| ObjectiveTake general flavonoid content contained in Modified Shoutai Wan as index, by single factor test and orthogonal design, we optimize the extraction process of flavonoid content contained in Modified Shoutai Wan. Applying UPLC-MS to study the pharmacokinetic parameters of hyperin and quercetin of flavonoid content contained in Modified Shoutai Wan inside rats, to prepare drug serum according to the time to drug peak. Cultivate primary human decidual cells and establish mifepristone injury human decidual cells models which act as carriers, to observe how the effect that medicated serum would bring on apoptosis model.Methods1. Optimize the extraction process of flavonoid content contained in Modified Shoutai Wan:with rutin as reference substance, applying colorimetric method to measure flavonoid content of extracting solution of Modified Shoutai Wan. By single factor design and orthogonal test, we optimize the technological parameters in reflux extraction of flavonoid content contained in Modified Shoutai Wan.2. Simultaneously, UPLC-MS should be applied to measure the content of hyperin and quercetin of Modified Shoutai Wan:by optimizing chromatographic condition and mass spectrum, we establish the UPLC-MS method and measure the content of hyperin and quercetin of Modified Shoutai Wan at the same time.3. The pharmacokinetics study of hyperin and quercetin of Modified Shoutai Wan contained in rats’blood:(1) Optimize hygroplasm combination condition, and establish the UPLC-MS method for measuring the content of hyperin and quercetin of Modified Shoutai Wan contained in rat plasma.(2) Randomly divide SD female rats into3groups with6rats in each group which was treated respectively by Modified Shoutai Wan, hyperin and quercetin. Then,5min,10min,20min,30min,45min,60min,90min,120min,240Min,360min after the perfusion, we take0.5ml blood sample each time from venous plexus around the orbital, and get plasma after separation. Applying extraction method with ethyl acetate, we conduct sample pretreatment, with diethylstilbestrol as internal standard. Optimized UPLC-MS method should be applied to measure the content of hyperin and quercetin of Modified Shoutai Wan contained in rats’ blood, to acquire drug-time curve and pharmacokinetic parameters.4. The establishment of apoptosis model of human decidual cells: Compound enzyme digestion method should be applied to separate of human decidual cells and differential adhesion method should be used to purify human decidual cells. Applying mifepristone of different concentration in human decidual cells, with index of cell inhibition rate, apoptosis rate and caspase-3gene expression, we can evaluate the apoptosis situation of human decidual cells and determine the appropriate concentration of mifepristone for modeling.5. The effect of Modified Shoutai Wan and its effective component drug serum on damage model of human decidual cells:Randomly divide SD female rats into6groups with5rats in each group which was treated with gavage respectively by dydrogesterone, Modified Shoutai Wan, dodder, hyperin, quercetin and normal saline. When the drug comes to peak, anesthetize rats with10%chloral hydrate and take blood sampling through aorta abdominalis, isolating drug serum. Selecting human decidual cells in good condition with90%purity, intervening them with mifepristone of molding concentration, then intervening model cells with prepared drug serum. We observe the effect of drug serum on cell model through MTT method, flow cytometry, PCR and Western blot detection index.Results1. Determination method of general flavonoid content contained in Modified Shoutai Wan is brief and reliable, and its standard curve is: y=1.205x, r=0.9998. Within the scope of0.112-0.56mg/mL, rutin has good linearity. By single factor investigation and orthogonal test, we obtain the best condition for extracting general flavonoid content contained in Modified Shoutai Wan:with60%ethanol concentration, the ratio of solid to liquid is1:10; reflux extracting twice, with0.5h of extraction each time.2. Establish the UPLC-MS method to detect simultaneously the content of hyper in and quercetin of Modified Shoutai Wan, with standard curve r>0.999; in the scope of0.25ug/ml-4ug/ml, it has good linearity, and its precision and recovery rate conform to the requirements. The test manifests that the content of hyperin and quercetin of Modified Shoutai Wan is respectively0.15mg/ml and1.98mg/ml.3. Respectively, in the scope of5-5000and1-1000ng/mL, the hyperin and quercetin of plasma have good linear relationship (R2=0.9954), with the Lowest Limit of Quantification (LLOQ)≤5ng/mL. The Relative Standard Deviation of Precision (RSD) of within-day precision and day to day precision are less than8.2%, and the Relative Error (RE) is-5%-6%. The average recovery rate was between71%-83%, and the mean value of matrix effects is between94%-99%. After oral gavage, the absorption of hyperin and quercetin are fastest for rats, with Tmax less than60min. From the drug-time curve, we can see the Cmax in compound group is higher than that of monomer groups, which indicates higher bioavailability.4. We can obtain human decidual cells through complex enzymatic method, and by differential adhesion method, we can purify them to the third generation. Cytokeratin7is negative, identified by fluorescence immunoassay. Vimentin is positive. And prolactin is positive, with its purity more than90%. By radioimmunoassay, supernatant of generation1-10cell all contain PRL which declines since eighth generations. During vigorous growth period, applying five mifepristone of different concentration, and detecting its growth inhibition rate with MTT method, we find that the half inhibitory concentration during24h is8X10-5mol/l. With the increase of concentration of mifepristone, apoptosis rate in early stage and caspase-3gene expression increase. Comprehensive speaking, we determine the apoptosis modeling concentration of human decidual cells is6×10-5mol/l, and the drug action time is24h.5. Affected by serum drug, the apoptosis models of human decidua cells compared with blank groups, and the result shows that the growth inhibitory rate, early apoptosis rate and Caspase-3apoptosis gene expression of progesterone serogroup, ShouTaiWan serogroup, dodder serogroup, Hyperoside serogroup and quercetin serogroup are in decrease. Conclusion:1. This study confirms the optimized reflux extraction process of general flavonoid content contained in Modified Shoutai Wan. This extraction process is simple, reasonable, and stable, with good repeatability and saving time and energy. It also provides reference for further study on Modified Shoutai Wan.2. It establishes the UPLC-MS method to detect simultaneously the content of hyperin and quercetin of Modified Shoutai Wan. Through validation, this method is simple and reliable, with its precision and recovery rates all meeting the requirements, which provides experimental basis for the quality control.3. It establishes the UPLC-MS analytical method to detect the content of hyperin and quercetin of rat plasma. This method is simple in sample pretreatment process, and is brief in analyzing time, with its specificity, linearity, precision, accuracy, recovery and matrix effects consistent with the requirements, and can be successfully applied to the pharmacokinetic study. The biological utilization of compound group increases which is better than that of single group, indicating that the compound Chinese medicine has synergistic effect, which promotes the effective absorption of hyperin and quercetin.4. Based on the primary cultural human decidual cells, it establishes the human decidual cells apoptosis models induced by mifepristone. Taken as carriers, it researches the effect of Modified Shoutai Wan and drug serm on the apoptosis of human decidual cells. The results show that Modified Shoutai Wan and its effective components can inhibit the apoptosis of human decidual cells in varying degrees. |
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