Biotransformation differences of ginsenoside compound K mediated by the gut microbiota from diabetic patients and healthy subjects / 中国天然药物

Many natural products can be bio-converted by the gut microbiota to influence pertinent efficiency. Ginsenoside compound K (GCK) is a potential anti-type 2 diabetes (T2D) saponin, which is mainly bio-transformed into protopanaxadiol (PPD) by the gut microbiota. Studies have shown that the gut microbiota between diabetic patients and healthy subjects are significantly different. Herein, we aimed to characterize the biotransformation of GCK mediated by the gut microbiota from diabetic patients and healthy subjects. Based on 16S rRNA gene sequencing, the results indicated the bacterial profiles were considerably different between the two groups, especially Alistipes and Parabacteroides that increased in healthy subjects. The quantitative analysis of GCK and PPD showed that gut microbiota from the diabetic patients metabolized GCK slower than healthy subjects through liquid chromatography tandem mass spectrometry (LC-MS/MS). The selected strain A. finegoldii and P. merdae exhibited a different metabolic capability of GCK. In conclusion, the different biotransformation capacity for GCK may impact its anti-diabetic potency.

Protopanaxadiol-type ginsenoside hydrolases and their application in the preparation of ginsenoside Compound K: a review / 生物工程学报

Ginsenoside Compound K (CK) has anti-cancer and anti-inflammatory pharmacological activities. It has not been isolated from natural ginseng and is mainly prepared by deglycosylation of protopanaxadiol. Compared with the traditional physicochemical preparation methods, the preparation of CK by hydrolysis with protopanaxadiol-type (PPD-type) ginsenoside hydrolases has the advantages of high specificity, environmental-friendliness, high efficiency and high stability. In this review, the PPD-type ginsenoside hydrolases were classified into three categories based on the differences in the glycosyl-linked carbon atoms of the hydrolase action. It was found that most of the hydrolases that could prepare CK were PPD-type ginsenoside hydrolase type Ⅲ. In addition, the applications of hydrolases in the preparation of CK were summarized and evaluated to facilitate large-scale preparation of CK and its development in the food and pharmaceutical industries.

Mechanism of ginsenoside CK inhibiting proliferation of human colon cancer SW480 cells / 中草药

Objective: To study the effects of ginsenoside CK on proliferation and apoptosis of human colon cancer cell line SW480, and further explore the mechanism. Methods: Cell viability was measured by CCK-8 assay. Cell cycle, apoptosis, reactive oxygen species (ROS) levels and changes in mitochondrial membrane potential were measured by flow cytometry. Hoechst staining further detected apoptosis. Western blotting was used to detect the release of cytochrome C and the expression of apoptosis-related proteins such as Bcl-2, Bax and cleaved Caspase-3. Results: Ginsenoside CK had a significant inhibitory effect on the proliferation of human colon cancer cell line SW480. Ginsenoside CK induced SW480 cells arrest in G0/G1 phase, promoted early apoptotic cells, significantly increased intracellular ROS levels and reduced the MMP level. Ginsenoside CK promoted the expression of Bax and cleaved-Caspase-3 and inhibited the expression of Bcl-2. In addition, ginsenoside CK released a large amount of cytochrome C in SW480 cells. Conclusion: Ginsenoside CK has a significant inhibitory effect on the proliferation of human colon cancer cell line SW480. The mechanism may be through the promotion of mitochondrial superoxide elevation, resulting in a significant increase in intracellular ROS levels and a significant decrease in MMP level, further leading to the release of cytochrome C, the up-regulated expression of Bax, the down-regulated expression of Bcl-2, and ultimately leading to apoptosis of cells.

Quantification of Panax notoginseng saponins metabolites in rat plasma with in vivo gut microbiota-mediated biotransformation by HPLC-MS/MS / 中国天然药物

Panax notoginseng saponins (PNS) are the major components of Panax notoginseng, with multiple pharmacological activities but poor oral bioavailability. PNS could be metabolized by gut microbiota in vitro, while the exact role of gut microbiota of PNS metabolism in vivo remains poorly understood. In this study, pseudo germ-free rat models were constructed by using broad-spectrum antibiotics to validate the gut microbiota-mediated transformation of PNS in vivo. Moreover, a high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) was developed for quantitative analysis of four metabolites of PNS, including ginsenoside F1 (GF1), ginsenoside Rh2 (GRh2), ginsenoside compound K (GCK) and protopanaxatriol (PPT). The results showed that the four metabolites could be detected in the control rat plasma, while they could not be determined in pseudo germ-free rat plasma. The results implied that PNS could not be biotransformed effectively when gut microbiota was disrupted. In conclusion, gut microbiota plays an important role in biotransformation of PNS into metabolites in vivo.

Effect of astragaloside IV on tumor cellular uptake and antitumor efficacy by ginsenoside compound K / 中草药

Objective: To investigate the effect of astragaloside IV on tumor cellular uptake and antitumor efficacy by ginsenoside compound K (GCK). Methods: The human lung adenocarcinoma cell line A549 was prepared as an antitumor model, the cytotoxicity of the mixtures of GCK and astragaloside IV to A549 cells was determined by MTT assay, the cellular uptake of GCK was detected by fluorescence microscopy, and the intracellular GCK was determined by HPLC. The enhancement of astragaloside IV in vivo efficacy by GCK was evaluated by nude mice bearing tumor model. Results: The effect of GCK on the inhibition of A549 cell proliferation was enhanced in the presence of astragaloside IV and astragaloside IV could increase the uptake rate of GCK in A549 cells, with the proportion of astragaloside IV increasing, the cytotoxicity was significantly stronger than GCK and the uptake rate was improved. In vivo antitumor assay of mice bearing tumor indicated that astragaloside IV could enhance the antitumor efficacy of GCK. Conclusion: Preliminary results indicate that the mixture of GCK and astragaloside IV have the effect of enhancing antitumor efficacy.

Determination of ginsenoside compound K in human plasma by liquid chromatography-tandem mass spectrometry of lithium adducts

Ginsenoside compound K (GCK), the main metabolite of protopanaxadiol constituents of Panax ginseng, easily produces alkali metal adduct ions during mass spectrometry particularly with lithium. Accordingly, we have developed a rapid and sensitive liquid chromatography-tandem mass spectrometric method for analysis of GCK in human plasma based on formation of a lithium adduct. The analyte and paclitaxel (internal standard) were extracted from 50 µL human plasma using methyl tert-butyl ether. Chromatographic separation was performed on a Phenomenex Gemini C18 column (50 mm×2.0 mm; 5 μm) using stepwise gradient elution with acetonitrile-water and 0.2 mmol/L lithium carbonate at a flow rate of 0.5 mL/min. Detection was performed in the positive ion mode using multiple reaction monitoring of the transitions at m/z 629→449 for the GCK-lithium adduct and m/z 860→292 for the adduct of paclitaxel. The assay was linear in the concentration range 1.00-1000 ng/mL (r (2)>0.9988) with intra- and inter-day precision of ±8.4% and accuracy in the range of -4.8% to 6.5%. Recovery, stability and matrix effects were all satisfactory. The method was successfully applied to a pharmacokinetic study involving administration of a single GCK 50 mg tablet to healthy Chinese volunteers.

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

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

Transformation of Compound K from Saponins in Leaves of Panax notoginseng by Immobilized β-Glucanase / 中草药·英文版

Objective To prepare an active anti-tumor component, compound K (C-K), from saponins in leaves of Panax notoginseng (SLPN) using immobilized β-glucanase. Methods Two entrapments, alginate gel-1 (Alg 1) and alginate gel-2 (Alg 2), were evaluated for their ability to immobilize β-glucanase. The amount and purity of C-K obtained from the transformation process were analyzed by HPLC, and the immobilizing parameters were optimized. Results β-Glucanase can be immobilized and reused with either of the entrapment. However, using AIg 1 resulted in higher enzyme activity than Alg 2. The optimal concentration of the immobilized enzyme was 10%; The optimal crosslinking time was 4-6 h; and the optimal concentration of the crosslinking agent was 6%-7%. Conclusion Immobilized β-glucanase shows sustained enzyme activity, good ethanol tolerance, and was reusable for the preparation of C-K from SLPN.

Microbial transformation on ginsenoside compound K from total saponins in fruit of Panax ginseng / 中草药

Objective To apply the microbial transformation to transforming the total saponins in the fruit of Panax ginseng (SFPG) and preparing ginsenoside compound K (C-K). Methods The four microbial strains m14, m3, m8, and m9 were screened and isolated from the soil in the botanic garden planted for P. ginseng and they were used for the microbial transformation of SFPG to optimize the strains. Taking C-K contents as index the microbial transformation was detected and analyzed by TLC and HPLC. Results The strain m14 was found to transform the SFPG efficiently to C-K at first. The optimal culturing and transformation conditions of m14 were obtained: time, 6 d; temperature, 30 ℃; revolution of cradle, 160 r/min; initial pH value, 5.5; substratum concentration, 120 mg/mL. Under the optimal condition, the content of C-K was 41.65 times as much as before transformation by m14. Conclusion The m14 is the most effective strain among the four fungal strains. It is the new way available for the C-K industrialized production.