Studies On The Quality Standard Of Plantaginis Semen And Pharmacokinetic Of Plantagoguanidinic Acid In Rat Plasma

Plantaginis Semen is one of the traditional Chinese medicine in China.We studied the chemical constituent of Plantaginis Semen,set up the quality standard,and determined the content of Plantaginis Semen in rat plasma.It is of great significance to clarify the chemical compositions and establish quality standard of Plantaginis Semen,and to explore the pharmacokinetics of PGA for the first time.And our study provided important scientific basis for making full use of toad skin of Plantaginis Semen.In this dissertation,the following results were achieved:1.Study on the Chemical Constituents of Plantaginis SemenThe dried seeds of Plantago asiatica L.were powdered and extracted with 75%EtOH.The extract with chloroform,ethyl acetate and n-butanol extraction in turn.The n-butanol part was systematically isolated and purified by multiple column chromatographies,reversed phase middle pressure liquid chromatography,TLC,and HPLC preparation.A total of seven compounds were isolated from the extract,and their structures were elucidated by spectroscopic method and physical and chemical properties.Finally,this compounds of them were identified as acteoside(1),5,7-dihydroxychromone(2),isovanillic acid(3),isoacteoside(4),aucubin(5),PGA(6),and geniposidic acid(7),and isovanillic acid was isolated from Plantaginis Semen for the first time.2.Study on the Quality Standard of Plantaginis SemenWe established a TLC method for the identification of PGA in Plantaginis Semen,using ethyl acetate-methanol-formic acid(10:0.5:2)as the mobile phase and bismuth potassium iodide-potassium iodide iodine(1:1)as the chromogenic agent.Results: PGA could be identified by TLC.Based on method validation,this method with good repeatability can be used for the TLC of Plantaginis Semen.We established an HPLC method to simultaneously determine three main kinds of active constituents,namely,PGA,acteoside and isoacteoside,before and after salt processing of Plantaginis Semen from different habitats.The CAPCELL PAK C18 MG ? S5 column(250 mm × 4.6 mm,5?m)was used with the mobile phase of methonal(A)-water(B)for gradient elution(0-45 min,25% A ? 42% A)at the flow rate of 1.0 mL/min.The detection wavelength was 210 nm,and the column temperature was at 30 ?.Results: The linear ranges of PGA,acteoside and isoacteoside were 0.04491-0.8982 ?g(r = 0.9999),0.2117-4.2338 ?g(r = 0.9999)and0.01688-0.3376 ?g(r = 1.0000),respectively.The average recoveries(n = 6)were99.3%(RSD = 0.52%),100.0%(RSD = 0.18%)and 99.1%(RSD = 1.9%),respectively.Based on method validation,this method with high separation efficiency and good repeatability can be used for the quality control of Plantaginis Semen.3.Study on Pharmacokinetic of PGA in Rat PlasmaAn UHPLC-MS/MS method for quantification of PGA in rat plasma was firstly applied to investigate the pharmacokinetic behavior in vivo,using protopine as internal standard.The chromatography was separated on a Phenomenex?Luna-C18column(2.1 mm × 150 mm,3.0 ?m)within 7.0 min by using a mobile phase consisting of acetonitrile-0.1% formic acid solution under gradient elution at a flow rate of 0.4 mL/min.Prepared samples were monitored by multiple reaction monitoring mode,with the target fragmentions m/z 226.2?84.2 for PGA and m/z354.2?188.9 for IS in positive electrospray ionization.The standard curves of PGA was ranged from 1 to 1000 ng/mL(r = 0.9962).the recovery was more than 88.6%,and the lower limit of quantitation in plasma for PGA was 0.1 ng/mL.Intra-and inter-day accuracy ranged from-8.6% to 4.9%.Furthermore,this validated method was successfully used for a pre-clinical pharmacokinetic study of PGA at a single dose of 20 mg/kg and 5 mg/kg in rats via oral and intravenous administration.The study showed that PGA was absorpted rapidly and eliminated gradually with a greater absolute oral bioavailability of 70.1% in rats.