RESUMO
Panax ginseng is processed to diversify efficacy. Four processed ginsengs containing white ginseng (WG), tae-geuk ginseng (TG), red ginseng (RG), and black ginseng (BG) were analyzed using nuclear magnetic resonance (NMR) spectroscopy for screening overall primary metabolites. There were significant differences in the sugar content among these four processed ginseng products. WG had a high sucrose content, TG had a high maltose content, and BG had high fructose and glucose content. In the multivariate analyses of NMR spectra, the PCA score plot showed significant discrimination between the four processed ginsengs. For effective clustering, orthogonal partial least squares discriminant analyses (OPLS-DA) with a 1:1 comparison were conducted and all OPLS models were validated using the permutation test, the root mean square error of estimation (RMSEE), and the root mean square error of prediction (RMSEP). All OPLS-DA score plots showed clear separations of processed ginseng products, and sugars such as sucrose and fructose mainly contributed to these separations.
Assuntos
Ginsenosídeos/análise , Metabolômica , Panax/química , Extratos Vegetais/análise , Açúcares/análise , Espectrometria de Massas , Ressonância Magnética Nuclear BiomolecularRESUMO
The commercial use of Panax ginseng berries is increasing as P. ginseng berries are known to contain large amounts of ginsenosides, and many pharmacological activities have been reported for the various ginsenosides. For the proper use of P.ginseng berries, it is necessary to study efficient and accurate quality control and the profiling of the overall composition of each cultivar. Ginseng berry samples from seven cultivars (Eumseung, Chung-buk Province, Republic of Korea) were analyzed using ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-QTOF/MS) for profiling of the ginsenosides, and high-resolution magic-angle-spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy for profiling of the primary metabolites. Comparing twenty-six ginsenoside profiles between the variant representatives and between the violet-stem variant, Kumpoong and Sunwon were classified. In the case of primary metabolites, the cultivars Kumpoong and Gopoong were classified. As a result of correlation analyses of the primary and secondary metabolites, in the Gopoong cultivar, the metabolism was found to lean toward energy metabolism rather than ginsenoside synthesis, and accumulation of osmolytes was low. The Gopoong cultivar had higher levels of most of the amino acids, such as arginine, phenylalanine, isoleucine, threonine, and valine, and it contained the highest level of choline and the lowest level of myo-inositol. Except for these, there were no significant differences of primary metabolites. In the Kumpoong cultivar, the protopanaxatriol (PPT)-type ginsenosides, ginsenoside Re and ginsenoside Rg2, were much lower than in the other cultivars, while the other PPT-type ginsenosides were inversely found in much higher amounts than in other cultivars. The Sunwon cultivar showed that variations of PPT-type ginsenosides were significantly different between samples. However, the median values of PPT-type ginsenosides of Sunwon showed similar levels to those of Kumpoong. The difference in primary metabolites used for metabolism for survival was found to be small in our results. Our data demonstrated the characteristics of each cultivar using profiling data of the primary and secondary metabolites, especially for Gopoong, Kumpoong, and Sunwon. These profiling data provided important information for further research and commercial use.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Frutas/química , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodos , Panax/química , Ginsenosídeos/análiseRESUMO
(1) Background: The ability to determine the age of ginseng is very important because the price of ginseng depends on the cultivation period. Since morphological observation is subjective, a new scientific and systematic method for determining the age of ginseng is required. (2) Methods: Three techniques were used for a metabolomics approach. High-resolution magic-angle-spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy was used to analyze powdered ginseng samples without extraction. Ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) and gas chromatography quadrupole time-of-fight mass spectrometry (GC-TOF/MS) were used to analyze the extracts of 4-, 5-, and 6-year-old ginseng. (3) Results: A metabolomics approach has the potential to discriminate the age of ginseng. Among the primary metabolites detected from NMR spectroscopy, the levels of fumarate and choline showed moderate prediction with an area under the curve (AUC) value of more than 0.7. As a result of UPLC-QTOF/MS-based profiling, 61 metabolites referring to the VIP (variable importance in the projection) score contributed to discriminating the age of ginseng. The results of GC×GC-TOF/MS showed clear discrimination of 4-, 5-, and 6-year-old ginseng using orthogonal partial least-squares discriminant analysis (OPLS-DA) to 100% of the discrimination rate. The results of receiver operating characteristic (ROC) analysis, 16 metabolites between 4- and 5-year-old ginseng, and 18 metabolites between 5- and 6-year-old ginseng contributed to age discrimination in all regions. (4) Conclusions: These results showed that metabolic profiling and multivariate statistical analyses can distinguish the age of ginseng. Especially, it is meaningful that ginseng samples from different areas had the same metabolites for age discrimination. In future studies, it will be necessary to identify the unknown variables and to collaboratively study with other fields the biochemistry of aging in ginseng.
Assuntos
Metabolômica/métodos , Panax/química , Extratos Vegetais/análise , Cromatografia Líquida , Análise Discriminante , Cromatografia Gasosa-Espectrometria de Massas , Espectroscopia de Ressonância Magnética , Panax/crescimento & desenvolvimento , Curva ROC , Espectrometria de Massas em TandemRESUMO
Red Liriope platyphylla (RLP) is a known herbal medicine used in the treatment of some chronic diseases including constipation, neurodegenerative disorders, diabetes and obesity. To determine and characterize putative biomarkers that predict the laxative effects induced by RLP treatment, alteration of endogenous metabolites was measured in the serum of loperamide (Lop)-induced constipation rats after administration of RLP extract (EtRLP) using 1H nuclear magnetic resonance (1H NMR) spectral data. The urine volume and amounts, and weights and water contents of stools were significantly recovered in the Lop + EtRLP treated group as compared to the No group, whereas body weight and food intake maintained constant levels. Also, significant recoveries in the thickness of mucosa and muscle were detected in the colon of the Lop + EtRLP treated group. Furthermore, pattern recognition showed absolutely different clustering of the serum analysis parameters when comparing the Lop treated group and Lop + EtRLP treated group. Of the 33 endogenous metabolites, 7 amino acids (alanine, arginine, glutamate, glutamine, glycine, threonine and valine) and 8 endogenous metabolites (betaine, creatine, glucose, taurine, ethanol, lactate, glycerol and succinate) were dramatically increased in the Lop + EtRLP treated SD rats. These results provide the first evidence pertaining to metabolic changes in the constipation rats treated with Lop + EtRLP. Additionally, these findings correlate with changes observed in 15 metabolites during the laxative effects of EtRLP.
RESUMO
The use of probiotics is considered an attractive biocontrol method. It is effective in growth promotion in aquaculture. However, the mode of action of probiotics in fish in terms of growth promotion remains unclear. The objective of the present study was to investigate growth promotion effect of dietary administration of host-derived probiotics, Lactococcus lactis WFLU12, on olive flounder compared to control group fed with basal diet by analyzing their intestinal and serum metabolome using capillary electrophoresis mass spectrometry with time-of flight (CE-TOFMS). Results of CE-TOFMS revealed that 53 out of 200 metabolites from intestinal luminal metabolome and 5 out of 171 metabolites from serum metabolome, respectively, were present in significantly higher concentrations in the probiotic-fed group than those in the control group. Concentrations of metabolites such as citrulline, tricarboxylic acid cycle (TCA) intermediates, short chain fatty acids, vitamins, and taurine were significantly higher in the probiotic-fed group than those in the control group. The probiotic strain WFLU12 also possesses genes encoding enzymes to help produce these metabolites. Therefore, it is highly likely that these increased metabolites linked to growth promotion in olive flounder are due to supplementation of the probiotic strain. To the best of our knowledge, this is the first study to show that dietary probiotics can greatly influence metabolome in fish. Findings of the present study may reveal important implications for maximizing the efficiency of using dietary additives to optimize fish health and growth.
RESUMO
Osteoarthritis (OA) is a degenerative joint disease that is characterized by irreversible articular cartilage destruction by inflammatory reaction. Among inflammatory stimuli, interleukin-1ß (IL-1ß) is known to play a crucial role in OA pathogenesis by stimulating several mediators that contribute to cartilage degradation. Recently, the marine brown alga Sargassum serratifolium has been reported to exhibit antioxidant and anti-inflammatory effects in microglial and human umbilical vein endothelial cell models using lipopolysaccharide and tumor necrosis factor-α, but its beneficial effects on OA have not been investigated. This study aimed to evaluate the anti-osteoarthritic effects of ethanol extract of S. serratifolium (EESS) in SW1353 human chondrocytes and, in parallel, primary rat articular chondrocytes. Our results showed that EESS effectively blocked the generation of reactive oxygen species in IL-1ß-treated SW1353 and rat primary chondrocytes, indicating that EESS has a potent antioxidant activity. EESS also attenuated IL-1ß-induced production of nitric oxide (NO) and prostaglandin E2, major inflammatory mediators in these cells, which was associated with the inhibition of inducible NO synthase and cyclooxygenase-2 expression. Moreover, EESS downregulated the level of gene expression of matrix metalloproteinase (MMP)-1, -3 and -13 in SW1353 chondrocytes treated with IL-1ß, resulting in their extracellular secretion reduction. In addition, the IL-1ß-induced activation of nuclear factor-kappa B (NF-κB) was restored by EESS. Furthermore, EESS reduced the activation of p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathways upon IL-1ß stimulation. These results indicate that EESS has the potential to exhibit antioxidant and anti-inflammatory effects through inactivation of the NF-κB, p38 MAPK, and PI3K/Akt signaling pathways. Collectively, these findings demonstrate that EESS may have the potential for chondroprotection, and extracts of S. serratifolium could potentially be used in the prevention and treatment of OA.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Condrócitos/efeitos dos fármacos , Interleucina-1beta/imunologia , Extratos Vegetais/farmacologia , Sargassum , Animais , Anti-Inflamatórios/química , Antioxidantes/química , Linhagem Celular , Células Cultivadas , Condrócitos/imunologia , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Masculino , NF-kappa B/imunologia , Osteoartrite/tratamento farmacológico , Osteoartrite/imunologia , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/imunologia , Extratos Vegetais/química , Proteínas Proto-Oncogênicas c-akt/imunologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/imunologia , Sargassum/química , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
Sargassum serratifolium C. Agardh is a marine brown alga that has long been used as an ingredient for food and medicine by many people living along Asian coastlines. Recently, various beneficial effects of extracts or compounds isolated from S. serratifolium have been reported, but their efficacies against bone destruction are unclear. Therefore, in this study, we investigated the inhibitory property of an ethanol extract of S. serratifolium (EESS) on osteoclast differentiation by focusing on the receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclastogenesis model using RAW 264.7 macrophages. Our results demonstrated that EESS reduced RANKL-induced osteoclast differentiation in RAW 264.7 cells, by inhibiting tartrate-resistant acid phosphatase (TRAP) activity and destroying the F-actin ring formation. EESS also attenuated RANKL-induced expressions of key osteoclast-specific genes, such as nuclear factor of activated T cells cytoplasmic 1 (NFATC1), TRAP, cathepsin K and matrix metalloproteinase-9. These effects were mediated by impaired nuclear translocation of nuclear factor (NF)-κB and suppression of IκB-α degradation. In addition, EESS effectively inhibited the production of reactive oxygen species (ROS) by RANKL, which was associated with enhanced expression of nuclear translocation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). Overall, our findings provide evidence that EESS suppresses RANKL-induced osteoclastogenesis and oxidative stress through suppression of NF-κB and activation of Nrf2/HO-1 signaling pathway, indicating that S. serratifolium has a potential application the prevention and treatment of osteoclastogenic bone disease.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Heme Oxigenase-1/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Osteoclastos/patologia , Estresse Oxidativo/efeitos dos fármacos , Ligante RANK/farmacologia , Sargassum/química , Actinas/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Etanol , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Extratos Vegetais/farmacologia , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Oxidative stress in chondrocytes plays a critical role in the pathogenesis of osteoarthritis as an important cause of articular cartilage degradation. Sargassum serratifolium C. Agardh, a marine brown algae, is known to have potent antioxidant activity. Nevertheless, no study has been conducted yet on the protective efficacy against oxidative stress in chondrocytes. Therefore, the aim of the current study is to investigate the mechanism of the antioxidative effect of ethanol extract of S. serratifolium (EESS) on DNA damage and apoptosis induced by hydrogen peroxide (H2O2) in SW1353 human chondrocytes. For this purpose, SW1353 cells exposed to H2O2 in the presence or absence of EESS were applied to cell viability assay, comet assay, immunoblotting and flow cytometry analyses. Our results showed that EESS effectively attenuated H2O2-induced cytotoxicity and DNA damage associated with the inhibition of reactive oxygen species (ROS) accumulation. EESS also weakened the mitochondria membrane permeabilization by H2O2, and recovered H2O2-induced decreased expression of anti-apoptotic Bcl-2 and pro-caspase-3, and degradation of poly (ADP-ribose) polymerase. In addition, EESS increased not only expression, but also phosphorylation of nuclear factor-erythroid 2 related factor 2 (Nrf2), and promoted the expression of heme oxygenase-1 (HO-1), a critical target enzyme of Nrf2, but decreased the expression of kelch-like ECH-associated protein-1; however, the inhibition of HO-1 activity by zinc protoporphyrin abolished the antioxidant potential induced by EESS against H2O2-mediated oxidative stress. Therefore, the results of this study suggest that the antioxidant efficacy of EESS in chondrocytes is at least involved in the Nrf2/HO-1 signaling pathway-dependent mechanism.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Dano ao DNA , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Sargassum/química , Transdução de Sinais/efeitos dos fármacos , Antioxidantes/química , Células Cultivadas , Humanos , Fator 2 Relacionado a NF-E2/fisiologia , Estresse Oxidativo/fisiologia , Extratos Vegetais/química , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Immunoregulatory elements have emerged as useful immunotherapeutic agents against cancer. In traditional medicine, Mori folium, the leaf of Morus alba L. (Moraceae), has been used for various medicinal purposes; however, the immunomodulatory effects have not been fully identified. We evaluated the immunoenhancing potential of water extract of Mori folium (WEMF) in murine RAW264.7 macrophages. METHODS: RAW264.7 cells were treated with WEMF for 24 hours and cell viability was detected by an MTT method. Nitric oxide (NO) levels in the culture supernatants were assayed using Griess reagent. The productions of prostaglandin E2 (PGE2) and immune-related cytokines was measured using ELISA detection kits. The mRNA and protein expression levels of Inducible NO synthase, COX-2, and cytokines were assayed by reverse transcription-PCR and Western blotting, respectively. The effect of WEMF on phagocytic activity was measured using a Phagocytosis Assay Kit. RESULTS: WEMF significantly stimulated the production of NO and PGE2 as immune response parameters at noncytotoxic concentrations, which was associated with the increased expression of inducible NO synthase and COX-2. The release and expression of cytokines, such as TNF-α, interleukin (IL)-1ß, IL-6, and IL-10, were also significantly increased in response to treatment with WEMF. Moreover, WEMF promoted the macrophagic differentiation of RAW264.7 cells and the resulting phagocytosis activity. CONCLUSIONS: WEMF has the potential to modulate the immune function by regulating immunological parameters. Further studies are needed to identify the active compounds and to support the use of WEMF as an immune stimulant.
RESUMO
The aim of this study was to investigate urinary metabolomic profiles associated with cadmium (Cd)-induced nephrotoxicity and their potential mechanisms. Metabolomic profiles were measured by high-resolution (1)H-nuclear magnetic resonance (NMR) spectroscopy in the urine of rats after oral exposure to CdCl2 (1, 5, or 25 mg/kg) for 6 wk. The spectral data were further analyzed by a multivariate analysis to identify specific urinary metabolites. Urinary excretion levels of protein biomarkers were also measured and CdCl2 accumulated dose-dependently in the kidney. High-dose (25 mg/kg) CdCl2 exposure significantly increased serum blood urea nitrogen (BUN), but serum creatinine (sCr) levels were unchanged. High-dose CdCl2 (25 mg/kg) exposure also significantly elevated protein-based urinary biomarkers including osteopontin, monocyte chemoattractant protein-1 (MCP-1), kidney injury molecules-1 (Kim-1), and selenium-binding protein 1 (SBP1) in rat urine. Under these conditions, six urinary metabolites (citrate, serine, 3-hydroxyisovalerate, 4-hydroxyphenyllactate, dimethylamine, and betaine) were involved in mitochondrial energy metabolism. In addition, a few number of amino acids such as glycine, glutamate, tyrosine, proline, or phenylalanine and carbohydrate (glucose) were altered in urine after CdCl2 exposure. In particular, the metabolites involved in the glutathione biosynthesis pathway, including cysteine, serine, methionine, and glutamate, were markedly decreased compared to the control. Thus, these metabolites are potential biomarkers for detection of Cd-induced nephrotoxicity. Our results further indicate that redox metabolomics pathways may be associated with Cd-mediated chronic kidney injury. These findings provide a biochemical pathway for better understanding of cellular mechanism underlying Cd-induced renal injury in humans.
Assuntos
Biomarcadores/urina , Cádmio/toxicidade , Rim/efeitos dos fármacos , Metaboloma , Animais , Moléculas de Adesão Celular/urina , Quimiocina CCL2/urina , Rim/metabolismo , Nefropatias/induzido quimicamente , Espectroscopia de Ressonância Magnética , Masculino , Análise Multivariada , Análise de Componente Principal , Ratos , Ratos Sprague-Dawley , Proteínas de Ligação a Selênio/urinaRESUMO
Human Ubc9 is homologous to ubiquitin-conjugating enzymes. However, instead of conjugating ubiquitin, it conjugates a ubiquitin homologue, small ubiquitin-like modifier 1 (SUMO-1), also known as UBL1, GMP1, SMTP3, PIC1, and sentrin. The SUMO-1 conjugation pathway is very similar to that of ubiquitin with regard to the primary sequences of the ubiquitin-activating enzymes (E1), the three-dimensional structures of the ubiquitin-conjugating enzymes (E2), and the chemistry of the overall conjugation pathway. The interaction of substrates with Ubc9 has been studied using NMR spectroscopy. Peptides with sequences that correspond to those of the SUMO-1 conjugation sites from p53 and c-Jun both bind to a surface adjacent to the active site Cys93 of human Ubc9, which has been previously shown to include residues that demonstrate the most significant dynamics on the microsecond to millisecond time scale. Mutations in this region, Q126A, Q130A, A131D, E132A, Y134A, and T135A, were constructed to evaluate the role of these residues in SUMO-1 conjugation. These alterations have significant effects on the conjugation of SUMO-1 with the target proteins p53, E1B, and promyelocytic leukemia protein and define a substrate binding site on Ubc9. Furthermore, the SUMO-1 conjugation site of p53 does not form any defined secondary structure when either free or bound to Ubc9. This suggests that a defined secondary structure at SUMO-1 conjugation sites in target proteins is not necessary for recognition and conjugation by the SUMO-1 pathway.