Conversion Of Ginsenosides Research Articles

Residue Effect-Guided Design: Engineering of S. Solfataricus β-Glycosidase to Enhance Its Thermostability and Bioproduction of Ginsenoside Compound K.

β-Glycosidase from Sulfolobus solfataricus (SS-BGL) is a highly effective biocatalyst for the synthesis of compound K (CK) from glycosylated protopanaxadiol ginsenosides. In order to improve the thermal stability of SS-BGL, molecular dynamics simulations were used to determine the residue-level binding energetics of ginsenoside Rd in the SS-BGL-Rd docked complex and to identify the top ten critical contributors. Target sites for mutations were determined using dynamic cross-correlation mapping of residues via the Ohm server to identify networks of distal residues that interact with the key binding residues. Target mutations were determined rationally based on site characteristics. Single mutants and then recombination of top hits led to the two most promising variants SS-BGL-Q96E/N97D/N302D and SS-BGL-Q96E/N97D/N128D/N302D with 2.5-fold and 3.3-fold increased half-lives at 95 °C, respectively. The enzyme activities relative to those of wild-type for ginsenoside conversion were 161 and 116%, respectively..

Effects of Puffing, Acid, and High Hydrostatic Pressure Treatments on Ginsenoside Profile and Antioxidant Capacity of Mountain-Cultivated Panax ginseng.

The purpose of this study was to investigate the effects of puffing, acid, and high hydrostatic pressure (HHP) treatments on the ginsenoside profile and antioxidant capacity of mountain-cultivated Panax ginseng (MCPG) before and after treatments. Puffing and HHP treatments decreased extraction yield and increased crude saponin content. The combination of puffing and HHP treatment showed significantly higher crude saponin content than each single treatment. Puffing treatment showed the highest ginsenoside conversion compared with HHP and acid treatments. Significant ginsenoside conversion was not observed in HHP treatment but was in acid treatment. When the puffing and acid treatments were combined, Rg3 and compound K content (1.31 mg and 10.25 mg) was significantly higher than that of the control (0.13 mg and 0.16 mg) and acid treatment (0.27 mg and 0.76 mg). No synergistic effect was observed between acid and HHP treatments. In the case of functional properties, the puffing treatment showed a significant increase in TFC (29.6%), TPC (1072%), and DPPH radical scavenging capacity (2132.9%) compared to the control, while acid and HHP combined treatments did not significantly increase; therefore, the synergistic effects of HHP/puffing and acid/puffing treatments were observed in crude saponin content and ginsenoside conversion, respectively. Consequently, puffing combined with acid or HHP treatments may provide new ways to produce high-value-added MCPG with a higher content of Rg3 and compound K or crude saponin compared to untreated MCPG.

Discovery of enzymes to biotransform ginsenoside Rd into ginsenosides F2 and CK using metagenomics and genomic mining.

Ginsenosides are the main active components of ginseng, including many types and different contents. Among them, minor ginsenosides have better biological functions and pharmacological activities than those of the major ginsenosides. However, minor ginsenosides cannot be obtained in large quantities, but by means of enzymatic transformation technology, some major ginsenosides can be de-glycosylated at a specific position to generate minor ginsenosides. In this study, we report two glycosidase genes associated with the conversion of ginsenoside Rd to ginsenosides F2 or CK. SWMU-CK-1 was identified among the total genes extracted from the feces of plum deer by local Blast screening for putative ginsenoside conversion function, which could cause the conversion of ginsenoside Rd → F2 → CK. The other gene was found in the Bifidobacterium breve 689b SGAir 0764 chromosome genome, which might have the same function as the β-glucosidase gene testified by the gene matching, named SWMU-F2-2, and can achieve the Rd → F2 transformation. This study reports two genes that enable achieving the biotransformation of rare ginsenosides, while it provides a new insight and a promising approach to explore new genes and develop new functions of existing genes.

Novel Split Intein-Mediated Enzymatic Channeling Accelerates the Multimeric Bioconversion Pathway of Ginsenoside.

Cascade reaction systems, such as protein fusion and synthetic protein scaffold systems, can both spatially control the metabolic flux and boost the productivity of multistep enzymatic reactions. Despite many efforts to generate fusion proteins, this task remains challenging due to the limited expression of complex enzymes. Therefore, we developed a novel fusion system that bypasses the limited expression of complex enzymes via a post-translational linkage. Here, we report a split intein-mediated cascade system wherein orthogonal split inteins serve as adapters for protein ligation. A genetically programmable, self-assembled, and traceless split intein was utilized to generate a biocatalytic cascade to produce the ginsenoside compound K (CK) with various pharmacological activities, including anticarcinogenic, anti-inflammatory, and antidiabetic effects. We used two types of split inteins, consensus atypical (Cat) and Rma DnaB, to form a covalent scaffold with the three enzymes involved in the CK conversion pathway. The multienzymatic complex with a size greater than 240 kDa was successfully assembled in a soluble form and exhibited specific activity toward ginsenoside conversion. Furthermore, our split intein cascade system significantly increased the CK conversion rate and reduced the production time by more than 2-fold. Our multienzymatic cascade system that uses split inteins can be utilized as a platform for regulating multimeric bioconversion pathways and boosting the production of various high-value substances.

Transformation Mechanism of Rare Ginsenosides in American Ginseng by Different Processing Methods and Antitumour Effects

The mechanism by which ginsenosides from Panax quinquefolium L. transform into rare saponins by different processing methods and their antitumour effects have yet to be fully elucidated. Our study aimed to detect the effect of amino acids and processing methods on the conversion of ginsenosides in American ginseng to rare ginsenosides, using 8 monomeric ginsenosides as substrates to discuss the reaction pathway and mechanism. S180 tumour-bearing mice were established to study the antitumour effects of American ginseng total saponins (AGS-Q) or American ginseng total saponins after transformation (AGS-H) synergistic CTX. The results showed that aspartic acid was the best catalyst, and the thermal extraction method had the best effect. Under the optimal conditions, including a reaction temperature of 110°C, an aspartic acid concentration of 5%, a reaction time of 2.5 h and a liquid-solid ratio of 30 mL/g, the highest conversion of Rk1 and Rg5 was 6.58 ± 0.11 mg/g and 3.74 ± 0.05 mg/g, respectively. In the reaction pathway, the diol group saponins participated in the transformation process, and the triol group saponins basically did not participate in the transformation process. AGS-Q or AGS-H synergistic CTX, or AGS-H synergistic CTX/2 could significantly increase the tumour inhibition rate, spleen index and white blood cell count, had a significant upregulation effect on IL-2 and IL-10 immune cytokines; significantly restored the ratio of CD4+/CD8+; and significantly inhibited the level of CD4+CD25+. AGS-Q or AGS-H synergistic with CTX or CTX/2 can significantly upregulate the expression of Bax and cleaved-Caspase-3 and inhibit the expression of antiapoptotic protein Bcl-2. AGS synergistic CTX in the treatment of S180 tumour-bearing mice can improve the efficacy and reduce toxicity.

Phnomibacter ginsenosidimutans gen. nov., sp. nov., a novel glycoside hydrolase positive bacterial strain with ginsenoside hydrolysing activity.

The conversion of major ginsenosides into minor ginsenosides attracts a lot of interest because of their biological and pharmaceutical activities. Therefore, for the conversion of ginsenosides, finding a novel competent glycoside hydrolase-producing bacterial strain is useful for future research studies and the mass production of minor ginsenosides. Wastewater samples were collected and screened for novel glycoside hydrolase bacterial strains using Reasoner's 2A+aesculin agar medium. As a result, a novel glycoside hydrolase positive bacterial strain (SB-02T) was identified and subjected to a polyphasic taxonomic analysis. Based on genome analysis, strain SB-02T was found to be affiliated with the family Chitinophagaceae and have less than 92.8 % sequence similarity to other members of the same family. Functional analysis indicated that SB-02T was able to hydrolyse the ginsenosides Rb1, Rc and Rd to F2 and C-K. Due to the conversion of ginsenosides, the strain's genome was sequenced and the genes were annotated by the NCBI. The average amino acid identity and average nucleotide identity values between SB-02T and the available reference genomes were 65.7 and 65.9 %, respectively. The novel isolate contained MK-7 as the predominant menaquinone, the major polyamine putrescine, and iso-C15 : 0, iso-C15 : 1 G and iso-C17 : 0 3-OH as major fatty acids. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Thus, based on the data presented here, strain SB-02T represents a novel species within a new genus in the family Chitinophagaceae, for which the name Phnomibacter ginsenosidimutans gen. nov., sp. nov. is proposed. The type strain of Phnomibacter ginsenosidimutans is SB-02T (=KACC 21266T=LMG 31707T). The genome annotation of SB-02T shows many glycoside hydrolase genes, which may be responsible for the efficient production of many kinds of minor ginsenosides and will be very helpful for future research (target gene cloning) and mass production of either F2 or C-K.

산삼 부정배양근의 진세노사이드 함량 증진과 성분 변환

Background: Culturing wild ginseng adventitious root using plant factory technology provides genetic safety and high productivity. This production technology is drawing attention in the fields of functional raw materials and product development. The cultivation method using elicitors is key technology for controlling biomass and increasing secondary metabolites.BRMethods and Results: Elicitor treatments using methyl jasmonate, pyruvic acid, squalene, β-sistosterol were performed to amplify total ginsenosides (Rb1, Rc, Rb2, Rb3, and Rd) of cultured wild ginseng adventitious root. Thereafter, fermentation and steaming processes were performed to convert total ginsenosides into minor molecular ginsenosides (Rg3, Rk1, and Rg5). The result indicated that methyl jasmonate minimizes the reduction in fresh weight of cultured wild ginseng adventitious root and maximizes total ginsenosides (sum of Rb1, Rc, Rb2, Rb3, and Rd). Ginsenoside conversion results showed a maximum degree of conversion of 131 ㎎/g.BRConclusions: In this study, we demonstrated that the optimal elicitor treatment method increased the content of total ginsenosides, while the steaming and fermentation processing method increased the content of minor ginsenosides.

Kinetic study of the thermal conversion of ginsenosides using lumped groups in steaming, hydrothermal reactions, and CO2-assisted hydrothermal reactions

Kinetic study of the thermal conversion of ginsenosides was carried out in steaming, hydrothermal reactions, and CO2-assisted hydrothermal reactions. Since these are complex reactions involving various ginsenosides, the components are divided into three lumped groups according to their structure. Meanwhile, the conventional series reaction model cannot explain the rapid increase of the final product over that of the intermediate. Therefore, a new model was proposed with a parallel reaction pathway that converts directly from the reactant to the final product. The pathway corresponded to E1 elimination that occurs competitively during deglycosylation by the SN1 mechanism. Consequently, the suggested model was able to explain the rapid increase of the final product and produced more accurate predictions. According to Arrhenius analysis, activation energy decreased in the order of steaming, hydrothermal reactions, and CO2-assisted hydrothermal reactions, which was due to the high ion product of subcritical water and in-situ generation of carbonic acid.

Exploration and Characterization of Novel Glycoside Hydrolases from the Whole Genome of Lactobacillus ginsenosidimutans and Enriched Production of Minor Ginsenoside Rg3(S) by a Recombinant Enzymatic Process.

Background: Several studies have reported that ginsenoside Rg3(S) is effective in treating metastatic diseases, obesity, and various cancers, however, its presence in white ginseng cannot be estimated, and only a limited amount is present in red ginseng. Therefore, the use of recombinant glycosidases from a Generally Recognized As Safe (GRAS) host strain is a promising approach to enhance production of Rg3(S), which may improve nutritional activity, human health, and quality of life. Method: Lactobacillus ginsenosidimutans EMML 3041T, which was isolated from Korean fermented pickle (kimchi), presents ginsenoside-converting abilities. The strain was used to enrich the production of Rg3(S) by fermenting protopanaxadiol (PPD)-mix-type major ginsenosides (Rb1, Rb2, Rc, and Rd) in four different types of food-grade media (1, MRS; 2, Basel Food-Grade medium; 3, Basel Food-Grade medium-I, and 4, Basel Food-Grade medium-II). Due to its tendency to produce Rg3(S), the presence of glycoside hydrolase in Lactobacillus ginsenosidimutans was proposed, the whole genome was sequenced, and the probable glycoside hydrolase gene for ginsenoside conversion was cloned. Results: The L. ginsenosidimutans EMML 3041T strain was whole genome sequenced to identify the target genes. After genome sequencing, 12 sets of glycoside hydrolases were identified, of which seven sets (α,β-glucosidase and α,β-galactosidase) were cloned in Escherichia coli BL21 (DE3) using the pGEX4T-1 vector system. Among the sets of clones, only one clone (BglL.gin-952) showed ginsenoside-transforming abilities. The recombinant BglL.gin-952 comprised 952 amino acid residues and belonged to glycoside hydrolase family 3. The enzyme exhibited optimal activity at 55 °C and a pH of 7.5 and showed a promising conversion ability of major ginsenoside Rb1→Rd→Rg3(S). The recombinant enzyme (GST-BglL.gin-952) was used to mass produce Rg3(S) from major ginsenoside Rb1. Scale-up of production using 50 g of Rb1 resulted in 30 g of Rg3(S) with 74.3% chromatography purity. Conclusion: Our preliminary data demonstrated that this enzyme would be beneficial in the preparation of pharmacologically active minor ginsenoside Rg3(S) in the functional food and pharmaceutical industries.

Cooperated biotransformation of ginsenoside extracts into ginsenoside 20(S)-Rg3 by three thermostable glycosidases.

The aim of this work was to transform ginsenoside extract into the pharmacologically active minor ginsenoside 20(S)-Rg3 by three thermostable glycosidases. The GH1 thermostable beta-glucosidase Tpebgl1 from Thermotoga petrophlia was found to have the ability to convert ginsenosides Rb1 and Rb2. Its properties concerning ginsenoside conversion were systematically investigated. It had high specific activity on pNPG (162·20Umg-1 ) and pNPArp (22·14Umg-1 ). The Km and Vmax of Tpebgl1 for pNPG were 0·28mmoll-1 and 470·2Umg-1 and for pNPArp were 17·30mmoll-1 and 74·28Umg-1 . Therefore, it could successfully convert ginsenosides Rb1 and Rb2 into ginsenoside Rd, which has been proven byexperiments in this paperthen. Tpebgl1 also had good tolerance to glucose and some organic solvents. These made Tpebgl1 a good catalyst candidate for industrial application. Finally, it was applied to convert ginsenoside extract into the pharmacologically active minor ginsenoside 20(S)-Rg3, combined with thermostable ginsenoside Rc converting α-1,6-l-arabinofranosidase Tt-Afs and ginsenoside Rd converting β-glucosidase Tpebgl3. A quantity of 10gl-1 of ginsenoside extract was transformed into 3·93gl-1 of Rg3 at 90°C, pH 5·0 for 3h, with a corresponding molar conversion of 98·19%. The thermostable enzyme Tpebgl1 was found to be a ginsenoside-converting enzyme and successfully applied in the preparation ofginsenoside 20(S)-Rg3 from ginsenoside extract. The three-stepcooperate transformation system of ginsenoside extract was established by using Tpebgl1,Tt-Afs (athermostable ginsenoside Rc converting α-1,6-l-arabinofranosidase) and Tpebgl3 (athermostableginsenoside Rb1 converting β-glucosidase). Converting all the major ginsenosides into protopanaxadiol-type ginsenoside extract would greatly reduce the cost of ginsenoside Rg3 preparation. Enzymes from thermophilic bacteria can meet the requirement of higher reaction temperatures in industrial reactions for substrate solubility promotion and bacterial contamination prevention.

Polysaccharide deriving from Ophiopogonis Radix promotes metabolism of ginsenosides in the present of human gut microbiota based on UPLC-MS/MS assay

The combined usage of Ginseng Radix et Rhizoma (ginseng) and Ophiopogonis Radix is common in oriental countries for thousands of years. The major active constituents of ginseng are ginsenosides, and the conversion of ginsenosides to different metabolites by gut microbiota has been reported. However, the effect of Ophiopogonis Radix, especially its polysaccharides, on the metabolism of ginsenosides by gut microbiota is not known. Here, an in vitro metabolism of ginseng extract, or ginsenosides, in combination with or without Ophiopogon polysaccharide was conducted. A sensitive and reliable UPLC-MS/MS approach using multiple reaction monitoring (MRM) in positive ion mode was developed simultaneously to quantify 22 ginsenosides in the broth of gut microbiota. After fermentation with the microbiota, 15 ginsenosides were detected and quantified, including 6 primary ginsenosides, i.e. Rb1, Rc, Rb2, Rb3, Rd and Re, and 9 metabolites, i.e. F2, Rg3, compound K, Rh2, PPD, Rg1, Rh1, Rg2 and PPT. The quantitative results therefore revealed the elimination of primary ginsenosides and the formation of their metabolites in time-dependent manners. Furthermore, Ophiopogon polysaccharide was shown to stimulate the metabolism of ginsenosides, triggered by gut microbiota. Our study can be extended to investigate the metabolism of different Panax species by gut microbiota when combining with other herbs.

Identification of novel glycoside hydrolases via whole genome sequencing of Niabella ginsenosidivorans for production of various minor ginsenosides.

In this study, many bacterial strains were screened for the production of minor ginsenosides, but based on conversion competence among the strains, the strain Niabella ginsenosidivorans BS26T has the good ginsenoside-transforming ability. Therefore, the strain BS26T was selected for complete genome sequence analysis to determine the target (glycoside hydrolase) functional genes. Whole genome analysis of strain BS26T showed 43 glycoside hydrolase genes in total. To determine the target functional gene, 12 sets of six different glycoside hydrolases (3 set of β-glucosidase; 3 set of trehalase; 3 set of arabinofuranosidase; 2 set of xylosidase; and one set of each α-galactosidase and α-fucosidase, respectively) were selected and cloned in E. coli BL21 (DE3) using the pGEX4T-1 vector and were characterized. Among these 12 sets of clones, only one, β-glucosidase (BglNg-767), showed ginsenoside conversion ability. The BglNg-767 comprised 767 amino acids and belonged to glycoside hydrolase family 3 (GH3). The recombinant GST-BglNg-767 was capable of altering the ginsenosides Rb1, Rd, and gypenoside XVII (Gyp-XVII) to F2; Rb2 to C-O; Rb3 to C-Mx1, and Rc to C-Mc1. Besides, complete genome sequence analysis of strain BS26T also indicates 30 endopeptidase genes, which may be responsible for self-hydrolysis of the proteins. Therefore, using SDS-PAGE analysis, we predict that the difference between the molecular weight of the expressed protein (around 90kDa) and the predicted amino-acid sequence (102.7kDa) is due to self-hydrolysis of the proteins.

Remarkable impact of amino acids on ginsenoside transformation from fresh ginseng to red ginseng

BackgroundAmino acids are one of the major constituents in Panax ginseng, including neutral amino acid, acidic amino acid, and basic amino acid. However, whether these amino acids play a role in ginsenoside conversion during the steaming process has not yet been elucidated. MethodsIn the present study, to elucidate the role of amino acids in ginsenoside transformation from fresh ginseng to red ginseng, an amino acids impregnation pretreatment was applied during the steaming process at 120°C. Acidic glutamic acid and basic arginine were used for the acid impregnation treatment during the root steaming. The ginsenosides contents, pH, browning intensity, and free amino acids contents in untreated and amino acid–treated P. ginseng samples were determined. ResultsAfter 2 h of steaming, the concentration of less polar ginsenosides in glutamic acid–treated P. ginseng was significantly higher than that in untreated P. ginseng during the steaming process. However, the less polar ginsenosides in arginine-treated P. ginseng increased slightly. Meanwhile, free amino acids contents in fresh P. ginseng, glutamic acid-treated P. ginseng, and arginine-treated P. ginseng significantly decreased during steaming from 0 to 2h. The pH also decreased in P. ginseng samples at high temperatures. The pH decrease in red ginseng was closely related to the decrease in basic amino acids levels during the steaming process. ConclusionAmino acids can remarkably affect the acidity of P. ginseng sample by altering the pH value. They were the main influential factors for the ginsenoside transformation. These results are useful in elucidating why and how steaming induces the structural change of ginsenoside in P. ginseng and also provides an effective and green approach to regulate the ginsenoside conversion using amino acids during the steaming process.

High efficiency production of ginsenoside compound K by catalyzing ginsenoside Rb1 using snailase

Abstract The rare ginsenoside Compound K (C-K) is attracting more attention because of its good physiological activity and urgent need. There are many pathways to obtain ginsenoside C-K, including chemical and biological methods. Among these, the conversion of PPD-type ginsenosides by enzymatic hydrolysis is a trend due to its high efficiency and mild conditions. For effectively extracting from the other panaxadiol saponins, the conversion process for ginsenoside C-K was investigated using snailases in this study. The univariate experimental design and response surface methodology were used to determine the optimal hydrolysis conditions for the conversion of ginsenoside Rb1 into ginsenoside C-K by snailases. The optimum conditions were as follows: pH 5.12, temperature 51 °C, ratio of snailase/substrate 0.21, and reaction time 48 h. On the basis of these parameters, the addition of 1.0 mmol·L− 1 ferric ion was found to significantly improve the enzymolysis of snailases for the first time. With the above conditions, the maximum conversion rate reached 89.7%, suggesting that the process can obviously increase the yield of ginsenoside C-K. The bioassay tests indicated that the ginsenoside C-K showed anti-tumor activity in a series of tumor cell lines. Based on these results, we can conclude that the process of rare ginsenoside C-K production by enzymolysis with snailase is feasible, efficient, and suitable for the industrial production and application.

Influence of organic acids and heat treatment on ginsenoside conversion

BackgroundHeat treatments are applied to ginseng products in order to improve physiological activities through the conversion of ginsenosides, which are key bioactive components. During heat treatment, organic acids can affect ginsenoside conversion. Therefore, the influence of organic acids during heat treatment should be considered. MethodsRaw ginseng, crude saponin, and ginsenoside Rb1 standard with different organic acids were treated at 130°C, and the chemical components, including ginsenosides and organic acids, were analyzed. ResultsThe organic acid content in raw ginseng was 5.55%. Organic acids were not detected in crude saponin that was not subjected to heat treatment, whereas organic acids were found in crude saponin subjected to heat treatment. Major ginsenosides (Rb1, Re, and Rg1) in ginseng and crude saponin were converted to minor ginsenosides at 130°C; the ginsenoside Rb1 standard was very stable in the absence of organic acids and was converted into minor ginsenosides in the presence of organic acids at high temperatures. ConclusionThe major factor affecting ginsenoside conversion was organic acids in ginseng. Therefore, the organic acid content as well as ginsenoside content and processing conditions should be considered important factors affecting the quality of ginseng products.

Fermentation of red ginseng extract by the probiotic Lactobacillus plantarum KCCM 11613P: ginsenoside conversion and antioxidant effects

BackgroundGinsenosides, which are bioactive components in ginseng, can be converted to smaller compounds for improvement of their pharmacological activities. The conversion methods include heating; acid, alkali, and enzymatic treatment; and microbial conversion. The aim of this study was to determine the bioconversion of ginsenosides in fermented red ginseng extract (FRGE). MethodsRed ginseng extract (RGE) was fermented using Lactobacillus plantarum KCCM 11613P. This study investigated the ginsenosides and their antioxidant capacity in FRGE using diverse methods. ResultsProperties of RGE were changed upon fermentation. Fermentation reduced the pH value, but increased the titratable acidity and viable cell counts of lactic acid bacteria. L. plantarum KCCM 11613P converted ginsenosides Rb2 and Rb3 to ginsenoside Rd in RGE. Fermentation also enhanced the antioxidant effects of RGE. FRGE reduced 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and reducing power; however, it improved the inhibition of β-carotene and linoleic acid oxidation and the lipid peroxidation. This suggested that the fermentation of RGE is effective for producing ginsenoside Rd as precursor of ginsenoside compound K and inhibition of lipid oxidation. ConclusionThis study showed that RGE fermented by L. plantarum KCCM 11613P may contribute to the development of functional food materials.

Remarkable Impact of Acidic Ginsenosides and Organic Acids on Ginsenoside Transformation from Fresh Ginseng to Red Ginseng

Panax ginseng contains many chemical components, including acidic ginsenosides and organic acids. However, whether these acidic substances play a role in ginsenoside transformation during steaming treatment has not yet been explored. In this paper, the content of neutral ginsenosides, acidic ginsenosides, and their degradation products in unsteamed and steamed P. ginseng were simultaneously quantified by high-performance liquid chromatography. We observed that neutral ginsenosides were converted to rare ginsenosides during the root steaming but not during the individual ginsenoside steaming. In contrast, acidic malonyl ginsenosides released malonic acid and acetic acid through demalonylation, decarboxylation, deacetylation reactions during the steaming at 120 °C. These malonyl ginsenosides not only were converted to rare ginsenosides but also promoted the degradation of neutral ginsenosides. Further studies indicated that a low concentration of organic acid was the determining factor for the ginsenoside conversion. The related mechanisms were deduced to be mainly acidic hydrolysis and dehydration. In summary, acidic ginsenosides and organic acids remarkably affected ginsenoside transformation during the steaming process. Our results provide useful information for precisely understanding the ginsenoside conversion pathways and mechanisms underlying the steaming process.

Microbial conversion of major ginsenosides in ginseng total saponins by Platycodon grandiflorum endophytes

BackgroundIn this study, we screened and identified an endophyte JG09 having strong biocatalytic activity for ginsenosides from Platycodon grandiflorum, converted ginseng total saponins and ginsenoside monomers, determined the source of minor ginsenosides and the transformation pathways, and calculated the maximum production of minor ginsenosides for the conversion of ginsenoside Rb1 to assess the transformation activity of endophyte JG09. MethodsThe transformation of ginseng total saponins and ginsenoside monomers Rb1, Rb2, Rc, Rd, Rg1 into minor ginsenosides F2, C-K and Rh1 using endophyte JG09 isolated by an organizational separation method and Esculin-R2A agar assay, as well as the identification of transformed products via TLC and HPLC, were evaluated. Endophyte JG09 was identified through DNA sequencing and phylogenetic analysis. ResultsA total of 32 β-glucosidase-producing endophytes were screened out among the isolated 69 endophytes from P. grandiflorum. An endophyte bacteria JG09 identified as Luteibacter sp. effectively converted protopanaxadiol-type ginsenosides Rb1, Rb2, Rc, Rd into minor ginsenosides F2 and C-K, and converted protopanaxatriol-type ginsenoside Rg1 into minor ginsenoside Rh1. The transformation pathways of major ginsenosides by endophyte JG09 were as follows: Rb1→Rd→F2→C-K; Rb2→C-O→C-Y→C-K; Rc→C-Mc1→C-Mc→C-K; Rg1→Rh1. The maximum production rate of ginsenosides F2 and C-K reached 94.53% and 66.34%, respectively. ConclusionThis is the first report about conversion of major ginsenosides into minor ginsenosides by fermentation with P. grandiflorum endophytes. The results of the study indicate endophyte JG09 would be a potential microbial source for obtaining minor ginsenosides.

Conversion of ginsenosides by Lactobacillus plantarum studied by liquid chromatography coupled to quadrupole trap mass spectrometry

Ginsenosides are the active components responsible for the pharmacological properties of ginseng, a commonly used medicinal plant and food ingredient. This study aimed to determine the changes of ginsenosides during fermentation of ginseng extract or reference ginsenosides with Lactobacillus plantarum. Chemically acidified ginseng extracts served as controls. High performance liquid chromatography coupled with quadrupole-trap (Q-TRAP) mass spectrometry method was employed for analysis and quantification of ginsenosides, and for identification of metabolites. A total of 14 metabolites were identified; the quantification of metabolites was achieved by tandem mass spectrometry in MRM mode. Metabolism of L. plantarum removed glucosyl moieties from ginsenosides Rb1, Rd, and Re at the C-20 position to produce a racemic mixture of products. Remarkably, removal of glycosyl residues occurred not only by hydrolysis but also by dehydration to produce racemic mixtures of Δ20(21) or Δ20(22) products. Biotransformation occurred more rapidly with the di-substituted ginsenoside Rb1 when compared to the mono-substituted ginsenoside Rd. This study thus extends the knowledge of biotransformation of ginsenosides to produce bioactive derivatives.

Improved anticancer effect of ginseng extract by microwave-assisted processing through the generation of ginsenosides Rg3, Rg5 and Rk1

In this study, we demonstrated the efficient structural conversion of ginsenosides, which enhances the anticancer activity of ginseng by microwave irradiation. The microwave-irradiated product of ginseng (MG) had a higher content of ginsenosides, Rg3+Rg5+Rk1, particularly Rg5 and Rk1, and, thus, has an increased medicinal effect. To confirm the anticancer effect of MG, it was tested in 5 human cancer cell lines (cervical cancer HeLa, gastric cancer AGS, colon cancer HCT-116, lung cancer A549, and liver cancer HepG2 cells). Anti-proliferation results for 5 human cancer cell lines treated with ginseng extracts indicate that HeLa cells treated with MG showed the highest activity with an IC50 value of 130.1 µg/mL. MG also suppressed the growth of human cervical cancer cell xenografts in athymic nude mice as an in vivo model. This growth suppression by MG is associated with the induction of cell death and autophagy. Moreover, there were no toxic sign or decrease in renal and hepatic function in mice administered with MG. Therefore, heat processing by microwave irradiation is a useful method to enhance the anticancer effect of ginseng as it increases the content of ginsenosides, Rg3, Rg5, and Rk1.