Rare Ginsenosides Research Articles

Glu@Zn‐Ni MOF Constructed Through Ligand Exchange for Bi‐Enzyme Immobilization With Improved Performance in Rare Ginsenoside Production

Glu@Zn‐Ni MOF Constructed Through Ligand Exchange for Bi‐Enzyme Immobilization With Improved Performance in Rare Ginsenoside Production

Green and Fast Synthesis of NiCo-MOF for Simultaneous Purification-Immobilization of Bienzyme to Catalyze the Synthesis of Ginsenoside Rh2.

Traditional metal-organic frameworks (MOFs) preparation is generally time-consuming, polluting, and lacking specificity for enzyme immobilization. This paper introduced a facile, rapid, and green method to produce three MOFs subsequently employed to purify and coimmobilize recombinant glycosyltransferase (UGT) and recombinant sucrose synthetase (SUSy) using histidine tag (His-tag) for the specific adsorption of Ni2+ and Co2+ from MOFs. This method simplified enzyme purification from crude extracts and enabled enzymes to be reused. The results demonstrated that NiCo-MOF exhibited a higher enzyme load (115.9 mg/g) than monometallic MOFs. Additionally, the NiCo-MOF@UGT&SUSy demonstrated excellent stability and efficiently produced the rare ginsenoside Rh2 by catalyzing a coupling reaction (95.6 μg/mL), solving the problem of the substrate cost of uridine diphosphate glucose (UDPG). The NiCo-MOF@UGT&SUSy retained 68.97% of the initial activity after 10 cycles. Finally, molecular docking studies elucidated the conversion mechanism of the target product Rh2. This technique is important in the industrialization of ginsenoside production and enzyme purification.

Biotransformation of ginsenoside compound K using β-glucosidase in deep eutectic solvents.

Ginsenoside compound K (CK) holds significant potential for application in the pharmaceutical industry, which exhibits numerous pharmacological activity such as cardioprotective and antidiabetic. However, the difficult separation technique and limited yield of CK hinder its widespread use. The study investigated the process of converting ginsenoside CK using β-glucosidase. It aimed to determine the specific site where the enzyme binds and the most favorable arrangement of the enzyme. Molecular docking was also employed to determine the interaction between β-glucosidase and ginsenosides, indicating a strong and spontaneous contact force between them. The effectiveness of the conversion process was further improved using a "green" deep eutectic solvent (DES). A univariate experimental design was used to determine the composition of DES and the optimal hydrolysis conditions for β-glucosidase to convert ginsenoside Rb1 into ginsenoside CK. The employment of β-glucosidase enzymatic hydrolysis in the synthesis of rare ginsenoside CK applying the environmentally friendly solvent DES is not only viable and effective but also appropriate for industrial use. The characterization methods confirmed that DES did not disrupt the structure and conformation of β-glucosidase. In ChCl:EG = 2:1 (30%, v/v), pH 5.0 of DES buffer, reaction temperature 50 ℃, enzyme substrate mass ratio 1:1, after 36h of reaction, the CK yield was 1.24 times that in acetate buffer, which can reach 86.2%. In this study, the process of using β-glucosidase enzymatic hydrolysis and producing rare ginsenoside CK in green solvent DES is feasible, efficient and suitable for industrial production and application.

The Antioxidant and Anti-Fatigue Effects of Rare Ginsenosides and γ-Aminobutyric Acid in Fermented Ginseng and Germinated Brown Rice Puree.

γ-aminobutyric acid (GABA) and rare ginsenosides are good antioxidant and anti-fatigue active components that can be enriched via probiotic fermentation. In this study, ginseng and germinated brown rice were used as raw materials to produce six fermented purees using fermentation and non-fermentation technology. We tested the chemical composition of the purees and found that the content of GABA and rare ginsenoside (Rh4, Rg3, and CK) in the puree made of ginseng and germinated brown rice (FGB) increased significantly after fermentation. The antioxidant activity of the six purees was determined using cell-free experiments, and it was found that FGB had better ferric-ion-reducing antioxidant power (FRAP) and 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging rates, exhibiting better antioxidant effects. We then evaluated the antioxidant effect of FGB in HepG2 cells induced by H2O2 and found that FGB can reduce the generation of reactive oxygen species (ROS) in HepG2 cells and increase the membrane potential level, thereby improving oxidative damage in these cells. In vivo experiments also showed that FGB has good antioxidant and anti-fatigue activities, which can prolong the exhaustive swimming time of mice and reduce the accumulation of metabolites, and is accompanied by a corresponding increase in liver glycogen and muscle glycogen levels as well as superoxide dismutase and lactate dehydrogenase activities. Finally, we believe that the substances with good antioxidant and anti-fatigue activity found in FGB are derived from co-fermented enriched GABA and rare ginsenosides.

Ginsenoside Rh4 prevents endothelial dysfunction as a novel AMPK activator.

The AMP-activated protein kinase (AMPK) signalling pathway is a desirable target for various cardiovascular diseases (CVD), while the involvement of AMPK-mediated specific downstream pathways and effective interventions in hyperlipidaemia-induced endothelial dysfunction remain largely unknown. Herein, we aim to identify an effective AMPK activator and to explore its efficacy and mechanism against endothelial dysfunction. Molecular docking technique was adopted to screen for the potent AMPK activator among 11 most common rare ginsenosides. In vivo, poloxamer 407 (P407) was used to induce acute hyperlipidaemia in C57BL/6J mice. In vitro, palmitic acid (PA) was used to induce lipid toxicity in HAEC cells. We discovered the strongest binding of ginsenoside Rh4 to AMPKα1 and confirmed the action of Rh4 on AMPK activation. Rh4 effectively attenuated hyperlipidaemia-related endothelial injury and oxidative stress both in vivo and in vitro and restored cell viability, mitochondrial membrane potential and mitochondrial oxygen consumption rate in HAEC cells. Mechanistically, Rh4 bound to AMPKα1 and simultaneously up-regulated AKT/eNOS-mediated NO release, promoted PGC-1α-mediated mitochondrial biogenesis and inhibited P38 MAPK/NFκB-mediated inflammatory responses in both P407-treated mice and PA-treated HAEC cells. The AMPK inhibitor Compound C treatment completely abrogated the regulation of Rh4 on the above pathways and weakened the lowering effect of Rh4 on endothelial impairment markers, suggesting that the beneficial effects of Rh4 are AMPK dependent. Rh4 may serve as a novel AMPK activator to protect against hyperlipidaemia-induced endothelial dysfunction, providing new insights into the prevention and treatment of endothelial injury-associated CVD.

Melanin resistance of heat-processed ginsenosides from Panax ginseng berry treated with citric acid through autophagy pathway

GFRS is the conversion product of Panax ginseng Meyer berry after citric acid heat treatment, which is rich in rare ginsenosides. However, the anti-melanin role of GFRS in the regulation of skin pigmentation and its material basis remains unclear. To compare the anti-melanin activity before and after citric acid heat treatment, we determined the effects of GFS and GFRS on tyrosinase activity and melanin lever under α-MSH stimulation and found the potential anti-melanin effect of GFRS. Further, Western blot and immunofluorescence methods were used to reveal the mechanism by which GFRS detects anti-melanin activity by promoting autophagy flux levels. In zebrafish models, GFRS inhibited endogenous melanin and tyrosinase better than arbutin and promoted the accumulation of autophagy levels in vivo. To determine the material basis of the anti-melanin effect of GFRS, HPLC was used to isolate and prepare 12 ginsenosides from GFRS, and their activity evaluation and structure–activity relationship analysis were performed. The results showed that the inhibitory effect of GFRS on melanin was Rg3 > Rg5 > Rk1 > Rd. Molecular docking showed that their docking fraction with mushroom tyrosinase was significantly better than that of arbutin, but the presence of C-20 glycosylation decreased the anti-melanin activity of Rd. To maximize the content of Rg3, Rg5, and Rk1, we optimized the process by using citric acid heat treatment of ginsenoside Rd and found that citric acid heat treatment at 100°C almost completely transformed Rd and obtained a high content of active ingredients. In summary, our data demonstrated that GFRS exerted anti-melanin effects by inducing autophagy. It was further revealed that Rg3, Rg5, and Rk1, as effective active components, could be enriched by the improved process of converting ginsenoside Rd by citric acid heat treatment.

Production of rare ginsenosides by biotransformation of Panax notoginseng saponins using Aspergillus fumigatus

Panax notoginseng saponins (PNS) are the main active components of Panax notoginseng. But after oral administration, they need to be converted into rare ginsenosides by human gut microbiota and gastric juice before they can be readily absorbed into the bloodstream and exert their effects. The sources of rare ginsenosides are extremely limited in P. notoginseng and other medical plants, which hinders their application in functional foods and drugs. Therefore, the production of rare ginsenosides by the transformation of PNS using Aspergillus fumigatus was studied in this research. During 50 days at 25 ℃ and 150 rpm, A. fumigatus transformed PNS to 14 products (1–14). They were isolated by varied chromatographic methods, such as silica gel column chromatography, Rp-C18 reversed phase column chromatography, semi-preparative HPLC, Sephadex LH-20 gel column chromatography, and elucidated on the basis of their 1H-NMR, 13C-NMR and ESIMS spectroscopic data. Then, the transformed products (1–14) were isolated and identified as Rk3, Rh4, 20 (R)-Rh1, 20 (S)-Protopanaxatriol, C-K, 20 (R)-Rg3, 20 (S)-Rg3, 20 (S)-Rg2, 20 (R)-R2, Rk1, Rg5, 20 (S)-R2, 20 (R)-Rg2, and 20 (S)-I, respectively. In addition, all transformed products (1–14) were tested for their antimicrobial activity. Among them, compounds 5 (C-K) and 7 [20 (S)-Rg3] showed moderate antimicrobial activities against Staphylococcus aureus and Candida albicans with MIC values of 6.25, 1.25 μg/mL and 1.25, 25 μg/mL, respectively. This study lays the foundation for production of rare ginsenosides.Graphical abstract

Engineering UDP-Glycosyltransferase UGTPg29 for the Efficient Synthesis of Ginsenoside Rg3 from Protopanaxadiol.

Rare ginsenosides Rg3 and Rh2, which exhibit diverse pharmacological effects, are derivatives of protopanaxadiol (PPD). UDP-glycosyltransferases, such as the M315F variant of Bs-YjiC (Bs-YjiCm) from Bacillus subtilis and UGTPg29 from Panax ginseng, can efficiently convert PPD into Rh2 and Rh2 into Rg3, respectively. In the present study, the N178I mutation of Bs-YjiCm was introduced, resulting in an increase in Rh2 production. UDP-glycosyltransferase UGTPg29 was then engineered to improve its robustness through semi-rational design. The variant R91M/D184M/A287V/A342L, which indicated desirable stability and activity, was utilized in coupling with the N178I variant of Bs-YjiCm and sucrose synthase AtSuSy from Arabidopsis thaliana to set up a "one-pot" three-enzyme reaction for the biosynthesis of Rg3. The influential factors, including the ratio and concentration of UDP-glycosyltransferases, pH, and the concentrations of UDP, sucrose, and DMSO, were optimized. On this basis, a fed-batch strategy was adopted to achieve a Rg3 yield as high as 12.38 mM (9.72 g/L) with a final yield of 68.78% within 24 h. This work may provide promising UDP-glycosyltransferase candidates for ginsenoside biosynthesis.

Integrated Analysis of Ginsenoside Content and Biomarker Changes in Processed Ginseng: Implications for Anti-Cancer Mechanisms.

Black ginseng is the processed product of ginseng, and it has been found that the content and types of rare ginsenosides increased after processing. However, there is limited research on the ginsenoside differences between cultivated and forest ginseng before and after processing and among various plant parts. This study investigated the effects of processing on ginsenosides in different parts of cultivated and forest ginseng. After processing, the contents of Re, Rg1, S-Rg3, Rg5, R-Rh1, Rk1, Rk3, and F4 were significantly increased or decreased, the growth age of forest ginseng was not proportional to the content of ginsenosides, and the differences in ginsenoside content in ginseng from different cultivation methods were relatively small. Chemometric analysis identified processing biomarkers showing varying percentage changes in different parts. Network pharmacology predicted the EGFR/PI3K/Akt/mTOR pathway as a potential key pathway for the anti-cancer effect of black ginseng.

Fermented Ginsenosides Alleviate Acute Liver Injury Induced by CCl4 in Mice by Regulating the AKT/mTOR Signaling Pathway.

This study investigated the alleviating effect of fermented ginsenosides obtained through yeast strain fermentation transformation on acute liver injury (ALI) induced by CCl4. Strains were screened for their ability to produce β-glucosidase, the transformation ability of the strain was verified by high-performance liquid chromatography, and the Saccharomyces cerevisiae strain F6 was obtained by 26S rRNA sequencing. After fermentation by F6 strain, it was found that the content of ginsenosides Re, Rb1, and Rb2 was significantly decreased (P < 0.05), and rare ginsenosides were detected, with the content of Rh4 and Rg5 reaching 2.65 mg·g-1 and 2.56 mg·g-1. We also explored the preventive effect of fermented ginsenoside extract (FGE) on ALI. Mice were evenly divided into 9 groups as follows: control group, ALI model group, positive drug bifendate group, and treatment group, which included 3 ginsenoside extract (GE) groups and 3 FGE groups (dosage of 150, 300, and 450 mg·kg-1 b.w.). The results showed that compared with the ALI model group, FGE significantly increased the levels of glutathione peroxidase, hydroperoxidase, and superoxide dismutase and also decreased the malondialdehyde level. The levels of alanine aminotransferase, aspartate aminotransferase, and total bilirubin markers were significantly reduced, and the levels of inflammatory cytokines TNF-α, IL-6, and IL-1β were significantly decreased. Bioinformatics analysis combined with Western blot validation explored the molecular mechanism of the effect of FGE. It was found that FGE could downregulate the expression of the p-AKT/AKT and the p-mTOR/mTOR ratios. These results suggested that FGE played an alleviative role in ALI by promoting autophagy to inhibit the AKT/mTOR signaling pathway.

Characterization of a glycosyltransferase from Paris polyphylla for application in biosynthesis of rare ophiopogonins and ginsenosides

Saponins are bioactive components of many medicinal plants, possessing complicated chemical structures and extensive pharmacological activities, but the production of high-value saponins remains challenging. In this study, a 6′-O-glucosyltransferase PpUGT7 (PpUGT91AH7) was functionally characterized from Paris polyphylla Smith var. yunnanensis (Franch.) Hand. -Mazz., which can transfer a glucosyl group to the C-6′ position of diosgenin-3-O-rhamnosyl-(1 → 2)-glucoside (1), pennogenin-3-O-rhamnosyl-(1 → 2)-glucoside (2), and diosgenin-3-O-glucoside (5). The KM and Kcat values of PpUGT7 towards the substrate 2 were 8.4 μM and 2 × 10−3 s−1, respectively. Through molecular docking and site-directed mutagenesis, eight residues were identified to interact with the sugar acceptor 2 and be crucial for enzyme activity. Moreover, four rare ophiopogonins and ginsenosides were obtained by combinatorial biosynthesis, including an undescribed compound ruscogenin-3-O-glucosyl-(1 → 6)-glucoside (10). Firstly, two monoglycosides 9 and 11 were generated using a known sterol 3-O-β-glucosyltransferase PpUGT80A40 with ruscogenin (7) and 20(S)-protopanaxadiol (8) as substrates, which were further glycosylated to the corresponding diglycosides 10 and 12 under the catalysis of PpUGT7. In addition, compounds 7–11 were found to show inhibitory effects on the secretion of TNF-α and IL-6 in macrophages RAW264.7. The findings provide valuable insights into the enzymatic glycosylation processes in the biosynthesis of bioactive saponins in P. polyphylla var. yunnanensis, and also serve as a reference for utilizing UDP-glycosyltransferases to construct high-value or rare saponins for development of new therapeutic agents.

Ameliorative effect of rare ginsenosides on reproductive injury induced by cyclophosphamide in female rats: based on metabonomics

Objective: To investigate the effect of rare ginsenosides (RGS) on reproductive injury induced by cyclophosphamide (CP) in female rats. Methods: Twenty-four female rats were divided into four groups [normal control (NC), RGS, CP, and CP+RGS group] with 6 rats in each group. CP group (the model group) and CP+RGS group (the treatment group) were intraperitoneally injected with CP 30 mg/kg for 5 days for modeling, and CP+RGS group was given RGS intragastric intervention. General growth status of rats in each group was observed, the organ index was calculated, and the pathological changes of ovary, uterus, liver and kidney were observed by hematoxylin-eosin staining. Serum levels of estradiol, follicle stimulating hormone (FSH), luteinizing hormone (LH), pro-inflammatory factors interleukin (IL) 6, IL-1β, tumor necrosis factor-α were detected. The urine samples were collected after RGS treatment for metabonomics analysis. Metabolomic profiling based on ultra performance liquid chromatography (UPLC) coupled with mass spectrometry (MS) was used to analyze and determine the urine metabolites of rats in each group. Results: Compared with NC group, the ovary index of CP group [(0.054±0.015) %] was significantly decreased (P<0.05), the uterus index [(0.293±0.036) %] and estradiol level [(62.9±6.4) pmol/L] were significantly decreased (all P<0.01), serum levels of FSH, LH, IL-6 and IL-1β [(20.4±1.0) U/L, (29.0±3.0) U/L, (185.4±28.6) ng/L, (72.9±2.0) ng/L, respectively] were significantly increased (all P<0.01). Compared with CP group, the ovary index in CP+RGS group [(0.075±0.010) %] was significantly increased (P<0.05), serum estradiol level [(122.1±16.2) pmol/L] was significantly increased (P<0.01), serum FSH, IL-1β and IL-6 levels [(16.7±1.0) U/L, (111.8±17.4) ng/L, (60.1±2.2) ng/L, respectively] were significantly decreased (all P<0.01). Metabonomics analysis results showed that, a total of 352 metabolites were detected in urine, of which 12 were found to be potential markers associated with reproductive injury according to the screening standard. After treatment with RGS, differential metabolites were improved in the direction of NC group. Pathway enrichment suggests that the therapeutic effect of RGS was related to multiple metabolic pathways, including purine metabolism and taurine and hypotaurine metabolism. Conclusion: RGS might reduce inflammation and thus ameliorate the damage caused by CP to the reproductive system of female rats by affecting purine metabolism and other pathways.

Heterologous synthesis of ginsenoside F1 and its precursors in Nicotiana benthamiana

Ginsenoside F1 has high medicinal values, which is a kind of rare triterpene saponin isolated from Panax plants. The extremely low content of ginsenoside F1 in herbs has limited its research and application in medical field. In this work, we constructed a pathway in tobacco for the biosynthesis of ginsenoside F1 by metabolic engineering. Four enzyme genes (PnDDS, CYP716A47, CYP716S1 and UGT71A56) isolated from Panax notoginseng were introduced into tobacco. Thus, a biosynthetic pathway for ginsenoside F1 synthesis was artificially constructed in tobacco cells; moreover, the four exogenous genes could be expressed in the roots, stems and leaves of transgenic plants. Consequently, ginsenoside F1 and its precursors were successfully synthesized in the transgenic tobacco, compared with Panax plants, the content of ginsenoside F1 in transgenic tobacco was doubled. In addition, accumulation of ginsenoside F1 and its precursors in transgenic tobacco shows organ specificity. Based on these results, a new approach was established to produce rare ginsenoside F1; meanwhile, such strategy could also be employed in plant hosts for the heterologous synthesis of other important or rare natural products.

Investigating the effects of rare ginsenosides on hyperuricemia and associated sperm damage via nontargeted metabolomics and gut microbiota

Ethnopharmacological relevanceIn ancient times, ginseng was used for hyperuricemia treatment as described in the classic traditional Chinese medical text Shang Han Lun. Recent studies have shown that common ginsenosides and rare ginsenosides (RGS) are the main active compounds in ginseng. RGS have higher activity and are less studied in the treatment of hyperuricemia. Aim of the studyTo determine whether RGS prevents and ameliorates potassium oxonate(PO)-induced hyperuricemia and concomitant spermatozoa damage in mice and the possible underlying mechanisms. Materials and methodsPotassium oxonate (PO, 300 mg/kg) induced hyperuricemia in mice via the oral administration of RGS (50, 100, or 200 mg/kg) or allopurinol (ALL, 5 mg/kg) for 35 days. Uric acid (UA) and xanthine oxidase (XO) levels were measured to assess the degree of histopathological damage in the liver, kidney, and testis, and renal creatinine (CRE), urea nitrogen (BUN), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and inflammatory factor (IL-1β) levels were measured to calculate the sperm density. Mechanisms were also explored based on blood and urine metabolomics and the gut microbiota. ResultsIn this study, we demonstrated that RGS containing Rg3, Rk1, Rg6, and Rg5 could reduce serum UA levels, inhibit serum and hepatic XO activity, reduce renal CRE and BUN levels, further restore renal SOD and GSH activities, reduce the accumulation of MDA in the kidneys, and attenuate the production of renal IL-1β. RGS was able to restore sperm density. Metabolomic analysis revealed that RGS improved sphingolipid metabolism, pyrimidine metabolism, and other metabolic pathways. 16S rDNA sequencing revealed that RGS could increase gut microbial diversity, restore the Firmicutes/Bacteroidetes (F/B) ratio, and adjust the intestinal microbial balance. Spearman's correlation analysis revealed a correlation between differentially metabolites and the gut microbiota. Lactobacillus and Akkermansia are the core genera. ConclusionRGS can be a candidate for the prevention and amelioration of hyperuricemia and concomitant sperm damage. Its mechanism of action is closely related to sphingolipid metabolism, pyrimidine metabolism, and the modulation of gut microbiota, such as Lactobacillus and Akkermansia.

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.

Ginseng fruit rare saponins (GFRS), a promising anti-wrinkle agent: Evidence of its antioxidant effect and its capacity to prevent matrix metalloproteinase (MMPs) expression in vitro and in vivo

Rare saponins show numerous significant biological activities, however, there has been no research on using ginseng fruit rare saponins (GFRS) to minimize skin wrinkles. This paper studied the anti-wrinkle effect, related mechanism, material basis of efficacy, and structure-activity relationship of GFRS in vivo and in vitro. The findings demonstrate that GFRS has potent collagenase and elastase inhibition capabilities in addition to superior ultraviolet absorption properties. Secondly, GFRS can strengthen the endogenous antioxidant defense system, thus lessening the UVB-induced ultraviolet damage that L929 cells and zebrafish experience. Matrix metalloproteinases (MMPs) studies have shown that GFRS could significantly inhibit the UVB-induced MMP1 and MMP9 proteins production. Finally, the main ginsenoside component in GFRS was identified by LC-MS, and 16 kinds of ginsenoside were analyzed, including rare ginsenoside such as Rg6, F4, and R/S-Rg3, which may be caused by the transformation of prototype saponins Re and Rd caused by citric acid heat treatment (CAHT). After doing a quantitative HPLC analysis on twelve of them, it was discovered that CAHT could enrich the content of rare saponins in GFS. By evaluating these ginsenoside’s anti-wrinkle properties. It is found that the main material basis for GFRS’s anti-wrinkle action may be ginsenoside Rg6 or F4. In summary, GFRS exhibits promising properties as an anti-wrinkle treatment because of its robust anti-oxidation, anti-collagenase, anti-elastase activity, favorable ultraviolet absorption effect, and capacity to regulate MMPs in vivo and in vitro models.

Targeted preparation of six rare ginsenosides using two β-glucosidases from Bifidobacterium adolescentis

In this study, six rare protopanaxadiol (PPD)-type ginsenosides, namely, F2, CK, C-Mc1, C-Mc, CO, and CY, were prepared using enzymatic hydrolysis. Firstly, major (PPD)-type ginsenosides, including Rb1, Rb2, Rc, and Rd, were biotransformed by employing single and combined β-glucosidases BaBgl1A and BaBgl3A. Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analyses revealed that ginsenosides F2, CO, and C-Mc1 could be produced by BaBgl1A, while rare ginsenosides CK, CY, and C-Mc could be produced by the combined use of BaBgl1A and BaBgl3A. Furthermore, optimal preparation conditions for these rare ginsenosides were elucidated, with yields exceeding 76.34%. Additionally, large quantities of rare ginsenosides were prepared to determine their structures. Characteristic absorption peaks in 13C NMR spectra and ion peaks in MALDI-TOF-MS suggested that the six high-purity rare ginsenosides (i.e., F2, CO and C-Mc1, CK, CY, and C-Mc) could be produced at high-efficiency. The findings of this study offer insights into novel enzymolysis methods for the targeted preparation of rare PPD-type ginsenosides, thus widening the range of raw materials that could be directed to in-depth biological activity research.

Enhanced recovery of rare ginsenosides and bioactive compounds from red ginseng marc using CO2-Assisted subcritical water

Subcritical water extraction (SWE) is a high-efficiency and environmentally sustainable technique for extracting bioactive compounds from red ginseng marc (RGM). In this study, CO2 was introduced into the SWE system as a green catalyst to enhance the extraction efficiency of biologically active compounds from RGM. A wide range of parameters, such as temperature (140–180 °C), pressure (10–20 MPa), and time (10–60 min), were optimized using response surface methodology to maximize the recovery of highly valuable bioactive components, including rare ginsenosides (RGs), total phenolic contents (TPCs), and Maillard reaction products (MRPs), as well as the antioxidant activity of the RGM extract. The extract obtained using CO2-assisted SWE (CO2-SWE) under the optimal conditions (172 °C, 10 MPa, and 10 min) was enriched in RGs (1.02–2.16 times), TPCs (0.98–4.68 times), and MRPs (1.08–2.20 times) and exhibited enhanced antioxidant activities (1.07–3.50 times) compared with the extracts produced by conventional SWE (N2 environment) and Soxhlet extraction with H2O or 80 vol% ethanol. Thus, CO2-SWE is an environmentally friendly and nontoxic technique with considerable potential for use in the pharmaceutical, cosmeceutical, and functional food sectors.

Novel glycosidase from Paenibacillus lactis 154 hydrolyzing the 28-O-β-d-glucopyranosyl ester bond of oleanane-type saponins

Oleanane-type ginsenosides are a class of compounds with remarkable pharmacological activities. However, the lack of effective preparation methods for specific rare ginsenosides has hindered the exploration of their pharmacological properties. In this study, a novel glycoside hydrolase PlGH3 was cloned from Paenibacillus lactis 154 and heterologous expressed in Escherichia coli. Sequence analysis revealed that PlGH3 consists of 749 amino acids with a molecular weight of 89.5 kDa, exhibiting the characteristic features of the glycoside hydrolase 3 family. The enzymatic characterization results of PlGH3 showed that the optimal reaction pH and temperature was 8 and 50 °C by using p-nitrophenyl-β-d-glucopyranoside as a substrate, respectively. The Km and kcat values towards ginsenoside Ro were 79.59 ± 3.42 µM and 18.52 s−1, respectively. PlGH3 exhibits a highly specific activity on hydrolyzing the 28-O-β-d-glucopyranosyl ester bond of oleanane-type saponins. The mechanism of hydrolysis specificity was then presumably elucidated through molecular docking. Eventually, four kinds of rare oleanane-type ginsenosides (calenduloside E, pseudoginsenoside RP1, zingibroside R1, and tarasaponin VI) were successfully prepared by biotransforming total saponins extracted from Panax japonicus. This study contributes to understanding the mechanism of enzymatic hydrolysis of the GH3 family and provides a practical route for the preparation of rare oleanane-type ginsenosides through biotransformation.Key points• The glucose at C-28 in oleanane-type saponins can be directionally hydrolyzed.• Mechanisms to interpret PlGH3 substrate specificity by molecular docking.• Case of preparation of low-sugar alternative saponins by directed hydrolysis.

Study on one-step purification and immobilization of glycosyltransferase by modified Fe3O4 for the synthesis of rare ginsenoside Rh2

Enzymatic catalysis is the most suitable method for synthesizing the ginsenoside Rh2. However, challenges in the purification and reuse of the enzyme limited the applications of this method. In this study, Fe3O4/PMG/NTA-Ni2+ magnetic nanospheres modified with nano-Fe3O4 were employed to achieve the one-step purification and immobilization of glycosyltransferase. The resulting assembly catalyzed ginsenoside protopanaxadiol (PPD) to more valuable Rh2. This material successfully immobilized the recombinant enzyme on the carrier. Moreover, the good dispersibility and superparamagnetism of the immobilized enzyme were maintained without any alteration in its structure, and the maximum immobilized enzyme amount was 180 mg/g. Notably, the pH value, temperature and storage stability of the immobilized enzyme were significantly improved compared with the free enzyme. After reusing the immobilized enzyme six times, the relative enzyme activity was still maintained at 67.8%. Thus, this study specifically adsorbed, purified, and immobilized free glycosyltransferase by modifying nanometer-sized Fe3O4 particles. This reduced operating costs and enhanced the properties of the enzyme.