Methanol-involved heterogeneous transformation of ginsenoside Rb1 to rare ginsenosides using heteropolyacids embedded in mesoporous silica with HPLC-MS investigation.

Background: The biological activity and pharmacological effects of rare ginsenosides have been proven to be superior to those of the major ginsenosides, but they are rarely found in ginseng. Methods: Ginsenoside Rb1 was chemically transformed with the involvement of methanol molecules by a synthesized heterogeneous catalyst 12-HPW@MeSi, which was obtained by the immobilization of 12-phosphotungstic acid on a mesoporous silica framework. High-performance liquid chromatography coupled with mass spectrometry was used to identify the transformation products. Results: A total of 18 transformation products were obtained and identified. Methanol was found to be involved in the formation of 8 products formed by the addition of methanol molecules to the C-24 (25), C-20 (21) or C-20 (22) double bonds of the aglycone. The transformation pathways of ginsenoside Rb1 involved deglycosylation, addition, elimination, cycloaddition, and epimerization reactions. These pathways could be elucidated in terms of the stability of the generated carbenium ion. In addition, 12-HPW@MeSi was able to maintain a 60.5% conversion rate of Rb1 after 5 cycles. Conclusion: Tandem and high-resolution mass spectrometry analysis allowed rapid and accurate identification of the transformation products through the characteristic fragment ions and neutral loss. Rare ginsenosides with methoxyl groups grafted at the C-25 and C-20 positions were obtained for the first time by chemical transformation using the composite catalyst 12-HPW@MeSi, which also enabled cyclic heterogeneous transformation and facile centrifugal separation of ginsenosides. This work provides an efficient and recyclable strategy for the preparation of rare ginsenosides with the involvement of organic molecules.

Biotransformation of Ginsenoside Rb1 to Ginsenoside Rd and 7 Rare Ginsenosides Using Irpex lacteus with HPLC-HRMS/MS Identification.

The biotransformation of ginsenosides using microorganisms represents a promising and ecofriendly approach for the production of rare ginsenosides. The present study reports on the biotransformation of ginsenoside Rb1 using the fungus Irpex lacteus, resulting in the production of ginsenoside Rd and seven rare ginsenosides with novel structures. Employing high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry, the identities of the transformation products were rapidly determined. Two sets of isomers with molecular weights of 980.56 and 962.55 were discovered among the seven rare ginsenosides, which were generated through the isomerization of the olefin chain in the protopanaxadiol (PPD)-type ginsenoside skeleton. Each isomer exhibited characteristic fragment ions and neutral loss patterns in their tandem mass spectra, providing evidence of their unique structures. Time-course experiments demonstrated that the transformation reaction reached equilibrium after 14 days, with Rb1 initially generating Rd and compound 5, followed by the formation of other rare ginsenosides. The biotransformation process catalyzed by I. lacteus was found to involve not only the typical deglycosylation reaction at the C-20 position but also hydroxylation at the C-22 and C-23 positions, as well as hydrogenation, transfer, and cyclization of the double bond at the C-24(25) position. These enzymatic capabilities extend to the structural modification of other PPD-type ginsenosides such as Rc and Rd, revealing the potential of I. lacteus for the production of a wider range of rare ginsenosides. The transformation activities observed in I. lacteus are unprecedented among fungal biotransformations of ginsenosides. This study highlights the application of a medicinal fungi-based biotransformation strategy for the generation of rare ginsenosides with enhanced structural diversity, thereby expanding the variety of bioactive compounds derived from ginseng.

Continuous-variable quantum key distribution robust against environmental disturbances.

Continuous variable quantum key distribution (CV-QKD) can guarantee that two parties share secure keys even in the presence of an eavesdropper. However, the polarization direction of the coherent state transmitted in CV-QKD is susceptible to environmental disturbances during channel transmission, making it difficult to share keys consistently over long periods of time. Therefore, a CV-QKD system that can resist environmental disturbance is very urgent. In this paper, we propose a new optical architecture for CV-QKD based on the Faraday-Michelson interference (FMI) structure, and finally form an all-single-mode (SM) fiber-based stable CV-QKD system which employs transmitted local oscillator (TLO) scheme and discrete modulation coherent state (DMCS) protocol. Specifically, since the Faraday mirror rotates the polarization direction of light by 90o, the birefringence effect of light can be effectively dealt with, thus ensuring the same polarization state of light before and after reflection. The final simulation results show that the theoretical secret key rate of this scheme can reach 139 kbps at 70 km, which can further improve the stability and robustness of CV-QKD in the real environment, and provide technical support for the next-generation high-stability QKD system.

Heterogeneous Transformation of Ginsenoside Rb1 with Ethanol Using Heteropolyacid-Loaded Mesoporous Silica and Identification by HPLC-MS.

Rare ginsenosides with major pharmacological effects are barely present in natural ginseng and are required to be obtained by transformation. In the current study, ginsenoside Rb1 was chemically transformed with the involvement of ethanol molecules to prepare rare ginsenosides using the synthesized heterogeneous catalyst 12-HPW@MeSi. A total of 16 transformation products were obtained and identified using high-performance liquid chromatography coupled with multistage tandem mass spectrometry and high-resolution mass spectrometry. Ethanol molecules were involved in the production of 6 transformation products by adding to the C-20(21), C-20(22), or C-24(25) double bonds on the aglycone to produce ethoxyl groups at the C-25 and C-20 positions. Transformation pathways of ginsenoside Rb1 are summarized, which involve deglycosylation, elimination, cycloaddition, epimerization, and addition reactions. In addition, 12-HPW@MeSi was recyclable through a simple centrifugation, maintaining an 85.1% conversion rate of Rb1 after 3 cycles. This work opens up an efficient and recycled process for the preparation of rare ginsenosides with the involvement of organic molecules.

Quantitative and Differential Analysis between Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd. Using HPLC-MS and GC-MS Coupled with Multivariate Statistical Analysis.

Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd. have different clinical efficacies, with the former typically used to treat typhoid fever and the latter mainly used to clear liver heat. The differences in their clinical efficacy are closely related to their complex chemical composition, especially the active components. In this study, the saponins and volatile oils in two varieties of Radix Bupleuri grown in different regions were extracted and analyzed using high-performance liquid chromatography (HPLC) and gas chromatography coupled with mass spectrometry (MS), and the absolute contents of five saikosaponins were accurately quantified using an established HPLC-MS method in the multiple reaction monitoring mode. Multivariate statistical analysis was performed to reveal the difference in the active components between the two varieties. The saikosaponin content was significantly affected by variety and growing region, with all five saikosaponins being significantly higher in Bupleurum chinense DC. than in Bupleurum scorzonerifolium Willd. The results of principal component analysis and hierarchical cluster analysis show a clear distinction between the two varieties in terms of both saponins and volatile oils. Twenty-one saponins, including saikosaponin b2 and b1, and fifty-two volatile oils, including 2-tetradecyloxirane and chloromethyl cyanide, were screened and identified as differential compounds contributing to the significant difference between the two varieties. These compounds may also be responsible for the difference in clinical efficacy between Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd. All the results suggest that the accumulation and diversity of active components in Radix Bupleuri are significantly affected by the variety. In contrast to previous reports, this study provides the absolute contents of five saikosaponins in Radix Bupleuri of different varieties and reduces the influence of the growing region on the analytical results by collecting samples from different regions. The results of this study may provide a reference for the identification and quality evaluation of different varieties of Radix Bupleuri.

Multi-rate and multi-protocol continuous-variable quantum key distribution.

Quantum key distribution (QKD) is an effective solution to ensure the secure transmission of information. However, for the large-scale application of QKD, the interoperability and flexibility of the transmitter and receiver are urgent issues to be solved. Here, for the first time, to the best of our knowledge, we experimentally verify the feasibility of one continuous-variable (CV) QKD system to achieve multiple protocols and rates. The flexibility of the system comes from the modulator realizing multiple protocols and a broadband coherent detector realizing multiple symbol rates. The results show that this system can switch between different rates and protocols to generate the secure key, and reveal its similarity to classical optical communication. Therefore, It can be adjusted according to user needs and provides a system-level solution for building a flexible quantum network.

Cloning and characterization of thermophilic endoglucanase and its application in the transformation of ginsenosides.

A novel endoglucanase (BcelFp) was identified from Fervidobaterium pennivorans DSM9078 which had biotransformation activity for protopanaxadiol (PPD)-type ginsenosides. Sequence analysis of BcelFp revealed that it could be classified into glycoside hydrolase family 5 (GH5). The gene encoding a 323-amino acid protein was cloned and expressed in Escherichia coli. The recombinant enzyme was purified, and its molecular weight was approximate 37 kDa. The recombinant BcelFp exhibited an optimal activity at 95 oC and pH 5.5 and showed high thermostability. The endoglucanase had high selectivity for cleaving the outer glucose moiety at the C3 carbon of ginsenoside Rb1, Rb2, Rc and Rd, which produced stronger pharmacologically active gypenoside XVII (GypXVII), Compound O (CO), Compound Mc1 (CMc1) and F2, respectively. The Km values for Rb1, Rb2, Rc and Rd were 3.66 ± 0.04 µM, 4.02 ± 0.12 µM, 5.95 ± 0.03 µM, 0.67 ± 0.006 µM, respectively. The kcat/Km value of BcelFp for ginsenoside Rd was 27.91 mM-1s-1, which was much higher than that of the previously enzymes. This study was the first report of the highly efficient and selective transformation of GypXVII, CO, CMc1 and F2 from Rb1, Rb2, Rc and Rd by a GH5-family thermophilic endoglucanase.

Characterization of a novel thermophilic beta-glucosidase from Thermotoga sp. and its application in the transformation of notoginsenoside R1.

A novel ß-glucosidase (Thglu3) was identified from Thermotoga sp. which had biotransformation activity for notoginsenoside R1 (NR-R1). Sequence analysis of Thglu3 revealed that it could be classified into glycoside hydrolase family 3 (GH3). The gene encoding a 719-amino acid protein was cloned and expressed in Escherichia coli. The recombinant enzyme was purified, and its molecular weight was approximately 81 kDa. The recombinant Thglu3 exhibited an optimal activity at 75 °C and pH 6.4. The ß-glucosidase had high selectivity for cleaving the outer glucose moiety at the C20 position of NR-R1, which produced the more pharmacologically active notoginsenoside R2 (NR-R2). Under the optimal reaction conditions for gram-scale production, 30 g NR-R1 was transformed to NR-R2 using 20 g crude enzyme at pH 6.4 and 75 °C within 1 h with a molar yield of 93%. This study was the first report of the highly efficient and selective gram-scale transformation of NR-R2 from NR-R1 by a thermophilic ß-glucosidase.

Quantitative Analysis and Differential Evaluation of Radix Bupleuri Cultivated in Different Regions Based on HPLC-MS and GC-MS Combined with Multivariate Statistical Analysis.

The quality of Radix Bupleuri is greatly affected by its growing environment. In this study, Radix Bupleuri samples that were harvested from seven different regions across northwest China were examined by high-performance liquid chromatography (HPLC) and gas chromatography (GC) coupled with mass spectrometry (MS) to reveal significant differences in quality contributed by the cultivation region. An HPLC-MS method was firstly established and used in the multiple reaction monitoring mode for the quantitative analysis of five saikosaponins in Radix Bupleuri so as to evaluate the difference in the absolute content of saikosaponins attributable to the cultivation region. The effect on the components of Radix Bupleuri was further investigated based on the profiles of the representative saponins and volatile compounds, which were extracted from the Radix Bupleuri samples and analyzed by HPLC-MS and GC-MS. Multivariate statistical analysis was employed to differentiate the Radix Bupleuri samples cultivated in different regions and to discover the differential compositions. The developed quantitative method was validated to be accurate, stable, sensitive, and repeatable for the determination of five saikosaponins. Further statistical tests revealed that the collected Radix Bupleuri samples were distinctly different from each other in terms of both saponins and volatile compounds, based on the provinces where they were grown. In addition, twenty-eight saponins and fifty-eight volatile compounds were identified as the differentially accumulated compositions that contributed to the discrimination of the Radix Bupleuri samples. The Radix Bupleuri samples grown in Shouyang county showed the highest content of saikosaponins. All of the results indicated that the cultivation region significantly affected the accumulation and diversity of the main chemical components of Radix Bupleuri. The findings of this research provide insights into the effect of the cultivation region on the quality of Radix Bupleuri and the differentiation of Radix Bupleuri cultivated in different regions based on the use of HPLC-MS and GC-MS combined with multivariate statistical analysis.

Enzymatic Biotransformation of Gypenoside XLIX into Gylongiposide I and Their Antiviral Roles against Enterovirus 71 In Vitro.

Biotransformation of specific saponins in the valuable medical plants to increase their bioavailability and pharmaceutical activities has attracted more and more attention. A gene encoding a thermophilic glycoside hydrolase from Fervidobaterium pennivorans DSM9078 was cloned and expressed in Escherichia coli. The purified recombinant enzyme, exhibiting endoglucanase cellulase activity, was used to transform gypenoside XLIX into gylongiposide I via highly selective and efficient hydrolysis of the glucose moiety linked to the C21 position in gypenoside XLIX. Under the optimal reaction conditions for large scale production of gylongiposide I, 35 g gypenoside XLIX was transformed by using 20 g crude enzyme at pH 6.0 and 80 °C for 4 h with a molar yield of 100%. Finally, 11.51 g of gylongiposide I was purified using a silica gel column with 91.84% chromatographic purity. Furthermore, inhibitory activities of gypenoside XLIX and gylongiposide I against Enterovirus 71 (EV71) were investigated. Importantly, the EC50 of gypenoside XLIX and gylongiposide I calculated from viral titers in supernatants was 3.53 µM and 1.53 µM, respectively. Moreover, the transformed product gylongiposide I has better anti-EV71 activity than the glycosylated precursor. In conclusion, this enzymatic method would be useful in the large-scale production of gylongiposide I, which would be a novel potent anti-EV71 candidate.

Simple continuous-variable quantum key distribution scheme using a Sagnac-based Gaussian modulator.

Continuous-variable quantum key distribution (CV-QKD) is a protocol that uses quantum mechanics to ensure that the distribution of an encryption key is secure even in the presence of eavesdroppers. The wide application of CV-QKD requires low cost, system simplicity, and system stability. However, owing to the particularity of Gaussian modulation in CV-QKD, an amplitude modulator (AM) and a bias controller are required, making the system structure complex and unstable. In this Letter, we achieve two-dimensional Gaussian modulation with only one phase modulator (PM) and a Sagnac ring structure, which significantly reduces the complexity of the system. We test the Gaussian modulation stability for 10 h, and the result shows that the expected secure key rate can be maintained at 80 kbit/s under a transmission distance of 50 km. This scheme opens up new, to the best of our knowledge, possibilities for a new generation of highly stable and simple CV-QKD systems.

Simultaneous determination and difference evaluation of 14 ginsenosides in Panax ginseng roots cultivated in different areas and ages by high-performance liquid chromatography coupled with triple quadrupole mass spectrometer in the multiple reaction-monitoring mode combined with multivariate statistical analysis.

BACKGROUND: Ginsenosides are not only the principal bioactive components but also the important indexes to the quality assessment of Panax ginseng Meyer. Their contents in cultivated ginseng vary with the growth environment and age. The present study aimed at evaluating the significant difference between 36 cultivated ginseng of different cultivation areas and ages based on the simultaneously determined contents of 14 ginsenosides. METHODS: A high-performance liquid chromatography (HPLC) coupled with triple quadrupole mass spectrometer (MS) method was developed and used in the multiple reaction-monitoring (MRM) mode (HPLC-MRM/MS) for the quantitative analysis of ginsenosides. Multivariate statistical analysis, such as principal component analysis and partial least squares-discriminant analysis, was applied to discriminate ginseng samples of various cultivation areas and ages and to discover the differentially accumulated ginsenoside markers. RESULTS: The developed HPLC-MRM/MS method was validated to be precise, accurate, stable, sensitive, and repeatable for the simultaneous determination of 14 ginsenosides. It was found that the 3- and 5-yr-old ginseng samples were differentiated distinctly by all means of multivariate statistical analysis, whereas the 4-yr-old samples exhibited similarity to either 3- or 5-yr-old samples in the contents of ginsenosides. Among the 14 detected ginsenosides, Rg1, Rb1, Rb2, Rc, 20(S)-Rf, 20(S)-Rh1, and Rb3 were identified as potential markers for the differentiation of cultivation ages. In addition, the 5-yr-old samples were able to be classified in cultivation area based on the contents of ginsenosides, whereas the 3- and 4-yr-old samples showed little differences in cultivation area. CONCLUSION: This study demonstrated that the HPLC-MRM/MS method combined with multivariate statistical analysis provides deep insight into the accumulation characteristics of ginsenosides and could be used to differentiate ginseng that are cultivated in different areas and ages.

Differentiation and identification of ginsenoside structural isomers by two-dimensional mass spectrometry combined with statistical analysis.

BACKGROUND: In the current phytochemical research on ginseng, the differentiation and structural identification of ginsenosides isomers remain challenging. In this paper, a two-dimensional mass spectrometry (2D-MS) method was developed and combined with statistical analysis for the direct differentiation, identification, and relative quantification of protopanaxadiol (PPD)-type ginsenoside isomers. METHODS: Collision-induced dissociation was performed at successive collision energy values to produce distinct profiles of the intensity fraction (IF) and ratio of intensity (RI) of the fragment ions. To amplify the differences in tandem mass spectra between isomers, IF and RI were plotted against collision energy. The resulting data distributions were then used to obtain the parameters of the fitted curves, which were used to evaluate the statistical significance of the differences between these distributions via the unpaired t test. RESULTS: A triplet and two pairs of PPD-type ginsenoside isomers were differentiated and identified by their distinct IF and RI distributions. In addition, the fragmentation preference of PPD-type ginsenosides was determined on the basis of the activation energy. The developed 2D-MS method was also extended to quantitatively determine the molar composition of ginsenoside isomers in mixtures of biotransformation products. CONCLUSION: In comparison with conventional mass spectrometry methods, 2D-MS provides more direct insights into the subtle structural differences between isomers and can be used as an alternative approach for the differentiation of isomeric ginsenosides and natural products.

A comparative study on chemical composition of total saponins extracted from fermented and white ginseng under the effect of macrophage phagocytotic function.

In this study, white ginseng was used as the raw material, which was fermented with Paecilomyces hepiali through solid culture medium, to produce ginsenosides with modified chemical composition. The characteristic chemical markers of the products thus produced were investigated using rapid resolution liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (RRLC-QTOF-MS). Chemical profiling data were obtained, which were then subjected to multivariate statistical analysis for the systematic comparison of active ingredients in white ginseng and fermented ginseng to understand the beneficial properties of ginsenoside metabolites. In addition, the effects of these components on biological activity were investigated to understand the improvements in the phagocytic function of macrophages in zebrafish. According to the established RRLC-QTOF-MS chemical profiling, the contents in ginsenosides of high molecular weight, especially malonylated protopanaxadiol ginsenosides, were slightly reduced due to the fermentation, which were hydrolyzed into rare and minor ginsenosides. Moreover, the facilitation of macrophage phagocytic function in zebrafish following treatment with different ginseng extracts confirmed that the fermented ginseng is superior to white ginseng. Our results prove that there is a profound change in chemical constituents of ginsenosides during the fermentation process, which has a significant effect on the biological activity of these compounds.

Genome sequencing of strain Cellulosimicrobium sp. TH-20 with ginseng biotransformation ability.

Biotransformation for increasing the pharmaceutical effect of ginsenosides is getting more and more attractions. Strain Cellulosimicrobium sp. TH-20 isolated from ginseng soil samples was identified to produce enzymes contributing to its excellent biotransformation activity against ginsenosides, the main active components of ginseng. Based on phylogenetic tree and homology analysis, the strain can be designated as Cellulosimicrobium sp. Genome sequencing was performed using the Illumina Miseq to explore the functional genes involved in ginsenoside transformation. The draft genome of Cellulosimicrobium sp. TH-20 encoded 3450 open reading frames, 51 tRNA, and 9 rRNA. All ORFs were annotated using NCBI BLAST with non-redundant proteins, Gene Ontology, Cluster of Orthologous Gene, and Kyoto Encyclopedia of Genes and Genomes databases. A total of 11 genes were selected based on the functional annotation analysis. These genes are relevant to ginsenoside biotransformation, including 6 for beta-glucosidase, 1 for alpha-N-arabinofuranosidase, 1 for alpha-1,6-glucosidase, 1 for endo-1,4-beta-xylanase, 1 for alpha-L-arabinofuranosidase, and 1 for beta-galactosidase. These glycosidases were predicted to catalyze the hydrolysis of sugar moieties attached to the aglycon of ginsenosides and led to the transformation of PPD-type and PPT-type ginsenosides.

Microbial transformation of ginsenoside Rb1, Re and Rg1 and its contribution to the improved anti-inflammatory activity of ginseng.

Microbial transformation of ginsenosides to increase its pharmaceutical effect is gaining increasing attention in recent years. In this study, Cellulosimicrobium sp. TH-20, which was isolated from soil samples on which ginseng grown, exhibited effective ginsenoside-transforming activity. After protopanaxadiol (PPD)-type ginsenoside (Rb1) and protopanaxatriol (PPT)-type ginsenosides (Re and Rg1) were fed to C. sp. TH20, a total of 12 metabolites, including 6 new intermediate metabolites, were identified. Stepwise deglycosylation and dehydrogenation on the feeding precursors have been observed. The final products were confirmed to be rare ginsenosides Rd, GypXVII, Rg2 and PPT after 96 h transformation with 38-96% yields. The four products showed improved anti-inflammatory activities by using lipopolysaccharide (LPS)-induced murine RAW 264.7 macrophages and the xylene-induced acute inflammatory model of mouse ear edema. The results indicated that they could dramatically attenuate the production of TNF-α more effectively than the precursors. Our study would provide an example of a unique and powerful microbial cell factory for efficiently converting both PPD-type and PPT-type ginsenosides to rare natural products, which extends the drug candidates as novel anti-inflammatory remedies.

Characterization and immunostimulating effects on murine peritoneal macrophages of oligosaccharide isolated from Panax ginseng C.A. Meyer.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax ginseng C.A. Meyer has been the most precious and renowned Chinese herb used in Asian countries for the treatment of various medical disorders. AIM OF THE STUDY: The aim of this work was to investigate the activation effect on murine peritoneal macrophages of oligosaccharide from the roots of P. ginseng. MATERIALS AND METHODS: In this work, the water-extracted oligosaccharide of P. ginseng was (WGOS) isolated and purified from the roots of P. ginseng by hot water extraction, ultrafiltration and gel-permeation chromatography. The monosaccharide composition and degree of polymerization (DP) of WGOS were determined by a combination of acid hydrolysis, high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Phagocytosis of macrophages was measured by uptake of the neutral red by macrophages, nitric oxide (NO) was determined by the Griess method, inducible NO synthase (iNOS) activity was determined by colorimetric method using a reagent kit, and tumor necrosis factor-α (TNF-α) was analyzed by enzyme linked immunosorbent assay (ELISA). The reactive species detection kit was used to measure the reactive oxygen species (ROS) level. RESULTS: WGOS was composed of glucose and the DP was ranging from 2 to 14. Immunological tests showed that treatment of WGOS significantly increased phagocytosis of macrophages, and promoted NO, TNF-α and ROS production. Furthermore, WGOS dose-dependently stimulated NO formation through the up-regulation of iNOS activity. CONCLUSIONS: Taken together, WGOS possessed high immunopotentiating activity and could be developed as a novel immnunostimulant.