| Background and objective:Functional dyspepsia is a syndrome defined by chronic or recurrent upper abdominal symptoms without any organic or biochemical abnormality. The clinical symptoms often include upper abdominal pain, upper abdominal fullness, early satiety, belching, losing of appetite, nausea, vomiting and other symptoms of upper abdominal discomfort. Chaihu-Shugan-San (CSS) is a popular traditional Chinese medicine formula to treat functional dyspepsia. It is widely accepted that the combined effect of multiple constituents of traditional Chinese medicine formula is considered to be crucial for treating diseases in clinical practice. The objective of the study are as follows:?To develop a simple, rapid and sensitive Ultra performance liquid chromatography coupled with photodiode array (UPLC-PDA) detection method for simultaneous determination of sixteen compounds in Chaihu-Shugan-San:gallic acid, oxypaeoniflora, albiflorin, paeoniflorin, liquiritin, ferulic acid (FA), narirutin, naringin, hesperidin, neohesperidin, meranzin hydrate (MH), liquiritigenin, quercimelin, benzoylpaeoniflorin, isoliquiritigenin, formononetin.?To develop a liquid chromatography-tandem mass spectrometry (LC-MS) method for the determination of FA and MH in plasmas of patients with functional dyspepsia after oral administration of Chaihu-Shugan-San decoction.?To investigated the effect of CSS, FA and MH on gastrointestinal motility in rats, partly elucidate the effective substance of CSS treating functional dyspepsia.?To develop a Ultra performance liquid churomatography coupled with photodiode array (UPLC-PDA) detection method to explore the pharmacokinetics of FA and MH in plasmas of patients with functional dyspepsia after oral administration of CSS decoction.Method:Part 1 The quantitative analysis of the sixteen active constituents in CSSThe analysis was performance on UPLC-PDA system with BEH C18 column (100×2.1 mm id,1.7?M). The mobile phase comprising acetonitrile (A) and 0.5% acetic acid (B) was used to elute the targets in gradient elution mode. Flow rate and detection wavelength were set at 0.5ml/min and 284nm. The sixteen constituents in CSS including gallic acid, oxypaeoniflorin, albiflorin, paeoniflorin, liquiritin, FA, narirutin, naringin, hesperidin, neohesperidin, MH, liquiritigenin, quercimelin, benzoylpaeoniflorin, isoliquiritigenin and formononetin were quantitative determinate, and the limit of determination (LOD), precision, recovery, sample stability and reproducibility of the sixteen constituents were examined in this study.Part 2 The qualitative analysis of FA and MH in the plasma of patients with functional dyspepsia after oral administration of CSS.The patients with functional dyspepsia were recuited and the plasmas were collected from the patients after oral administration of CSS. The plasma samples were precipitated with methanol. FA and MH were determined by LC-MS. Electrospray ionization (ESI) source was applied and operated in the positive ion mode. Selected reaction monitoring (SRM) mode with the transition of m/z 194.8-m/z 177.0 and m/z 261.1-m/z243.0 were used to detect FA and MH. Waters Xterra C18 column (150 mm×2.1 mm,5?m) was used in the experiment. The column temperature was set at 40?.10 mmol·L-1 ammonium formate-acetonitrile (40:60, v/v) was used as mobile phase and the flow rate was 0.2mL·min-1 The sample room temperature was set at 5?and the injection volume was 20?L.Part 3 The effects of CSS, FA and MH on promoting gastrointestinal motility.Gastric emptying (GE) and intestinal transit (IT) were used to study the effects of CSS, FA and MH on promoting gastrointestinal motility in vivo in rat. The intestinal muscle strips test in vitro was used to investigate the effects of FA and MH on Intestinal contraction.Part 4 The pharmacokinetics of FA and MH in plasmas of patients with functional dyspepsia after oral administration of CSS decoction.A total of 7 patients with functional dyspepsia were recruited in this study. The plasma samples were preprocessed by protein precipitation method. The UPLC system was used to determine the contents of FA and MH in the blood samples of 7 volunteers with functional dyspepsia, and the blood was collected through the vein at 5,10,15,30,45,60,90, 120,180,240,300,360,480 min. The dates of plasma concentration-time were fitted by using the 3P97 analytical program of pharmacokinetics. The internal standard (IS) was sulfamethoxazole. The detection wavelengths of FA and MH were set at 321 nm and 324 nm respectively.Result:Part1(1) A simple, rapid and sensitive Ultra performance liquid chromatography coupled with photodiode array (UPLC-PDA) detection method was established for the simultaneous determination of sixteen compounds in CSS. The 16 marker constituents were sufficiently separated successfully in less than 17.5 min. There were no interference peaks in the vicinity of the target compounds in the chromatograms of CSS decoction. All calibration curves showed good linear regression (r2> 0.9992) within test ranges of the sixteen targets. The relative standard deviations (RSD) of intra-and inter-day precision were from 1.87% to 5.61% indicating good precision. The average recoveries of investigated sixteen targets ranged from 96.13% to 102.46%, with RSD values<6.74%, showing good recoveries. The sample of CSS decoction has good stability in 24 h, with RSD values from 0.78% to 2.98%. The RSD of the sixteen components in the reproducibility test fluctuates between 1.94% and 5.36%, indicating good reproducibility. (2) Quantitative determination of the sixteen marker constituents in CSS decoction was made by UPLC-PDA system.Part 2According to the mass spectrometry analysis of main chromatogram peaks, FA and MH were successfully detected in the plasmas of patients with functional dyspepsia after oral administration of CSS.Part 3Compared with the control group, CSS (20g/kg), FA (100 mg/kg) and MH (14,28 mg/kg) significantly increase the rate of GE and IT in rats (P<0.05) FA (100?mol/L) and MH (10?mol/L) increased the mean amplitude of contraction in the longitudinal and circular strip compared with untreated controls (P<0.05). in addition, MH (30,100?mol/L) increased the mean amplitude of contraction in the longitudinal and circular strip compared with untreated controls (P<0.01)Part 4After oral administration of CSS, FA and MH were separated completely in the plasma of patients with functional dyspepsia and no other interfering peaks were found in the spectrum of the chromatograms. The retention times of FA and MH were 6.5 and 15.7 min, and that of sulfamethoxazole was 8,0 min. The recovery rates of FA and MH were from 95.35% to 102.56% with RSD< 2.81% and from 95.58% to102.09% with RSD<3.86%, respectively. The calibration curve for FA was linear over the concentration range from 10 to 320 ng/ml. The low detection limit of FA based on a signal-to-noise ratio of 3 was 1.5 ng/mL and the low concentration limit was 4.5 ng/ml. The calibration curve for MH was linear over the concentration range from 7.5 to 720 ng/mL. The low detection limit was 2.5 ng/mL and the low concentration limit was 7.5 ng/mL. The pharmacokinetics of FA and MH in patient plasma was fitted with a two-compartment model. Time to reach peak concentration of FA (0.199 mg/L) and MH (0.371 mg/L) was 27.50 min and 23.57 min, respectively. The elimination half-life and area under the concentration-time curve from t= 0 to the last time of FA and MH were 131.27 min and 14.835?g·min/mL and 139.53 min and 31.445?g·min/mL, respectively.Conclusion:1. A rapid, sensitive, and reliable UPLC-PDA method for the simultaneous determination of sixteen compounds in CSS was developed. The method was validated for specificity, accuracy, precision, and limits of detection and was successfully applied to determine the contents of sixteen components in CSS.2. LC-MS is a convenient, rapid and effective method to separate and quality FA and MH in plasmas of patients with functional dyspepsia after oral administration of CSS. 3. CSS and its absorbed compound FA and MH have the significant effect on promoting gastrointestinal motility in rats. FA and MH are the effective substance of CSS to treat functional dyspepsia.4. FA and MH in the plasmas of patients with functional dyspepsia were simultaneously determined by the UPLC method after oral administration of CSS. The pharmacokinetics of prokinetic CSS and its compounds are useful for monitoring CSS formulas in clinical practice and for understanding therapeutic mechanisms. |
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