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In this study, we propose a novel strategy utilizing deep eutectic solvents (DESs) as both the extraction solvent and dispersing liquid, with nanometer zinc oxide (ZnO) serving as the adsorbent. This method incorporates ultrasound-assisted matrix solid phase dispersion (UA-MSPD) for the extraction of six active components (salidroside, echinacoside, acteoside, specnuezhenide, nuezhenoside G13, and oleanolic acid) from Ligustri Lucidi Fructus samples. The extracts were then analyzed using high-performance liquid chromatography equipped with a diode array detector. The effects of various parameters such as dispersant dosage, DESs volume, grinding time, ultrasonication duration, and eluent volume on extraction recovery were investigated and optimized using a central composite design under response surface methodology. The optimized conditions yielded detection limits ranging from 0.003 to 0.01 mg/g and relative standard deviations of 8.7% or lower. Extraction recoveries varied between 93% and 98%. The method demonstrated excellent linearity for the analytes (R2 ≥ 0.9997). The simple, green, and efficient DESs/ZnO-UA-MSPD technique proved to be rapid, accurate, and reliable for extracting and analyzing the six active ingredients in Ligustri Lucidi Fructus samples.
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Ligustrum , Extracción en Fase Sólida , Ondas Ultrasónicas , Óxido de Zinc , Extracción en Fase Sólida/métodos , Óxido de Zinc/química , Ligustrum/química , Disolventes Eutécticos Profundos/química , Cromatografía Líquida de Alta Presión , Frutas/química , Extractos Vegetales/química , Extractos Vegetales/análisis , Tamaño de la Partícula , Solventes/químicaRESUMEN
Inulin-type fructan (ITF) defined as a polydisperse carbohydrate consisting mainly of ß-(2-1) fructosyl-fructose links exerts potential prebiotics properties by selectively stimulating the growth of Bifidobacterium and Lactobacillus. This study reported the modulation of human gut microbiota in vitro by ITF from Codonopsis pilosula roots using 16S ribosomal RNA gene sequencing. The microbiota community structure analysis at genus levels showed that 50 mg/mL ITF significantly stimulated the growth of Prevotella and Faecalibacterium. LEfSe analysis showed that ITF at 25 and 50 mg/mL primarily increased the relative abundance of genera Parabacteroides and Alistipes (LDA Score > 4), and genera Prevotella and Faecalibacterium (LDA Score > 4) as well as Acidaminococcus, Megasphaera, Bifidobacterium and Megamonas (LDA Score > 3.5), respectively. Meanwhile, ITF at 25 and 50 mg/mL exhibited the effects of lowering pH values of samples after 24 h fermentation (p < 0.05). The results indicated that ITF likely has potential in stimulating the growth of Prevotella and Faecalibacterium as well as Bifidobacterium of human gut microbiota.
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Inulin-type fructan CP-A, a predominant polysaccharide in Codonopsis pilosula, demonstrates regulatory effects on immune activity and anti-inflammation. The efficacy of CP-A in treating ulcerative colitis (UC) is, however, not well-established. This study employed an in vitro lipopolysaccharide (LPS)-induced colonic epithelial cell model (NCM460) and an in vivo dextran sulfate sodium (DSS)-induced colitis mouse model to explore CP-A's protective effects against experimental colitis and its underlying mechanisms. We monitored the clinical symptoms in mice using various parameters: body weight, disease activity index (DAI), colon length, spleen weight, and histopathological scores. Additionally, molecular markers were assessed through enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence (IF), immunohistochemistry (IHC), and Western blotting assays. Results showed that CP-A significantly reduced reactive oxygen species (ROS), tumor necrosis factor-alpha (TNF-α), and interleukins (IL-6, IL-1ß, IL-18) in LPS-induced cells while increasing IL-4 and IL-10 levels and enhancing the expression of Claudin-1, ZO-1, and occludin proteins in NCM460 cells. Correspondingly, in vivo findings revealed that CP-A administration markedly improved DAI, reduced colon shortening, and decreased the production of myeloperoxidase (MPO), malondialdehyde (MDA), ROS, IL-1ß, IL-18, and NOD-like receptor protein 3 (NLRP3) inflammasome-associated genes/proteins in UC mice. CP-A treatment also elevated glutathione (GSH) and superoxide dismutase (SOD) levels, stimulated autophagy (LC3B, P62, Beclin-1, and ATG5), and reinforced Claudin-1 and ZO-1 expression, thereby aiding in intestinal epithelial barrier repair in colitis mice. Notably, the inhibition of autophagy via chloroquine (CQ) diminished CP-A's protective impact against colitis in vivo. These findings elucidate that CP-A's therapeutic effect on experimental colitis possibly involves mitigating intestinal inflammation through autophagy-mediated NLRP3 inflammasome inactivation. Consequently, inulin-type fructan CP-A emerges as a promising drug candidate for UC treatment.
Asunto(s)
Codonopsis , Colitis Ulcerosa , Colitis , Ratones , Animales , Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Inulina/metabolismo , Inulina/farmacología , Inulina/uso terapéutico , Interleucina-18 , Codonopsis/metabolismo , Proteínas NLR/metabolismo , Fructanos/metabolismo , Fructanos/farmacología , Fructanos/uso terapéutico , Especies Reactivas de Oxígeno/metabolismo , Lipopolisacáridos/farmacología , Claudina-1/metabolismo , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/patología , Autofagia , Sulfato de Dextran , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Colon/metabolismo , Colon/patologíaRESUMEN
Codonopsis pilosula, a traditional Chinese medicinal and edible plant, contains several bioactive components. However, the biosynthetic mechanism is unclear because of the difficulties associated with functional gene analysis. Therefore, it is important to establish an efficient genetic transformation system for gene function analysis. In this study, we established a highly efficient Agrobacterium-mediated callus genetic transformation system for C. pilosula using stems as explants. After being pre-cultured for 3 days, the explants were infected with Agrobacterium tumefaciens strain GV3101 harboring pCAMBIA1381-35S::GUS at an OD600 value of 0.3 for 15 min, followed by co-cultivation on MS induction medium for 1 day and delayed cultivation on medium supplemented with 250 mg l-1 cefotaxime sodium for 12 days. The transformed calli were selected on screening medium supplemented with 250 mg l-1 cefotaxime sodium and 2.0 mg l-1 hygromycin and further confirmed by PCR amplification of the GUS gene and histochemical GUS assay. Based on the optimal protocol, the induction and transformation efficiency of calli reached a maximum of 91.07%. The establishment of a genetic transformation system for C. pilosula calli lays the foundation for the functional analysis of genes related to bioactive components through genetic engineering technology.
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Codonopsis Radix (CR) is an important traditional Chinese medicine used for the treatment of spleen deficiency syndrome (SDS). Codonopsis pilosula polysaccharides (CPP) in CR are considered to be responsible for tonifying the spleen function; however, the mechanisms of the polysaccharides have remained unclear. This study aimed to investigate the treatment mechanisms of CPP in SDS mice using a combinational strategy of 16S rRNA gene sequencing and targeted metabolomics. Here, studies demonstrated that CPP had invigorating effect in vivo in Sennae Folium-induced SDS in mice by organ indexes, D-xylose determination, gastrointestinal hormones levels and goblet cells observation. Antibiotic treatment revealed that the intestinal microbiota was required for the invigorating spleen effect of CPP. Furthermore, gut microbiota analysis found that CPP significantly enriched probiotic Lactobacillus and decreased the abundance of some opportunistic pathogens, such as Enterococcus and Shigella. The metabolic profile analysis of the colonic content revealed that 25 chemicals were altered significantly by CPP, including amino acids, organic acids, fatty acids, carbohydrates and carnitine etc., which are mainly related to "energy conversion" related processes such as amino acids metabolism, tricarboxylic acid cycle, and nitrogen metabolism. Spearman's correlation assays displayed there were strong correlations among biochemical indicators-gut microbiota-metabolomics. In summary, these results provided a new perspective for CPP improving SDS by regulating energy metabolism related bacteria and pathways.
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ETHNOPHARMACOLOGICAL RELEVANCE: Although the incidence of ulcerative colitis (UC) increases every year, there is still a lack of satisfactory treatment options. Anemone chinensis Bunge (AB), a traditional Chinese herb, is a potent compound that can be prepared as a decoction, and then administered as an enema to relieve UC symptoms. However, the therapeutic effect and mechanisms of aqueous AB on UC are still unknown. AIM OF THE STUDY: This study investigates the potential therapeutic value and mechanism of AB aqueous enema for UC. MATERIALS AND METHODS: First, the practical components in aqueous AB were extracted and identified by UPLC-MS/MS. Second, the potential active targets and target genes related to UC were predicted, mapped, and analyzed by network pharmacology. Then, the effects of AB aqueous enema on UC were assessed using the dextran sulfate sodium (DSS)-induced colitis model with mice. Finally, the level of inflammation, the expression level of proteins associated with the colonic mucosal barrier, and the microbiota associated with the intestinal mucosal were investigated. RESULTS: Fourteen active ingredients in AB were identified. The network pharmacology-based analysis demonstrated that the active ingredients possibly affected ten key targets, such as IL-6, TNF, and PTGS2. They are also related to the tight junction proteins ZO-1, occludin, and claudin-1. Furthermore, mice treated with DSS developed severe mucosal colitis. AB aqueous enema decreased the disease activity index (DAI), significantly inhibited colonic damage, and greatly decreased colon length shortening (p < 0.05). AB also significantly restored tight junction proteins and the associated mucin proteins mucin-2 (MUC2) and mucin-3A (MUC3A). In addition, the diversity of the gut microbiota after administration of DSS was significantly decreased. However, the diversity was entirely restored after AB treatment. Recovery of the abundance of colonic mucosal bacteria, especially Lactobacillus reuteri and Lactobacillus gasseri, occurred at the species level after AB treatment. In vitro, AB can be utilized by the two bacteria, especially under glucose deficiency. CONCLUSIONS: Our study demonstrated that the AB aqueous enema alleviated colitis by restoring intestinal barrier proteins and regulating the gut microbiota.
Asunto(s)
Anemone/química , Colitis Ulcerosa/tratamiento farmacológico , Medicamentos Herbarios Chinos/farmacología , Microbioma Gastrointestinal/efectos de los fármacos , Animales , Cromatografía Líquida de Alta Presión , Colitis Ulcerosa/patología , Sulfato de Dextran , Modelos Animales de Enfermedad , Medicamentos Herbarios Chinos/administración & dosificación , Enema , Femenino , Inflamación/tratamiento farmacológico , Inflamación/patología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Espectrometría de Masas en Tándem , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
OBJECTIVE: To evaluate the prebiotic effects of Codonopsis pilosula polysaccharide (CPP) on human gut bacteria in vitro. METHODS: Codonopsis Radix was extracted with water at 100°C, and the extract was precipitated by 80% ethanol to obtain CPP. Human fresh fecal samples were collected from three healthy adults and used to ferment CPP. The fermented samples were collected to be analyzed by 16S rRNA sequencing. RESULTS: The results showed that CPP exhibited significantly the stimulation on the growth of genus Bifidobacterium of human gut bacteria (Padj < 0.05). Although CPP also exhibited regulative trends on the genera including Acidaminococcus, Bilophila, Dorea, and Eggerthella, no significant differences were observed (Padj > 0.05), which was likely associated with the limited samples (n = 3). CONCLUSION: CPP has the potential to stimulate the growth of Bifidobacterium of the human gut bacteria and to be benefit to human health.
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To reduce the amount of greenhouse gas emissions and remedy related environmental damage, the research on carbon capture and storage (CCS) is gaining momentum and so is the search for a more effective way to control corrosion of pipeline steel used to transport impure supercritical (SC) CO2. Herein, we prepared an electroless high-phosphorus Ni-P coating and, for the first time, systematically explored the underlying mechanism of the interfacial process in applying Ni-P coating to protect pipeline steel that transports impure SC CO2. It is found that, benefiting from the formation of a protective surface film, Ni-P coating significantly mitigates the corrosion effects from SC CO2 and impurities (e.g., O2 and NO2), especially the synergistic effect of impurities. Concurrently, it effectively avoids the localized corrosion resulting from nonuniform adsorption of the aqueous phase. Although O2 and NO2 can degrade the coating through boosting water precipitation, deteriorating the water chemistry, and reducing the surface film protectiveness, the corrosion inhibition efficiency of Ni-P coating is invariably higher than 80%, independent of the varying causticity of SC CO2 streams, demonstrating that the coating has a superior stability toward corrosion attack. The as-prepared Ni-P coating undoubtedly holds great potential as an alternative for corrosion control of CO2 transport pipeline in the CCS industry. This work provides a new, feasible method to ensure the safe and efficient operation of CCS.
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Bioactive metabolites in Codonopsis pilosula are of particular interest as an immunostimulant. Methyl jasmonate (MeJA) plays an important role in the elicitation of metabolite biosynthesis. Here, we explored the response of metabolites to MeJA elicitation in C. pilosula adventitious roots and multiple shoots. The results showed that the biomass, polysaccharide, and lobetyolin content of adventitious roots exhibited the highest increases with 100 µmol·L-1 MeJA at the 16th day of subculture, whereas the atractylenolide III (a terpenoid) content increased extremely with 50 µmol·L-1 MeJA treatment at the 7th day of subculture. In addition, the biomass and lobetyolin content significantly increased at the 4th day after treatment. Similarly, the polysaccharide and lobetyolin content increased in multiple shoots. Further identification of different metabolites responding to MeJA by ¹H-NMR showed an extremely significant increase of the lobetyolinin level, which coincided with lobetyolin. Accordingly, the precursor, fatty acids, showed a highly significant decrease in their levels. Furthermore, a significant increase in ß-d-fructose-butanol glycoside was detected, which was accompanied by a decrease in the sucrose level. Accordingly, the enzyme genes responsible for terpenoid and carbohydrate biosynthesis, CpUGPase, and CpPMK, were up regulated. In conclusion, MeJA promoted culture growth and accelerated bioactive metabolite accumulation by regulating the expression of the metabolite biosynthesis related genes, CpUGPase and CpPMK in C. pilosula.
Asunto(s)
Acetatos/farmacología , Codonopsis/efectos de los fármacos , Ciclopentanos/farmacología , Regulación de la Expresión Génica de las Plantas , Oxilipinas/farmacología , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas de Plantas/genética , Biomasa , Codonopsis/genética , Codonopsis/crecimiento & desarrollo , Codonopsis/metabolismo , Ácidos Grasos/biosíntesis , Lactonas/metabolismo , Redes y Vías Metabólicas/efectos de los fármacos , Redes y Vías Metabólicas/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Proteínas de Plantas/metabolismo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/genética , Brotes de la Planta/crecimiento & desarrollo , Brotes de la Planta/metabolismo , Poliinos/metabolismo , Sesquiterpenos/metabolismo , UTP-Glucosa-1-Fosfato Uridililtransferasa/genética , UTP-Glucosa-1-Fosfato Uridililtransferasa/metabolismoRESUMEN
OBJECTIVE: To isolate the hypoglycemic bioactive components from leaves of Psidium guajava and evaluate their AMP-activated protein kinase (AMPK) activities. METHODS: A variety of column chromatography was used for the isolation of compounds, and nuclear magnetic resonance (NMR) and mass spectrum (MS) were used for the structure identification of compounds. AMP-activated protein kinase (AMPK) activity of compounds obtained from leaves of Psidium guajava was evaluated in L6 rat myoblast cells and L02 human hepatic cells by western blot. RESULTS: Six principal flavonoids largely present in the leaves of Psidium guajava, quercetin (1), quercetin-3-O-α-L-arabinofuranoside (2), quercetin-3-O-α-L-arabinopyranoside (3), quercetin-3-O-ß-D-galactopyranoside (4), quercetin-3-O-ß-D-glucopyranoside (5), and quercetin-3-O-ß-D-xylopyranoside (6), were obtained and compound 1-6 exhibited significant activity on AMPK activation both in L6 cells and L02 cells (p < 0.01) compared with Control. In particular, the effects of quercetin on AMPK activation were extremely significant compared with Control (p < 0.001). CONCLUSIONS: These findings demonstrated that these flavonoids had potential for the activation of AMPK and hypoglycemic activity.
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Radix Codonopsis, derived from the roots of Codonopsis pilosula (Franch.) Nannf., Codonopsis pilosula (Franch.) Nannf. Var. modesta (Nannf.) L.T. Shen and Codonopsis tangshen Oliv., has been used as traditional Chinese medicine for improving poor gastrointestinal function, treating gastric ulcers and chronic gastritis in China. Inulin-type fructans are carbohydrates consisting mainly of ß (2â1) fructosyl-fructose links in chemical structure and exhibit a range of properties such as prebiotic activity, fat substitutes in low-calorie foods and disease-modifying effects. The prebiotic effects of inulin-type fructans are hypothesized to improve gastrointestinal function through alterations to gut microbiota composition and metabolism. In the present study, three inulin-type fructans with high degree of polymerization (DP = 16, 22, and 31) were isolated from the roots of Codonopsis pilosula (Franch.) Nannf. and their structures were confirmed by MALDI-TOF-MS, 1D- and 2D-NMR. The prebiotic activity of these fructans was evaluated by detecting growth stimulation on Bifidobacterium longum. The results demonstrated that three fructans at a concentration of 2.0 g/L exhibited significant growth stimulation on Bifidobacterium longum in a time-dependent manner (p < 0.01). The data indicated that inulin-type fructans in Radix Codonopsis could be used as potential prebiotics.
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Bifidobacterium longum/efectos de los fármacos , Codonopsis/química , Inulina/farmacología , Raíces de Plantas/química , Prebióticos , Bifidobacterium longum/crecimiento & desarrollo , Espectroscopía de Resonancia Magnética con Carbono-13 , Espectroscopía de Protones por Resonancia Magnética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The present study is to study the chemical constituents from ethanol extract of Psidium guajava leaves. The constituents were separated and purified by silica gel column chromaiographios over, macroporous resin D-101, Sephadex LH-20, and ODS. Six flavonoids compounds were isolated and identified as quercetin(1), quercetin-3-O-α-D-arabinopyranoside(2), quercetin-3-O-α-D-ribopyranoside(3), quercetin-3-O-ß-D-galactopyranoside(4), quercetin-3-O-α-D-glucopyranoside(5), and quercetin-3-O-α-D-xylpyranoside(6). The antioxidant effects of six flavonoids was evaluated by scavenging ability of DPPH, superoxide anion, ABTS·âº, and reducing effect of Fe³âº as well as total antioxidant capacity(FRAP). Vitamin C was used as positive control. The results indicated that six flavonoids exhibited significant antioxidant effects.
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Antioxidantes/química , Flavonoides/química , Psidium/química , Fitoquímicos/análisis , Hojas de la Planta/química , Quercetina/químicaRESUMEN
Radix Codonopsis has been used in traditional Chinese medicine for strengthening the immune system, improving poor gastrointestinal function, treating gastric ulcers and chronic gastritis and so on. In the present study, an inulin-type fructan CP-A was obtained from the roots of Codonopsis pilosula (Franch.) Nannf. and its structure was confirmed by MS and NMR as (2 â 1) linked-ß-d-fructofuranose. The protective effects of CP-A against ethanol-induced acute gastric ulcer in rats were intensively investigated. A Lacy assay demonstrated that CP-A-treated group (50 mg/kg) showed the gastric damage level 1, which was similar to the positive control group, while the model group exhibited the gastric damage level 3. The Guth assay demonstrated that the mucosa ulcer index for CP-A groups at the doses of 50 mg/kg and 25 mg/kg significantly decreased compared with that in the model group (p < 0.05). Meanwhile, CP-A significantly increased the activities of SOD and GSH-Px, and decreased the contents of MDA and NO, and the activity of MPO in gastric tissue in a dose-dependent manner (p < 0.05). The present research reported for the first time that inulin-type fructan CP-A were likely the potential component in Radix Codonopsis for treatment of acute gastric ulcers.
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Codonopsis/química , Fructanos/administración & dosificación , Raíces de Plantas/química , Úlcera Gástrica/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Etanol/efectos adversos , Femenino , Fructanos/química , Fructanos/farmacología , Masculino , Estructura Molecular , Óxido Nítrico/metabolismo , Ratas , Úlcera Gástrica/inducido químicamente , Úlcera Gástrica/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
The vascular endothelium is important in the physiological homeostasis of blood vessels. Increasing evidence demonstrates that oxidative stressinduced endothelial damage is involved in the pathogenesis of several cardiovascular diseases, including atherosclerosis. Hyperoside, one of major active components from Apocynum venetum L. (LuoBuMa), which is a traditional Chinese herbal medicine commonly used for the prevention of cardiovascular diseases, exhibits diverse bioactivities, including antiinflammatory and antioxidant effects. In the present study, the protective effects of hyperoside against hydrogen peroxide (H2O2)induced apoptosis of human umbilical vein endothelial cells (HUVECs) were investigated. The results demonstrated that hyperoside significantly prevented the loss of cell viability, the increase of endothelial Ca2+ content and apoptosis in H2O2induced HUVECs. Additionally, reverse transcription-polymerase chain reaction and western blot analysis revealed that hyperoside significantly decreased the mRNA expression levels of Bcell lymphoma (Bcl)2 associated X protein (Bax), cleaved caspase3 and phosphorylatedp38, while increasing the mRNA expression of Bcl2 in H2O2induced HUVECs. The present findings suggested that hyperoside has protective effects against H2O2induced apoptosis in HUVECs and serves a key role in the prevention of cardiovascular diseases.
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Antioxidantes/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Peróxido de Hidrógeno/farmacología , Quercetina/análogos & derivados , Antioxidantes/química , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Calcio/metabolismo , Caspasa 3/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Quercetina/química , Quercetina/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Oxidative stress plays a critical role in endothelial injury and the pathogenesis of diverse cardiovascular diseases, including atherosclerosis. Isoquercitrin (quercetin-3-glucoside), a flavonoid distributed widely in plants, exhibits many biological activities, including anti-allergic, anti-viral, anti-inflammatory, and anti-oxidative effects. In the present study, the inhibitory effect of isoquercitrin on H2O2-induced apoptosis of EA.hy926 cells was evaluated. MTT assays showed that isoquercitrin significantly inhibited H2O2-induced loss of viability in EA.hy926 cells. Hoechst33342/PI and Annexin V-FITC/PI fluorescent double staining indicated that isoquercitrin inhibited H2O2-induced apoptosis of EA.hy926 cells. Western blotting demonstrated that isoquercitrin prevented H2O2-induced increases in cleaved caspase-9 and cleaved caspase-3 expression, while increasing expression of anti-apoptotic protein Mcl-1. Additionally, isoquercitrin significantly increased the expression of p-Akt and p-GSK3ß in a dose-dependent manner in EA.hy926 cells. LY294002, a PI3K/Akt inhibitor, inhibited isoquercitrin-induced GSK3ß phosphorylation and increase of Mcl-1 expression, which indicated that regulation of isoquercitrin on Mcl-1 expression was likely related to the modulation of Akt activation. These results demonstrated that the anti-apoptotic effect of isoquercitrin on H2O2-induced EA.hy926 cells was likely associated with the regulation of isoquercitrin on Akt/GSK3ß signaling pathway and that isoquercitrin could be used clinically to interfere with the progression of endothelial injury-associated cardiovascular disease.
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Apoptosis/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Quercetina/análogos & derivados , Supervivencia Celular/efectos de los fármacos , Cromonas/farmacología , Células Endoteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta/biosíntesis , Humanos , Peróxido de Hidrógeno/toxicidad , Morfolinas/farmacología , Proteína Oncogénica v-akt/biosíntesis , Fosfatidilinositol 3-Quinasas/biosíntesis , Inhibidores de las Quinasa Fosfoinosítidos-3 , Quercetina/química , Quercetina/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Endothelial injury has been implicated in the pathogenesis of many cardiovascular diseases, including thrombotic disorders. Hyperin (quercetin-3-O-galactoside), a flavonoid compound and major bioactive component of the medicinal herb Apocynum venetum L., is commonly used to prevent endothelium dysfunction. However, its mode of action remains unclear. To the best of our knowledge, we have for the first time investigated the protective effect hyperin exerts against H2O2-induced injury in human endothelium-derived EA.hy926 cells using isobaric tags for relative and absolute quantitation (iTRAQ)based quantitative proteomic analysis. The results showed that H2O2 exposure induced alterations in the expression of 250 proteins in the cells. We noted that the expression of 52 proteins associated with processes such as cell apoptosis, cell cycle and cytoskeleton organization, was restored by hyperin treatment. Of the proteins differentially regulated following H2O2 stress, the anti-apoptotic protein, myeloid cell leukemia-1 (Mcl-1), and the pro-apoptotic protein, BH3-interacting domain death agonist (Bid), exhibited marked changes in expression. Hyperin increased Mcl-1 expression and decreased that of Bid in a dose-dependent manner. In addition, flow cytometric analysis and western blot analysis of the apoptosis-related proteins, truncated BID (tBid), cleaved caspase-3, cleaved caspase-9, Fas, FasL and caspase-8, demonstrated that the rate of apoptosis and the pro-apoptotic protein levels were decreased by hyperin pretreatment. In the present study we demonstrate that hyperin effectively prevents H2O2induced cell injury by regulating the Mcl1 and Bid-mediated antiapoptotic mechanism, suggesting that hyperin is a potential candidate for use in the treatment of thrombotic diseases.
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Apoptosis/efectos de los fármacos , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Endotelio/efectos de los fármacos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Estrés Oxidativo/efectos de los fármacos , Sustancias Protectoras/farmacología , Quercetina/análogos & derivados , Apocynum/química , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Caspasa 9/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular , Endotelio/citología , Endotelio/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Sustancias Protectoras/química , Proteómica , Quercetina/química , Quercetina/farmacologíaRESUMEN
In this paper, the RP-HPLC specific chromatography was adopted, with DIKMA-C18 (4.6 mm x 250 mm, 5 µm) as the chromatographic column, with a gradient elution compose of acetonitrile and 0.1% phosphoric acid at flow rate of 0.8 mL · min(-1), the detection wavelength was 220 nm. The difference of the HPLC specific chromatograms between the Lu Dangshen and other different base sources and different producing area of Codonopsis Radix was compared, involved in the similarities and differences of the number and the relative peak area of characteristic peaks in the HPLC specific chromatograms. The HPLC specific chromatograms of Lu Dangshen was established and the relative retention times of seven peaks was determined, and the peaks of codonopyrrolidium B, syringin, lobetyolin, tangshenoside I and atractylenoide III were identified; The HPLC specific chromatograms of Lu Dangshen provided a method for scientific evaluation and effective control the quality of Lu Dangshen from Shanxi famous-region.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Codonopsis/química , Medicamentos Herbarios Chinos/análisis , Raíces de Plantas/química , Glucósidos/análisis , Fenilpropionatos/análisis , Control de CalidadRESUMEN
Vascular endothelial cells produce reactive oxygen species (ROS) during the process of energy metabolism in aerobic respiration. A growing body of evidence indicates that excessive ROS is implicated in the pathogenesis of cardiovascular diseases including atherosclerosis. The newly synthesized halophenol, 2,4',5'-trihydroxyl-5,2'-dibromo diphenylmethanone (TDD), exhibits antioxidative and cytoprotective activities in vitro. In this study, the protective effect of TDD against hydrogen peroxide (H2O2)-induced oxidative injury of EA.hy926 cells was investigated. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-dephenyltetrazolium bromide (MTT) assay, while the effect of TDD on the transcription profile of EA.hy926 cells subjected to H2O2-induced oxidative injury was evaluated by microarray analysis. Several signaling pathways, including apoptosis, were significantly associated with TDD. Flow cytometric analysis was used to evaluate anti-apoptotic effect of TDD. Subsequently, RT-PCR and Western blot were used to detect the expressions of the apoptosis-associated protein, Bcl-2 and Bax. Meanwhile the expression of cleaved caspase-3, an executioner of apoptosis, was also detected by Western blot. The results showed that pretreatment of EA.hy926 cells with TDD prevented the decrease of cell viability induced by H2O2, and attenuated H2O2-induced elevation of Bax and cleaved caspase-3 while increased Bcl-2 expressions. In summary, TDD inhibited H2O2-induced oxidative injury of EA.hy926 cells through negative regulation of apoptosis. These findings suggest that TDD is a potential candidate for therapeutic intervention in oxidative stress-associated cardiovascular diseases.
Asunto(s)
Benzofenonas/farmacología , Células Endoteliales/patología , Peróxido de Hidrógeno/toxicidad , Estrés Oxidativo/efectos de los fármacos , Fenoles/farmacología , Sustancias Protectoras/farmacología , Apoptosis/efectos de los fármacos , Benzofenonas/química , Western Blotting , Caspasa 3/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenoles/química , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Endothelial tight junction is a crucial intracellular junctional structure that controls paracellular permeability across vascular endothelium. Oxidative stress-mediated elevation in endothelial permeability is associated with pathogenesis of several cardiovascular diseases. In the present research, the regulation of farrerol on occludin, a transmembrane proteins associated with endothelial tight junction, was investigated in hydrogen peroxide-induced human endothelium-derived EA.hy926 cells. Western blot analysis demonstrated that H2O2 exposure caused a significant decrease in occludin expression, but had little effect on ZO-1 expression, and the decrease of occludin expression was significantly attenuated by farrerol in a dose-dependent manner. Meanwhile, immunofluorescent staining assay also demonstrated that the loss of occludin expression induced by H2O2 exposure was restored by farrerol pretreatment. Further investigations showed that farrerol prevented H2O2-induced activation of extracellular signal-regulated kinase (ERK) 1/2 in a dose-dependent manner. The use of U0126, a specific inhibitor of MEK1/2, proved that H2O2-induced decrease of occludin in EA.hy926 cells was likely associated with activation of ERK1/2, which indicated that the regulation of farrerol on occludin expression in H2O2-induced EA.hy926 cells was likely related to the modulation of ERK1/2 activation. In conclusion, the present study demonstrates for the first time that farrerol has potential effects on oxidative stress-induced endothelial tight junction disruption and suggests that farrerol is a potential candidate for the intervention of endothelial permeability-associated cardiovascular diseases.
Asunto(s)
Cromonas/farmacología , Peróxido de Hidrógeno/farmacología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Ocludina/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Permeabilidad/efectos de los fármacos , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismoRESUMEN
Four new limonoids (1-4), together with five known limonoids (5-9), were isolated from the fruits of Melia toosendan. Their structures were elucidated based on extensive spectroscopic analyses (1D- and 2D-NMR, HRESIMS, IR, [α](D)). The isolated compounds were evaluated for their neurite outgrowth-promoting activities. Compounds 2 and 6 significantly enhanced NGF-mediated neurite outgrowth in PC12 cells at concentrations ranging from 0.1 to 50.0 µM.