In Silico and In Vitro Study of Isoquercitrin against Kidney Cancer and Inflammation by Triggering Potential Gene Targets.

Kidney cancer has emerged as a major medical problem in recent times. Multiple compounds are used to treat kidney cancer by triggering cancer-causing gene targets. For instance, isoquercitrin (quercetin-3-O-ß-d-glucopyranoside) is frequently present in fruits, vegetables, medicinal herbs, and foods and drinks made from plants. Our previous study predicted using protein-protein interaction (PPI) and molecular docking analysis that the isoquercitrin compound can control kidney cancer and inflammation by triggering potential gene targets of IGF1R, PIK3CA, IL6, and PTGS2. So, the present study is about further in silico and in vitro validation. We performed molecular dynamic (MD) simulation, gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, cytotoxicity assay, and RT-PCR and qRT-PCR validation. According to the MD simulation (250 ns), we found that IGF1R, PIK3CA, and PTGS2, except for IL6 gene targets, show stable binding energy with a stable complex with isoquercitrin. We also performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the final targets to determine their regulatory functions and signaling pathways. Furthermore, we checked the cytotoxicity effect of isoquercitrin (IQ) and found that 5 µg/mL and 10 µg/mL doses showed higher cell viability in a normal kidney cell line (HEK 293) and also inversely showed an inhibition of cell growth at 35% and 45%, respectively, in the kidney cancer cell line (A498). Lastly, the RT-PCR and qRT-PCR findings showed a significant decrease in PTGS2, PIK3CA, and IGF1R gene expression, except for IL6 expression, following dose-dependent treatments with IQ. Thus, we can conclude that isoquercitrin inhibits the expression of PTGS2, PIK3CA, and IGF1R gene targets, which in turn controls kidney cancer and inflammation.

Potential of Gut Microbial Metabolites in Treating Osteoporosis and Obesity: A Network Pharmacology and Bioinformatics Approach.

BACKGROUND The gut microbial metabolites demonstrate significant activity against metabolic diseases including osteoporosis (OP) and obesity, but active compounds, targets, and mechanisms have not been fully identified. Hence, the current investigation explored the mechanisms of active metabolites and targets against OP and obesity by using network pharmacology approaches. MATERIAL AND METHODS The gutMGene database was used to collect gut microbial targets-associated metabolites; DisGeNET and OMIM databases were used to identify targets relevant to OP and obesity. A total of 63 and 89 overlapped targets were considered the final OP and obesity targets after creating a Venn diagram of metabolites-related targets and disease-related targets. Furthermore, the top 20% of degrees, betweenness, and closeness were used to form the sub-network of protein-protein interaction of these targets. Finally, the biotransformation-increased receptors and biological mechanisms were identified and validated using ADMET properties analysis, molecular docking, and molecular dynamic simulation. RESULTS GO, KEGG pathway analysis, and protein-protein interactions were performed to establish metabolites and target networks. According to the enrichment analysis, OP and obesity are highly linked to the lipid and atherosclerosis pathways. Moreover, ADMET analysis depicts that the major metabolites have drug-likeliness activity and no or less toxicity. Following that, the molecular docking studies showed that compound K and TP53 target have a remarkable negative affinity (-8.0 kcal/mol) among all metabolites and targets for both diseases. Finally, the conformity of compound K against the targeted protein TP53 was validated by 250ns MD simulation. CONCLUSIONS Therefore, we summarized that compound K can regulate TP53 and could be developed as a therapy option for OP and obesity.

In silico and in vitro inhibition of host-based viral entry targets and cytokine storm in COVID-19 by ginsenoside compound K.

SARS-CoV-2 is a novel coronavirus that emerged as an epidemic, causing a respiratory disease with multiple severe symptoms and deadly consequences. ACE-2 and TMPRSS2 play crucial and synergistic roles in the membrane fusion and viral entry of SARS-CoV-2 (COVID-19). The spike (S) protein of SARS-CoV-2 binds to the ACE-2 receptor for viral entry, while TMPRSS2 proteolytically cleaves the S protein into S1 and S2 subunits, promoting membrane fusion. Therefore, ACE-2 and TMPRSS2 are potential drug targets for treating COVID-19, and their inhibition is a promising strategy for treatment and prevention. This study proposes that ginsenoside compound K (G-CK), a triterpenoid saponin abundant in Panax Ginseng, a dietary and medicinal herb highly consumed in Korea and China, effectively binds to and inhibits ACE-2 and TMPRSS2 expression. We initially conducted an in-silico evaluation where G-CK showed a high affinity for the binding sites of the two target proteins of SARS-CoV-2. Additionally, we evaluated the stability of G-CK using molecular dynamics (MD) simulations for 100 ns, followed by MM-PBSA calculations. The MD simulations and free energy calculations revealed that G-CK has stable and favorable energies, leading to strong binding with the targets. Furthermore, G-CK suppressed ACE2 and TMPRSS2 mRNA expression in A549, Caco-2, and MCF7 cells at a concentration of 12.5 µg/mL and in LPS-induced RAW 264.7 cells at a concentration of 6.5 µg/mL, without significant cytotoxicity.ACE2 and TMPRSS2 expression were significantly lower in A549 and RAW 264.7 cells following G-CK treatment. These findings suggest that G-CK may evolve as a promising therapeutic against COVID-19.

Comparison of In Vitro Estrogenic Activity of Polygoni multiflori Radix and Cynanchi wilfordii Radix via the Enhancement of ERα/ß Expression in MCF7 Cells.

Postmenopausal women experience several symptoms, including inflammation and a sharp rise in oxidative stress caused by estrogen deprivation. Although estrogen replacement therapy (ERT) is generally regarded as an effective treatment for menopause, it has been used less frequently due to some adverse effects and high costs. Therefore, there is an immediate need to develop an effective herbal-based treatment that is affordable for low-income populations. Acordingly, this study explored the estrogen-like properties of methanol extracts from Cynanchum wilfordii (CW) and Poligonum multiflorum (PM), two important medicinal plants in Republic of Korea, Japan, and China. Due to the similar names and morphologies of these two radixes, they are frequently confused in the marketplace. Our previous colleagues discriminated between these two plants. In this study, we investigated the estrogenic activity of PM and CW using several in vitro assays with their possible mechanism of action. First, their phytochemical contents, such as gallic acid, 2,3,5,4'-tetrahydroxystilbene-2-O-glucoside (TSG) and emodin, were quantified using high-performance liquid chromatography (HPLC). Secondly, estrogen-like activity was assessed utilizing the well-known E-screen test and gene expression analysis in estrogen receptor (ER)-positive MCF7 cells. ROS inhibition and anti-inflammatory effects were analyzed using HaCaT and Raw 264.7 cells, respectively. Our findings demonstrate that PM extracts significantly increased the expression of the estrogen-dependent genes (ERα, ERß, pS2) and boosted MCF7 cell proliferation in comparison to CW extracts. Additionally, PM extract demonstrated a significant reduction in reactive oxygen species (ROS) production as well as an enhanced antioxidant profile compared to the CW extract. Further, the PM extract treatment significantly reduced the generation of nitric oxide (NO) in RAW 264.7 cells, a murine macrophage cell line, demonstrating the anti-inflammatory properties of the extract. Finally, this research offers an experimental foundation for the use of PM as a phytoestrogen to minimize menopausal symptoms.

A review on Impact of dietary interventions, drugs, and traditional herbal supplements on the gut microbiome.

The gut microbiome is the community of healthy, and infectious organisms in the gut and its interaction in the host gut intestine (GI) environment. The balance of microbial richness with beneficial microbes is very important to perform healthy body functions like digesting food, controlling metabolism, and precise immune function. Alternately, this microbial dysbiosis occurs due to changes in the physiochemical condition, substrate avidity, and drugs. Moreover, various categories of diet such as "plant-based", "animal-based", "western", "mediterranean", and various drugs (antibiotic and common drugs) also contribute to maintaining microbial flora inside the gut. The imbalance (dysbiosis) in the microbiota of the GI tract can cause several disorders (such as diabetes, obesity, cancer, inflammation, and so on). Recently, the major interest is to use prebiotic, probiotic, postbiotic, and herbal supplements to balance such microbial community in the GI tract. But, there has still a large gap in understanding the microbiome function, and its relation to the host diet, drugs, and herbal supplements to maintain the healthy life of the host. So, the present review is about the updates on the microbiome concerns related to diet, drug, and herbal supplements, and also gives research evidence to improve our daily habits regarding diet, drugs, and herbal supplements. Because our regular dietary plan and traditional herbal supplements can improve our health by balancing the bacteria in our gut.

Siderophore-producing rhizobacteria reduce heavy metal-induced oxidative stress in Panax ginseng Meyer.

BACKGROUND: Panax ginseng is one of the most important medicinal plants and is usually harvested after 5 to 6 years of cultivation in Korea. Heavy metal (HM) exposure is a type of abiotic stress that can induce oxidative stress and decrease the quality of the ginseng crop. Siderophore-producing rhizobacteria (SPR) may be capable of bioremediating HM contamination. METHODS: Several isolates from ginseng rhizosphere were evaluated by in vitro screening of their plant growth-promoting traits and HM resistance. Subsequently, in planta (pot tests) and in vitro (medium tests) were designed to investigate the SPR ability to reduce oxidative stress and enhance HM resistance in P. ginseng inoculated with the SPR candidate. RESULTS: In vitro tests revealed that the siderophore-producing Mesorhizobium panacihumi DCY119T had higher HM resistance than the other tested isolates and was selected as the SPR candidate. In the planta experiments, 2-year-old ginseng seedlings exposed to 25 mL (500 mM) Fe solution had lower biomass and higher reactive oxygen species level than control seedlings. In contrast, seedlings treated with 108 CFU/mL DCY119T for 10 minutes had higher biomass and higher levels of antioxidant genes and nonenzymatic antioxidant chemicals than untreated seedlings. When Fe concentration in the medium was increased, DCY119T can produce siderophores and scavenge reactive oxygen species to reduce Fe toxicity in addition to providing indole-3-acetic acid to promote seedling growth, thereby conferring inoculated ginseng with HM resistance. CONCLUSIONS: It was confirmed that SPR DCY119T can potentially be used for bioremediation of HM contamination.

Diversity of Ginsenoside Profiles Produced by Various Processing Technologies.

Ginseng is a traditional medicinal herb commonly consumed world-wide owing to its unique family of saponins called ginsenosides. The absorption and bioavailability of ginsenosides mainly depend on an individual's gastrointestinal bioconversion abilities. There is a need to improve ginseng processing to predictably increase the pharmacologically active of ginsenosides. Various types of ginseng, such as fresh, white, steamed, acid-processed, and fermented ginsengs, are available. The various ginseng processing methods produce a range ginsenoside compositions with diverse pharmacological properties. This review is intended to summarize the properties of the ginsenosides found in different Panax species as well as the different processing methods. The sugar moiety attached to the C-3, C-6, or C-20 deglycosylated to produce minor ginsenosides, such as Rb1, Rb2, Rc, Rd→Rg3, F2, Rh2; Re, Rf→Rg1, Rg2, F1, Rh1. The malonyl-Rb1, Rb2, Rc, and Rd were demalonylated into ginsenoside Rb1, Rb2, Rc, and Rd by dehydration. Dehydration also produces minor ginsenosides such as Rg3→Rk1, Rg5, Rz1; Rh2→Rk2, Rh3; Rh1→Rh4, Rk3; Rg2→Rg6, F4; Rs3→Rs4, Rs5; Rf→Rg9, Rg10. Acetylation of several ginsenosides may generate acetylated ginsenosides Rg5, Rk1, Rh4, Rk3, Rs4, Rs5, Rs6, and Rs7. Acid processing methods produces Rh1→Rk3, Rh4; Rh2→Rk1, Rg5; Rg3→Rk2, Rh3; Re, Rf, Rg2→F1, Rh1, Rf2, Rf3, Rg6, F4, Rg9. Alkaline produces Rh16, Rh3, Rh1, F4, Rk1, ginsenoslaloside-I, 20(S)-ginsenoside-Rh1-60-acetate, 20(R)-ginsenoside Rh19, zingibroside-R1 through hydrolysis, hydration addition reactions, and dehydration. Moreover, biological processing of ginseng generates the minor ginsenosides of Rg3, F2, Rh2, CK, Rh1, Mc, compound O, compound Y through hydrolysis reactions, and synthetic ginsenosides Rd12 and Ia are produced through glycosylation. This review with respect to the properties of particular ginsenosides could serve to increase the utilization of ginseng in agricultural products, food, dietary supplements, health supplements, and medicines, and may also spur future development of novel highly functional ginseng products through a combination of various processing methods.

Advances in Saponin Diversity of Panax ginseng.

Ginsenosides are the major bioactive constituents of Panax ginseng, which have pharmacological effects. Although there are several reviews in regards to ginsenosides, new ginsenosides have been detected continually in recent years. This review updates the ginsenoside list from P. ginseng to 170 by the end of 2019, and aims to highlight the diversity of ginsenosides in multiple dimensions, including chemical structure, tissue spatial distribution, time, and isomeride. Protopanaxadiol, protopanaxatriol and C17 side-chain varied (C17SCV) manners are the major types of ginsenosides, and the constitute of ginsenosides varied significantly among different parts. Only 16 ginsenosides commonly exist in all parts of a ginseng plant. Protopanaxadiol-type ginsenoside is dominant in root, rhizome, leaf, stem, and fruit, whereas malonyl- and C17SCV-type ginsenosides occupy a greater proportion in the flower and flower bud compared with other parts. In respects of isomeride, there are 69 molecular formulas corresponding to 170 ginsenosides, and the median of isomers is 2. This is the first review on diversity of ginsenosides, providing information for reasonable utilization of whole ginseng plant, and the perspective on studying the physiological functions of ginsenoside for the ginseng plant itself is also proposed.

Silicon confers protective effect against ginseng root rot by regulating sugar efflux into apoplast.

Root rot caused by Ilyonectria mors-panacis is a devastating fungal disease leading to defect in root quality and causes reduced yield during the perennial life cycle of Panax ginseng Meyer. This indicates the imperative need to understand the molecular basis of disease development and also to enhance tolerance against the fungus. With this idea, the protective effect of silicon (supplied as silica nanoparticles) in P. ginseng root rot pathosystem and its molecular mechanism was investigated in the current study. We have tested different concentrations of silicon (Si) to disease-infected ginseng and found that long term analysis (30 dpi) displayed a striking 50% reduction in disease severity index upon the treatment of Si. Expectedly, Si had no direct degradative effect against the pathogen. Instead, in infected roots it resulted in reduced expression of PgSWEET leading to regulated sugar efflux into apoplast and enhanced tolerance against I. mors-panacis. In addition, under diseased condition, both protopanaxadiol (PPD) and protopanaxatriol (PPT) type ginsenoside profile in roots were higher in Si treated plants. This is the first report indicating the protective role of Si in ginseng-root rot pathosystem, thereby uncovering novel features of ginseng mineral physiology and at the same time, enabling its usage to overcome root rot.

Rhizobium panacihumi sp. nov., an isolate from ginseng-cultivated soil, as a potential plant growth promoting bacterium.

A novel bacterial strain designated DCY116T was isolated from ginseng-cultivated soil in Gochang-gun, Republic of Korea. Strain DCY116T, belongs to the genus Rhizobium, and is closely related to Rhizobium yantingense H66T (98.3%), Neorhizobium huautlense S02T (98.2%), Rhizobium soli DS-42T (98.1%), Rhizobium smilacinae PTYR-5T (97.9%), and Neorhizobium alkalisoli CCBAU 01393T (97.9%) based on 16S rRNA gene sequence analysis. Analysis of the housekeeping genes atpD, recA, and glnII showed low levels of sequence similarity (96.8%) between strain DCY116T and other closely related species. Strain DCY116T was Gram-stain negative, motile by peritrichous flagella, rod-shaped, strictly aerobic, catalase- and oxidase-positive. Q-10 was the predominant ubiquinone. The major cellular fatty acids were identified as C16:0 and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, and an unknown lipid (L1-3). Genomic DNA G + C content of strain DCY116T was determined to be 57.2 mol%. DNA-DNA homology values between strain DCY116T and closely related species of the genus Rhizobium were lower than 40%. Strain DCY116T produced indole-3-acetic acid, siderophores, and was able to solubilize phosphate as a potential plant growth promoting bacterium. In conclusion, the results of this study support strain DCY116T as a novel species of the genus Rhizobium, for which the name Rhizobium panacihumi is proposed. The type strain is DCY116T (= KCTC 62017T = JCM 32251T).

Ornithinimicrobium panacihumi sp. nov., Antagonistic Bacteria Against Root Rot Fungal Pathogens, Isolated from Cultivated Ginseng Soil.

A Gram-positive bacterium (DCY118T) was isolated from ginseng-cultivated soil in Gochang-gun, Republic of Korea. This isolate was assigned to the genus Ornithinimicrobium and is closely related to Ornithinimicrobium kibberense K22-20T (98.8%), O. pekingense DSM 21552T (98.5%), O. algicola JC311T (98.2%), and O. humiphilum DSM 12362T (97.9%) based on 16S rRNA gene sequence analysis. However, strain DCY118T showed < 55% DNA-DNA homology with closely related reference strains. Cells were non-motile, non-sporulating, catalase- and oxidase-positive, aerobic, short rods, and cocci, and produced light-yellow, circular, and smooth colonies on TSA medium. MK-8(H4) was the predominant menaquinone. The major cellular fatty acids were iso-C15:0, anteiso-C15:0, and C16:0. The polar lipid profile consisted of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylinositol (PI), an unknown phospholipid (PL1), an unknown amino lipid (AL1), and unidentified polar lipids (L1-5). The genomic DNA G+C content was 71.1 mol%. The peptidoglycan contained L-ornithine as the diagnostic diamino acid. Whole-cell sugars were composed of glucose, arabinose, and xylose. Overall, data collected from phenotypic and genotypic tests during this study indicated that strain DCY118T could not be assigned to a recognized species. Strain DCY118T showed antagonistic activity against the fungal pathogens causing root rot in ginseng, i.e., Fusarium solani (KACC 44891T) and Cylindrocarpon destructans (KACC 44660T). The results from this study confirm the DCY118T strain as a new species within the genus Ornithinimicrobium, for which the name Ornithinimicrobium panacihumi is proposed. The type strain is DCY118T (=KCTC 39962T=JCM 32156T).

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.

Development of a single-nucleotide-polymorphism marker for specific authentication of Korean ginseng (Panax ginseng Meyer) new cultivar "G-1".

BACKGROUND: Korean ginseng (Panax ginseng) is a well-known medicinal plant of Oriental medicine that is still in practice today. Until now, a total of 11 Korean ginseng cultivars with unique features to Korean ginseng have been developed based on the pure-line-selection method. Among them, a new cultivar namely G-1 with different agricultural traits related to yield and content of ginsenosides, was developed in 2012. METHODS: The aim of this study was to distinguish the new ginseng cultivar G-1 by identifying the unique single-nucleotide polymorphism (SNP) at its 45S ribosomal DNA and Panax quinquefolius region than other Korean ginseng cultivars using multiplex amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR). RESULTS: A SNP at position of 45S ribosomal DNA region between G-1, P. quinquefolius, and the other Korean ginseng cultivars was identified. By designing modified allele-specific primers based on this site, we could specifically identified G-1 and P. quinquefolius via multiplex PCR. The unique primer for the SNP yielded an amplicon of size 449 bp in G-1 cultivar and P. quinquefolius. This study presents an effective method for the genetic identification of the G-1 cultivar and P. quinquefolius. CONCLUSION: The results from our study shows that this SNP-based approach to identify the G-1 cultivar will be a good way to distinguish accurately the G-1 cultivar and P. quinquefolius from other Korean ginseng cultivars using a SNP at 45S ribosomal DNA region.

Microbacterium rhizomatis sp. nov., a ß-glucosidase-producing bacterium isolated from rhizome of Korean mountain ginseng.

A novel Gram-staining-positive, rod-shaped bacterium, designated DCY100(T), was isolated from rhizome of mountain ginseng root in Hwacheon mountain, Gangwon province, Republic of Korea. The 16S rRNA gene sequence analysis showed that strain DCY100(T) belonged to the genus Microbacterium and was most closely related to Microbacterium ginsengisoli KCTC 19189(T) (97.9%), Microbacterium lacus JCM 15575(T) (97.2%) and Microbacterium invictum DSM 19600(T) (97.1%). The major menaquinones were MK-11 and MK-12. The major polar lipids were found to be diphosphatidylglycerol, phosphatidylglycerol and one unidentified glycolipid. The major fatty acids (>10.0%) were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The cell-wall peptidoglycan contained the amino acids ornithine, alanine, glutamic acid and glycine; whole-cell sugars consisted of glucose, galactose, rhamnose and ribose. The DNA G+C content was 63.6 ± 0.7 mol%. The DNA-DNA hybridization relatedness values between strain DCY100(T) and Microbacterium ginsengisoli KCTC 19189(T), Microbacterium lacus JCM 15575(T) and Microbacterium invictum DSM 19600(T) were 36.2 ± 0.4, 22.0 ± 3.0 and 15.3 ± 1.8%, respectively. On the basis of phenotypic, chemotaxonomic and genotypic analyses, the isolate is classified as a representative of a novel species in the genus Microbacterium, for which the name Microbacterium rhizomatis DCY100(T) is proposed. The type strain is DCY100(T) ( = KCTC 39529(T) = JCM 30598(T)).

Stimulative effect of ginsenosides Rg5:Rk1 on murine osteoblastic MC3T3-E1 cells.

Panax ginseng C.A. Meyer (P. ginseng), hereafter referred to as P. ginseng, is known to exert a wide range of pharmacological effects both in vitro and in vivo; however, few studies have investigated the effects of ginseng on bone metabolism. We therefore investigated the potential antiosteoporotic properties of ginseng on the growth and differentiation of murine MC3T3-E1 cells. Rg5:Rk1 is a mixture of protopanaxadiol-type ginsenosides, isolated from fresh P. ginseng root, via a repetitive steaming and drying process. In this study, we examined the stimulatory effects of Rg5:Rk1 on the differentiation and mineralization of MC3T3-E1 cells. Undifferentiated cells were treated with a range of concentrations of Rg5:Rk1 (1-50 µg/mL), and cell viability was measured with the 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Treatment with Rg5:Rk1 significantly increased cell viability in a dose-dependent manner. To investigate the possible mechanisms by which Rg5:Rk1 affects the early differentiation phase of MC3T3-E1 cells, the cells were treated with Rg5:Rk1 for 14-24 days before assessing the levels of multiple osteoblastic markers. The markers examined included alkaline phosphatase (ALP) activity type I collagen content (Coll-I), calcium deposition (by Alizarin Red S staining), extracellular mRNA expression of bone morphogenetic protein-2 (BMP-2), and the level of Runt-related transcription factor 2 (Runx2). Rg5:Rk1 treatment also increased the activities of proteins associated with osteoblast growth and differentiation in a dose-dependent manner. Overall, we found that the Rg5:Rk1 mixture of ginsenosides improved the osteoblastic function of MC3T3-E1 cells by increasing their proliferative capacity. This improvement is due to the action of Rg5:Rk1 on BMP-2, which is mediated by Runx2-dependent pathways.

Ginseng saponins and the treatment of osteoporosis: mini literature review.

The ginseng plant (Panax ginseng Meyer) has a large number of active ingredients including steroidal saponins with a dammarane skeleton as well as protopanaxadiol and protopanaxatriol, commonly known as ginsenosides, which have antioxidant, anticancer, antidiabetic, anti-adipocyte, and sexual enhancing effects. Though several discoveries have demonstrated that ginseng saponins (ginsenosides) as the most important therapeutic agent for the treatment of osteoporosis, yet the molecular mechanism of its active metabolites is unknown. In this review, we summarize the evidence supporting the therapeutic properties of ginsenosides both in vivo and in vitro, with an emphasis on the different molecular agents comprising receptor activator of nuclear factor kappa-B ligand, receptor activator of nuclear factor kappa-B, and matrix metallopeptidase-9, as well as the bone morphogenetic protein-2 and Smad signaling pathways.

Enzymatic biotransformation of ginsenoside Rb1 to 20(S)-Rg3 by recombinant ß-glucosidase from Microbacterium esteraromaticum.

Microbacterium esteraromaticum was isolated from ginseng field. The ß-glucosidase gene (bgp1) from M. esteraromaticum was cloned and expressed in Escherichia coli BL21 (DE3). The bgp1 gene consists of 2,496 bp encoding 831 amino acids which have homology to the glycosyl hydrolase family 3 protein domain. The recombinant ß-glucosidase enzyme (Bgp1) was purified and characterized. The molecular mass of purified Bgp1 was 87.5 kDa, as determined by SDS-PAGE. Using 0.1 mg ml(-1) enzyme in 20 mM sodium phosphate buffer at 37°C and pH 7.0, 1.0 mg ml(-1) ginsenoside Rb1 was transformed into 0.444 mg ml(-1) ginsenoside Rg3 within 6 h. The Bgp1 sequentially hydrolyzed the outer and inner glucose attached to the C-20 position of ginsenosides Rb1. Bgp1 hydrolyzed the ginsenoside Rb1 along the following pathway: Rb1 → Rd → 20(S)-Rg3. This is the first report of the biotransformation of ginsenoside Rb1 to ginsenoside 20(S)-Rg3 using the recombinant ß-glucosidase.

Molecular docking studies of anti-apoptotic BCL-2, BCL-XL, and MCL-1 proteins with ginsenosides from Panax ginseng.

Anti-apoptotic proteins such as BCL-2, BCL-XL and MCL-1 bind with pro-apoptotic proteins to induce apoptosis mechanism. BCL-2 family proteins are key regulators of apoptosis process. Over expression of these anti-apoptotic proteins lead to several cancers by preventing apoptosis. A number of studies revealed that ginseng derivatives reduce tumor growth. Ginseng, the most valuable medicinal herb found in eastern Asia belongs to Araliaceae family. In this study, docking simulations were performed for anti-apoptotic proteins with several ginsenosides from Panax ginseng. Our finding shows ginsenosides Rf, Rg1, Rg3 and Rh2 have more binding affinity with BCL-2, BCL-XL and MCL-1 and other ginsenosides also interact with each anti-apoptotic proteins. Therefore, ginseng derivatives represent a novel class of potent inhibitors and could be used for cancer chemotherapy.

Classification and characterization of putative cytochrome P450 genes from Panax ginseng C. A. Meyer.

In plants heme containing cytochrome P450 (P450) is a superfamily of monooxygenases that catalyze the addition of one oxygen atom from O2 into a substrate, with a substantial reduction of the other atom to water. The function of P450 families is attributed to chemical defense mechanism under terrestrial environmental conditions; several are involved in secondary and hormone metabolism. However, the evolutionary relationships of P450 genes in Panax ginseng remain largely unknown. In the present study, data mining methods were implemented and 116 novel putative P450 genes were identified from Expressed Sequence Tags (ESTs) of a ginseng database. These genes were classified into four clans and 22 families by sequence similarity conducted at amino acid level. The representative putative P450 sequences of P. ginseng and known P450 family from other plants were used to construct a phylogenetic tree. By comparing with other genomes, we found that most of the P450 genes from P. ginseng can be found in other dicot species. Depending on P450 family functions, seven P450 genes were selected, and for that organ specific expression, abiotic, and biotic studies were performed by quantitative reverse transcriptase-polymerase chain reaction. Different genes were found to be expressed differently in different organs. Biotic stress and abiotic stress transcript level was regulated diversely, and upregulation of P450 genes indicated the involvement of certain genes under stress conditions. The upregulation of the P450 genes under methyl jasmonate and fungal stress justifies the involvement of specific genes in secondary metabolite biosynthesis. Our results provide a foundation for further elucidating the actual function and role of P450 involved in various biochemical pathways in P. ginseng.

Bioconversion of ginsenoside Rb1 into compound K by Leuconostoc citreum LH1 isolated from kimchi

About 40 different types of ginsenoside (ginseng saponin), a major pharmacological component of ginseng, have been identified along with their physiological activities. Among these, compound K has been reported to prevent the development of and the metastasis of cancer by blocking the formation of tumors and suppressing the invasion of cancerous cells. In this study, ginsenoside Rb1 was converted into compound K via interaction with the enzyme secreted by ¥â-glucosidase active bacteria, Leuconostoc citreum LH1, extracted from kimchi. The optimum time for the conversion of Rb1 to compound K was about 72 hrs at a constant pH of 6.0 and an optimum temperature of about 30¨¬C. Under optimal conditions, ginsenoside Rb1 was decomposed and converted into compound K by 72 hrs post-reaction (99 percent). Both TLC and HPLC were used to analyze the enzymatic reaction. Ginsenoside Rb1 was consecutively converted to ginsenoside Rd, F2, and compound K via the hydrolyses of 20-C ¥â-(1 ¡æ 6)-glucoside, 3-C ¥â-(1 ¡æ 2)glucoside, and 3-C ¥â-glucose of ginsenoside Rb1.