Evolving the 3-O/6-O regiospecificity of a microbial glycosyltransferase for efficient production of ginsenoside Rh1 and unnatural ginsenoside.

Glycosyltransferase is a popular and promising enzyme to produce high-value-added natural products. Rare ginsenoside Rh1 and unnatural ginsenoside 3ß-O-Glc-PPT are promising candidates for drugs. Herein, the microbial glycosyltransferase UGTBL1 was able to catalyze the 20(S)-protopanaxatriol (PPT) 3-O/6-O-glycosylation with poor 6-O-regiospecificity. A structure-guided strategy of mutations involving loop engineering, PSPG motif evolution, and access tunnel engineering was proposed to engineer the enzyme UGTBL1. The variant I62R/M320H/P321Y/N170A from protein engineering achieved a great improvement in 6-O regioselectivity which increased from 10.98 % (WT) to 96.26 % and a booming conversion of 95.57 % for ginsenoside Rh1. A single mutant M320W showed an improved 3-O regioselectivity of 84.83 % and an increased conversion of 98.13 % for the 3ß-O-glc-PPT product. Molecular docking and molecular dynamics (MD) simulations were performed to elucidate the possible molecular basis of the regiospecificity and catalytic activity. The unprecedented high titer of ginsenoside Rh1 (20.48 g/L) and 3ß-O-Glc-PPT (18.04 g/L) was attained with high regioselectivity and yields using fed-batch cascade reactions from UDPG recycle, which was the highest yield reported to date. This work could provide an efficient and cost-effective approach to the valuable ginsenosides.

How to More Effectively Obtain Ginsenoside Rg5: Understanding Pathways of Conversion.

Ginsenoside Rg5, a relatively uncommon secondary ginsenoside, exhibits notable pharmacological activity and is commonly hypothesized to originate from the dehydration of Rg3. In this work, we compared different conversion pathways using Rb1, R-Rg3 and S-Rg3 as the raw material under simple acid catalysis. Interestingly, the results indicate that the conversion follows this reaction activity order Rb1 > S-Rg3 > R-Rg3, which is contrary to the common understanding of Rg5 obtained from Rg3 by dehydration. Our experimental results have been fully confirmed by theoretical calculations and a NOESY analysis. The DFT analysis reveals that the free energies of S-Rg3 and R-Rg3 in generating carbocation are 7.56 mol/L and 7.57 mol/L, respectively, which are significantly higher than the free energy of 1.81 mol/L when Rb1 generates the same carbocation. This finding aligns with experimental evidence suggesting that Rb1 is more prone to generating Rg5 than Rg3. The findings from the nuclear magnetic resonance (NMR) analysis suggest that the fatty chains (C22-C27) in R-Rg3 and S-Rg3 adopt a Gauche conformation and an anti conformation with C16-C17 and C13-C17, respectively, due to the relatively weak repulsive van der Waals force. Therefore, the configuration of R-Rg3 is more conducive to the formation of intramolecular hydrogen bonds between 20C-OH and 12C-OH, whereas S-Rg3 lacks this capability. Consequently, this also explains the fact that S-Rg3 is more prone to dehydration to generate Rg5 than R-Rg3. Additionally, our research reveals that the synthetic route of Rg5 derived from protopanaxadiol (PPD)-type ginsenosides (including Rb1, Rb2, Rb3, Rc and Rd) exhibits notable advantages in terms of efficacy, purity and yield when compared to the pathway originating from Rg3. Moreover, this study presents a highly effective and practical approach for the extensive synthesis of Rg5, thereby facilitating the exploration of its pharmacological properties and potential application in drug discovery.

Effects of rare ginsenoside on idiopathic pulmonary fibrosis / 中国药科大学学报

@#To investigate whether rare ginsenosides could alleviate idiopathic pulmonary fibrosis (IPF), C57BL/6 mice were randomly divided into control group, bleomycin (BLM)-induced IPF group, rare ginsenoside Rk1 group, rare ginsenoside Rk3 group, rare ginsenoside Rh4 group and rare ginsenoside Rg5 group.All mice except those in the control group were given bleomycin injection.The IPF model was established by BLM for 28 days.The treatment group was given ginsenoside intragastrically at the same time.After the experiment, the lung tissues of mice were collected and the pathological changes of the mice lungs were observed.The content of hydroxyproline (HYP) in mouse lung tissue was measured.The expression of IPF-related genes in mouse lung tissues was detected.In in vitro experiments, Medical Research Council cell strain-5 (MRC-5) was used to induce IPF cell model using transforming growth factor-β1 (10 ng/mL).The effects of four saponins on the expression of IPF-related genes were analyzed by MTT assay, HYP content determination and RT-qPCR.All four rare ginsenosides could effectively alleviate the pathological process such as alveolar structure destruction caused by IPF, reduce the content of HYP, and down-regulate the expression of IPF-related genes, indicating that rare ginsenosides can effectively alleviate IPF.

Gram-Scale Production of Ginsenoside F1 Using a Recombinant Bacterial ß-Glucosidase.

Naturally occurring ginsenoside F1 (20-O-ß-D-glucopyranosyl-20(S)-protopanaxatriol) is rare. Here, we produced gram-scale quantities of ginsenoside F1 from a crude protopanaxatriol saponin mixture comprised mainly of Re and Rg1 through enzyme-mediated biotransformation using recombinant ß-glucosidase (BgpA) cloned from a soil bacterium, Terrabacter ginsenosidimutans Gsoil 3082T. In a systematic step-by-step process, the concentrations of substrate, enzyme, and NaCl were determined for maximal production of F1. At an optimized NaCl concentration of 200 mM, the protopanaxatriol saponin mixture (25 mg/ml) was incubated with recombinant BgpA (20 mg/ml) for 3 days in a 2.4 L reaction. Following octadecylsilyl silica gel column chromatography, 9.6 g of F1 was obtained from 60 g of substrate mixture at 95% purity, as assessed by chromatography. These results represent the first report of gramscale F1 production via recombinant enzyme-mediated biotransformation.

Explore of synthesis method of different configuration pseudo-ginsenoside Rg2, pseudo-ginsenoside Rh1, and pseudo-PPT / 中草药

Objective To explore an efficient preparation method of pseudo-ginsenoside Rg2, pseudo-ginsenoside Rh1, and pseudo-PPT, as to provide theoretical basis for the preparation of pseudo-ginsenosides and pseudo-PPT. Methods Ginsenosides Re, Rh1, and PPT as raw material, via a simple three-step called acetylation, elimination-addition and saponification achieve the preparation of 20 (E/Z)-pseudo-ginsenoside Rg2, 20 (E/Z)-pseudo-ginsenoside Rh1, and 20 (E/Z)-pseudo-PPT. The detailed structure elucidation of the compounds were obtained by NMR, HR-ESI-MS, and IR. Results The production rates of 20 (E/Z)-pseudo-ginsenoside Rg2, 20 (E/Z)-pseudo-ginsenoside Rh1, and 20 (E/Z)-pseudo-PPT were 41%/13%, 43%/11%, and 56%/15%, respectively. Among them, 20 (Z)-pseudo-PPT was identified as new triterpenoid. Conclusion The method through the price relatively cheap and easy gain reactants ginsenoside Re, ginsenoside Rh1, and PPT prepared active better pseudo-ginsenoside Rg2, pseudo-ginsenoside Rh1, and pseudo-PPT, the method for the preparation of other types of pseudo-ginsenoside provides a new train of thought. At the same time, the method is simple and the yield is high.

20(R)-Ginsenoside-Rh19, a novel ginsenoside from alkaline hydrolysates of total saponins in stems-leaves of Panax ginseng / 中草药

Objective: To study the chemical constituents of alkaline hydrolysates of total saponins from the stems and leaves of Panax ginseng. Methods: The chemical constituents were isolated and purified by various chromatographic methods, and the chemical structures were identified by NMR and MS spectra analyses. Results: A total of 30 compounds were isolated and identified. Among them, 28 were determined as 20(S)-protopanaxadiol (1), 20(R)-protopanaxadiol (2), dammar-20(21),24-diene-3β,6α,12β-triol (3), dammar-20(22)E,24-diene-3β,6α,12β-triol (4), 20(S)-protopanaxatriol (5), 20(R)-protopanaxatriol (6), 20(S)-ginsenoside Rh2 (7), 20(R)-ginsenoside Rh2 (8), ginsenoside Rh16 (9), isoginsenoside Rh3 (10), 20(S)-dammar-3β,6α,12β,20,25-pentol (11), 20(R)-dammar-3β,6α,12β,20,25-pentol (12), ginsenoside Rk3 (13), 20(S)-ginsenoside Rh1 (14), 20(R)-ginsenoside Rh1 (15), ginsenoside F1 (16), ginsenoside Rh19 (17), 20(R)-ginsenoside Rh19 (18), dammar-20(22)E-ene-3β,6α,12β,25-tetrol (19), notoginsenoside T2 (20), ginsenoside Rg6 (21), 20(22)E-ginsenoside F4 (22), ginsenoside Rk1 (23), 20(S)-ginsenoside Rg3 (24), 20(R)-ginsenoside Rg3 (25), 20(S)-ginsenoside Rg2 (26), 20(R)-ginsenoside Rg2 (27), and 3β,6α,12β,25-tetrahydroxy-dammar-20(22)E-ene-6-O-α-L-rhamno- pyranosyl-(1→2)-β-D-glucopyranoside (28). Conclusion: Compound 18 is a new saponin. Compounds 3, 4, 11, 12, and 19 are rare dammarane-type triterpenes, and 7-10, 13-18, and 20-28 are rare ginsenosides.

Preparation of rare ginsenoside by transformation of ginsenoside Re catalyzed with aspartic acid / 中草药

Objective: A new, environment-friendly and efficient method for the preparation of rare ginsenoside Rg6, F4, Rk3, and Rh4 was established, which provides a theoretical basis for preparing rare ginsenosides. Methods: Rare ginsenoside was prepared by hydrolyzing ginsenoside Re using aspartic acid as the catalyst, through semi preparative HPLC, the target compounds Rg6, F4, Rk3, and Rh4 were rapidly separated from the degradation products, quantitative analysis, and structure identification by HPLC and NMR. Results: Ginsenoside Re was hydrolyzed by aspartic acid according to the ratio 10∶1 at 120℃ for 1 h, the conversion rate of ginsenoside Re was 100%, the yields of rare ginsenoside Rg6, F4, Rk3, and Rh4 were 11.2%, 13.1%, 20.6%, and 24.3%, respectively, and the purity of the four compounds were all above 99%. Conclusion: The method is simple, low-cost, and non-pollution for environment, the research has important application value for the development of green environmental protection of rare ginsenosides drugs and health food.

20(S)-Ginsenoside-Rf2, a novel triterpenoid saponin from stems and leaves of Panax ginseng / 中草药

Objective: To study the chemical constituents of saponins in the stems and leaves of Panax ginseng. Methods: The chemical constituents were isolated and purified by various chromatographic methods, and the chemical structures were identified by NMR and MS data analyses. Results: A total of 39 compounds were isolated and identified. Among them, 17 compounds were determined as ginsenoside Re (1), 20(S)-ginsenoside Rh1 (2), 20(R)-ginsenoside Rh1 (3), ginsenoside Rh5 (4), 20(E)-ginsenoside F4 (5), ginsenoside F2 (6), 20(S)-ginsenoside Rg3 (7), 20(R)-ginsenoside Rg3 (8), 20(S)-ginsenoside Rf2 (9), 20(R)-ginsenoside Rf2 (10), 20(S)- protopanaxadiol (11), 20(R)-protopanaxadiol (12), 20(S)-ginsenoside Rh2 (13), 20(R)-ginsenoside Rh2 (14), 20(S)-protopanaxatriol (15), 20(R)-protopanaxatriol (16), and ginsenoside Rd (17). Conclusion: Compound 9 is a new saponin. Compounds 2-10, 13, and 14 are rare ginsenosides.

Transformation of rare ginsenoside Compound K from ginsenoside Rb1 catalyzed by snailase immobilization onto microspheres / 中草药

Objective: To prepare snailase immobilization onto microspheres and to optimize the process conditions for the transformation of rare ginsenoside Compound K (CK) from ginsenoside Rb1 (Rb1) catalyzed by snailase immobilization onto microspheres. Methods: Considering the recovery rate of enzyme activity as the target, crosslink-embedding method was used for preparing the snailase immobilization onto microspheres and optimizing the preparation technology by orthogonal test. Furthermore, the enzyme characterization of temperature, enzymatic properties of pH value, thermal stability, pH stability, and storage stability was studied, and the effectiveness of temperature, concentration reaction time, and transformational times on the bioconversion rate was studied to optimize the preparation conditions. Results: The best process was achieved at 2% sodium alginate, 2% CaCl2, SiO2 and snail enzyme mass ratio of 1:1, with the above conditions, the enzyme activity recovery rate was 81.94%, immobilization snailase and free snailase exhibit different properties about thermal stability and pH stability, the optimum temperature was 60℃, and the optimum pH was 5.0. Under these conditions, the snailase immobilization onto microspheres remained 55.17% enzyme activity when storaged at 15℃ for 30 d. The best process was achieved at 55℃, the substrate concentration was 1.0 mg/mL, the conversion time was 36 h, the effective continuous transformational times were five rounds and the average transformational ratio for rare ginsenoside CK was up to 36.79%. Conclusion: The results concluded from the experiments indicate that the immobilization procedure could promote the resistance of enzyme against temperature, pH shift, and some other tough reaction conditions, meanwhile prolong the enzymatic lifetime for storage. The bioconversion rate is impoved and it is feasible to prepare rare ginsenoside CK by enzymolysis with snailase immobilization onto microspheres. Besides, the condition is moderate and it is suitable for industrialization.