Ginsenoside Rh2 Research Articles

Ginsenoside Rh3 Inhibits Proliferation and Induces Apoptosis of Colorectal Cancer Cells

Background: Colorectal cancer is a common malignant tumor of the digestive tract, the morbidity rate of which is rising in recent years. Ginsenoside Rh3 was reported to have anticancer activity; however, the underlying mechanism still needs to be explored in depth. Methods: Rabbit blood was used to test hemolytic effects of ginsenoside protopanaxadiol (PPD), Rh2, Rh3, and Rg3. Human colorectal cancer SW1116 cells were treated with different concentration of ginsenoside PPD, Rh2, Rh3, and Rg3 in vitro. MTT and TUNEL assay were used to examine cell proliferation and apoptosis. Semi quantitative RT-PCR, immunocytochemistry assay and flow cytometry assay were used to detect the expression of caspase3. Results: The results showed that the inhibiting effects on SW1116 cells of PPD and Rh2 were stronger than those of Rh3 (p < 0.01), but Rh3 had better solubility and slighter hemolytic effects on blood cells than those ginsenosides. Ginsenoside Rh3 inhibited the proliferation of SW1116 cells at 60 μg/mL (p < 0.01), the inhibition effect was increased sharply when the dose of Rh3 was increased from 60 to 120 μg/mL, the inhibition rate was 62.1% at 120 μg/mL, the inhibition appeared at 9 h, and the peak activity occurred at 12 h and maintained until 48 h (p < 0.01). Compared to the control group, the ratio of apoptotic cells, the expression level of mRNA and protein of caspase3 increased in 120 μg/mL Rh3 treated group. Conclusion: As a potential anticancer medicine, ginsenoside Rh3 could inhibit the proliferation of colorectal cancer cells in a dose- and time-dependent manner and induce cell apoptosis through upregulating the expression of caspase3.

Photoluminescent And Self-Assembled Hyaluronic Acid-Zinc Oxide-Ginsenoside Rh2 Nanoparticles And Their Potential Caspase-9 Apoptotic Mechanism Towards Cancer Cell Lines.

BackgroundZinc oxide nanoparticles (ZnO NPs) are used in modern cancer therapy based on their specific target, efficacy, low toxicity and biocompatibility. The photocatalytic performance of Zinc oxide (ZnO) nanocomposites with hyaluronic acid (HA) was used to study anticancer properties against various human cancer cell lines.MethodsZinc oxide (ZnO) nanocomposites functionalized by hyaluronic acid (HA) were prepared by a co-precipitation method (HA-ZnONcs). The submicron-flower-shaped nanocomposites were further functionalized with ginsenoside Rh2 by a cleavable ester bond via carbodiimide chemistry to form Rh2HAZnO. The physicochemical behaviors of the synthesized ZnO nanocomposites were characterized by various analytical and spectroscopic techniques. We carried out 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay to evaluate the toxicity of Rh2HAZnO in various human cancer cells (A549, MCF-7, and HT29). Furthermore, to confirm the apoptotic effects of Rh2HAZnO and to determine the role of the Caspase-9/p38 MAPK pathways by various molecular techniques such as RT-PCR and Western blotting. Furthermore, Rh2HAZnO induced morphological changes of these cell lines, mainly intracellular reactive oxygen species (ROS) were observed by ROS staining and nucleus by Hoechst staining.ResultsWe confirmed that Rh2HAZnO exhibits the anti-cancer effects on A549 lung cancer, HT29 colon cancer, and MCF7 breast cancer cells. Moreover, intracellular reactive oxygen species (ROS) were observed in three cancer cell lines. Rh2HAZnO induced apoptotic process through p53-mediated pathway by upregulating p53 and BAX and downregulating BCL2. Specifically, Rh2HAZnO induced activation of cleaved PARP (Asp214) in A549 lung cancer cells and upregulated Caspase-9/phosphorylation of p38 MAPK in other cell lines (HT29 and MCF-7). Furthermore, Rh2HAZnO induced morphological changes in the nucleus of these cell lines.ConclusionThese results suggest that the potential anticancer activity of novel Rh2HAZnO nanoparticles might be linked to induction of apoptosis through the generation of ROS by activation of the Caspase-9/p38 MAPK pathway.

Synthesis and anti-tumor activity of ginsenoside Rh_2 caprylic acid monoester

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.

Dose-dependent exposure profile and metabolic characterization of notoginsenoside R1 in rat plasma by ultra-fast liquid chromatography-electrospray ionization-tandem mass spectrometry.

Notoginsenoside R1 (NGR1 ), a diagnostic protopanaxatriol-type (ppt-type) saponin in Panax notoginseng, possesses potent biological activities including antithrombotic, anti-inflammatory, neuron protection and improvement of microcirculation, yet its pharmacokinetics and metabolic characterization as an individual compound remain unclear. The aim of this study was to investigate the exposure profile of NGR1 in rats after oral and intravenous administration and to explore the metabolic characterization of NGR1 . A simple and sensitive ultra-fast liquid chromatographic-tandem mass spectrometric method was developed and validated for the quantitative determination of NGR1 and its major metabolites, and for characterization of its metabolic profile in rat plasma. The blood samples were precipitated with methanol, quantified in a negative multiple reaction monitoring mode and analyzed within 6.0 min. Validation parameters (linearity, precision and accuracy, recovery and matrix effect, stability) were within acceptable ranges. After oral administration, NGR1 exhibited dose-independent exposure behaviors with t1/2 over 8.0 h and oral bioavailability of 0.25-0.29%. A total of seven metabolites were characterized, including two pairs of epimers, 20(R)-notoginsenoside R2 /20(S)-notoginsenoside R2 and 20(R)-ginsenoside Rh1 /20(S)-ginsenoside Rh1 , with the 20(R) form of saponins identified for the first time in rat plasma. Five deglycometabolites were quantitatively determined, among which 20(S)-notoginsenoside R2 , ginsenoside Rg1 , ginsenoside F1 and protopanaxatriol displayed relatively high exploration, which may partly explain the pharmacodynamic diversity of ginsenosides after oral dose.

Systematic exploration of Astragalus membranaceus and Panax ginseng as immune regulators: Insights from the comparative biological and computational analysis

BackgroundImmune system plays a decisive role for defending various pathogenic microorganisms. Astragalus membranaceus (AM) and Panax ginseng (PG) are two tonic herbs used in traditional Chinese medicine (TCM) as immune booster and help to control diseases with their healthy synergistic effect on immune system. PurposeThis study was aimed to investigate the promote effect and molecular mechanisms of AM and PG on immune system as booster and to control the target diseases using animal and computational systematic study. MethodsComputational models including absorption, distribution, metabolism, and elimination (ADME) with weighted ensemble similarity (WES) algorithm-based models and ClueGo network analysis were used to find the potential bioactive compounds targets and pathways, which were responsible for immune regulation. Viscera index analysis, proliferation activity of splenic lymphocytes and cytotoxic activity of NK cells assays were performed to validate the effect of AM and PG on immune system of long-term administrated mice. Metabonomic study of mice plasma was conducted to investigate effect of AM and PG on the endogenous metabolic perturbations, together with correlation analysis. ResultsAM and PG simultaneously showed the ability to strengthen the immune system function including enhancement of spleen and thymus index, proliferation of splenic lymphocytes and cytotoxic activity of NK cells. Besides, the different molecular mechanisms of AM and PG on immune regulation were also investigated by analyzing the potential bioactive compounds, enzymes actions and pathways. Quercetin, formononetin and kaempferol were the main immune-related compounds in AM, while ginsenoside Ra1, ginsenoside Rh1 and kaempferol in PG. About 10 target proteins were found close to immune regulation, including acetylcholinesterase (ACHE, common target in AM and PG), sphingosine kinase 1(SPHK1), cytidine deaminase (CDA), and Choline O-acetyltransferase (CHAT). Glycerophospholipid metabolism was regulated in both AM and PG groups. Pyrimidine metabolism and sphingolipid metabolism were considered as the special pathway in AM groups. Energy metabolism and glycerolipid metabolism were the special pathways in PG groups. ConclusionA novel comprehensive molecular mechanism analysis method was established and applied to clarify the scientific connotation of AM and PG as immune regulation, with similar herbal tonic effect provided in clinical practice of TCM, which can provide a new line of research for drug development (immune booster) using AM and PG.

Ginsenoside Rh1 inhibits colorectal cancer cell migration and invasion in vitro and tumor growth in vivo.

Colorectal cancer (CRC) is the third leading cause of cancer-associated mortality worldwide. Ginsenoside Rh1 (Rh1) is a traditional medicine monomer with antitumor activity; however, the effects of Rh1 in CRC remain to be determined. In the present study, SW620 cells were treated with different concentrations of Rh1. Cell Counting Kit-8, wound healing and Transwell assays were performed to measure cell viability and proliferation, migration and invasion, respectively. Subsequently, the mRNA expression levels of matrix metallopeptidase (MMP)1, MMP3 and tissue inhibitor of metalloproteinases 3 (TIMP3) were detected by reverse transcription-quantitative PCR analysis. In addition, the protein expression levels of MMP1, MMP3, TIMP3, and total or phosphorylated (p-)ERK1/2, P38, JNK were detected by western blotting. Furthermore, tumor growth was examined in a nude mouse xenograft model. The results of the present study indicated that Rh1 was not toxic to CRC cells at various concentrations (0, 50 or 100 µM) and treatment durations (24 or 48 h). However, cell proliferation was suppressed by Rh1 in a dose-dependent manner. Rh1 (100 µM) significantly inhibited cell migration and invasion in vitro. Additionally, Rh1 suppressed the mRNA and protein expression of MMP1 and MMP3, and promoted TIMP3 expression. Rh1 decreased the ratios of p-P38/P38, p-ERK1/2/ERK1-2 and p-JNK/JNK in vitro and in vivo, which suggested that Rh1 inactivated the mitogen-activated protein kinase (MAPK) signaling pathway. Notably, Rh1 markedly decreased tumor volume and weight in vivo. In conclusion, the present study demonstrated that Rh1 inhibited the proliferation, migration and invasion of CRC cells in vitro and tumor growth in vivo. This inhibition was at least partially due to the inhibition of MMP1 and MMP3 expression, the increase in TIMP3 expression level and the MAPK signaling pathway inactivation. Therefore, Rh1 may effectively inhibit the development of CRC as an anticancer drug, and may have a supporting effect during CRC treatment.

Apoptosis-Inducing Effect of Ginsenoside Rh2 on Human Acute T Lymphoblastic Leukemia Jurkat Cells and Its Mechanism

To investigate the apoptosis-inducing effect of Ginsenoside (Rh2) on human acute T lymphoblastic leukemia Jurkat cells and it mechamism. The effects of different concentration of Rh2 (0, 10 , 20, 40 and 80 µg/ml) on the proliferation activity of Jurkat cells were detected by methyl thiazolyl tetrazolium (MTT) method, and the semi-inhibitory concentration (IC50) of Rh2 on Jurkat cells at 48 h was calculated. Microscopy and Hoechst 33258 fluorescence staining were used to observe the apoptosis of Jurkat cells treated with IC50 Rh2 for 48 h. And then, the cell experiment was divided into 4 groups: control, Rh2 (IC50), PI3K inhibitor LY294002 (50 µmol/l) and Rh2 (IC50) + LY294002 (50 µmol/l). After synchronous culture for 48 h, the apoptosis and cycle changes of Jurkat cells were detected by using PI single staining and Annexin V-FITC/PI double staining, respectively. Western blot was used to detect the expression level of apoptosis-related protein BAX, BCL-2, Cleaved-Caspasase 3, cell cycle-related protein Cyclin D1 and PI3K/AKT signaling pathway-related protein AKT and p-AKT. Rh2 (10-80 µg/ml) inhibited the Jurkat cell proliferation in a dose-time dependent manner (r48h = 0.999, P＜0.01; r80 µg/ml = 0.991; P＞0.05), accompanied by obvious morphological changes of apoptosis cells. Flow cytometry showed that compared with the control group, the cell apoptosis rate in Rh2 or LY294002 group significantly increased, and the cell cycle was mostly blocked in G0/G1 phase. However, the cell apoptosis and cell cycle block in Rh2+LY294002 group were more significant than that in Rh2 and LY294002 group. Western blot showed that compared with the control group, Rh2 significantly promoted the expression of BAX and Cleaved-Caspasase 3, inhibited the expression of BCL-2, Cyclin D1 and p-AKT, furthermore LY294002 significantly promoted this effect. Rh2 can induce the apoptosis of Jurkat cells in time-dose dependent manner, moreover, Rh2 also can result in an obvious block of Jurkat cells at G0/G1, that may be closely related to a series of apoptotic signaling cascades mediated by Rh2 inhibiting PI3K/AKT pathway.

20(S)-Ginsenoside Rh2 displays efficacy against T-cell acute lymphoblastic leukemia through the PI3K/Akt/mTOR signal pathway

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) is a kind of aggressive hematological cancer, and the PI3K/Akt/mTOR signaling pathway is activated in most patients with T-ALL and responsible for poor prognosis. 20(S)-Ginsenoside Rh2 (20(S)-GRh2) is a major active compound extracted from ginseng, which exhibits anti-cancer effects. However, the underlying anticancer mechanisms of 20(S)-GRh2 targeting the PI3K/Akt/mTOR pathway in T-ALL have not been explored. MethodsCell growth and cell cycle were determined to investigate the effect of 20(S)-GRh2 on ALL cells. PI3K/Akt/mTOR pathway–related proteins were detected in 20(S)-GRh2–treated Jurkat cells by immunoblotting. Antitumor effect of 20(S)-GRh2 against T-ALL was investigated in xenograft mice. The mechanisms of 20(S)-GRh2 against T-ALL were examined by cell proliferation, apoptosis, and autophagy. ResultsIn the present study, the results showed that 20(S)-GRh2 decreased cell growth and arrested cell cycle at the G1 phase in ALL cells. 20(S)-GRh2 induced apoptosis through enhancing reactive oxygen species generation and upregulating apoptosis-related proteins. 20(S)-GRh2 significantly elevated the levels of pEGFP-LC3 and autophagy-related proteins in Jurkat cells. Furthermore, the PI3K/Akt/mTOR signaling pathway was effectively blocked by 20(S)-GRh2. 20(S)-GRh2 suppressed cell proliferation and promoted apoptosis and autophagy by suppressing the PI3K/Akt/mTOR pathway in Jurkat cells. Finally, 20(S)-GRh2 alleviated symptoms of leukemia and reduced the number of white blood cells and CD3 staining in the spleen of xenograft mice, indicating antitumor effects against T-ALL invivo. ConclusionThese findings indicate that 20(S)-GRh2 exhibits beneficial effects against T-ALL through the PI3K/Akt/mTOR pathway and could be a natural product of novel target for T-ALL therapy.

Production of Rare Ginsenosides Rg3 and Rh2 by Endophytic Bacteria from Panax ginseng.

The ginsenosides Rh2 and Rg3 induce tumor cell apoptosis, inhibit tumor cell proliferation, and restrain tumor invasion and metastasis. Despite Rh2 and Rg3 having versatile pharmacological activities, contents of them in natural ginseng are extremely low. To produce ginsenosides Rh2 and Rg3, the saponin-producing capacity of endophytic bacteria isolated from Panax ginseng was investigated. In this work, 81 endophytic bacteria isolates were taken from ginseng roots by tissue separation methods. Among them, strain PDA-2 showed the highest capacity to produce the rare ginsenosides; the concentrations of rare ginsenosides Rg3 and Rh2 reached 62.20 and 18.60 mg/L, respectively. On the basis of phylogenetic analysis, it was found that strain PDA-2 belongs to the genus Agrobacterium and was very close to Agrobacterium rhizogenes.

Ginsenoside Rh2 reverses cyclophosphamide-induced immune deficiency by regulating fatty acid metabolism.

Ginsenoside Rh2 (G-Rh2) has well-established potent antitumor activity; yet, the effects of G-Rh2 on immune and metabolism regulation in cancer treatment, especially non-small cell lung cancer (NSCLC) remain unclear. We showed that G-Rh2 had a synergistic antitumor effect with cyclophosphamide (CY) on mice with NSCLC, and improved the immune deficiency caused by CY. Consistently, G-Rh2 exhibited no inhibitory effect on tumor growth of T cells-deficient nude mice. Furthermore, G-Rh2 treatment triggered the oxidative decomposition of fatty acid (FA), suppressed FA synthesis, increased ketone level, and decreased glucocorticoid (CORT) secretion. G-Rh2 significantly down-regulated the expression of fatty acid synthase (FASN). Of note, in liver-specific FASN knockout mice G-Rh2 failed to show the same immune enhancement effects. Further mechanistic exploration revealed that G-Rh2 suppressed the expression and nuclear translocation of sterol regulatory element binding protein 1 (SREBP-1), and disturbed the SREBP-1-FASN interaction in vitro.

Reversal effect of ginsenoside Rh2 on oxaliplatin-resistant colon cancer cells and its mechanism.

Chemotherapy is an important treatment modality for colon cancer, however, drug resistance is the main factor leading to treatment failure. Ginsenoside Rh2 (G-Rh2), the main bioactive metabolite of ginseng, is known to possess the ability to potently induce cell apoptosis, inhibit cell proliferation and reverse multidrug resistance in a variety of cancer cells. The present study examined the effect of G-Rh2 on oxaliplatin (L-OHP)-resistant colon cancer cells and its potential mechanism. L-OHP-resistant colon cancer cells (LoVo/L-OHP) and LoVo cells were used in the present study. The effect of G-Rh2 on LoVo/L-OHP and LoVo cell proliferation was measured using a 3-(4,5 dimethylthiazol-z-yl)-3,5-diphenyltetrazolium bromide assay. The effects of G-Rh2 on LoVo/L-OHP and LoVo cell apoptosis were detected by flow cytometry. The mRNA and protein expression of apoptosis-related genes Bax, Bcl-2 and caspase-3, drug resistance-related genes P-glycoprotein (P-gp) and Smad4, were determined in LoVo/L-OHP and LoVo cells treated with G-Rh2 by reverse transcription-quantitative polymerase chain reaction and western blot analyses. G-Rh2 treatment significantly inhibited the proliferation and induced the apoptosis of LoVo/L-OHP and LoVo cells. In addition, G-Rh2 treatment resulted in a significant increase in pro-apoptotic factors, Bax and caspase-3, and decrease in anti-apoptotic factor Bcl-2 in the LoVo/L-OHP and LoVo cells. Furthermore, G-Rh2 treatment significantly decreased the levels of P-gp and increased the levels of Smad4 in the LoVo/L-OHP and LoVo cells. It was found that L-OHP had no significant effects on LoVo/L-OHP cell proliferation or apoptosis, whereas G-Rh2 + L-OHP treatment significantly inhibited LoVo/L-OHP cell proliferation and induced apoptosis. L-OHP had no significant effects on the expression of P-gp, Smad4, Bcl-2, Bax or caspase-3 in LoVo/L-OHP cells. Treatment with G-Rh2 + L-OHP significantly reduced the expression of P-gp and Bcl-2, and enhanced the expression levels of Smad4, Bax and caspase-3. These findings demonstrated that G-Rh2 reversed the drug resistance of LoVo/L-OHP cells to L-OHP, and this may be mediated by inhibiting cell proliferation and promoting apoptosis and regulating the expression of drug resistance genes. These results suggest that G-Rh2 may function as a potent anticancer drug for drug resistance in colon cancer treatment.

The activity of the saponin ginsenoside Rh2 is enhanced by the interaction with membrane sphingomyelin but depressed by cholesterol

The membrane activity of some saponins, such as digitonin or alpha-hederin, is usually attributed to their interaction with membrane cholesterol (Chol). This contrasts with our recent publication showing that Chol, contrary to sphingomyelin (SM), can delay the cytotoxicity of the saponin ginsenoside Rh2, challenging the usual view that most saponins mediate their membrane effects through interaction with Chol. The aim of the present study was to elucidate the respective importance of Chol and SM as compared to phosphatidylcholine (PC) species in the membrane-related effects of Rh2. On simple lipid monolayers, Rh2 interacted more favorably with eggSM and DOPC than with Chol and eggPC. Using Large Unilamellar Vesicles (LUVs) of binary or ternary lipid compositions, we showed that Rh2 increased vesicle size, decreased membrane fluidity and induced membrane permeability with the following preference: eggSM:eggPC > eggSM:eggPC:Chol > eggPC:Chol. On Giant Unilamellar Vesicles (GUVs), we evidenced that Rh2 generated positive curvatures in eggSM-containing GUVs and small buds followed by intra-luminal vesicles in eggSM-free GUVs. Altogether, our data indicate that eggSM promotes and accelerates membrane-related effects induced by Rh2 whereas Chol slows down and depresses these effects. This study reconsiders the theory that Chol is the only responsible for the activity of saponins.

Ginsenoside Rh2 Inhibits Angiogenesis in Prostate Cancer by Targeting CNNM1.

To explore the molecular mechanism by which ginsenoside Rh2 (G-Rh2) inhibits prostate cancer by regulating vascular growth. Different concentrations of G-Rh2 with three prostate cancer cell lines (LNCaP, PC3 and DU145) were transplanted in nude mice, and tumor mass volume was measured over time. LNCaP, PC3 and DU145 were co-cultured with vascular endothelial cells to determine the optimal concentration of G-Rh2 by MTT assay. LNCaP, PC3 and DU145 were cultured under the selected concentration (0, 0.01, 0.05, 0.1, 0.5 and 1 mg/mL) of G-Rh2, and the expression levels of CD31, VEGF, PDGF and CNNM1 detected by qRT-PCR and western blot. The expression pattern of CD31 was detected in CNNM1 overexpressed and knockout LNCaP, PC3 and DU145 cells under G-Rh2. G-Rh2 significantly inhibited the growth of all three prostate cancer cell lines in the dorsum of nude mice (P <0.05), and the increment rate of vascular endothelial cells co-cultured with LNCaP, PC3 and DU145 (P <0.05). The expression of CD31, VEGF, PDGF and CNNM1 genes in LNCaP, PC3 and DU145 cells was inhibited by G-Rh2. Overexpression of CNNM1 reversed the inhibitory effect of G-Rh2 on the expression of CD31 in these cells (P <0.05), while the function of knockout of CNNM1 and the inhibitory effect of G-Rh2 appeared to be similar (P <0.05). In conclusion, G-Rh2 inhibited prostate cancer growth by inhibiting its angiogenesis through decreasing the expression of CNNM1 in the cancer cells.

Langerhans cell-like dendritic cells treated with ginsenoside Rh2 regulate the differentiation of Th1 and Th2 cells in vivo

AbstractGinsenoside Rh2 is one of the rare ginsenosides extracted from Panax ginseng C. A. Mey. The anti-allergic activity of ginsenoside Rh2 has been documented in some literature. In this work, an anti-allergic mechanism of ginsenoside Rh2 was investigated by focusing on the differentiation of T cells through Langerhans cells (LCs). Langerhans cell-like dendritic cells (LDCs) were generated in vitro and were used as substitute for LCs.In vivo the mRNA expression for IFN-γ and CXCR3 of T cells was increased after being injected with ginsenoside Rh2-treated LDCs thereby increasing the concentration of IFN-γ in the culture supernatants of CD3+/CD28+ T lymphocytes. However,in vitro, the expression of mRNA for CD40 and CD80 on ginsenoside Rh2-treated LDCs was up-regulated significantly and the endocytic activity of LDCs was down-regulated slightly. These findings indicate that T cells differentiation could be regulated by ginsenoside Rh2 through LDCs in vivo by altering the antigen presenting capacity, maturation and phagocytosis of LDCs.

Neuroprotective Effects of Ginsenosides against Cerebral Ischemia.

Ginseng has been used worldwide as traditional medicine for thousands of years, and ginsenosides have been proved to be the main active components for their various pharmacological activities. Based on their structures, ginsenosides can be divided into ginseng diol-type A and ginseng triol-type B with different pharmacological effects. In this study, six ginsenosides, namely ginsenoside Rb1, Rh2, Rg3, Rg5 as diol-type ginseng saponins, and Rg1 and Re as triol-type ginseng saponins, which were reported to be effective for ischemia-reperfusion (I/R) treatment, were chosen to compare their protective effects on cerebral I/R injury, and their mechanisms were studied by in vitro and in vivo experiments. It was found that all ginsenosides could reduce reactive oxygen species (ROS), inhibit apoptosis and increase mitochondrial membrane potential in cobalt chloride-induced (CoCl2-induced) PC12 cells injury model, and they could reduce cerebral infarction volume, brain neurological dysfunction of I/R rats in vivo. The results of immunohistochemistry and western blot showed that the expression of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), silencing information regulator (SIRT1) and nuclear transcription factor P65 (NF-κB) in hippocampal CA1 region of some ginsenoside groups were also reduced. In general, the effect on cerebral ischemia of Rb1 and Rg3 was significantly improved compared with the control group, and was the strongest among all the ginsenosides. The effect on SIRT1 activation of ginsenoside Rb1 and the inhibition effect of TLR4/MyD88 protein expression of ginsenoside Rb1 and Rg3 were significantly stronger than that of other groups. The results indicated that ginsenoside Rg1, Rb1, Rh2, Rg3, Rg5 and Re were effective in protecting the brain against ischemic injury, and ginsenoside Rb1 and Rg3 have the strongest therapeutic activities in all the tested ginsenosides. Their neuroprotective mechanism is associated with TLR4/MyD88 and SIRT1 activation signaling pathways, and they can reduce cerebral ischemic injury by inhibiting NF-κB transcriptional activity and the expression of proinflammatory cytokines, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6).

Efficient separation determination of protopanaxatriol ginsenosides Rg1, Re, Rf, Rh1, Rg2 by HPLC

An efficient and flexible protocol was developed for simultaneous detection of the ginsenosides Rg1, Re, Rf, Rh1, and Rg2 in ginseng extract. In the analysis of white ginseng that contains no ginsenoside Rf, separation of the remaining ginsenosides was achieved within just 10 min under isocratic chromatographic conditions. For the analysis of red ginseng that contains ginsenoside Rf as a characteristic constituent, the gradient elution conditions were optimized. The method is based on high-pressure liquid chromatography employing a Shiseido UG 80 Capcell Pak NH2(4.6 mm I.D. × 250 mm, 5 μm)column and isocratic elution using acetonitrile (A) and water(B) in a ratio of 76: 24 (v/v), The optimal gradient elution conditions are as follows: 0–3 min, 89% A, 3–25 min, 89–84% A, 25–30 min, 84–82% A, 30–35 min, 82-76% A, then returning to 89% solvent A in 5 min. The flow rate was 0.80 mL min−1. The column temperature was set at 25℃ and the detection wavelength was at 203 nm. The working concentration ranges for ginsenoside Re, Rh1, Rg2, Rg1, and Rf were 0.23−1450.0 mg• L−1, 0.05−1130.0 mg• L−1, 0.11−687.0 mg• L−1, 0.051−1325 mg• L−1, and 0.55−800.0 mg• L−1, respectively. The method was validated for linearity, precision, and accuracy. And the further confirmation of five ginsenosides was conducted by QTOF-MS. Analysis of raw extracts of ginseng, white ginseng, and red ginseng for the five components showed satisfactory recovery.

Pro-angiogenic Ginsenosides F1 and Rh1 Inhibit Vascular Leakage by Modulating NR4A1

Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis, but VEGF-induced angiogenesis is often accompanied by a vascular permeability response. Ginsenosides are triterpenoid saponins from the well-known medicinal plant, ginseng, and have been considered a candidate for modulating angiogenesis. Here, we systemically investigated the effects of 10 different ginsenosides on human umbilical vein endothelial cells and newly identified that two PPT-type ginsenosides, F1 and Rh1 induce the migration and proliferation of endothelial cells. Interestingly, RNA transcriptome analysis showed that gene regulation induced by VEGF in endothelial cells is distinct from that of ginsenoside F1 and Rh1. In addition, F1 and Rh1 significantly inhibited vascular leakage both in vitro and in vivo, which are induced by vascular endothelial growth factor. Furthermore, comparative transcriptome analysis revealed that these effects of F1 and Rh1 on vascular leakage restoration are mainly caused by changes in VEGF-mediated TNFα signaling via NFκB, particularly by the suppression of expression and transcriptional activity of NR4A1 by F1 and Rh1, even in the presence of VEGF. These findings demonstrate that ginsenosides F1 and Rh1 can be a promising herbal remedy for vessel normalization in ischemic disease and cancer and that NR4A1 is the key target.

Quantitative Proteomics Combined with Affinity MS Revealed the Molecular Mechanism of Ginsenoside Antitumor Effects.

Ginsenosides have previously been demonstrated to effectively inhibit cancer cell growth and survival in both animal models and cell lines. However, the specific ginsenoside component that is the active ingredient for cancer treatment through interaction with a target protein remains unknown. By an integrated quantitative proteomics approach via affinity mass spectrum (MS) technology, we deciphered the core structure of the ginsenoside active ingredient derived from crude extracts of ginsenosides and progressed toward identifying the target protein that mediates its anticancer activity. The Tandem Mass Tag (TMT) labeling quantitative proteomics technique acquired 55620 MS/MS spectra that identified 5499 proteins and 3045 modified proteins. Of these identified proteins, 224 differentially expressed proteins and modified proteins were significantly altered in nonsmall cell lung cancer cell lines. Bioinformatics tools for comprehensive analysis revealed that the Ras protein played a general regulatory role in many functional pathways and was probably the direct target protein of a compound in ginsenosides. Then, affinity MS screening based on the Ras protein identified 20(s)-protopanaxadiol, 20(s)-Ginsenoside Rh2, and 20(s)-Ginsenoside Rg3 had affinity with Ras protein under different conditions. In particular, 20(s)-protopanaxadiol, whose derivatives are the reported antitumor compounds 20(s)-Ginsenoside Rh2 and 20(s)-Ginsenoside Rg3 that have a higher affinity for Ras via a low KD of 1.22 μM and the mutation sites of G12 and G60, was demonstrated to play a core role in those interactions. Moreover, the molecular mechanism and bioactivity assessment results confirmed the identity of the chemical ligand that was directly acting on the GTP binding pocket of Ras and shown to be effective in cancer cell bioactivity profiles.

Ginsenoside Rh2 Ameliorates Doxorubicin-Induced Senescence Bystander Effect in Breast Carcinoma Cell MDA-MB-231 and Normal Epithelial Cell MCF-10A.

The anthracycline antibiotic doxorubicin is commonly used antineoplastic drug in breast cancer treatment. Like most chemotherapy, doxorubicin does not selectively target tumorigenic cells with high proliferation rate and often causes serve side effects. In the present study, we demonstrated the cellular senescence and senescence associated secretory phenotype (SASP) of both breast tumor cell MDA-MB-231 and normal epithelial cell MCF-10A induced by clinical dose of doxorubicin (100 nM). Senescence was confirmed by flattened morphology, increased level of beta galactose, accumulating contents of lysosome and mitochondrial, and elevated expression of p16 and p21 proteins. Similarly, SASP was identified by highly secreted proteins IL-6, IL-8, GRO, GM-CSF, MCP-1, and MMP1 by antibody array assay. Reciprocal experiments, determined by cell proliferation and apoptosis assays and cell migration and cell invasion, indicated that SASP of MDA-MB-231 cell induces growth arrest of MCF-10A, whereas SASP of MCF-10A significantly stimulates the proliferation of MDA-MB-231. Interestingly, SASP from both cells powerfully promotes the cell migration and cell invasion of MDA-MB-231 cells. Treatment with the natural product ginsenoside Rh2 does not prevent cellular senescence or exert senolytic. However, SASP from senescent cells treated with Rh2 greatly attenuated the above-mentioned bystander effect. Altogether, Rh2 is a potential candidate to ameliorate this unwanted chemotherapy-induced senescence bystander effect.

RETRACTED: Ginsenoside Rh2 inhibits proliferation but promotes apoptosis and autophagy by down-regulating microRNA-638 in human retinoblastoma cells

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. The journal was initially contacted by the corresponding author to report that the article was submitted without the consent of an author. Given the comments of Dr Elisabeth Bik regarding this article “… the Western blot bands in all 400+ papers are all very regularly spaced and have a smooth appearance in the shape of a dumbbell or tadpole, without any of the usual smudges or stains. All bands are placed on similar looking backgrounds, suggesting they were copy/pasted from other sources, or computer generated ”, the journal requested the authors to provide the raw data. However, the authors were not able to fulfil this request and therefore the Editor-in-Chief decided to retract the article.