RESUMO
An efficient method was established by high-speed countercurrent chromatography (HSCCC) for the separation and purification of three flavonoids from Oroxylum indicum. Optimized by single-factor and orthogonal experiments, the optimal extraction conditions were an extraction temperature of 50°C, a solid-to-liquid ratio of 1:50 (g/ml), an ethanol concentration of 75% and an extraction time of 45 min. Using a two-phase solvent system composed of chloroform-methanol-water (6:10:5, v/v/v), the preparative separation was successfully performed by HSCCC in head-to-tail elution mode. Totals of 12.63 mg of oroxin A at a purity of 97.61% with 96.46% recovery, 10.96 mg oroxin B at a purity of 98.32% with 98.81% recovery, and 9.34 mg baicalein at a purity of 98.64% with 97.87% recovery were obtained in one-step separation from 200 mg crude extract. Their chemical structures were confirmed by melting points, HPLC, UV, FTIR, MS, 1 H and 13 C NMR data. Furthermore, they were efficient scavengers of 1,1-diphenyl-2-picrylhydrazyl and hydroxyl free radicals in a concentration-dependent manner.
Assuntos
Distribuição Contracorrente , Flavonoides , Flavonoides/química , Distribuição Contracorrente/métodos , Extratos Vegetais/química , Solventes , Cromatografia Líquida de Alta PressãoRESUMO
A highly sensitive LC-MS/MS method was developed to measure oroxin B in rat plasma and tissue homogenates. The analyte and IS were isolated from biological matrices by a simple protein precipitation followed by centrifugation. Detection was conducted by electrospray negative-ionization mass spectrometry in selected-reaction monitoring mode. The assay was linear in the concentration range 4.52-904 ng/mL with intra- and inter-day precision of <14.41%. It was successfully applied to the pharmacokinetics and tissue distribution studies of oroxin B after an intravenous dose of 1.0 mg/kg in rats. The results would be useful for further development of oroxin B.
Assuntos
Cromatografia Líquida/métodos , Dissacarídeos/análise , Dissacarídeos/farmacocinética , Flavonas/análise , Flavonas/farmacocinética , Espectrometria de Massas em Tandem/métodos , Animais , Dissacarídeos/química , Estabilidade de Medicamentos , Flavonas/química , Modelos Lineares , Masculino , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Distribuição TecidualRESUMO
This study aims to investigate the anticancer effect of Oroxin B (OB) both in vitro and in vivo, and the molecular mechanism involved in microRNA-221 and the PI3K/Akt/PTEN pathway through modulation of apoptosis in Hepatocellular carcinoma (HCC). DEN-induced rats and HepG2 cells based on the microfluidic chip were employed, while the mRNA and protein expression of microRNA-221, PI3K, p-Akt and PTEN were evaluated by RT-PCR and Western blot analysis. Based on Microfluidic Chip and DEN-induced rat model, OB effectively exerts anti-liver cancer effect both in vitro and in vivo, and the expression of miR-221 in OB treated groups was significantly lower than that in the control group (** p < 0.01). The RT-PCR and Western blot results suggested the PI3K mRNA and protein in OB treated groups were both lower than those in control group and indicated the overexpression of PTEN. Therefore, OB effectively exerts anticancer effects by positively regulating the PTEN gene and then inactivating the PI3K/Akt signaling pathway through down-regulating the expression of the microRNA-221, thereby inducing apoptosis of liver cancer cells. This study offers a theoretical evidence for further development and clinical guidance of OB as an anti-tumor agent.
Assuntos
Apoptose/efeitos dos fármacos , Dissacarídeos/farmacologia , Flavonas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , MicroRNAs/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Biomarcadores , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Histocitoquímica , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , RatosRESUMO
Cancer cells have both tumor-adaptive and -suppressive endoplasmic reticulum (ER) stress machineries that determine cell fate. In malignant tumors including lymphoma, constant activation of tumor-adaptive ER stress and concurrent reduction of tumor-suppressive ER stress favors cancer cell proliferation and tumor growth. Current ER stress-based anti-tumor drugs typically activate both tumor-adaptive and -suppressive ER stresses, resulting in low anti-cancer efficacy; hence, selective induction of tumor-suppressive ER stress and inhibition of tumor-adaptive ER stress are new strategies for novel anti-cancer drug discovery. Thus far, specific tumor-suppressive ER stress therapeutics have remained absent in clinical settings. In this study, we explored unique tumor-suppressive ER stress agents from the traditional Chinese medicinal herb Oroxylum indicum, and found that a small molecule oroxin B selectively induced tumor-suppressive ER stress in malignant lymphoma cells, but not in normal cells, effectively inhibited lymphoma growth in vivo, and significantly prolonged overall survival of lymphoma-xenografted mice without obvious toxicity. Mechanistic studies have revealed that the expression of key tumor-adaptive ER-stress gene GRP78 was notably suppressed by oroxin B via down-regulation of up-stream key signaling protein ATF6, while tumor-suppressive ER stress master gene DDIT3 was strikingly activated through activating the MKK3-p38 signaling pathway, correcting the imbalance between tumor-suppressive DDIT3 and tumor-adaptive GRP78 in lymphoma. Together, selective induction of unique tumor-suppressive ER stress and concurrent inhibition of tumor-adaptive ER stress in malignant lymphoma are new and feasible approaches for novel anti-lymphoma drug discovery and anti-lymphoma therapy.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Dissacarídeos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Flavonas/farmacologia , Linfoma de Células B/tratamento farmacológico , Fator 6 Ativador da Transcrição/genética , Fator 6 Ativador da Transcrição/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Chaperona BiP do Retículo Endoplasmático , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , MAP Quinase Quinase 3/metabolismo , Camundongos , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Metabolic-associated fatty liver disease (MAFLD) has become a common chronic liver disease, but there is no FDA-approved drug for MAFLD treatment. Numerous studies have revealed that gut microbiota dysbiosis exerts a crucial effect on MAFLD progression. Oroxin B is a constituent of the traditional Chinese medicine Oroxylum indicum (L.) Kurz. (O. indicum), which has the characteristics of low oral bioavailability but high bioactivity. However, the mechanism through which oroxin B improves MAFLD by restoring the gut microbiota balance remains unclear. To this end, we assessed the anti-MAFLD effect of oroxin B in HFD-fed rats and investigated the underlying mechanism. Our results indicated that oroxin B administration reduced the lipid levels in the plasma and liver and lowered the lipopolysaccharide (LPS), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) levels in the plasma. Moreover, oroxin B alleviated hepatic inflammation and fibrosis. Mechanistically, oroxin B modulated the gut microbiota structure in HFD-fed rats by increasing the levels of Lactobacillus, Staphylococcus, and Eubacterium and decreasing the levels of Tomitella, Bilophila, Acetanaerobacterium, and Faecalibaculum. Furthermore, oroxin B not only suppressed Toll-like receptor 4-inhibitor kappa B-nuclear factor kappa-B-interleukin 6/tumor necrosis factor-α (TLR4-IκB-NF-κB-IL-6/TNF-α) signal transduction but also strengthened the intestinal barrier by elevating the expression of zonula occludens 1 (ZO-1) and zonula occludens 2 (ZO-2). In summary, these results demonstrate that oroxin B could alleviate hepatic inflammation and MAFLD progression by regulating the gut microbiota balance and strengthening the intestinal barrier. Hence, our study suggests that oroxin B is a promising effective compound for MAFLD treatment.
Assuntos
Microbioma Gastrointestinal , Hepatopatia Gordurosa não Alcoólica , Ratos , Animais , Camundongos , Fator de Necrose Tumoral alfa/metabolismo , Interleucina-6/metabolismo , Dieta Hiperlipídica/efeitos adversos , Disbiose/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fígado , NF-kappa B/metabolismo , Inflamação/tratamento farmacológico , Camundongos Endogâmicos C57BLRESUMO
Background: Osteoarthritis (OA) is a common aging-related degenerative joint disease with chronic inflammation as its possible pathogenesis. Oroxin B (OB), a flavonoid isolated from traditional Chinese herbal medicine, possesses anti-inflammation properties which may be involved in regulating the pathogenesis of OA, but its mechanism has not been elucidated. Our study was the first to explore the potential chondroprotective effect and elucidate the underlying mechanism of OB in OA. Methods: In vitro, primary mice chondrocytes were stimulated with IL-1ß along with or without the administration of OB or autophagy inhibitor 3-methyladenine (3-MA). Cell viability assay was measured with a cell counting kit-8 (CCK-8). The phenotypes of anabolic-related (Aggrecan and Collagen II), catabolic-related (MMP3, MMP13, and ADAMTS5), inflammation-related (iNOS, COX-2, TNF-α, IL-6, and IL-1ß), and markers of related signaling pathways in chondrocytes with different treatment were detected through western blot, RT-qPCR, and immunofluorescent staining. In vivo, the destabilized medial meniscus (DMM) operation was performed to establish the OA mice model. After knee intra-articular injection with OB for 8 weeks, the mice's knee joints were obtained for subsequent histological staining and analysis. Results: OB reversed the expression level of anabolic-related proteins (Aggrecan and Collagen II) and catabolic-related (MMP3, MMP13, and ADAMTS5) in IL-1ß-induced chondrocytes. Mechanistically, OB suppressed the inflammatory response stimulated by IL-1ß, as the inflammation-related (iNOS, COX-2, TNF-α, IL-6, and IL-1ß) markers were downregulated after the administration of OB in IL-1ß-induced chondrocytes. Besides, the activation of PI3K/AKT/mTOR signaling pathway induced by IL-1ß could be inhibited by OB. Additionally, the autophagy process impaired by IL-1ß could be rescued by OB. What's more, the introduction of 3-MA to specifically inhibit the autophagic process impairs the protective effect of OB on cartilage. In vivo, histological staining revealed that intra-articular injection of OB attenuated the cartilage degradation, as well as reversed the expression level of anabolic and catabolic-related proteins such as Aggrecan, Collagen II, and MMP13 induced in DMM-induced OA models. Conclusions: The study verified that OB exhibited the chondroprotective effect by anti-inflammatory, inhibiting the PI3K/AKT/mTOR signaling pathway, and enhancing the autophagy process, indicating that OB might be a promising agent for the treatment of OA.
Assuntos
Osteoartrite , Fosfatidilinositol 3-Quinases , Camundongos , Animais , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/farmacologia , Metaloproteinase 3 da Matriz/uso terapêutico , Agrecanas/metabolismo , Agrecanas/farmacologia , Agrecanas/uso terapêutico , Ciclo-Oxigenase 2/metabolismo , Ciclo-Oxigenase 2/farmacologia , Ciclo-Oxigenase 2/uso terapêutico , Osteoartrite/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , NF-kappa B/metabolismo , Interleucina-6 , Condrócitos , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Anti-Inflamatórios/uso terapêutico , Autofagia/fisiologia , Colágeno/metabolismoRESUMO
BACKGROUND: Osteoclasts are the major players in bone resorption and have always been studied in the prevention and treatment of osteoporosis. Previous studies have confirmed that a variety of flavonoids inhibit osteoporosis and improve bone health mainly through inhibiting osteoclastogenesis. Oroxin B (OB) is a flavonoid compound extracted from traditional Chinese herbal medicine Oroxylum indicum (L.) Vent, exerts potent antitumor and anti-inflammation effect, but its effect on osteoclastogensis remains unknown. METHODS: We comprehensively evaluated the effect of OB on the formation and function of osteoclasts and the underling mechanism by bone marrow-derived macrophage in vitro. In vivo, we used mice ovariectomized model to verify the protective effect of OB. RESULTS: OB was found to inhibit osteoclast formation and bone resorption function in vitro, in a dose-dependent manner and the increased osteoclastic-related genes induced by RANKL (NFATc1, c-fos, cathepsin K, RANK, MMP9 and TRAP) were also attenuated following OB treatment. Mechanistical investigation showed OB abrogated the increased phosphorylation level of MAPK and NF-κB pathway, and diminished the expression of the vital transcription factors for osteoclastogenesis. OB also prevented ovariectomy (OVX)-induced bone loss by inhibiting osteoclast formation and activity in mice. CONCLUSION: Our study demonstrated that OB may act as an anti-osteoporosis agent by inhibiting osteoclast maturation and attenuating bone resorption.
Assuntos
Reabsorção Óssea/tratamento farmacológico , Dissacarídeos/farmacologia , Flavonas/farmacologia , Osteoclastos/efeitos dos fármacos , Ovariectomia , Animais , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacosRESUMO
Oroxin B, a flavonoid, is a major bioactive component form Oroxylum indicum (L.) Vent. with enormous anti-hepatoma effects. To data, the oroxin B metabolism studies remain underexplored. This study was designed to characterize oroxin B metabolism in vivo and in vitro by ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS). Consequently, 30 metabolites in rats, 8 metabolites in liver microsomes and 18 metabolites in intestinal bacteria were identified, and 9 metabolites were recognized by comparison with standards. The biotransformation processes involved ketone, acetylation, loss of C12H20O10, and loss of C6H10O5. And baicalein and oroxin A were generated after loss of C12H20O10, and loss of C6H10O5, respectively, and further went through some other reactions, such as oxidation, methylation, internal hydrolysis, hydrogenation, loss of O, ketone, glycine conjugation, glucuronide conjugation and their composite reactions. The results provide valuable evidence for elucidation the potential mechanism of oroxin B pharmacological action, and offer reasonable guidelines for further investigations of oroxin B safety and efficacy.
Assuntos
Flavonoides , Espectrometria de Massas em Tandem , Animais , Cromatografia Líquida de Alta Pressão , Dissacarídeos , Flavonas , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Oroxin B (OB) is one of flavonoids isolated from traditional Chinese herbal medicine Oroxylum indicum (L.) Vent. Recent studies suggest that flavonoids have obvious anti-liver tumors effect, but the precise molecular mechanism is still unclear. OBJECTIVE: The current study was performed to investigate the antitumor effects of OB on human hepatoma cell line SMMC-772 and explore the part of molecular mechanisms in this process. MATERIALS AND METHODS: MTT method, terminal deoxynucleotidyl transferase dUTP nick end labeling assay and flow cytometry were utilized to detect the inhibition of proliferation and the apoptosis after treating OB in of SMMC-7721 cells. The mRNA and proteins expressions of COX-2, vascular endothelial growth factor (VEGF), phosphatidylinositol-3-kinase (PI3K), p-AKT, and PTEN were measured by a real-time polymerase chain reaction and Western Blot method. RESULTS: The results showed that OB inhibited proliferation of SMMC-7721 cell in a dose-dependent manner, and induced its apoptosis. Moreover, OB unregulated PTEN and downregulated COX-2, VEGF, p-AKT, and PI3K. CONCLUSION: Our results demonstrated that OB significantly inhibits proliferation and induce apoptosis, which may be strongly associated with the inhibiting COX-2/VEGF and PTEN/PI3K/AKT pathway signaling pathway in SMMC-7721 cells, OB potentially be used as a novel therapeutic agent for liver cancer. SUMMARY: OB (Oroxin B) is one of the effective flavonoid components of traditional Chinese medicine O. indicum (L.)OB can inhibite the proliferation and promoted apoptosis of the human hepatoma cell line SMMC 7721OB plays a role of antitumor effect may to regulate COX 2/VEGF and PTEN/PI3K/AKT pathways directly or indirectly. Abbreviations used: OB: Oroxin B; MTT: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; COX-2: cyclooxygenase-2; PI3K: phosphatidylinositol 3 kinase; PTEN: Phosphatase and tensin homolog deleted on chromosome ten; VEGF: Vascular endothelial growth factor; RT-PCR: Reverse transcription polymerase chain reaction; DAPI: Diamidino 2 phenylindole; PBS: Phosphate buffer saline; FITC: Fluorescein isothiocyanate; PI: Propidium Iodide; RIPA: Radio immunoprecipitation assay lysis buffer; PMSF: Phenylmethanesulfonyl fluoride; PAGE: Polyacrylamide gel electrophoresis.