| Trollius chinensis, dried flowers of Trollius chinensis Bunge, hasbeen used as an important traditional Chinese medicine in Chinese folk fora long history, which widely grows in the southwest, northwest, northeast.\Bencao Gangmu Shiyi\ said that it was"bitter in taste, cold-natured,asepsis"and could be used for the treatment of aphtha, larynxboss, lightfever atrophy of the gum, ear ache, ophthalmalgia, and that it had theeffects of eyesight improvement and anti-miasma. Modern pharmacolo-gical studies show that it possesses antimicrobial and antiviral actions andhas been used widely to treat cold, fever, chronic tonsillitis, acute tympan-itis, urinary tract infection and other inflammations.Flavonoids are the principal components of Trollius chinensis. Flavo-noids and flavonoid monomer separation and purification can be done inmany ways. Traditional methods include a variety of column chromatogra-phy, preparative TLC and solvent extraction methods. PK of medicine res-earch studied the drug in the body's absorption, distribution, metabolismand excretion, which use mathematical models to quantitatively describethe dynamic process of drug in the body. Pharmacology and clinical eve-lopment of medicine has an important significance. There are two reportsthat studied the PK of vitexin in rats and Beagle dogs. PK research has notbeen reported that vitexin was administered to rabbits in different ways.Therefore, the evaluation of the absorption, distribution, metabolismand excretion characteristics of this potentially important medical constit-uent is a major prerequisite for the interpretation of the pharmacologicaltesting and the use of clinical drug aright guidance. In this study, wecompared the pharmacological characteristics of vitexin in the three different administration forms to rabbits with HPLC. We also investigatedthe tissue distribution metabolismcharacteristics.1. Extraction of vitexin from Trollius chinensisFirst, HPLC method was developed for determineation of vitexin.The column was an Hypersil BDS C18 ( 4.6×150 mm, 5μm ) with a mixture of acetonitrile-acetic acid ( 15: 85 ) solution as the mobile phase,at a flow rate of 1.0 ml·min-1. The column temperature was at 30℃. Thedetection wavelength was at 340 nm. Second, PHPLC method wasestablished for purifying the substances of vitexin. The column wasZORBAX SB-C18: ( 21.2 mm×250 mm, 7μm ) with a mixture ofacetonitrile-acetic acid ( 15: 85 ) solution as the mobile phase, at a flowrate of 20 ml·min-1. The column temperature was at 25℃. The detectionwavelength was at 340 nm. Fraction was collected: based on the peak, thethreshold being Min: 2.2. TLC is used for the qualitation analysis ofvitexin. Both NMR and HPLC are used for the structure and quantitativeanalysis of vitexin.The results showed that vitexin was used column chromatographyand PHPLC to purify. TLC showed that vitexin had the same Rf value withthe reference. The Rf value is 0.6. NMR analysis showed that the structurewas vitexin. HPLC analysis showed that the purity of vitexin was 98.8%.It obtained high purity. It can be used as reference substance.2. Pharmacokinetic study of vitexin on rabbitsRabbits were divided into three different groups randomly, with sixrabbits at a group. The rabbits in each group were treated with i.v., i.p., i.m.at a certain dose which was corresponded to approximately 20 mg·kg-1 ofvitexin. Blood samples were obtained from carotid according to thespecific schedule and were collected and centrifuged at 11000 rpm for 10minutes. After centrifugation, 0.1 ml plasma was transferred to tubes and0.2 ml of methanol was added into it. Then the tube was votex mixed for 1 minute and the mixture was centrifuged at 11000 rpmfor 10 minutes. A20μl of the supernatant layer was injected into the HPLC system. Theoriginal data was analyzed with the 3P97 pharmacokinetics software; Thebest compartment modle was selected according to the least AIC valuesand the largest correlation coefficient was obtained from real concentra-tion. The pharmacokinetic characteristics of the three different administ-rations forms of vitexin to rabbits were in compliance with the opentwo-compartment model. The parameters were as follows underintravenous injection, T?αis 5.052±0.231 min, T?β114.291±3.462 min,AUC(0-t) 716.916±43.786μg·min·ml-1, MRT(0-t) is 17.361±0.245 min;under intraperitoneal injection, T?αis 25.234±2.329 min, T?βis137.982±6.091 min, AUC(0-t) is 89.085±4.179μg·min·ml-1, MRT(0-t)156.570±0.211 min; under intramuscular injection T?αis 31.684±5.432min, T?β154.098±6.974, AUC(0-t) 65.759±3.854μg·min·ml-1, MRT(0-t) is196.684±0.087 min. The pharmacokinetic researches showed that the T?βof the three different of administrations of vitexin in rabbits were allrelatively short.Rabbits were divided into twelve different groups randomly, with sixrabbits being each group. The rabbits in each group were treated with i.v.at a certain dose which was corresponded to approximately 20 mg·kg-1 ofvitexin. The tissues ( kidney, liver, lung, spleen, heart, brain ) werecollected after 5, 10, 15, 30, 45, 60, 75, 90, 120, 180, 240 and 300 minafter administration respectively and saline was used to flush the blood onthe surface of tissues. 20μl of the supernatant layer was injected into theHPLC system. Then the concentration of vitexin was determined indifferent tissues; the content of vitexin per gramme tissue calculated incomply with standard curves prepared in relevant tissues. Time taken toreach the highest concentration ( Tmax ) ranged from 10min to 40min formost tissues, which showed rapid distribution of drug fromcirculation intothe tissues or rapid uptake of vitexin. The AUC of each tissue have a sequence as follows: kidney>liver>lung>spleen>heart>brain. Thatshowed that vitexin was hard to penetrate through blood brain barrier. |
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