrolyzed the ginsenoside Rb2 along the following pathway: Rb2 → Rd → 20(S)-Rg3. This is the first report of the biotransformation of ginsenoside Rb2 to ginsenoside 20(S)-Rg3 using the recombinant glycosidase.     

Enzymatic preparation of genuine prosapogenin, 20(S)-ginsenoside Rh1, from ginsenosides Re and Rg1

It was found that a lactase preparation from Penicillium sp. nearly quantitatively hydrolyzed ginsenosides Re and Rg1, which are major saponins in roots of Panax ginseng, to a minor saponin, 20(S)-ginsenoside Rh1 [6-O-beta-D-glucopyranosyl-20(S)-protopanaxatriol]. This is the first report on the enzymatic preparation of ginsenoside Rh1 with a high efficiency. This enzyme also readily hydrolyzed ginsenoside Rg2 to ginsenoside Rh1.     

Production of ginsenoside aglycons and Rb1 deglycosylation pathway profiling by HPLC and ESI-MS/MS using Sphingobacterium multivorum GIN723

Using enrichment culture, Sphingobacterium multivorum GIN723 (KCCM80060) was isolated as having activity for deglycosylation of compound K and ginsenoside F1 to produce ginsenoside aglycons such as S-protopanaxadiol (PPD(S)) and S-protopanaxatriol (PPT(S)). Through BLAST search, purified enzyme from S. multivorum GIN723 was revealed to be the outer membrane protein. The purified enzyme from S. multivorum GIN723 has unique specificity for the glucose moiety. However, it has activity with PPD and PPT group ginsenosides such as ginsenosides Rb1, Rb2, Rb3, Rc, F2, CK, Rh2, Re, and F1. From these results, it was predicted that the enzyme has activity on several ginsenosides. Therefore, the biotransformation pathway from Rb1, which is a major, highly glycosylated compound of ginseng, was analyzed using high-performance liquid chromatography and electrospray ionization mass spectrometry/mass spectrometry. The dominant biotransformation pathway from Rb1 to PPD(S) was determined to be Rb1 → Gp-XVII → Gp-LXXV → CK → PPD(S). S. multivorum GIN723 can be used as a whole cell biocatalyst because its activity as whole cells is nine times higher than its activity as cell extracts. The specific activity of whole cells is 2.89 nmol/mg/min in the production of PPD(S). On the other hand, the specific activity of cell extracts is 0.32 nmol/mg/min. The productivity of this enzyme in whole cell form is 500 mg/1 l of cultured cell. Its optimum reaction condition is 10 mM of calcium ions added to a phosphate buffer with a pH of 8.5.