Comprehensive Profiling and Quantification of Ginsenosides in the Root, Stem, Leaf, and Berry of Panax ginseng by UPLC-QTOF/MS.

The effective production and usage of ginsenosides, given their distinct pharmacological effects, are receiving increasing amounts of attention. As the ginsenosides content differs in different parts of Panax ginseng, we wanted to assess and compare the ginsenosides content in the ginseng roots, leave, stems, and berries. To extract the ginsenosides, 70% (v/v) methanol was used. The optimal ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-QTOF/MS) method was used to profile various ginsenosides from the different parts of P. ginseng. The datasets were then subjected to multivariate analysis including principal component analysis (PCA) and hierarchical clustering analysis (HCA). A UPLC-QTOF/MS method with an in-house library was constructed to profile 58 ginsenosides. With this method, a total of 39 ginsenosides were successfully identified and quantified in the ginseng roots, leave, stem, and berries. PCA and HCA characterized the different ginsenosides compositions from the different parts. The quantitative ginsenoside contents were also characterized from each plant part. The results of this study indicate that the UPLC-QTOF/MS method can be an effective tool to characterize various ginsenosides from the different parts of P. ginseng.

Ethanol extract of Lithospermum erythrorhizon Sieb. et Zucc. promotes osteoblastogenesis through the regulation of Runx2 and Osterix.

Bone remodeling and homeostasis are largely the result of the coordinated action of osteoblasts and osteoclasts. Osteoblasts are responsible for bone formation. The differentiation of osteoblasts is regulated by the transcription factors, Runx2 and Osterix. Natural products of plant origin are still a major part of traditional medicinal systems in Korea. The root of Lithospermum erythrorhizon Sieb. et Zucc. (LR), the purple gromwell, is an herbal medicine used for inflammatory and infectious diseases. LR is an anti-inflammatory and exerts anticancer effects by inducing the apoptosis of cancer cells. However, the precise molecular signaling mechanisms of osteoblastogenesis as regards LR and osteoblast transcription are not yet known. In this study, we investigated the effects of ethanol (EtOH) extract of LR (LES) on the osteoblast differentiation of C2C12 myoblasts induced by bone morphogenetic protein 4 (BMP4) and the potential involvement of Runx2 and Osterix in these effects. We found that the LES exhibited an ability to induce osteoblast differentiation. LES increased the expression of the osteoblast marker, alkaline phosphatase (ALP), as well as its activity, as shown by ALP staining and ALP activity assay. LES also increased mineralization, as shown by Alizarin Red S staining. Treatment with LES increased the protein levels (as shown by immunoblotting), as well as the transcriptional activity of Runx2 and Osterix and enhanced osteogenic activity. These results suggest that LES modulates osteoblast differentiation at least in part through Runx2 and Osterix.

A direct protein kinase B-targeted anti-inflammatory activity of cordycepin from artificially cultured fruit body of Cordyceps militaris.

BACKGROUND: Cordyceps militaris is one of well-known medicinal mushrooms with anti-inflammatory, anti-cancer, anti-diabetic, and anti-obesity activities. OBJECTIVE: The objective of the following study is to isolate chemical components from the ethanol extract (Cm-EE) from Cordyceps militaris and to evaluate their anti-inflammatory activities. MATERIALS AND METHODS: Column chromatographic separation was performed and anti-inflammatory roles of these compounds were also examined by using NO production and protein kinase B (AKT) activity assays. RESULTS: From Cm-EE, 13 constituents, including trehalose (1), cordycepin (2), 6-hydroxyethyladenosine (3), nicotinic amide (4), butyric acid (5), ß-dimorphecolic acid (6), α-dimorphecolic acid (7), palmitic acid (8), linoleic acid (9), cordycepeptide A (10), 4-(2-hydroxy-3-((9E,12E)-octadeca-9,12-dienoyloxy)propoxy)-2-(trimethylammonio)butanoate (11), 4-(2-hydroxy-3-(palmitoyloxy)propoxy)-2-(trimethylammonio)butanoate (12), and linoleic acid methyl ester (13) were isolated. Of these components, compound 2 displayed a significant inhibitory effect on NO production in lipopolysaccharide (LPS)-activated RAW264.7 cells. Furthermore, this compound strongly and directly suppressed the kinase activity of AKT, an essential signalling enzyme in LPS-induced NO production, by interacting with its ATP binding site. CONCLUSION: C. militaris could have anti-inflammatory activity mediated by cordycepin-induced suppression of AKT.

Glycosyl glycerides from hydroponic Panax ginseng inhibited NO production in lipopolysaccharide-stimulated RAW264.7 cells.

BACKGROUND: Although the aerial parts of hydroponic Panax ginseng are reported to contain higher contents of total ginsenosides than those of roots, the isolation and identification of active metabolites from the aerial parts of hydroponic P. ginseng have not been carried out so far. METHODS: The aerial parts of hydroponic P. ginseng were applied on repeated silica gel and octadecylsilane columns to yield four glycosyl glycerides (Compounds 1-4), which were identified based on nuclear magnetic resonance, infrared, fast atom bombardment mass spectrometry, and gas chromatography/mass spectrometry data. Compounds 1-4 were evaluated for inhibition activity on NO production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. RESULTS AND CONCLUSION: The glycosyl glycerides were identified to be (2S)-1-O-7(Z),10(Z),13(Z)-hexadecatrienoyl-3-O-ß-d-galactopyranosyl-sn-glycerol (1), (2S)-1-O-linolenoyl-3-O-ß-d-galactopyranosyl-sn-glycerol (2), (2S)-1-O-linolenoyl-2-O-linolenoyl-3-O-ß-d-galactopyranosyl-sn-glycerol (3), and 2(S)-1-O-linoleoyl-2-O-linoleoyl-3-O-ß-d-galactopyranosyl-sn-glycerol (4). Compounds 1 and 2 showed moderate inhibition activity on NO production in LPS-stimulated RAW264.7 cells [half maximal inhibitory concentration (IC50): 63.8 ± 6.4µM and 59.4 ± 6.8µM, respectively] without cytotoxicity at concentrations < 100µM, whereas Compounds 3 and 4 showed good inhibition effect (IC50: 7.7 ± 0.6µM and 8.0 ± 0.9µM, respectively) without cytotoxicity at concentrations < 20µM. All isolated compounds showed reduced messenger RNA (mRNA) expression of interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α in LPS-induced macrophage cells with strong inhibition of mRNA activity observed for Compounds 3 and 4.

Chemical constituents of Hericium erinaceum associated with the inhibitory activity against cellular senescence in human umbilical vascular endothelial cells.

Hericium erinaceum is an edible and medicinal mushroom widely used in Korea, Japan, and China. On the search for biologically active compounds supporting the medicinal usage, the MeOH extract of the fruiting bodies of H. erinaceum was investigated for its chemical constituents. Six compounds were isolated and identified as hericenone D (1), (22E,24R)-5α,8α-epidioxyergosta-6,22-dien-3ß-ol (2), erinacerin B (3), hericenone E (4), hericenone F (5) and isohericerin (6) by comparing their spectroscopic data with previously reported values. The inhibitory effects on adriamycin-induced cellular senescence in human dermal fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVECs) of the isolates (1-6) were studied. Among the isolated compounds, ergosterol peroxide (2) reduced senescence associated ß-galactosidase (SA-ß-gal) activity increased in HUVECs treated with adriamycin. According to experimental data obtained, the active compound may inspire the development of a new pharmacologically useful substance to be used in the treatment and prevention of age-related diseases.

The potential of minor ginsenosides isolated from the leaves of Panax ginseng as inhibitors of melanogenesis.

Three minor ginsenosides, namely, ginsenoside Rh6 (1), vina-ginsenoside R4 (2) and vina-ginsenoside R13 (3), were isolated from the leaves of hydroponic Panax ginseng. The chemical structures were determined based on spectroscopic methods, including fast atom bombardment mass spectroscopy (FAB-MS), 1D-nuclear magnetic resonance (NMR), 2D-NMR, and, infrared (IR) spectroscopy. The melanogenic inhibitory activity of compounds 1, 2 and 3 was 23.9%, 27.8% and 35.2%, respectively, at a concentration of 80 µM. Likewise, the three compounds showed inhibitory activity on body pigmentation on a zebrafish model, which is commonly used as a model for biomedical or cosmetic research. These results from in vitro and in vivo systems suggest that the three aforementioned compounds isolated from Panax ginseng may have potential as new skin whitening compounds.

2-Hydroxy-3,4-naphthochalcone (2H-NC) inhibits TNFα-induced tumor invasion through the downregulation of NF-κB-mediated MMP-9 gene expression.

The control of tumor metastasis is important for the successful prevention and treatment of cancer. Emerging evidence indicates that various natural and synthetic chalcones exhibit antimetastatic activity through the inhibition of nuclear factor-κB (NF-κB), although the precise mechanism by which this occurs is currently unclear. In this study, 2-hydroxy-3,4-naphthochalcone (2H-NC) was found to reduce tumor necrosis factor alpha (TNFα)-induced MMP-9 mRNA expression and gelatinolytic enzyme activity. These actions were associated with inhibition of RelA/p65 NF-κB activity. In addition, 2H-NC inhibited TNFα-induced invasion of MDA-MB-231 breast cancer cells, as assessed using a three-dimensional spheroid invasion assay. Taken together, these data demonstrate that 2H-NC prevents TNFα-induced tumor cell invasion through downregulation of NF-κB-mediated MMP-9 gene expression, and thereby identify naphthochalcones as a potentially effective class of molecules to use as a platform for the development of antimetastatic agents.

Benzyl alcohol derivatives from the mushroom Hericium erinaceum attenuate LPS-stimulated inflammatory response through the regulation of NF-κB and AP-1 activity.

On the search for anti-inflammatory compounds from natural Korean medicinal sources, a bioassay-guided fractionation and chemical investigation of the MeOH extract from the fruiting bodies of Hericium erinaceum resulted in the isolation and identification of five benzyl alcohol derivatives (1-5). In this study, their anti-inflammatory effects on lipopolysaccharide (LPS)-induced production of pro-inflammatory mediators were examined using RAW 264.7 macrophage cells. The structures of isolates were identified by comparing their spectroscopic data with previously reported values. The analysis of their inhibitory activities on LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in RAW 264.7 macrophage cells showed that erinacerin B (2) and hericenone E (4) decreased the levels of NO and PGE2 production in a concentration-dependent manner. Next, this study was performed to examine their mechanism of action on the regulation of NO and PGE2 production. Compounds 2 and 4 were found to block the LPS-induced phosphorylation of two major inflammatory transcription factors, NF-κB (p65/p50) and AP-1 (c-Jun and c-Fos). Taken together, these results suggest that down-regulation of LPS-induced NO and PGE2 production by compounds 2 and 4 is mediated through the modulation of NF-κB and AP-1 activation in macrophage cells. These results impact the development of potential health products for preventing and treating inflammatory diseases.

Quality evaluation of Panax ginseng roots using a rapid resolution LC-QTOF/MS-based metabolomics approach.

Korean ginseng (Panax ginseng C.A. Meyer) contains several types of ginsenosides, which are considered the major active medicinal components of ginseng. The types and quantities of ginsenosides found in ginseng may differ, depending on the location of cultivation, making it necessary to establish a reliable method for distinguishing cultivation locations of ginseng roots. P. ginseng roots produced in different regions of Korea, China, and Japan have been unintentionally confused in herbal markets owing to their complicated plant sources. PCA and PLS-DA using RRLC-QTOF/MS data was able to differentiate between ginsengs cultivated in Korea, China, and Japan. The chemical markers accountable for such variations were identified through a PCA loadings plot, tentatively identified by RRLC-QTOF/MS and partially verified by available reference standards. The classification result can be used to identify P. ginseng origin.

A comparison of antioxidant activity of Korean White and Red Ginsengs on H2O2-induced oxidative stress in HepG2 hepatoma cells.

The aim of this study was to determine and compare the preventive effect of Korean White Ginseng and Red Ginseng on oxidative stress in H2O2-treated HepG2 cells. The roots of ginseng were extracted with 70% methanol and partitioned with butanol to obtain saponin fractions, which have been known as bioactive constituents of ginseng. 2',7'-Dichlorofluorescein diacetate (DCF-DA) assay and malondialdehyde (MDA) content were measured for evaluating intracellular reactive oxygen species (ROS) generation. Also, mRNA expressions and activities of antioxidant enzymes were analyzed to determine the antioxidant activity of saponin or non-saponin fractions of ginsengs. According to DCF-DA assay, H2O2-induced MDA release and ROS generation were significantly reduced by treatment with saponin fractions of white and red ginseng roots. Also, saponin fractions increased effectively intracellular antioxidant enzyme activities including catalase, glutathione peroxidase and superoxide dismutase in H2O2- treated HepG2 hepatoma cells. In general, red ginseng was more effective than white ginseng for reducing oxidative stress. These results indicate that administration of red ginseng may certainly contribute relatively stronger than white ginseng to prevent from damaging liver function by oxidative stress.

Anti-inflammatory cycloartane-type saponins of Astragalus membranaceus.

A new cycloartane-type triterpene glycoside, agroastragaloside V (1) was isolated from the roots of Astragalus membranaceus. The structure was identified as 3-O-ß-(2'-O-acetyl)-D-xylopyranosyl-6-O-ß-D-glucopyranosyl-(24S)-3ß,6α,24α,25-tetrahydroxy- 9,19-cyclolanostane, by means of spectroscopic methods, including HR-FAB/MS, 1D NMR (1H, 13C, DEPT), 2D NMR (gCOSY, gHSQC, gHMBC, NOESY), and IR spectroscopy. Four known cycloartane glycosides, namely, agroastragaloside I (2), agroastragaloside II (3), isoastragaloside II (4) and astragaloside IV (5) were also isolated. All isolated compounds were tested for the ability to inhibit LPS-induced nitric oxide production in RAW264.7 macrophages.

Anti-inflammatory lignans from the fruits of Acanthopanax sessiliflorus.

A new lignan, named acanthosessilin A (1), as well as eight known lignan and lignan glycosides 2-9 were isolated from an ethanolic extract of Acanthopanax sessiliflorus fruits. The chemical structures were determined by spectroscopic methods, including HR-EIMS, 1D NMR (1H, 13C, DEPT), 2D NMR (gCOSY, gHSQC, gHMBC, NOESY), and IR spectroscopy. All isolated compounds were tested for the ability to inhibit LPS-induced nitric oxide production in RAW264.7 macrophages.

Association between ABCB1 Polymorphisms and Ischemic Stroke in Korean Population.

Neuronal expression of ATP-binding cassette, sub-family B (MDR/TAP), member 1 (ABCB1) has been demonstrated after brain ischemia. To investigate whether ABCB1 polymorphisms are associated with the development, risk factors (hypertension, dyslipidemia, and diabetes mellitus), severity (National Institutes of Health Stroke Scale, NIHSS), and sequelae (Modified Barthel Index, MBI) of ischemic stroke (IS), four single nucleotide polymorphisms (SNPs) of the ABCB1 gene [rs4148727, promoter, -154T>C; rs3213619, 5'-untranslation region (5'UTR), -129T>C); rs1128503, synonymous, Gly412 (C>T); rs3842, 3'UTR, A>G] were analyzed in 121 IS patients and 291 control subjects. SNPStats and SPSS 18.0 were used to obtain odds ratios (OR), 95% confidence intervals (CI), and p values. Multiple logistic regression models (codominant1, codominant2, dominant, recessive, and log-additive models) were applied to analyze the genetic data. The rs3842 SNP was weakly associated with the development of IS (p=0.020 in codominant1 model and p=0.028 in dominant model). In the analysis of clinical phenotypes, ABCB1 polymorphisms were nominally associated with hypertension (rs3213619 and rs3842, p<0.05), dyslipidemia (rs1128503, p<0.05), diabetes (rs3842, p<0.05), and NIHSS (rs4148727, p<0.05). Interestingly, rs3842 showed statistically strong association between IS with hypertension and IS without hypertension (Fisher's exact p=0.003, OR=0.11, 95% CI=0.03-0.51 in recessive model). These results suggest that the ABCB1 gene may be associated with the development and clinical phenotypes of IS in Korean population.

Effects of Natural Bioactive Products on the Growth and Ginsenoside Contents of Panax ginseng Cultured in an Aeroponic System.

This study was conducted to evaluate the effects of natural bioactive products such as Manda enzyme (T1), Yangmyeongwon (T2), effective microorganisms (T3), and Kelpak (T4) on the growth and ginsenoside contents of Panax ginseng cultured in an aeroponic system using a two-layer vertical type of nutrient bath under natural light conditions. The growth of ginseng plants showed specific characteristics according to the positions in which they were cultured due to the difference of light transmittance and temperature in the upper and lower layers during aeroponic culture in a two-layer vertical type of system. The growth of the aerial part of the leaves and stems of ginseng plants cultured in the lower layer (4,000 to 6,000 lx, 23℃ to 26℃) of the nutrient bath was observed to be superior to that of the ginseng plants cultured in the upper layer (12,000 to 15,000 lx, 25℃ to 28℃). The leaf area was significantly larger in the treatment of T2 and T4 (46.70 cm(2)) than with other treatments. Conversely, the values of the root weight and root diameter were higher in ginseng plants cultured in the upper layer of the nutrient bath. The root weight was significantly heavier in the treatment of T4 (6.46 g) and T3 (6.26 g) than with other treatments. The total ginsenoside content in the leaves and roots was highest in the ginseng plants cultured by the treatment of T1, at 16.20%, while the total ginsenoside content obtained by other treatments decreased in the order of T4, T5 (control), T2, and T3, at 13.21%, 12.30%, 14.84%, and 14.86%, respectively. The total ginsenoside content of the ginseng leaves was found to be significantly higher in the treatment of T1 in the lower layer of the nutrient bath, at 15.30%, while the content of the ginseng roots in the treatments of T3 and T4, at 1.27% and 1.23%, respectively, was significantly higher than in other treatments in the upper layer of the nutrient bath.