The use of a microwave-assisted extraction (MAE) method for the extraction ofphlorotannins from Saccharinajaponica Aresch (S.japonica) has been evaluated with particular emphasis on the influential parameters, inc...The use of a microwave-assisted extraction (MAE) method for the extraction ofphlorotannins from Saccharinajaponica Aresch (S.japonica) has been evaluated with particular emphasis on the influential parameters, including the ethanol concentration, solid/liquid ratio, extraction time, extraction temperature, and microwave power. The MAE procedure was optimized using single-factor design and orthogonal array design (OAD). The content of total phlorotannins in S. japonica was determined using a Folin-Ciocalteu (FC) assay. A maximum total phlorotannin content of 0.644 mg of phloroglucinol equivalent per gram of dry weight plant (mg PGE/g DW) was obtained using the optimized model, which included an ethanol concentration of 55%, solid/liquid ratio of 1:8, extraction time of 25 min, irradiation power of 400 W, and temperature of 60~C. Under similar conditions, the application of a conventional extraction method led to a lower phlorotarmin yield of 0.585 mg PGE/g WD. These results demonstrated that the MAE approach provided better results for the extraction ofphlorotarmins from S.japonica and was a promising technique for the extraction of phenolic compounds from S. japonica and other materials. In addition, screening tests for the inhibitory activity showed that the phlorotannin-containing extracts significantly inhibited the growth of human hepatocellular carcinoma cells (HepG2) by inducing their apoptosis. The morphological changes that occurred during cell apoptosis were characterized using Hoechst33258 staining.展开更多
AIM: To investigate the effects of Chrysanthemum indicum extract (CIE) on inhibition of proliferation and on apoptosis, and the underlying mechanisms, in a human hepatocellular carcinoma (HCC) MHCC97H cell line. ...AIM: To investigate the effects of Chrysanthemum indicum extract (CIE) on inhibition of proliferation and on apoptosis, and the underlying mechanisms, in a human hepatocellular carcinoma (HCC) MHCC97H cell line. METHODS: Viable rat hepatocytes and human endothelial ECV304 cells were examined by trypan blue exclusion and MTT assay, respectively, as normal controls. The proliferation of MHCC97H cells was determined by MTT assay. The cellular morphology of MHCC97H cells was observed by phase contrast microscopy. Flow cytometry was performed to analyze cell apoptosis with annexin V/propidium iodide (PI), mitochondrial membrane potential with rhodamine 123 and cell cycle with PI in MHCC97H cells. Apoptotic proteins such as cytochrome C, caspase-9, caspase-3 and cell cycle proteins, including P21 and CDK4, were measured by Western blotting. RESULTS: CIE inhibited proliferation of MHCC97H cells in a timeand dose-dependent manner without cytotoxicity in rat hepatocytes and human endothelial ceils. CIE induced apoptosis of MHCC97H cells in a concentration-dependent manner, as determined by flow cytometry. The apoptosis was accompanied by a decrease in mitochondrial membrane potential, release of cytochrome C and activation of caspase-9 and caspase-3. CIE arrested the cell cycle in the S phase by increasing P21 and decreasing CDK4 protein expression. CONCLUSION: CIE exerted a significant apoptotic effect through a mitochondrial pathway and arrested the cell cycle by regulation of cell cycle-related proteins in MHCC97H cells without an effect on normal cells. The cancer-specific selectivity shown in this study suggests that the plant extract could be a promising novel treatment for human cancer.展开更多
Objective: Porcine liver extract has been shown to be effective in the clinical treatment of severe hepatitis. The aim of the present study was to study its antifibrotic as well as immune regulatory effect in vitro. ...Objective: Porcine liver extract has been shown to be effective in the clinical treatment of severe hepatitis. The aim of the present study was to study its antifibrotic as well as immune regulatory effect in vitro. Methods: Hepat'ocytes, hepatic stellate cells (HSCs), hepatoma cell line (HepG2) and human peripheral blood mononuclear cells (PMNCs) were studied with respect to proliferation, extracellular matrix production and apoptotic activities by proliferation assay, radioimmunoassay, gene transfection, reporter gene analysis and flow cytometry, respectively. Results: A strong stimulatory proliferation effect was observed in hepatocytes, and an inhibitory effect was found in HSCs. Hyaluronic acid (HA) production and reporter gene activities driven by various etl(I) procollagen gene promoters in HSC-T6 were significantly decreased after treatment with the extract. Fluo-Anexin V binding apoptotic HepG2 cells were more prominent in the presence of 60ug/ml extract. More CD4^+/CD69^+ positive T lymphocytes existed in the presence of the extract. Conclusion: Porcine liver extract is effective for antifibrogenesis via hepatocyte regeneration, HSC and hepatoma cell inhibition in vitro. The elevation of active T lymphocytes is helpful for immune surveillance. Fine mapping of the extract is necessary in order to get definite molecules which are essential in all described functions.展开更多
OBJECTIVE: To investigate the apoptotic effects and underlying molecular mechanisms of Celastrus orbiculatus (C. orbiculatus) extract in human hepa- tocellular carcinoma cells. METHODS: Human hepatocellular carcin...OBJECTIVE: To investigate the apoptotic effects and underlying molecular mechanisms of Celastrus orbiculatus (C. orbiculatus) extract in human hepa- tocellular carcinoma cells. METHODS: Human hepatocellular carcinoma cells (HCCLM6) were treated with C. orbiculatus extract (COE) at different nontoxic concentrations (10, 20, 40, 80, and 160 IJg/mL). The effect of COE on HC-CLM6 viability was examined using 3-(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. Cellular apoptosis following COE treatment was assessed by flow cytometry and western blot analysis. RESULTS: COE significantly inhibited cell viability and induced apoptosis of HCCLM6 cells in a dose-dependent manner. Apoptosis was accompa- nied by increased Bax expression and decreased Bcl-2 expression. In addition, COE treatment led to the release of cytochrome c, activation of cas- pase-3, and cleavage of poly (ADP-ribose) poly- merase (PARP). Furthermore, activation of extracel- lular signal-regulated kinase (ERK), p38 kinase, and c-Jun N-terminal kinase (JNK) phosphorylation, and down-regulation of Akt phosphorylation was ob- served. CONCLUSION: COE induces mitochondrial-mediat- ed, caspase-dependent apoptosis in HCCLM6 cells, which might be attributed to the activation of mito- gen-activated protein kinase (MAPK) and inhibition of Akt signaling pathways. These data suggest that COE may be a potential treatment for human hepa- tocellular carcinoma.展开更多
We present a more accurate method for the quantification of superoxide anion (02-) and hydrogen peroxide (H202) simulta- neously in human HepG2 cell extracts. After the xanthine/xanthine oxidase system was added i...We present a more accurate method for the quantification of superoxide anion (02-) and hydrogen peroxide (H202) simulta- neously in human HepG2 cell extracts. After the xanthine/xanthine oxidase system was added into cell extract which was devoid of O2*- and H202, steady-state and in-situ produced O2*- and H202 by xanthine/xanthine oxidase system was labeled by fluorescent probes and subsequently separated by microchip electrophoresis. Based on this method, two differential equations with the calibration coefficients were established for O2 and H202, respectively. Using the established dual-calibration coefficients, we obtained the calibrated concentrations of 02* and H202 that produced in human HepG2 cells, which were lower (0.66±0.03 and 0.82±0.04 lamol/L for 02*-and H202, respectively) than that (0.85±0.03 and 0.96±0.03 gmol/L for O. and H202, respectively) obtained from statutory working curve. The proposed dual-calibration coefficient protocol takes into account both the complex matrix effect of the biological system and real time decaying of O2 *- and H202, providing a method with higher accuracy.展开更多
基金Supported by the National Natural Science Foundation of China(No.51203028)Rural Science and Technology Correspondent and Technology Service System Construction Projects,Guangdong Province(No.2010A020507001-91)Science and Technology Project of Panyu District,Guangzhou City(No.2010-12-08)
文摘The use of a microwave-assisted extraction (MAE) method for the extraction ofphlorotannins from Saccharinajaponica Aresch (S.japonica) has been evaluated with particular emphasis on the influential parameters, including the ethanol concentration, solid/liquid ratio, extraction time, extraction temperature, and microwave power. The MAE procedure was optimized using single-factor design and orthogonal array design (OAD). The content of total phlorotannins in S. japonica was determined using a Folin-Ciocalteu (FC) assay. A maximum total phlorotannin content of 0.644 mg of phloroglucinol equivalent per gram of dry weight plant (mg PGE/g DW) was obtained using the optimized model, which included an ethanol concentration of 55%, solid/liquid ratio of 1:8, extraction time of 25 min, irradiation power of 400 W, and temperature of 60~C. Under similar conditions, the application of a conventional extraction method led to a lower phlorotarmin yield of 0.585 mg PGE/g WD. These results demonstrated that the MAE approach provided better results for the extraction ofphlorotarmins from S.japonica and was a promising technique for the extraction of phenolic compounds from S. japonica and other materials. In addition, screening tests for the inhibitory activity showed that the phlorotannin-containing extracts significantly inhibited the growth of human hepatocellular carcinoma cells (HepG2) by inducing their apoptosis. The morphological changes that occurred during cell apoptosis were characterized using Hoechst33258 staining.
基金Supported by Grants From the National Natural Science Foundation of China,No.30672766Science and Technology Developing Foundation of Shaanxi Province,China,No.2006K16-G4(1)
文摘AIM: To investigate the effects of Chrysanthemum indicum extract (CIE) on inhibition of proliferation and on apoptosis, and the underlying mechanisms, in a human hepatocellular carcinoma (HCC) MHCC97H cell line. METHODS: Viable rat hepatocytes and human endothelial ECV304 cells were examined by trypan blue exclusion and MTT assay, respectively, as normal controls. The proliferation of MHCC97H cells was determined by MTT assay. The cellular morphology of MHCC97H cells was observed by phase contrast microscopy. Flow cytometry was performed to analyze cell apoptosis with annexin V/propidium iodide (PI), mitochondrial membrane potential with rhodamine 123 and cell cycle with PI in MHCC97H cells. Apoptotic proteins such as cytochrome C, caspase-9, caspase-3 and cell cycle proteins, including P21 and CDK4, were measured by Western blotting. RESULTS: CIE inhibited proliferation of MHCC97H cells in a timeand dose-dependent manner without cytotoxicity in rat hepatocytes and human endothelial ceils. CIE induced apoptosis of MHCC97H cells in a concentration-dependent manner, as determined by flow cytometry. The apoptosis was accompanied by a decrease in mitochondrial membrane potential, release of cytochrome C and activation of caspase-9 and caspase-3. CIE arrested the cell cycle in the S phase by increasing P21 and decreasing CDK4 protein expression. CONCLUSION: CIE exerted a significant apoptotic effect through a mitochondrial pathway and arrested the cell cycle by regulation of cell cycle-related proteins in MHCC97H cells without an effect on normal cells. The cancer-specific selectivity shown in this study suggests that the plant extract could be a promising novel treatment for human cancer.
基金Supported by the National Natural Science Foundation of China (30270605)
文摘Objective: Porcine liver extract has been shown to be effective in the clinical treatment of severe hepatitis. The aim of the present study was to study its antifibrotic as well as immune regulatory effect in vitro. Methods: Hepat'ocytes, hepatic stellate cells (HSCs), hepatoma cell line (HepG2) and human peripheral blood mononuclear cells (PMNCs) were studied with respect to proliferation, extracellular matrix production and apoptotic activities by proliferation assay, radioimmunoassay, gene transfection, reporter gene analysis and flow cytometry, respectively. Results: A strong stimulatory proliferation effect was observed in hepatocytes, and an inhibitory effect was found in HSCs. Hyaluronic acid (HA) production and reporter gene activities driven by various etl(I) procollagen gene promoters in HSC-T6 were significantly decreased after treatment with the extract. Fluo-Anexin V binding apoptotic HepG2 cells were more prominent in the presence of 60ug/ml extract. More CD4^+/CD69^+ positive T lymphocytes existed in the presence of the extract. Conclusion: Porcine liver extract is effective for antifibrogenesis via hepatocyte regeneration, HSC and hepatoma cell inhibition in vitro. The elevation of active T lymphocytes is helpful for immune surveillance. Fine mapping of the extract is necessary in order to get definite molecules which are essential in all described functions.
基金Supported by the National Natural Science Foundation of China(No.81173603 and No.81274141)State Administration of Traditional Chinese Medicine of China(No.0405ZP35)+3 种基金Jiangsu Provincial Social Development Project(No. BE2011738)the Natural Science Foundation of Jiangsu Province(No.BK2012686)Administration of Traditional Chinese Medicine of Jiangsu Province(No.LZ11210)and the Project of Cooperation between Yangzhou University and Yangzhou City(No.YZ2010157)
文摘OBJECTIVE: To investigate the apoptotic effects and underlying molecular mechanisms of Celastrus orbiculatus (C. orbiculatus) extract in human hepa- tocellular carcinoma cells. METHODS: Human hepatocellular carcinoma cells (HCCLM6) were treated with C. orbiculatus extract (COE) at different nontoxic concentrations (10, 20, 40, 80, and 160 IJg/mL). The effect of COE on HC-CLM6 viability was examined using 3-(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. Cellular apoptosis following COE treatment was assessed by flow cytometry and western blot analysis. RESULTS: COE significantly inhibited cell viability and induced apoptosis of HCCLM6 cells in a dose-dependent manner. Apoptosis was accompa- nied by increased Bax expression and decreased Bcl-2 expression. In addition, COE treatment led to the release of cytochrome c, activation of cas- pase-3, and cleavage of poly (ADP-ribose) poly- merase (PARP). Furthermore, activation of extracel- lular signal-regulated kinase (ERK), p38 kinase, and c-Jun N-terminal kinase (JNK) phosphorylation, and down-regulation of Akt phosphorylation was ob- served. CONCLUSION: COE induces mitochondrial-mediat- ed, caspase-dependent apoptosis in HCCLM6 cells, which might be attributed to the activation of mito- gen-activated protein kinase (MAPK) and inhibition of Akt signaling pathways. These data suggest that COE may be a potential treatment for human hepa- tocellular carcinoma.
基金supported by the National Basic Research Program of China(2013CB933800)the National Natural Science Foundation of China(21227005,21390411,91313302,21205074)
文摘We present a more accurate method for the quantification of superoxide anion (02-) and hydrogen peroxide (H202) simulta- neously in human HepG2 cell extracts. After the xanthine/xanthine oxidase system was added into cell extract which was devoid of O2*- and H202, steady-state and in-situ produced O2*- and H202 by xanthine/xanthine oxidase system was labeled by fluorescent probes and subsequently separated by microchip electrophoresis. Based on this method, two differential equations with the calibration coefficients were established for O2 and H202, respectively. Using the established dual-calibration coefficients, we obtained the calibrated concentrations of 02* and H202 that produced in human HepG2 cells, which were lower (0.66±0.03 and 0.82±0.04 lamol/L for 02*-and H202, respectively) than that (0.85±0.03 and 0.96±0.03 gmol/L for O. and H202, respectively) obtained from statutory working curve. The proposed dual-calibration coefficient protocol takes into account both the complex matrix effect of the biological system and real time decaying of O2 *- and H202, providing a method with higher accuracy.