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Objective:To establish the fingerprints of Microctis Folium by ultra high performance liquid chromatography (UPLC); To determine the contents of three flavonoids in the Microctis Folium; To evaluate the quality difference of Microctis Folium from different producing areas. Methods:The fingerprints were performed on Agilent ZORBAX SB C18 column (2.1 mm×150 mm,1.8 μm). The mobile phase was acetonitrile - 0.1 % acetic acid solution with gradient elution at a flow rate of 0.30 ml/min. The column temperature was 30 ℃ and the detection wavelength was 315 nm. The common fingerprint peaks were identified by UPLC-mass spectrometry, and the identification results were confirmed by comparison of reference materials. Waters Cortecs T3 C18 chromatographic column (2.1 mm × 100 mm,1.6 μm) was used for content determination. The mobile phase was methanol-0.1 % formic acid solution with gradient elution at a flow rate of 0.35 ml/min. The column temperature was 30 ℃ and the detection wavelength was 339 nm. The contents of vitexin, isovitexin and narcissoside in 15 batches of Microctis Folium from different habitats were determine. Results:There were 11 common peaks in the fingerprint of Microctis Folium. Identified by mass spectrometry and confirmed by reference substance,10 chemical components were identified, including caffeic acid, p-hydroxycinnamic acid, ferulic acid, vitexin, isovitexin, kaempferol-3-O-rutoside, astragaloside, narcissoside, isorhamnetin-3-O-glucoside and linden glycoside. The similarity between the fingerprints of 15 batches of Microctis Folium and the control fingerprint was greater than 0.95, indicating that the overall similarity of the fingerprints of Microctis Folium from different producing areas was high. The total contents of three active components were 3.23-5.64 mg/g in Yangjiang City, Guangdong, 3.60-5.78 mg/g in Zhanjiang City, Guangdong, 4.68-5.73 mg/g in Yulin City, Guangxi and 3.87-5.21 mg/g in Wuzhishan City, Hainan . There was no significant difference in the content of three active components in different producing areas. Conclusion:The fingerprints and the determination method established in the study are stable and feasible, which can be used for the quality evaluation of Microctis Folium.
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Objective:To establish the quality evaluation method of Perillae caulis formula granules based on the three kind of quality indexes of standard decoction. Methods:Eighteen batches of Perillae caulis were collected from different habitats according to different technical requirements, eighteen batches of standard decoction and three batches of formula granules were prepared and the paste-forming rates were calculated. The content of Caffeic acid and Rosmarinic acid were determined and calculated by Ultra High Performance Liquid Chromatography (UPLC). Then the fingerprints of standard decoction of and formula granules of Perillae caulis were established by UPLC . The similarity values of fingerprints between formula granules and standard decoction were calculated. Results:The average paste-forming rate of standard decoction was (7.16±1.97)%. The paste-forming rates of three batches of formula granules were 5.52%, 5.25% and 5.34%, respectively. The average content of Caffeic acid and Rosmarinic acid in standard decoction was (12.06±3.37)mg/g. The contents of three batches of formula granules were 5.52, 5.82, 5.77 mg/g, respectively. Seven common fingerprint peaks were identified in the fingerprints of standard decoction and formula granules, three of which were identified as Caffeic acid, N-Feruloyl Octopus amine and Rosmarinic acid by comparison of reference substance. The fingerprints similarity of Perillae caulis dispensing granules and standard decoction were 1.000, 0.995 and 0.997, respectively. Conclusions:The quality indexes of three batches of formulation granules are consistent with standard decoction. This method can provide basis for the establishment of quality standard of Perillae caulis dispensing granules.
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OBJECTIVE: To investigate the effects of combined exposure to di(2-ethylhexyl) phthalate(DEHP) and bisphenol A(BPA) on glucose metabolism in female rats during gestational and lactation periods, and its possible mechanism. METHODS: Twenty-four specific pathogen free pregnant SD rats were randomly divided into control group, DEHP group, BPA group, and combined exposure group, with 6 rats in each group. From the 5 th day of gestation to the 21 st day after birth of the offspring, the rats in the DEHP group were treated with DEHP 600 mg/kg body weight(bw); rats in BPA group were treated with 80 mg/kg bw BPA, and rats in combined exposure group were treated with 600 mg/kg bw DEHP and 80 mg/kg bw BPA by intragastric perfusion, while the rats in the control group were given the same amount of corn oil, once per day. After exposure, maternal rats were sacrificed immediately. The levels of glucose metabolism related indicators in liver tissues and serum were examined, and the mRNA and protein expression of phosphatidylinositol 3-kinase(PI3 K)/protein kinase B(AKT) signaling pathway related factors in liver tissues were detected by real-time fluorescence quantitative polymerase chain reaction and Western blot. RESULTS: Except for the activity of phosphoenolpyruvate carboxykinase(PEPCK) in BPA group, the levels of liver glycogen and serum high density lipoprotein cholesterol(HDL-C) in rats of the 3 exposure groups decreased(P<0.05), while the activity of serum PEPCK and the level of low density lipoprotein cholesterol(LDL-C) increased(P<0.05) compared with rats in the control group. The levels of liver glycogen and serum HDL-C in the combined exposure group were lower than that in the BPA group(P<0.05), while the level of serum LDL-C were lower than that in DEHP group and BPA group(P<0.05). The levels of serum glycosylated serum protein, total cholesterol and triglyceride in the 4 groups were not statistically different when compared with each other(P>0.05). Except for the PI3 K protein in DEHP group, the mRNA and protein expression of PI3 K, AKT, and glucose transporter 4 in liver tissues of rats in the 3 exposure groups decreased(P<0.05), and the mRNA expression of forkhead box protein 1(Foxo1) decreased(P<0.05), but the protein expression of FOXO1 increased(P<0.05) compared with the control group. CONCLUSION: Exposure to DEHP or BPA during pregnancy and lactation can cause glucose metabolism disorders in rats. The combined exposure of DEHP and BPA has certain synergistic effect. This process may be achieved through the PI3 K/AKT signaling pathway.
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OBJECTIVE: To investigate the effect of bisphenol A exposure on the expression of DNA methyltransferase(Dnmt) gene in mouse Leydig cell line TM3 cells. METHODS: TM3 cells were randomly divided into 0, 20, 50, 125 and 300 μmol/L dose group(0 μmol/L dose group was the control group). Cells were treated with various concentration of bisphenol A solution for 24 hours.The viability of TM3 cells was determined by CCK-8 method, and the mRNA expression of Dnmt1, Dnmt3 a and Dnmt3 b was detected by real-time fluorescence quantitative polymerase chain reaction. RESULTS:The viability of TM3 cells decreased with the increasing doses of bisphenol A(P<0.01). The relative mRNA expression of Dnmt1 in TM3 cells decreased with the increase of doses of bisphenol A(P<0.01).The relative mRNA expression of Dnmt3 b in TM3 cells in the 20, 50, 125, 300 μmol/L dose group was lower than that in the control group(P<0.05).There was no statistical significant difference in the relative mRNA expression of Dnmt3 a in TM3 cells in these 5 groups(P>0.05). CONCLUSION: Bisphenol exposure is cytotoxic to TM3 cells. This toxic effect may be related to changes in Dnmt mRNA expression.
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OBJECTIVE: To investigate the mechanism of calcium ion(Ca~(2+))/calcineurin(CaN) signaling pathway in bisphenol A(BPA)-induced interleukin-6(IL-6) secretion in macrophages. METHODS: Raw 264.7 cells in logarithmic growth phase were divided into control group, activator group, BPA low-, medium-and high-dose groups and inhibitor group. The cells in control group were treated with 0.10% dimethyl sulfoxide. The activator group was treated with lipopolysaccharide at mass concentration of 2 mg/L. The 3 BPA groups were treated with BPA at final concentrations of 1, 10, or 100 μmol/L. Two sets of verapamil or tacrolimus(FK506) groups were given verapamil at the final concentration of 10 or 30 μmol/L; or final concentration of FK506 at 250 or 500 nmol/L. Then the cells were treated with final concentration of 100 μmol/L BPA. The cells were collected at 4, 12, and 24 hours. The mRNA expression of IL-6 and CaN at 4 and 12 hours were detected by real-time fluorescence quantitative polymerase chain reaction. The relative expression of IL-6 and CaN protein was detected at 24 hours by enzyme-linked immunosorbent assay, and the intracellular Ca~(2+) level was detected at 4 hours using a single-tube multi-function detector. RESULTS: At 12 hours, the mRNA expression of IL-6 in the 100 μmol/L BPA group was higher than that in control group, activator group and the 1 and 10 μmol/L BPA groups(P<0.05), and higher than that in the 10 and 30 μmol/L verapamil groups, and in the 250 and 500 nmol/L FK506 groups(P<0.05). The mRNA expression of CaN in the 100 μmol/L BPA group was higher than that in control group, activator group and 1 and 10 μmol/L BPA groups(P<0.05), and higher than in 10 and 30 μmol/L verapamil groups(P<0.05). The relative expression of IL-6 protein in the 100 μmol/L BPA group was higher than that in control group, activator group and 1 and 10 μmol/L BPA groups(P<0.05). The relative expression of CaN protein in 100 μmol/L BPA group and 10 and 30 μmol/L verapamil groups were higher than that in control group and activator group(P<0.05). The relative expression of CaN protein in the 10 and 30 μmol/L verapamil groups were lower than that in the 100 μmol/L BPA group(P<0.05). The intracellular Ca~(2+) level in the 100 μmol/L BPA group was higher than that in control group and activator group(P<0.05). The intracellular Ca~(2+) level in the 10 μmol/L verapamil group was lower than that in the 100 μmol/L BPA group(P<0.05). CONCLUSION: BPA might promote the secretion of IL-6 through Ca~(2+)/CaN signaling pathway in macrophages.
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OBJECTIVE: To observe the hepatic injury induced by combined exposure to diethylhexyl phthalate( DEHP) and bisphenol A( BPA) in rats and explore the mechanism of oxidative stress. METHODS: Thirty-two specific pathogen free healthy male SD rats were randomly divided into control group,DEHP(750 mg/kg body weight) group,BPA(100 mg/kg body weight) group and combined exposure group,with 8 rats in each group. The rats were gavaged once per day,7 days per week,for 6 weeks. The changes of liver organ coefficient and histopathology were observed. The activities of superoxide dismutase( SOD), glutathione peroxidase( GSH-Px) and the levels of hydrogen peroxide( H2 O2),malondialdehyde( MDA) were detected by spectrophotometry. The relative mRNA expression of antioxidant gene nuclear factor erythroid-2 related factor 2( Nrf2),heme oxygenase-1( HO-1),glutamate cysteine ligase catalytic subunit( Gclc),thioredoxin reductase( Txnrd),superoxide dismutase 3( Sod3) and glutathione peroxidase 1( Gpx1) in liver tissue were examined by real-time fluorescent quantitative polymerase chain reaction. RESULTS: The body weight of DEHP exposure group was lower than that of control group from the beginning of the 2 nd week( P < 0. 05),and the body weight of combined exposure group was lower than control group from the beginning of the 3 rd week( P < 0. 05). The liver mass and organ coefficients in DEHP group and combined exposure group were significantly higher than that of control group( P <0. 05). The results of pathology examination showed that there was necrosis of liver cells in DEHP group,vacuolar degeneration in cytoplasm of BPA group,and severe inflammatory cell infiltration in combined exposure group. The activity of SOD and GSH-Px of each exposure group was reduced( P < 0. 05),the H2 O2 level of each exposure group was increased(P < 0. 05),meanwhile the MDA level in the liver tissue of the BPA group and the combined exposure group increased compared with the control group( P < 0. 05). The relative mRNA expression of Nrf2,HO-1 and Gpx1 in each exposure group were decreased( P < 0. 05),the relative mRNA expression of Gclc,Txnrd and Sod3 in DEHP group and mixed exposure group were decreased compared with the control group( P < 0. 05). The relative mRNA expression of Nrf2,HO-1,Gclc,Txnrd and Sod3 in combined exposure group were decreased compared with the BPA group( P < 0. 05).CONCLUSION: Under the conditions of this study,DEHP and BPA alone or in combination could cause hepatic injury. The combined effect was greater than single effect. The effect of DEHP was greater than that of BPA. The liver injury induced by DEHP and BPA was related to Nrf2 signaling pathway.