Studies On Bacteriostatic Therapeutic Material Basis And Pharmacokinetics Of Wikstroemia Indica

Wikstroemia indica(L.)C.A.Mey.is a member of thymelaeaceae family and mainly distributes in middle and southeast part of China.Its root and rhizome could be used as medicine.As a traditional Chinese herb,it has been widely used for the treatment of bronchitis,tonsillitis,pneumonia,mastitis,lymphnoditis,rheumatalgia and many types of cancer.The chemical compenents,bacteriostatic activity,quality control method and pharmacokinetics of W.indica had been studied in this paper.Four chemical reference substances(daphnoretin,genkwanin,apigenin and triumbelletin)from 75%ethanol extraction of W.indica were isolated by silica gel,Sephadex LH-20,thin layer chromatography and recrystallization.BPC chromatogram of chemical constituents in W.indica was established by UHPLC-Q-TOF-MS/MS.There were twenty-one chromatographic peaks identified.Comparing with reference substances,rutin,scopoletin,umbelliferone,myricetin,arctiin,luteolin,quercetin,apigenin,triumbelletin,kaempferol,daphnoretin,arctigenin,aloe-emodin,genkwanin and emodin were affirmed.Other chemical constituents were speculated based on molecular weight,MS/MS datas and relevant literature.The bacteriostasis of W.indica extract on four fungus(Candida albicans,Candida tropicalis,Aspergillus fumigatus and Cryptococcus neoformans var.gattii)and four bacteria(Escherichia coli,Bacillus subtilis,Pseudomonas aeruginosa and Staphylococcus aureus)had been studied by trace liquid culture medium dilution method.The results showed that the bacteriostasis of W.indica was stronger on bacteria than on fungus.On this basis,the action of drug serum of W.indica bacteriostatic on Bacillus subtilis and Staphylococcus aureus were researched by cup-plate method.The drug serum was effective for two bacteria and there were time-effect and dose-effect relationship between the drug serum and bacteria.By bivariate correlation analysis between the chromatographic peak area and pharmacologic datas,it was found that flavonoid,coumarin,lignan and anthraquinone constituents were negative correlation with pharmacodynamic index.So those four type components can be defined as bacteriostatic therapeutic material basis of W.indica.An UPLC-MS/MS method was developed to determine seven constituents(umbelliferone,apigenin,triumbelletin,daphnoretin,arctigenin,genkwanin and emodin)in W.indica extract.The chromatographic analysis was performed on an ACQUITY UPLC? BEH C18 column(2.1 × 50mm,1.7μm)by gradient elution with the mobile phase of 0.05%formic acid aqueous solution and acetonitrile.Multiple reaction monitoring mode with positive and negative electrospray ionization interface was carried out to detect the components.This method was examined and found to be satisfied in terms of specificity,linearity,precision,repeatability,accuracy and stability for all analytes.The established method was applied to determine the amounts of seven compounds in eighteen W.indica samples from different geographical origins.The results indicated that obvious difference existed in the contents of seven components and the developed method could be a helpful tool for the quality control of W.indica.An UPLC-MS/MS method was developed for simultaneous determination of umbelliferone,apigenin,daphnoretin and genkwanin in rat plasma using psoralen as internal standard.Plasma samples were protein precipitated with acetonitrile followed by liquid-liquid extracted with ethyl acetate.Four ingredients were separated on an ACQUITY UPLC? BEH C18 column(2.1×50 mm,1.7μm)using gradient elution with the mobile phase consisting of 0.1%formic acid aqueous solution and acetonitrile,and detected by positive ion electrospray ionization in multiple reaction monitoring mode.The method was linear for all analytes over investigated ranges with all correlation coefficients greater than 0.99.The validated lower limit of quantification was 3.1 ng/mL for umbelliferone,2.6 ng/mL for apigenin,11.7 ng/mL for daphnoretin and 2.4 ng/mL for genkwanin,respectively.The mean absolute extraction recoveries of analytes and IS from rat plasma were all more than 75.0%.The validated method was successfully applied to investigate the pharmacokinetics of four chemical ingredients after oral administration of W.indica extract to rats.Cmax were 152.5±12.63,83.80±9.16,605.7±30.71 and 29.08±3.61 μg/L,Tmax were 0.83±0.25,1.15±0.12,2.42±1.09 and 6.46±2.07 h and t1/2 were 13.29±4.15,14.50±3.02,15.79±5.13 and 36.24±9.11 h for umbelliferone,apigenin,daphnoretin and genkwanin,respectively.