Majonoside-R2 extracted from Vietnamese ginseng protects H9C2 cells against hypoxia/reoxygenation injury via modulating mitochondrial function and biogenesis.

Vietnamese ginseng has a therapeutic effect on various diseases; however its bioactivity against cardiac hypoxia/reoxygenation (HR) injury remains unclear. In this study, we evaluated the protective roles of total saponin extract (TSE) and majonoside-R2 (MR2) targeting mitochondria in HR-induced rat cardiomyocyte H9C2 cells. The results showed that both TSE and MR2 effectively protected the cells from HR damage. Particularly, 9 µM of MR2 significantly increased the viability of HR-induced cells (p < 0.05). Interestingly, MR2 treatment markedly prevented the loss of mitochondrial membrane potential and cardiolipin content, and an increase in reactive oxygen species production in HR-treated H9C2 cells. Moreover, MR2 treatment altered the mRNA expression of genes involved in mitochondrial biogenesis under HR conditions. The present study documented for the first time the cardioprotective effects of MR2 against HR injury by maintaining mitochondrial function and modulating mitochondrial biogenesis.

Monoclonal antibody-based enzyme-linked immunosorbent assay for quantification of majonoside R2 as an authentication marker for Nngoc Linh and Lai Chau ginsengs.

Background: Recent years have witnessed increasing interest in the high amount of ocotillol-type saponin in Panax vietnamensis, particularly in relation to majonoside R2 (MR2). This unique 3%-5% MR2 content impart Ngoc Linh and Lai Chau ginsengs with unique pharmacological activities. However, in the commercial domain, unauthentic species have infiltrated and significantly hindered access to the authentic, efficacious variety. Thus, suitable analytical techniques for distinguishing authentic Vietnamese ginseng species from others is becoming increasingly crucial. Therefore, MR2 is attracting considerable attention as a target requiring effective management measures. Methods: An enzyme-linked immunosorbent assay (ELISA) was developed by producing monoclonal antibodies against MR2 (mAb 16E11). The method was thoroughly validated, and the potential of the immunoassay was confirmed by high-performance liquid chromatography with ultraviolet spectroscopy. Furthermore, ELISA was applied to the assessment of the MR2 concentrations of various Panax spp., including Korean, American, and Japanese ginsengs. Results and conclusions: An icELISA using mAb 16E11 exhibited linearity between 3.91 and 250 ng/mL of MR2, with detection and quantification limits of 1.53 and 2.50 - 46.6 ng/mL, respectively. Based on this study, the developed icELISA using mAb 16E11 could be a valuable tool for analyzing MR2 level to distinguish authentic Ngoc Linh and Lai Chau ginsengs from unauthentic ones. Furthermore, the analysis of the samples demonstrated that Ngoc Linh and Lai Chau ginsengs exhibit a notably higher MR2 value than all other Panax spp. Thus, MR2 might be their ideal marker compound, and various bioactivities of this species should be explored.

Majonoside-R2 Postconditioning Protects Cardiomyocytes Against Hypoxia/Reoxygenation Injury by Attenuating the Expression of HIF1α and Activating RISK Pathway.

Reoxygenation of hypoxic cardiac myocytes can paradoxically induce myocardial injury and affect the recovery processes. Pharmacological postconditioning is an efficient strategy used in clinical practice that protects cardiomyocytes from hypoxia/reoxygenation (HR) injury. Natural products or foods have been known to possess effective cardioprotective properties. Majonoside-R2 (MR2) is a dominant saponin component of Vietnamese ginseng that has several biological effects. In this study, we evaluated the protective effect of MR2 on HR-stimulated cardiomyocytes and investigated the related molecular mechanisms. H9C2 cardiomyocytes were exposed to HR conditions with or without MR2 supplementation. Samples from experimental groups were used to analyze the expression of apoptosis- and activating reperfusion injury salvage kinase (RISK)-related factors in response to HR injury by using enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and Western blotting. Post-treatment, MR2 enhanced cell viability under HR conditions. We found that MR2 suppressed the expression of hypoxia-inducible factor 1-alpha (HIF1α) and transforming growth factor beta 1 (TGFß1), modulated Akt/GSK3ß/cAMP response element-binding signaling, and regulated gene expression related to apoptosis (B cell lymphoma-extra-large [Bcl-xl], Bcl-2 homologous killer [Bak], Bcl-2 associated X [Bax], and connexin 43 [Cnx43]). Thus, the present findings demonstrate that MR2 protects cardiomyocytes against HR injury by suppressing the expression of HIF1α and activating the RISK pathway.

Ocotillol, a Majonoside R2 Metabolite, Ameliorates 2,4,6-Trinitrobenzenesulfonic Acid-Induced Colitis in Mice by Restoring the Balance of Th17/Treg Cells.

In a preliminary experiment, majonoside R2 (MR2), isolated from Vietnamese ginseng (Panax vietnamensis Ha et Grushv.), inhibited differentiation to Th17 cells and was metabolized to ocotillol via pseudoginsenoside RT4 (PRT4) by gut microbiota. Therefore, we examined the inhibitory effects of MR2 and its metabolites PRT4 and ocotillol against Th17 cell differentiation. These ginsenosides significantly suppressed interleukin (IL)-6/tumor growth factor beta-induced differentiation of splenic CD4(+) T cells into Th17 cells and expression of IL-17 in vitro. Among these ginsenosides, ocotillol showed the highest inhibitory effect. We also examined the anti-inflammatory effect of ocotillol in mice with 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. Oral administration of ocotillol significantly suppressed TNBS-induced colon shortening, macroscopic score, myeloperoxidase activity, and production of nitric oxide and prostaglandin E2. Ocotillol treatment increased TNBS-suppressed expression of tight junction proteins ZO-1, occludin, and claudin-1 in the colon. Treatment with ocotillol inhibited TNBS-induced expression of tumor necrosis factor (TNF)-α and IL-1ß, as well as activation of NF-κB and MAPKs. Moreover, treatment with ocotillol inhibited TNBS- induced differentiation to Th17 cells in the lamina propria of colon, as well as expression of T-bet, RORγt, IL-17, and IL-23. Ocotillol treatment also increased Treg cell differentiation and Foxp3 and IL-10 expression. These findings suggest that orally administered MR2 may be metabolized to ocotillol in the intestine by gut microbiota and the transformed ocotillol may ameliorate inflammatory diseases such as colitis by restoring the balance of Th17/Treg cells.

Anti-inflammatory effects of vina-ginsenoside R2 and majonoside R2 isolated from Panax vietnamensis and their metabolites in lipopolysaccharide-stimulated macrophages.

Panax vietnamensis Ha et Grushv., with its main constituents vina-ginsenoside R2 (VR2) and majonoside R2 (MR2), is used in traditional folk medicine in the hill tribes of Vietnam for anti-fatigue, anti-inflammatory, and life-saving purposes. In a preliminary study, VR2 and MR2 were shown to be metabolized to pseudoginsenoside RT4 (PRT4) and ocotillol by human gut microbiota. Therefore, we measured the anti-inflammatory effects of VR2, MR2, and their metabolites in lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages. Among these ginsenosides, only VR2 exhibited cytotoxicity against peritoneal macrophages. MR2, PRT4, and ocotillol inhibited LPS-stimulated transcription factor (NF)-κB activation, and expression of the proinflammatory cytokines tumor necrosis factor-α and interleukin (IL)-1. However, these ginsenosides did not inhibit peptidoglycan-induced NF-κB activation in the macrophages. These three ginsenosides also inhibited LPS-stimulated cyclooxygenase-2 and inducible NO synthase expression, and phosphorylation of NF-κB signal molecules IL-1 receptor-associated kinase 1 and tumor growth factor-ß-activated kinase 1 in peritoneal macrophages. Treatment with either PRT4 or ocotillol inhibited the Alexa Fluor 488-conjugated LPS-mediated shift of macrophages, as observed by flow cytometry. They also potently inhibited the binding of LPS to TLR4 on peritoneal macrophages, both with and without transfected MyD88 siRNA. Among the tested ginsenosides, ocotillol exhibited the strongest inhibitory effect on inflammation in LPS-stimulated macrophages via the NF-κB signaling pathway. Based on these findings, orally administered VR2 and MR2 of P. vietnamensis may be metabolized to ocotillol via PRT4, and the metabolites, particularly ocotillol, may inhibit inflammation by inhibiting the binding of LPS to TLR4 on macrophages.