Effects and mechanism of ginsenoside Rh2 on the proliferation and apoptosis of human glioma cells / 中国药房

ABSTRACT OBJECTIVE To investigate the effects and mechanism of ginsenoside Rh2 on the proliferation and apoptosis in human glioma U87 and U251 cells. METHODS Using human glioma U87 and U251 cells as subjects， the proliferation and apoptosis， as well as the expression of histone deacetylase 1（HDAC1） protein and apoptosis-related proteins [B cell lymphoma-2（Bcl-2）， Bcl-2-associated X protein （Bax） and cleaved caspase-3] were detected after being treated with different concentrations of ginsenoside Rh2. RESULTS The concentrations of 10，20，30，40，50，60，70，80 μmol/L ginsenoside Rh2 could generally significantly increase the proliferation inhibition rate of U87 and U251 cells （P＜0.05 or P＜0.01）， and the half inhibitory concentrations of this component after 48 hours of action were 51.34 and 55.84 μmol/L， respectively；30，50 μmol/L ginsenoside Rh2 could increase the total apoptotic rate of both types of cells， reduced the protein expressions of HDAC1 and Bcl-2， and increased the protein expressions of Bax and cleaved caspase-3 significantly （P＜0.05 or P＜0.01）. CONCLUSIONS Ginsenoside Rh2 has a significant inhibitory effect on the proliferation of glioma cells and promotes the apoptosis of cells， which may be through reducing the expression of HDAC1 protein and activating the Bcl-2 family protein-mediated apoptosis pathway.

c-MYC-mediated TRIB3/P62+ aggresomes accumulation triggers paraptosis upon the combination of everolimus and ginsenoside Rh2

The mammalian target of rapamycin (mTOR) pathway is abnormally activated in lung cancer. However, the anti-lung cancer effect of mTOR inhibitors as monotherapy is modest. Here, we identified that ginsenoside Rh2, an active component of Panax ginseng C. A. Mey., enhanced the anti-cancer effect of the mTOR inhibitor everolimus both in vitro and in vivo. Moreover, ginsenoside Rh2 alleviated the hepatic fat accumulation caused by everolimus in xenograft nude mice models. The combination of everolimus and ginsenoside Rh2 (labeled Eve-Rh2) induced caspase-independent cell death and cytoplasmic vacuolation in lung cancer cells, indicating that Eve-Rh2 prevented tumor progression by triggering paraptosis. Eve-Rh2 up-regulated the expression of c-MYC in cancer cells as well as tumor tissues. The increased c-MYC mediated the accumulation of tribbles homolog 3 (TRIB3)/P62+ aggresomes and consequently triggered paraptosis, bypassing the classical c-MYC/MAX pathway. Our study offers a potential effective and safe strategy for the treatment of lung cancer. Moreover, we have identified a new mechanism of TRIB3/P62+ aggresomes-triggered paraptosis and revealed a unique function of c-MYC.

Construction of cell factories for high production of ginsenoside Rh_2 in Saccharomyces cerevisiae / 中国中药杂志

Ginsenoside Rh_2 is a rare active ingredient in precious Chinese medicinal materials such as Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma, and Panacis Quinquefolii Radix. It has important pharmacological activities such as anti-cancer and improving human immunity. However, due to the extremely low content of ginsenoside Rh_2 in the source plants, the traditional way of obtaining it has limitations. This study intended to apply synthetic biological technology to develop a cell factory of Saccharomyces cerevisiae to produce Rh_2 by low-cost fermentation. First, we used the high protopanaxadiol(PPD)-yielding strain LPTA as the chassis strain, and inserted the Panax notoginseng enzyme gene Pn1-31, together with yeast UDP-glucose supply module genes[phosphoglucose mutase 1(PGM1), α-phosphoglucose mutase(PGM2), and uridine diphosphate glucose pyrophosphorylase(UGP1)], into the EGH1 locus of yeast chromosome. The engineered strain LPTA-RH2 produced 17.10 mg·g~(-1) ginsenoside Rh_2. This strain had low yield of Rh_2 while accumulated much precursor PPD, which severely restricted the application of this strain. In order to further improve the production of ginsenoside Rh_2, we strengthened the UDP glucose supply module and ginsenoside Rh_2 synthesis module by engineered strain LPTA-RH2-T. The shaking flask yield of ginsenoside Rh_2 was increased to 36.26 mg·g~(-1), which accounted for 3.63% of the dry weight of yeast cells. Compared with those of the original strain LPTA-RH2, the final production and the conversion efficiency of Rh_2 increased by 112.11% and 65.14%, respectively. This study provides an important basis for further obtaining the industrial-grade cell factory for the production of ginsenoside Rh_2.

Screening potential Chinese materia medica and their monomers for treatment diabetic nephropathy based on caspase-1-mediated pyroptosis / 南方医科大学学报

OBJECTIVE@#To screen potential traditional Chinese medicine and their active monomer ingredients for treatment of diabetic nephropathy (DN) through the mechanism of caspase-1-mediated pyroptosis.@*METHODS@#Using the Chinese Medicine System Pharmacology Analysis Platform (TCMSP), we screened traditional Chinese drugs and their active monomer components targeting caspase-1, and searched for the potential gene targets of the monomer components using GeneCards database. Cytoscape was used to construct the monomer compound-gene target network. Gene ontology (GO) functional enrichment analysis and Kyoto Gene and Gene Encyclopedia (KEGG) pathway enrichment analysis were used to predict the molecular mechanism of the screened traditional Chinese medicine and monomers. In SD rat models of diabetic mellitus (DM), we tested the therapeutic effect of ginsenoside Rh2 (daily dose of 20 mg/kg for 12 weeks) by examining renal pathology with HE staining and detecting the expressions of pyroptosis marker proteins caspase-1, GSDMD, IL-1β and IL-18 in the renal tissues using Western blotting, the serum levels of IL-1β and IL-18 and activities of cathepsin B and cathepsin L.@*RESULTS@#Ginsenoside Rh2 could effectively dock with caspase-1 molecule. Fourteen targets were identified in ginsenoside Rh2 target network. GO function enrichment analysis revealed 27 GO terms associated with molecular function (4 terms), cell component (10 terms) and biological process (13 terms). KEGG pathyway enrichment analysis identified 4 signaling pathways involving lysosomes, glycosaminoglycan degradation, galactose metabolism, and sphingolipid metabolism pathways. In the animal experiment, treatment with ginsenoside Rh2 significantly alleviated renal pathologies and down-regulated the expressions of pyroptosis marker proteins (cleaved caspase-1, GSDMD-N, IL-1β and IL-18) ( < 0.05 or 0.01), lowered serum levels of IL-1β and IL-18 ( < 0.01), and enhanced the activities of cathepsin B and cathepsin L in the serum of the diabetic rats.@*CONCLUSIONS@#Ginsenoside Rh2 may inhibit caspase-1-mediated pyroptosis through the lysosome pathway to improve kidney damages in rat models of DN.

Mechanism of Ginsenoside Rh2 Against Liver Cancer / 中国实验方剂学杂志

Ginsenoside Rh2 is a tetracyclic triterpenoid saponin monomer containing a dammarane-type skeleton, with advantages of low toxicity, low molecular weight, good fat solubility and strong anticancer effect, and is the main anticancer effective in ginseng. In recent years, there have been emerging findings on ginsenoside Rh2, indicating an obvious anticancer activity against a variety of cancers with a high morbidity and mortality. Particularly, ginsenoside Rh2 has a significant anti-hepatocarcinoma effect, so the studies on the mechanism of action have gradually been given attention. In this paper, we have reviewed more than 100 domestic and foreign relevant literatures in Chinese and English databases in the past 20 years, such as CNKI, Wanfang Data, VIP Data, Pub Med, and conducted detailed collection, analysis and summary for the contents of the anti-hepatocarcinoma mechanism of ginsenoside Rh2. According to the findings, although there are many reports on the anti-hepatocarcinoma effect of ginsenoside Rh2, the mechanism of action of ginsenoside Rh2 against liver cancer has not been systematically elaborated. Therefore, this paper comprehensively discusses the anti-hepatocarcinoma effect of ginsenoside Rh2, clarifies that the mechanism of action of ginsenoside Rh2 against liver cancer may be related with the inhibition of the proliferation of hepatoma cells, the induction of differentiation of hepatoma cells, the promotion of apoptosis of hepatoma cells, the inhibition of invasion and metastasis of hepatoma cells, the reduction of drug resistance of liver cancer cells, and the improvement of immunity. For the first time, the mechanism of action of ginsenoside Rh2 against liver cancer was comprehensively summarized, which provided reference for researches on ginsenoside Rh2 against liver cancer, evidence and ideas for further researches on ginsenoside Rh2, new research directions for the treatment of liver cancer and new hope to patients with liver cancer.

Synthesis and anti-tumor activity of ginsenoside Rh_2 caprylic acid monoester / 中国中药杂志

Ginsenoside Rh_2,firstly isolated from red ginseng,is protopanaxadiol type of steroidal saponin. Rh_2 possessed variety of activities,but bioavailability of oral administration Rh_2 was extremely low due to poor absorption. Moreover,ginsenoside Rh_2 exhibited toxicity on human normal cells. Therefore,to improve stronger anti-tumor activity and attenuate toxicity,it was essential to design and optimize chemical structure of ginsenoside Rh_2. Through n-octanoylchloride modifications,a novel ester derivative of ginsenoside Rh_2 named caprylic acid monoester of Rh_2( C-Rh_2) was designed and synthesized. Structure of novel ginsenoside derivative was identified by1 D and 2 D NMR,as well as ESI-MS analyses. Anti-tumor effect of C-Rh_2 was tested on H22 tumor bearing mice. C-Rh_2 displayed certain anti-tumor activities and exhibited less toxicity than Rh_2. In the present study,C-Rh_2 as ester form of ginsenoside Rh_2 showed better anti-tumor activity and less toxicity,but the specific mechanism needs further investigation.

Enhancement of oral bioavailability and immune response of Ginsenoside Rh2 by co-administration with piperine / 中国天然药物

Ginsenoside Rh2 (Rh2) is one of the major bioactive ginsenosides in Panax ginseng. However, the oral bioavailability of Rh2 is low, with P-glycoprotein (P-gp) and CYP3A4 being reported to be the main factors. The purpose of the present study was to determine the enhancing effect of piperine on the oral bioavailability as well as bioactivity of Rh2. The inhibitory effect of piperine on P-gp and CYP3A4 was determined using a Caco-2 monolayer model and a recombinant CYP3A4 metabolic system, respectively. The pharmacokinetics of oral Rh2 (10 mg·kg) administered alone or in combination with piperine (10 and 20 mg·kg) was performed in rats. The immune boosting effect of Rh2 was assessed in rats by measuring IL-12 level after treated by Rh2 alone or co-administered with piperine. The results indicated that piperine significantly increased the permeability of Rh2 and inhibited the metabolism of Rh2. The pharmacokinetic study results showed that the AUC of Rh2 was significantly increased in combination with piperine at high dose (20 mg·kg) when compared to the control group, with relative bioavailability of 196.8%. The increase of Rh2 exposure led to increased serum levels of IL-12. In conclusion, piperine may be used as a bioenhancer to improve pharmacological effect of Rh2 when given orally.

Enhancement of oral bioavailability and immune response of Ginsenoside Rh2 by co-administration with piperine / 中国天然药物

Ginsenoside Rh2 (Rh2) is one of the major bioactive ginsenosides in Panax ginseng. However, the oral bioavailability of Rh2 is low, with P-glycoprotein (P-gp) and CYP3A4 being reported to be the main factors. The purpose of the present study was to determine the enhancing effect of piperine on the oral bioavailability as well as bioactivity of Rh2. The inhibitory effect of piperine on P-gp and CYP3A4 was determined using a Caco-2 monolayer model and a recombinant CYP3A4 metabolic system, respectively. The pharmacokinetics of oral Rh2 (10 mg·kg) administered alone or in combination with piperine (10 and 20 mg·kg) was performed in rats. The immune boosting effect of Rh2 was assessed in rats by measuring IL-12 level after treated by Rh2 alone or co-administered with piperine. The results indicated that piperine significantly increased the permeability of Rh2 and inhibited the metabolism of Rh2. The pharmacokinetic study results showed that the AUC of Rh2 was significantly increased in combination with piperine at high dose (20 mg·kg) when compared to the control group, with relative bioavailability of 196.8%. The increase of Rh2 exposure led to increased serum levels of IL-12. In conclusion, piperine may be used as a bioenhancer to improve pharmacological effect of Rh2 when given orally.

Active ingredients screening by cell membrane chromatography and simultaneous quantitation of ginsenosides in bulk drug of secondary ginsenoside H dripping pills / 中草药

To establish a HPLC-MS/MS method for simultaneous determination and active ingredients screening of pseudoginsenoside RT5, 20(S)-ginsenoside Rh1 and 20(S)-ginsenoside Rh2 by cell membrane chromatography (CMC) in secondary ginsenoside H dripping pills (SGHDP). Methods The samples were separated on Century SIL BDS C18 column (250 mm × 4.6 mm, 5 μm) eluted with 0.2% formic acid aqueous solution-acetonitrile in a gradient mode, and the target compounds were analyzed by positive ion multiple reaction monitoring (MRM) mode, and active ingredients of SGHDP obtained in solid-phase of biomembrane by CMC technology were determined at the same time. Results The linear ranges of pseudoginsenoside RT5, 20(S)-ginsenoside Rh1, and 20(S)-ginsenoside Rh2 were 0.095-0.235, 0.042-0.168, and 0.105-0.419 mg/mL; the extraction recoveries were 99.95%, 100.12%, and 100.06%; and RSD were 1.06%, 0.96%, and 0.91%, respectively. The contents of pseudoginsenoside RT5, 20(S)-ginsenoside Rh1, and 20(S)-ginsenoside Rh2 in SGHDP were 21.24%, 21.42%, and 29.70%, respectively. 20(S)-Ginsenoside Rh2 was the active ingredient obtained by biomembrane using as a new quality control maker for SGHDP. Conclusion The developed method is accurate and reliable for the determination of three ginsenosides in SGHDP, and provides a new reference for quality control of SGHDP. 20(S)-Ginsenoside Rh2 is a immobilization component of red cell membrane, speculated to be the active ingredient of SGHDP, which is in consistent with previous studies on antitumor and antidepression.

Determination of content changes of ginsenoside Re, Rg1, Rb1, Rg3, Rh1, and Rh2 before and after pulping of mountain cultivated ginseng by HPLC / 中草药

Objective To analyze the content changes of six kinds of ginsenosides Re, Rg1, Rb1, Rg3, Rh1, and Rh2 after pulping of mountain cultivated ginseng. Methods The HPLC-UV method was performed on an Innoval ODS-2 chromatographic column (250 mm × 4.6 mm, 5 μm), gradient elution of acetonitrile and water with column temperature 30 ℃, at a flow rate of 1.0 mL/min, and detected at 203 nm with injection volume as 20.0 μL. Results The content of six kinds of ginsenosides Re, Rg1, Rb1, Rg3, Rh1 and Rh2 were changed from 0.651, 0.506, 0.363, 0.014, 0.023, 0.031 mg/g to 0.517, 0.413, 0.105, 0.122, 0.214, 0.098 mg/g after pulping of mountain cultivated ginseng. The calibration curve was liner within 2.5-100 mg/L for ginsenoside Re, Rg1, Rb1, Rg3, Rh1, and Rh2, respectively, with the correlation r2 > 0.999 5 and perfect precision, stability, and repeatability. The average recoveries ranged from 95% to 105%, and RSD values varied from 1.25% to 3.5%. Conclusion The content of six kinds of ginsenosides Re, Rg1, Rb1, Rg3, Rh1, and Rh2 in mountain cultivated ginseng were changed after the pulping. The content of ginsenoside Re, Rg1, and Rb1 was reduced and rare ginsenoside Rg3, Rh1, and Rh2 was increased by 8.7 times, 9.3 times, and 3.2 times respectively after the pulping. The HPLC method for simultaneous determination of six kinds of ginsenosides has good accuracy and reliability and can provide scientific basis for the quality evaluation of mountain cultivated ginseng pulp.

Effect of ginsenoside Rh2 on drug resistance sensitivity of human gastric cancer SGC7901/ADR cells / 中草药

Objective To observe the effect of ginsenoside Rh2 (G-Rh2) on the proliferation, cell cycle and chemotherapy sensitivity of human gastric cancer cell line SGC7901/ADR. Methods MTT assay was used to detected the effects of G-Rh2 and adriamycin (ADR) on the proliferation of drug-resistant SGC7901/ADR cells and to calculate the reversal fold (RF). Flow cytometry was selected to detect The effects of G-Rh2 on cell cycle. the effects of G-Rh2 on the expression of P-gp and Bcl-2 proteins in drug-resistant SGC7901/ADR cells was detected by Western blotting. Results Compared with the IC50 value (54.52 μmol/L) after the ADR single drug treatment, the IC50 value (30.14 μmol/L) of the cells treated with G-Rh2 and ADR decreased significantly, and RF was 1.81. G-Rh2 combined with ADR arrested the cell cycle in G2/M phase and significantly decreased the protein expression of P-gp and Bcl-2(P < 0.05). Conclusion G-Rh2 combined with ADR could increase the chemosensitivity of SGC7901/ADR cells, which may inhibit the proliferation of gastric cancer cells by blocking cell cycle and increasing apoptosis.

Effect of ginsenoside Rh2(S) on apoptosis of human leukemia cell by inhibiting HDAC6 / 中草药

Objective To explore the specific mechanism of ginsenoside Rh2(S) inducing the apoptosis of leukemia cells. Methods The effect of Rh2(S) on proliferation of leukemia cells K562 and KG1a was measured by cell counting kit-8 assay (CCK-8 assay). The growth states of cells were observed under the inverted phase microscope, and cell cycle distribution, and apoptosis were determined by flow cytometry (FCM). The expression levels of HDAC6, HSP90, Akt, GSK-3β, β-catenin, and cell cycle apoptosis related proteins were ascertained by Western blotting. Results The results of CCK-8 showed that Rh2(S) had the most obvious inhibitory effect on the proliferation of leukemia cells. Rh2(S) significantly induced apoptosis and led to cell cycle arrest at G0/G1 phase of leukemia cells by FCM. While the microscope observation showed that the number of cells was decreased and normal cell morphology changed. Rh2(S) decreased the expression of Bcl-2, Cyclin D1, HDAC6, HSP90, p-Akt, and β-catenin and increased the expression of Bax, Ac-α-tubulin, and GSK-3β by Western blotting. Conclusion Rh2(S) can effectively inhibit the proliferation and promote the apoptosis of K562 and KG1a cells, its specific mechanism may relate to inhibitting the expression of HDAC6, resulting in a decline in the expression of HSP90, so as to further inhibit the activity of Akt activation of GSK-3, and finally inhibit Wnt/β-catenin pathway.

Effects of ginsenoside RH2 on neovascularization of rats with middle cere-bral artery occlusion / 中国病理生理杂志

AIM:To investigate the effects of ginsenoside RH 2(GS-RH2 )on neovascularization of rats with middle cerebral artery occlusion(MCAO)and its potential mechanisms.METHODS:SPF Sprague-Dawley rats were ran-domly divided into sham operation(sham)group,MCAO model(MCAO)group and GS-RH2 group,with 18 rats in each group.After surgery,the general condition and neurological function score of the rats were assessed.At the 1st day,3rd day and 7th day after intervention,the microvessel density(MVD),the content of malondialdehyde(MDA)and the activ-ity of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)were examined.The protein expression of kelch-like ECH-associated protein 1(Keap1),nuclear factor E2-related factor 2(Nrf2)and heme oxygenase-1(HO-1)was de-termined by Western blot.RESULTS:Compared with sham group ,the rats in MCAO group showed significant neurobe-havioral obstacles and ischemic brain infarction with higher neurological function score ,while treatment with GS-RH2 sig-nificantly improved behavioral impairment and reduced the infarction volume with lower neurological function score.The MVD score in GS-RH2 group was increased as the animal survival time prolonged ,while the MVD score in MCAO group was decreased.After intervention for 7 d,the MVD score in GS-RH2 group was significantly higher than that in MCAO group(P<0.05).Compared with sham group,the content of MDA was increased and the activities of SOD and GSH-Px were decreased in MCAO group at each time point.After intervention for 7 d,the MDA content was decreased and the SOD and GSH-Px activities were increased in GS-RH2 group compared with MCAO group.After intervention for 7 d,the protein expression of Nrf2 and HO-1 was increased,while the protein expression of Keap1 was decreased in GS-RH2 group com-pared with MCAO group(P<0.05).CONCLUSION:Ginsenoside RH2 promotes neovascularization of MCAO model rats.The mechanism may be related to the activation of Keap 1/Nrf2 signaling pathway ,promotion of the antioxidant enzyme activity and inhibition of oxidative stress.

Study on the drug-resistant reversal effects of ginsenoside Rh2 in human hepatocellular carcinoma HepG2/ADM cells and its mechanism / 医学研究生学报

Objective Ginsenoside Rh2 can inhibit the proliferation of a variety of malignant tumor cells.However, little research has been done on the sensitivity of Rh2 in human hepatocellular carcinoma HepG2/ADM cells with multidrug resistance(MDR).This study aimed to explore the reversing effects of ginsenoside Rh2 on the MDR of human hepatocellular carcinoma HepG2/ADM cells and its potential mechanism.Methods MTT assay was applied to detect the effect of Rh2(0-250 μg/mL) on the viability of HepG2/ADM cells and screen out the optimum drug-resistant reversal concentration of Rh2.Cells were divided into 3 groups: HepG2/ADM group (without any medicine treatment), ADM group(ADM treatment for 48 h), ADM+40 μg/mL Rh2 group (pretreatment of 40μg/mL Rh2 for 30 min followed by ADM treatment for 48 h).Flow cytometry was applied to detect the effect of Rh2 on the fluorescence intensity of cellular Rh-123.RT-PCR was used to measure the expression of MDR1 gene.Western blot was used to detect the protein levels of P-gp, Bax, Bcl-2 and cleaved caspase-3.Results 40 μg/mL ginsenoside Rh2 significantly reversed the MDR of HepG2/ADM cells by a 2.55-to-3.70-fold increase in sensitivity.Furthermore, compared with ADM group, the efflux of Rh-123 in HepG2/ADM cells were remarkably inhibited by Rh2 in ADM+40 μg/mL Rh2 group (65.83±1.78 vs 78.21±1.26, P<0.01), along with the down-regulated expressions of MDR1 (0.48±0.02 vs 0.86±0.05, P<0.05), P-gp(0.97±0.04 vs 1.91±0.03,P<0.01), Bcl-2(1.25±0.05 vs 1.86±0.03, P<0.05) and the up-regulated protein level of Bax (1.76±0.04 vs 1.25±0.02,P<0.05) and cleved caspase-3(0.42±0.04 vs 38.26±5.45,P<0.05).Conclusion Ginsenoside Rh2 can effectively reverse the MDR of HepG2/ADM cells, and the potential mechanism is related to the decreased expressions of MDR1 and P-gp, the increasing drug concentration inside the cells and the Bax/Bcl-2 signaling pathway.

Ginsenoside Rh2 induced human colorectal cancer cell apoptosis through PI3K/AKT/GSK-3βpathway / 中国药理学通报

Aim To investigate the effect of Ginsen-oside Rh2 on apoptosis in human colorectal cancer cell SW480,and to explore the possible mechanism of it. Methods The proliferation activity of SW480 treated with Ginsenoside Rh2 was measured CCK-8 assay.Ap-optosis rates were evaluated by FCM.Hoechst 33258 staining was used to observe cell nucleus morphologi-cal;change SW480 cells were treated with Ginsenoside Rh2,and the protein expressions of Bcl-2,Bax,p53, cleaved caspase-3 ,PI3 K,AKT,P-AKT,GSK-3β,P-GSK-3βwere detected by Western blot;SW480 cells were treated with LY294002,Rh2,LY294002+Rh2, the expressions of PI3 K,AKT,P-AKT,GSK-3β,P-GSK-3βwere detected by Western blot.Results The proliferation of SW480 cells was significantly inhibited by Ginsenoside Rh2 in dose-dependent and time-de-pendent manner.FCM showed the inducing apoptosis effect of Ginsenoside Rh2 was significantly different from that of control group.Hoechst 33258 staining in-dicated clearly cell apoptosis in Ginsenoside Rh2 treat-ment groups.Western blot showed Ginsenoside Rh2 decreased expression of Bcl-2,increased expression of Bax,p53 and cleaved caspase-3,PI3K/AKT/GSK-3βpathway proteins PI3 K,P-AKT,P-GSK-3βdecreased obviously,AKT and GSK-3βwere not changed signifi-cantly in SW480.SW480 cells were separately treated with LY294002,Rh2,LY294002 +Rh2,there were no significant difference in AKT and GSK-3βprotein a-mong all groups,and the expression of PI3 K,P-AKT, P-GSK-3βdecreased more obviously in LY294002 +Rh2 group compared with LY294002 and Rh2 alone. Conclusion Rh2 induces colorectal cancer cell apop-tosis through PI3 K/AKT/GSK-3βpathway,which ac-tivates p53 and cleaved caspase-3,and destroys the balance of Bcl-2/Bax.

Protopanaxadiol ginsenosides dynamics in enzymatic reaction and preparation of rare ginsenosides / 中草药

Objective: To cheaply prepare the rare ginsenosides by biotransformation, ginsenosides C-K, C-Mc, F2, and Rh2 from commercially available Protopanaxadiol (PPD) ginsenoside mixture were prepared using a crude enzyme of Aspergillus g.848 strain. Methods: The rare ginsenosides were obtained from PPD ginsenosides by enzyme reaction; The composition of PPD raw materials and ginsenoside products was measured by HPLC. The monomer ginsenosides and Rh2 group of enzyme reaction product were separated by a silica gel column; The produced monomer ginsenosides were identified by NMR; Rh2 group was identified by UPLC-MS. Results: The raw material of PPD ginsenosides was consisted of ginsenoside Rb1, Rd, Rb2, Rc, and Rg3 groups with four kinds of isomers. During the reaction of enzyme, the best reaction time for the ginsenoside F2 production was 1.5 h to 2 h; The best reaction time for the ginsenoside C-K production was 24 h to 30 h; If producing the Rh2 group, when reacted to 6 h to 12 h, the content of Rg3 group was low, and Rh2 group was high. In the production of C-K, 20 g of crude products were obtained from 30 g of PPD ginsenosides by enzyme reaction, and 8.16 g of C-K, 1.01 g of C-MC, 0.45 g of F2, and 0.19 g of Rh2 group were separated using silica gel column. The rare ginsenosides were identified by NMR, and the Rh2 group was identified using UPLC-MS method. Conclusion: The high activity of monomer ginsenoside C-K, C-Mc, F2, and Rh2 group are successfully prepared from the PPD ginsenosides by enzymatic conversion.

Effect of ginsenoside Rh2 on proliferation and apoptosis of KG1α cells by autophagy pathway / 中草药

Objective: To investigate the antitumour activity of ginsenoside Rh2 against human leukemia KG1α cells through apoptosis and autophagy pathway. Methods: CCK-8 assay was used to screen the most effective ingredient on the proliferations among ginsenoside Rh2 in leukemia KG1α cell line; FCM detected cell apoptosis; Hoechst staining observed the cell morphological changes of apoptosis; Acridine staining detected Rh2 effected on autophagy; Western blotting and RT-PCR detected the expression levels of the proteins closely associated with autophagy and apoptosis. After joining autophagy inhibitors, using CCK-8 to test the proliferation activity of cells, cell apoptosis was measured by FCM. Results: CCK-8 indicated that Rh2 could inhibit the proliferation of KG1α cells significantly with dose- and time-dependent manners; FCM indicated that Rh2 induced apoptosis; Hoechest staining showed that KG1α cells had typical apoptotic morphological changes by treated Rh2; Acridine staining revealed that Rh2 cause increase in the number of acidic autophagy vesicles in cells, causing cell autophagy levels increased; Western blotting and RT-PCR results showed that Rh2 increased the expression of Beclin-1, LC3A, and LC3B, activated Caspase-3 and Bax/Bcl-2 rates, and MAPK, ATK, and ERK signaling pathway; After using autophagy inhibitors (3-MA), autophagy crippled that Rh2 inhibited the proliferation and induced apoptosis in KG1α cells. Conclusion: Ginsenoside Rh2 could significantly enhance autophagy through activated MAPK, ATK, and ERK signaling pathway, and then inhibit the proliferation and induce apoptosis in KG1α cells.

Ginsenoside Rh₂ induces apoptosis and autophagy of K562 cells by activating p38 / 中国中药杂志

To study the effect of ginseng saponin Rh₂ in inducing apoptosis of human leukemia K562 cells, and explore its mechanism from the aspect of autophagy pathway. CCK-8 assay was used to examine the growth inhibition of human leukemia cell lines K562 treated with ginsenoside Rh₂; flow cytometry (FCM) was used to detect cell apoptosis; Hoechst staining was used to observe the changes of cell morphological apoptosis; Acridine and MDC staining were used to detect the effects of the Rh₂ on autophagy; Western blot and RT-PCR were used to detect the expression levels of the proteins closely associated with autophagy and apoptosis. In order to study the effect of autophagy in proliferation and apoptosis, we used the autophagy inhibitor (3-MA).CCK-8 indicated that Rh₂ at low concentration could effectively inhibit the proliferation of leukemia cellsin dose- and time-dependent manners in K562 cells; FCM indicated that Rh₂ induced apoptosis; Hoechest staining showed that K562 cells had typical apoptotic morphological changes by treated Rh₂; Acridine and MDC staining showed that Rh₂ enhanced the green fluorescence and a large number of acidic autophagy vesicles were present; Western blot and RT-PCR results showed that Rh₂ increased the expression levels of Beclin-1, LC3A, LC3B, activated Caspase-3 and p-p38 in K562 cells; application of autophagy inhibitors(3-MA) could weaken the inhibition effect of Rh₂ on proliferation and induction effect on apoptosis in K562 cells. Ginsenoside Rh₂ inhibited the proliferation and induced apoptosis probably through activating p-p38, and inducing cell autophagy signaling pathway in K562 cells.

Study on anti-hepatoma effect of ginsenoside Rh2 and mechanism of degradation of β-catenin through activating GSK-β / 中草药

Objective: To investigate the anticancer effect and the mechanism of ginsenoside Rh2 on animal model of HepG2 liver carcinoma. Methods: HE staining was used to observe cell morphology. Immunohistochemical staining was used to detect the expression of GSK-3β, β-catenin, and MMP-3 in isolated single cells. The activity of GSK-3β was checked by ELISA kit. The expression levels of GSK-3β, β-catenin, Bax, Bcl-2, CyclinD1, and MMP-3 genes were measured by qRT-PCR. The expression of β-catenin and GSK-3β proteins were determined by Western blotting. Results: HE staining showed that the nucleus was atypia and account for a large proportion of the whole cell in HepG2 group and HepG2-β-catenin group. But nucleus atypia in HepG2-β-catenin group was more obvious. Condensation nuclei and a lot of broken cells were observed in HepG2-β-catenin + ginsenoside Rh2 group and HepG2 + ginsenoside Rh2 group. However, condensation nuclei and broken cells in HepG2 + ginsenoside Rh2 group were more obvious. Immunohistochemical results indicated the expression of GSK-3β increased, while β-catenin and MMP-3 expression decreased in HepG2-β-catenin + ginsenoside Rh2 group, compared with HepG2-β-catenin group. The expression of β-catenin and MMP-3 in HepG2 + ginsenoside Rh2 group was lower than that in HepG2-β-catenin + ginsenoside Rh2 group, while GSK-3β was no significant difference. The ELISA results indicated that the activity of GSK-3β was increased in HepG2 + ginsenoside Rh2 group and HepG2-β-catenin + ginsenoside Rh2 group. Compared with HepG2-β-catenin + ginsenoside Rh2 group, the expression of Bax gene in HepG2 + ginsenoside Rh2 group increased significantly, and the expression levels of Bcl-2, CycliD1, and MMP-3 genes were also significantly lower, the difference was statistically significant (P <0.01). The Western blotting results showed that compared with HepG2-β-catenin + ginsenoside Rh2 group, the expression of β-catenin protein in HepG2 + ginsenoside Rh2 group was also significantly lower, the difference was statistically significant (P < 0.01). Conclusion: In vivo experiment shows that weight of tumor is decreased by ginsenoside Rh2 through activating GSK-3β to degrade β-catenin and could inhibit the ability of HepG2 cells metastasis.

Pharmacokinetic study on ginsenoside Rg3 and ginsenoside Rh2 in gut microbiota dysbiosis rats / 中草药

Objective: The aim of this study was to explore the pharmacokinetics of ginsenoside Rg3 and its deglycosylated metabolite, ginsenoside Rh2 in lincomycin-induced gut microbiota dysbiosis rats after ig administration of ginsenoside Rg3. Methods: An LC-MS/MS analytical method was developed and validated to detect ginsenoside Rg3 and Rh2 in plasma of rats. The method was validated by specificity, linearity, lower limits of quantification (LLOQ), precision, accuracy, matrix effect, recovery, and stability. Lincomycin (orally, 5 000 mg/kg, 7 d) was selected to induce gut microbiota dysbiosis. The fecal moisture contents and the β-D-glucosidase activity were also assessed in this study. And the plasma samples were collected and analyzed after ig administration of ginsenoside Rg3 (20 mg/kg). Results: The results indicated that this method could be used for the determination of the concentration of ginsenoside Rg3 and Rh2 in plasma of rats. The fecal moisture content in rats treated with lincomycin was significantly increased (P < 0.01) and the β-D-glucosidase activity was decreased (P < 0.01) compared with the control rats. The AUC0~∞ and Cmax in gut microbiota dysbiosis rats were increased, while the AUC0~t and Cmax of its active metabolite, ginsenoside Rh2 were significantly decreased (P < 0.01) compared with normal rats. Conclusion: The pharmacokinetic profile of ginsenoside Rg3 and Rh2 is changed in gut microbiota dysbiosis rats, which partly relates to the decreased β-D-glucosidase activity.