Inhibition of Osteoclast Differentiation by Ginsenoside Rg3 in RAW264.7 Cells via RANKL, JNK and p38 MAPK Pathways Through a Modulation of Cathepsin K: An In Silico and In Vitro Study.

Various studies have demonstrated that overexpression of cathepsin K (Cat-K) causes excessive bone loss, which ultimately leads to a variety of bone diseases including osteoporosis. Therefore, inhibition of Cat-K signifies a potential therapeutic target in osteoporosis treatment. Ginsenoside Rg3 is one of the most promising compound of Panax ginseng Meyer (P. ginseng) with numerous biological activities. Thus, in recent study the inhibitory effect of Rg3 isolated from P. ginseng was investigated in order to impede the osteoclast activity by an in silico approach followed by in vitro study validation using RAW264.7 cells through the investigation of different biological activity prediction such as absorption distribution metabolism and excretion (ADMET) properties against Cat-K protein. The docking results of our study showed that Rg3 is a non-toxic compound and may act as a drug-like molecule. Additionally, the molecular interaction of Rg3 with the active residues of Cat-K markedly describes its inhibitory effects on osteoclastogenesis. Findings of the present study exhibited that Rg3 significantly reduced receptor activator of nuclear factor kappa B ligand (RANKL)-induced tartrate-resistant acid phosphatase (TRAP) activity, pit formation (actin rings), and TRAP-positive multinucleated cells development in RAW264.7 cells. Furthermore, Rg3 dose-dependently reduced the mRNA expression levels of osteoclast-specific markers such as RANK, TRAP, and Cat-K induced by RANKL through the down regulation of p38, extracellular signal-regulated kinase, and c-Jun N-terminal kinase (JNK) pathways. In conclusion, in silico docking study and in vitro validation together suggested that Rg3 inhibits osteoclastogenesis and reduces bone resorption through the inhibition of Cat-K. Therefore, Rg3 might be a useful therapeutic agent for the treatment of osteoporosis and proper bone formation. Copyright © 2015 John Wiley & Sons, Ltd.

Effect of white, red and black ginseng on physicochemical properties and ginsenosides.

A systematic comparison of the ginsenosides and physicochemical properties of white ginseng (WG), red ginseng (RG) and black ginseng (BG) was performed. The purpose of the present study was to identify the effects of the physicochemical properties by steaming process. During the steaming process, ginsenosides transform into specific ginsenosides by hydrolysis, dehydration and isomerization at C-3, C-6 or C-20. Steaming ginseng led to a significant increase in reducing sugar, acidic polysaccharide and phenolic compounds content. Antioxidative properties were investigated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity, compared with BHA (Butylated hydroxyanisole). RG and BG exhibited higher antioxidant activity than WG. The maximum residue level for Benzo(a)pyrene was established to 5 µg/kg in food products. The levels of benzo(a)pyrene in WG and RG were not detected. Benzo(a)pyrene was detected in the BG, the content was 0.17 µg/kg. The scientific achievements of the present study could help consumers to choose different type of ginseng products available on the market.

In vitro evaluation of the potential therapeutic role of Dendropanax morbifera extract in ameliorating osteoporosis and resultant bone impairment using MC3T3-E1 cells.

Osteoporosis is a widespread musculoskeletal deformity that affects thousands of older people every year, leading to bone abnormalities and ultimately increasing the risk of bone fractures in both genders. It is considered a lethal disease causing death in thousands of people at the late stage of life. Dendropanax morbifera Leveille is a subtropical broad-leaved prevalent species in Korea. Extracts of the leaves, stems, roots, and seeds of D. morbifera have been used in traditional medicine for the treatment of numerous diseases such as diabetes, atherogenesis, skin disorders, and headaches. However, the anti-osteoporosis effects of D. morbifera have not been examined. The primary objectives of this study were to elucidate the anti-osteoporosis effect of D. morbifera extract through an in vitro study using pre-osteoblastic MC3T3-E1 cells. We found that D. morbifera strongly increased the expression of bone metabolic markers such as alkaline phosphatase (ALP) activity, type I collagen (Col-I) level, and mineralization. Additionally, D. morbifera extract also upregulated the mRNA expression levels of osteogenic genes including ALP, osteocalcin (OCN), osterix (Osx), and runt-related transcription factor 2 (Runx2) in MC3T3-E1 cells via upregulation of bone morphogenetic protein 2 (BMP-2)/p38 MAPK/JNK and Smad1/5/8 signaling pathways. Moreover, addition of D. morbifera significantly suppressed the inhibitory effect of SB203580 (p38 inhibitor). In conclusion, the current study demonstrated that D. morbifera extract significantly increased osteoblast differentiation and mineralization in MC3T3-E1 cells by regulating BMP-2/p38/JNK and Smad1/5/8. Our study might be helpful in the discovery and development of new anti-osteoporosis therapeutic agents.

Computational Investigation of Ginsenoside F1 from Panax ginseng Meyer as p38 MAP Kinase Inhibitor: Molecular Docking and Dynamics Simulations, ADMET Analysis, and Drug Likeness Prediction.

Ginsenoside F1 (G-F1) is biologically an active compoud isolated from Korean Panax ginseng Meyer. In the present study, the potential therapeutic effect of G-F1 were investigated by computational target fishing approaches including ADMET prediction, biological activity prediction from chemical structure, molecular docking, and molecular dynamics methods. Results were suggested to express the biological activity of G-F1 against p38 MAP kinase protein. The p38 MAP kinase protein is an important signal transducing enzyme involved in many cellular regulations, including signaling pathways, pain and inflammation. Numerous studies are shown that an abnormal activation of p38 MAP kinase leads to variety of diseases. The pharmacokinetic result proves that G- F1 can act non-toxic drug like molecule. In addition, molecular level interaction results of G- F1 with p38 MAP kinase active (binding) sites residues clearly defines its inhibitory action on p38 MAP kinase. Further, molecular dynamics study also supported p38 MAP kinase and G-F1 structural stability. Findings from out study will assist to discover the active drug like molecules from Panax ginseng with help of molecular modeling techniques.

Therapeutic potential of compound K as an IKK inhibitor with implications for osteoarthritis prevention: an in silico and in vitro study.

Ginsenosides have been used traditionally as an oriental medicine. However, the anti-osteoarthritic effect of ginsenoside compound K (hereafter referred to as CK) has not been reported. Therefore, in this study, the protective effects of CK were evaluated in silico and in vitro using H2O2-stimulated MC3T3-E1 cells by measuring the levels of proinflammatory cytokines responsible for articular cartilage degradation. In silico results demonstrated that, among the selected ginsenosides, CK is a non-toxic drug-like molecule with strong binding affinity for selected cytokine-activated kinase such as IkBα kinase (IKK). The molecular binding energy of CK with the active sites of IKK suggests anti-osteoarthritic functions. Cultured H2O2-stimulated MC3T3-E1 cells that were exposed to CK showed dramatically increased expression of osteoblast differentiation markers such as alkaline phosphatase (ALP) activity, type I collagen (Col-I) content, and mineralization. During aging, H2O2 also leads to the production of reactive oxygen species (ROS) and nitric oxide (NO), which play important roles in the development of osteoarthritis (OA). Therefore, the effect of CK on ROS and NO generation was also examined. Our results showed that CK dose-dependently inhibited H2O2-induced ROS and NO production in MC3T3-E1 cells. Moreover, qRT-PCR data showed that CK increased expression of osteogenic markers such as ALP and Col-I but decreased expression of inflammatory-related genes including IKK and interleukin 1ß (IL-1ß) in a dose-dependent manner in H2O2-stimulated MC3T3-E1 cells. The findings of this study suggest the use of CK as a novel protective and therapeutic agent in AO.

Amelioration of insulin resistance by Rk1 + Rg5 complex under endoplasmic reticulum stress conditions.

BACKGROUND: Diabetes mellitus is a metabolic syndrome exaggerated by stress conditions. Endoplasmic reticulum stress (ERS) impairs the insulin signaling pathway making the diabetic conditions worsen. Pharmacological agents are supplied externally to overcome this malfunction. Ginsenosides from Panax ginseng C.A Meyer possesses many pharmacological properties and are used for the treatment of diabetes. OBJECTIVE: To investigate the effects of the Rk1 +Rg5 complex on the amelioration of insulin resistance in 3T3-L1 cells under endoplasmic reticulum stress conditions. MATERIALS AND METHODS: Heat-processed ginseng extracts are found to contain many pharmacologically active ginsenosides. Among them Rk1 +Rg5 is found to be present in higher concentrations than the other minor ginsenosides. The Rk1 +Rg5 complex was tested for its effect in the 3T3-L1 insulin-resistant model and subjected to the MTT assay, glucose oxidase assay and gene expression studies using RT-PCR and real-time PCR under endoplasmic reticulum stress conditions. RESULTS: Rk1 +Rg5 treatment is found to increase the glucose uptake into the cells when compared to that of a positive control (tunicamycin treatment group, TM). Further we have analyzed the role at gene expression level. The Rk1 +Rg5 complex was found to show an effect on the IGF 2R receptor, CHOP-10, and C/EBP gene at a particular treated concentration (50 µM). Moreover, stress condition (about 50% decreases) was overcome by the ginsenoside treatments at 50 µM. CONCLUSION: The present results showed that under endoplasmic reticulum stress conditions Rk1 +Rg5 complex exhibits a potential protective role in insulin-resistant 3T3-L1 cells.

Molecular characterization of two glutathione peroxidase genes of Panax ginseng and their expression analysis against environmental stresses.

Glutathione peroxidases (GPXs) are a group of enzymes that protect cells against oxidative damage generated by reactive oxygen species (ROS). GPX catalyzes the reduction of hydrogen peroxide (H2O2) or organic hydroperoxides to water or alcohols by reduced glutathione. The presence of GPXs in plants has been reported by several groups, but the roles of individual members of this family in a single plant species have not been studied. Two GPX cDNAs were isolated and characterized from the embryogenic callus of Panax ginseng. The two cDNAs had an open reading frame (ORF) of 723 and 681bp with a deduced amino acid sequence of 240 and 226 residues, respectively. The calculated molecular mass of the matured proteins are approximately 26.4kDa or 25.7kDa with a predicated isoelectric point of 9.16 or 6.11, respectively. The two PgGPXs were elevated strongly by salt stress and chilling stress in a ginseng seedling. In addition, the two PgGPXs showed different responses against biotic stress. The positive responses of PgGPX to the environmental stimuli suggested that ginseng GPX may help to protect against environmental stresses.