ABSTRACT
ObjectiveThis study investigated the mechanism of Wenjingtang in the prevention and treatment of endometriosis （EMT） from the perspective of regulating hypoxia stress and mitochondrial function. MethodPrimary human endometrial stromal cells （ESCs） form ectopic endometrial tissues were isolated and cultured， the cells were divided into control group （Control）， 5% control serum group （5% KBXQ）， 10% control serum group （10% KBXQ）， 5% Wenjingtang serum group （5% WJTXQ） and 10% Wenjingtang serum group （10% WJTXQ）. ESCs in different groups were detected for proliferation by methyl thiazolyl tetrazolium （MTT） assay， mRNA and protein expression of hypoxia inducible factor-1α （HIF-1α） by real-time fluorescent quantitative polymerase chain reaction （Real-time PCR） and Western blot analysis， mitochondrial ultrastructure by transmission electron microscope， mitochondrial function ［mitochondrial membrane potential， mitochondrial membrane potential（MMP） and cytochrome C（Cyt C） content］ and apoptosis （cell membrane permeability， nuclear fluorescence intensity， nuclear size and cell counts） by high content screening （HCS） assay， apoptosis rate by flow cytometry， and proteins of B-cell lymphoma-2 （Bcl-2） associated X （Bax）， Bcl-2 and cleaved cysteinyl aspartate specific proteinase-3 （cleaved Caspase-3） by Western blot. ResultCompared with Control group， the 5% KBXQ and 10% KBXQ groups showed increased cell viability （P<0.01）， there was no significant change in HIF-1α mRNA and protein expression， transmission electron microscopy showed that the mitochondrial cristae were obvious and the inner and outer membranes of mitochondria were clear， HCS multichannel fluorescence staining showed that there were no significant changes in the expression of MMP， Cyt C and cell membrane permeability， and the nuclei showed uniform light staining， there were no significant changes in apoptosis rate， cleaved Caspase-3 protein expression and Bax/Bcl-2 ratio. Compared with Control group and corresponding concentration KBXQ group， the 5% WJTXQ and 10% WJTXQ group showed decreased cell viability （P<0.01） and HIF-1α mRNA and protein levels （P<0.05，P<0.01）， the ultrastructure of mitochondria was destroyed， some mitochondria were swollen and the cristae were blurred， moreover， decreased MMP and up-regulated Cyt C release （P<0.05，P<0.01）， increased cell membrane permeability （P<0.01）， and apoptosis characteristics included nuclear pyknosis， DNA agglutination in nucleus and decrease of cell numbers were observed （P<0.05，P<0.01）， increased apoptosis rate （P<0.01）， which was consistent with the results of HCS analysis， and up-regulated expression levels of cleaved Caspase-3 protein and Bax/Bcl-2 ratio （P<0.05，P<0.01）. ConclusionIn conclusion， the results suggest that Wenjingtang can improve hypoxia stress via down-regulating HIF-1α expression in ectopic ESCs， and inhibit cell proliferation， reduce mitochondrial biological activity and induce apoptosis， which might be the internal mechanism of Wenjingtang in preventing and treating EMT.
ABSTRACT
Paeoniae Radix Rubra is a traditional Chinese medicine commonly used in clinical practice， it is mostly wild and widely distributed in different areas of China. In addition， the plant of Paeoniae Radix Rubra also has ornamental value. Modern phytochemical researches showed that the chemical constituents of Paeoniae Radix Rubra were complex. Up to now， more than 300 chemical constituents have been found， mainly including monoterpene glycosides， triterpenoids， flavonoids， tannins， phenolic acids， saccharides， steroids， volatile oils and so on. Among them， the content of monoterpene glycosides was the highest， and the types of volatile oil were the most. Paeoniae Radix Rubra has a wide range of pharmacological effects， exerting different curative effects in multiple systems such as blood， cardiovascular， nervous and digestive system. It can protect myocardial cells and nerve cells， stabilize microcirculation， anti-endotoxin， anti-atherosclerosis， reduce pulmonary hypertension， anti-depression， protect liver， anti-gastric ulcer， anti-tumor， slow down aging， treat Parkinson's syndrome and diabetes and its complications， anti-radiation， anti-inflammatory， anti-virus and so on. Through reviewing the literature on chemical constituents and pharmacological effects of Paeoniae Radix Rubra， it was found that total glycosides and monomers such as paeoniflorin， albiflorin， benzoylpaeoniflorin and gallic acid may be the main active components of Paeoniae Radix Rubra. At present， the research on Paeoniae Radix Rubra mainly focused on monoterpene glycosides， while the research on flavonoids and volatile oil in Paeoniae Radix Rubra was less. It is suggested that research on these two components should be strengthened in the future.
ABSTRACT
To establish the method for determining non-volatile ingredients of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid A, rosmarinic acid, ferulic acid, rutin, luteoloside, isoquercitrin, hesperidin, diosmin, diosmetin, luteolin, acacetin and linarin in Menthae Haplocalycis Herba formula granules and traditional herbal pieces by UPLC-MS/MS, and analyze the correlation of non-volatile ingredients in Menthae Haplocalycis Herba formula granules and traditional herbal pieces. Shim-pack GIST C_(18) column(2.1 mm×100 mm, 2 μm) was adopted with acetonitrile-0.1% formic acid aqueous solution as the mobile phase for gradient elution at the flow rate of 0.4 mL·min~(-1). The column temperature was set at 35 ℃. The quantitative analysis was performed using the electrospray ionization source and the multiple reaction monitoring. The linear relationship, resolution, repeatability and recovery of the 16 chemical components all met the requirements. The 16 non-volatile ingredients in traditional herbal pieces of Menthae Haplocalycis Herba could be tracked in formula granules. There were certain differences of the 16 chemical components among Menthae Haplocalycis Herba formula granules of different manufacturers and traditional herbal pieces of different producing areas. The UPLC-MS/MS method was simple, rapid and accurate, and could be used for the quality control of non-volatile ingredients in Menthae Haplocalycis Herba formula granules and traditional herbal pieces.
Subject(s)
Chromatography, High Pressure Liquid , Chromatography, Liquid , Correlation of Data , Drugs, Chinese Herbal , Tandem Mass SpectrometryABSTRACT
This present study aims to establish a UPLC method for simultaneously determining eleven components such as new chlorogenic acid,chlorogenic acid,caffeic acid,cryptochlorogenic acid,artichoke,isochlorogenic acid A,isochlorogenic acid B,isochlorogenic acid C,rutin,hibisin and loganin in Lonicerae Japonicae Flos,Lonicerae Japonicae Caulis and leaves of Lonicera japonica and comparing the differences in the contents of phenolic acids,flavonoids and iridoid glycosides of Lonicerae Japonicae Flos,Lonicerae Japonicae Caulis and leaves of Lonicera japonica.The method was carried out on an ACQUITY UPLC BEH C18column(2.1 mm×100 mm,1.7 μm) by a gradient elution using acetonitrile and 0.1% phosphoric acid.The flow rate was 0.3 mL·min-1.The column temperature was maintained at 30 ℃.The sample room temperature was 8 ℃.The wavelength was set at 326 nm for new chlorogenic acid,chlorogenic acid,caffeic acid,cryptochlorogenic acid,artichoke,isochlorogenic acid A,isochlorogenic acid B and isochlorogenic acid C,352 nm for rutin and lignin,and 238 nm for loganin.The injection volume was 1 μL.The eleven components has good resolution and was separated to baseline.Each component had a wide linear range and a good linear relationship(r≥0.999 6),the average recovery rate(n=9) was 98.96%,100.7%,97.24%,97.06%,99.53%,96.78%,98.12%,95.20%,95.12%,100.2%,98.61%and with RSD was 2.5%,1.4%,1.9%,2.1%,1.7%,1.9%,1.6%,2.0%,1.4%,2.2%,2.0%,respectively.Based on the results of the content determination,the chemometric methods such as cluster analysis and principal component analysis were used to compare the Lonicerae Japonicae Flos,Lonicerae Japonicae Caulis and leaves of Lonicera japonica.The results showed that Lonicerae Japonicae Flos and leaves of Lonicera japonica were similar in the chemical constituents,but both showed chemical constituents difference compored to Lonicerae Japonicae Caulis.The established multi-component quantitative analysis method can provide a reference for the quality control of Lonicerae Japonicae Flos,Lonicerae Japonicae Caulis and leaves of Lonicera japonica.
Subject(s)
Chromatography, High Pressure Liquid , Drugs, Chinese Herbal , Chemistry , Flavonoids , Flowers , Chemistry , Hydroxybenzoates , Iridoid Glycosides , Lonicera , Chemistry , Phytochemicals , Plant Leaves , Chemistry , Quality ControlABSTRACT
Objective To establish a QAMS method for content determination of six compositions (chlorogenic acid, caffeic acid, cryptochlorogenin acid, isochlorogenic acid A, isochlorogenic acid C and loganin) from Lonicerae Japonicae Caulis; To verify the feasibility and applicability of this method in quality control of Lonicerae Japonicae Caulis. Methods Chlorogenic acid was set as internal reference substance. The HPLC analysis was performed on a Waters Symmetry C18 column (4.6 mm × 250 mm, 5 μm) with a mobile phase consisted of acetonitrile and 0.4% phosphoric acid solution in gradient elution manner at a flow rate of 1 mL/min. The column temperature was maintained at 35 ℃, and the detection wavelength was set at 327 nm for chlorogenic acid, caffeic acid, cryptochlorogenin acid, isochlorogenic acid A, isochlorogenic acid C and 236 nm for loganin. Results The relative correction factors of caffeic acid, cryptochlorogenin acid, isochlorogenic acid A, isochlorogenic acid C and loganin were established; there was no obvious difference between calculated value of QAMS and measured value of external standard method. Conclusion The quality control mode of QAMS can be used for multi-index synchronization quality evaluation of the six compositions from Lonicerae Japonicae Caulis.