| Trollius chinensis, belonging to dried flowers of Trollius chinensisBunge, is a kind of perennial herb. It grows over the north and southwestregions in China. It has a long history as a medicinal in China. It could beused for the treatment of tonsillitis, mouth throat swelling, acute enteritisand so on. in Chinese folk. Modern pharmaceutical research shows thattrollius has antibacteria, anti-virus, anti-oxidation and otherpharmacological activity. Trollius mainly contains flavonoids, alkaloids,organic acids, volatile oil composition, in which flavonoids compounds areits main active ingredient. The study show that flavonoids mainly containorientin, vitexin, orientin-2′′-O-β-L-galactopyranosyl, quercetin and other substances. The content of orientin is the highest . The study show orientinhas a good pharmacological effection on antibacteria, anti-virusanti-thrombotic, anti-oxidation and protecting acute myocardial ischemia.Currently, The Study on orientin was focused on its pharmacologicaleffects. About its vivo study is very rarely. The pharmacokinetics plays animportant role in clarifying the effectiveness of traditional Chinesemedicine, and exploring the material basis of traditional Chinese medicineand the role of the mechanism, etc. It is a necessity for the activeingredients in traditional Chinese medicine to research their absorption,distribution, metabolismand excretion in the body.In this experiment, Orientin monomer was extracted from Trolliuschinensis. Pharmacokinetic characteristics of orientin was studied in NewZealand white rabbits by three kinds of adminastrtion, intheveins, muscles,intraperitoneal injection. The study made a meaningful exploration formechanisms of orientin and the material basis of pharmacodynamic study. It will provide a scientific basis for clinical rational application of the drugand the evaluation of the quality of preparation and the development of thenew drug.1 The extraction and separation of orientin fromTrollius chinensis.In the experiment, a HPLC method for determineation of orientin wasestablished. At the same time, a preparative HPLC method was establishedfor purifying orientin monomer. Experimental procedures are follows: (1)The extraction of total flavonoids in Trollius: the crushed dry Trollius wasweighed. The pigment in Trollius was removed with acetone refluxextraction twice. The dried residue was extracted twice by 70% ethanol(solid-liquid ratio 1:20), at 80℃water bath, with the time of extractionbeing 1.5h. And then filtration was combined. In the end, the totalflavonoids was obtained. Ethaol was recovery and the flavonoid wasdiluted by water. the diluted flavonoid was concentrted by centrifugal10000 r·min-1. (2) Purification of flavonoids: The 300ml concentratedflavonoid liquid was added to the AB-8 macroporous resin column and itwas adsorbed staticly by the resin column in about 20min. First theimpurities was washed away by the distilled water four times as large thecolumn. And then the column was eluted with 70% with the flowing speedof eluation. Being 5 mL·min-1. Orientin was detected by HPLC at any time.(3) Orientin monomer separation and preparation: The evaporated solidwas dissolved by the mobile phase and filtered by 0.45μm organ-micropore.The solution was injected to PHPLC. The requirement fraction wasassembled and freezed. In the end the orientin monomer was obtained. (4)The content of orientin samples was determined by high performanceliquid chromatography. Both orientin standard and thin-layerchromatography are used for the qualitation and quantitative analysis oforientin. Orientin was identify by1H-NMR.The results show that content of orientin was above 98%, orientinrecovery 99.8%, RSD 0.47%. It can be seen, That method is quite simple. Orientin monomer wasused to control quality of drugs in pharmacokinetic study.2 Studies on pharmacokinetic of orientin in rabbitIn this experiment, the HPLC was used to determine theconcentrations of the orientin in plasma and tissues of New Zealand whiterabbits. Pharmacokinetic characteristics of orientin in NewZealand whiterabbits was compared by three kinds of administration. Such as theveins,muscle and intraperitoneal injection. After intravenous administration,distribution of the tissue of orientin glycosides in New Zealand whiterabbits was researched.(1) Study on pharmacokinetic of orientin in rabbit18 rabbits were divided into three groups, with one dose 15mg·kg-1,After administrating of orientin, blood was collected at different timepoints iv (2.5, 5, 10,15, 20, 25, 30, 35, 45, 55, 65, 80, 95, 110, 125min). im(5, 10, 15, 20, 25, 30, 35, 45, 55, 65, 80, 95, 110, 120, 160, 220, 280min),ip (5, 10, 15, 20, 25, 30, 35, 45, 55, 65, 80, 95, 110, 120min).The drugconcentrations were determined by HPLC. At last, according to theprinciple of‘AIC least and R biggest’, 3P97software was used to analyzethe drug concentration-time data and gain the best compartment model andthe pharmacokinetic parameters.(2)Tissue distribution study of orientin42 New Zealand white rabbits were prepared with 12-hour overnightfast, and freely drinking water. They were randomly divided into 6 groups,ear vein injection of orientin (20mgkg-1). After 5, 10, 15, 30, 45, 60, 75 min,they were killed by bleeding respectively. Heart, liver, brain, kidney, lungand spleen tissues were collected immediately. Surface of various tissueswas flushed by saline and surface moisture was dried by filter paper. Andthen it was cut to pieces after precisely weighing. And the saline was addedaccording to 1g : 1ml ratio, and 50% of the tissue fluid was prepared withelectrical homogenizer under ice bath. And it was stirred by 4000r·min-1 centrifuge for 10 min. And then the supernatant was obtained. Afteradding twice as much as the volume of acetonitrile to the supernatant,protein was removed by 10000r·min-1 centrifuge for 10 minutes, thesupernatant was opreservated at -20℃. the drug content in various tissueswas determined by HPLC.The experiment results include that the pharmacokinetics of orientinfit two–compartment model. The main pharmacokinetic parameters at onedose of 15 mg·kg-1. iv, ip, im are as follows: T1/2?: 4.61, 8.19, 6.39 min;T1/2β:40.76, 109.34, 59.55min; V 1.54, 8.66, 7.33 L·kg-1; AUC(0-t):264.36,93.78, 67.95μg·min·mL-1; AUC(0-∞): 310.82, 170.71,101.07; MRT: 28.47,54.29, 48.38min;CL(S): 0.106, 0.1190, 223L·(kg·min) -1. Orientin wasabsorbed and eliminated immediately in rabbit. After administration of 20mg·kg-1iv of Orientin on rabbit, AUC of heart, liver, brain, kidney, lung,spleen was 26, 285, 15, 552, 163, 38μg·min·mL-1. The content of orientin inkidney was the highest after 15 minutes. The research illuminated there wasno accumulation of loganin in rabbit tissues.The above results show that the pharmacokinetics of orientin in vivoof New Zealand white rabbits fit two-compartment open model by i.v, i.m,i.p. Orientin was absorbed and right away in rabbit. Orientin wasdistributed extensively in tissues such as kidney, liver and lung. It wasdifficult for orientin in crossing the blood brain barrier. |
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