| 2, 3, 5, 4＇-tetrahydroxystilbene-2-O-β-D-glycoside (stilbene glycoside), is one of the water-soluble bioactive constituents in Radix polygoni multiflori. In order to clarify the absorption,distribution and excretion characteristics of stilbene glycoside and to guide the use of clinical drug aright, we separated the stilbene glycoside from Radix polygoni multiflori and studied pharmacokinetics with reversed-phase HPLC in rats by given drug intravenously and orally. Objective: To develop a method to separate stilbene glycoside from Radix polygoni multiflori which meets the requirements of pharmacokinetic study; To establish a reversed-phase HPLC method for determination of stilbene glycoside in plasma and tissue sample; To study the absorption,tissue distribution and excretion characteristics of stilbene glycoside in rats with HPLC. Methods: (1)To separate stilbene glycoside by using silica gel column chromatography with gradient elution consisting of ethyl acetate and petroleum ether. (2)The SD rats, ♀♂, weighing 300g were used in the experiments. (3)HPLC method was established to determine the concentration of stilbene glycoside. (4)Chromatographic separation was optimized by choosing appropriate stationary phase and adjusting the composition, proportion and flow rate to separate the peak of stilbene glycoside thoroughly. (5)Pretreatment of sample: Transfer 0.2 mL of the plasma or tissue samples, add two times volume of methanol to deposit protein, centrifugate, transfer the upper clear liquid, filter, inject 20μL of each sample into the instrument, record the peak area of stilbene glycoside and calculate concentration of stilbene glycoside in plasma or tissue. (6)The selectivity of the method: Take the blank plasma sample,stilbene glycoside reference solution and blank plasma sample spiked with reference solution into the instrument,and then record chromatogram respectively. (7)Preparation of standard curve in plasma: transfer 0.2mL of blank plasma samples, spiked with two times volume of a series of reference solution respectively; operate as same as the steps of item (3) from "centrifugate…", record the stilbene glycoside peak area, and then regression equation was obtained with concentration of stilbene glycoside as abscissa, the corresponding peak area as ordinate. (8)Confirmation of the lowest detection concentration: Dilute a series of low concentration reference solution spiked into blank plasma until the value of S/N was more than 3. Under this circumstance, the corresponding concentration was the lowest detection limitation. (9)In the experiment of recovery, transfer 0.2mL of blank plasma sample, spiked with different concentration of reference solution, inject it into the instrument for five times, determine the peak areas of stilbene glycoside and calculate the mean concentration of stilbene glycoside in comply with the same standard curve in the same day, and then calculated the recovery. (10)With-in day precision and between-day precision: Transfer blank plasma sample, spiked with three different concentration of stilbene glycoside reference solution, each concentration for five shares, determine the samples at the different time in the same day(with-in day precision) or in different day of a week(between-day precision). (11)In the experiment of stability, transfer the same plasma sample, determine the relevant peak areas at 0, 2, 4, 6, 8, 10 and 12 hour, respectively, and then calculate the RSD. (12)The experiment design of absorption: Rats were divided into four different groups randomly, six rats each group (iv 20mg·kg-1,iv 10mg·kg-1,ig 100mg·kg-1,ig 50mg·kg-1,respectively). Collect blood samples from carotid after given drug at the prearranged time (0,2,5,10,15,20,30,45,60,90,120min, intravenous injection groups; 0,5,15,30,45,60,90,120,150,180min, oral administration groups), and draw into tubes containing heparin sodium respectively. The plasma samples were separated by centrifugation at 4000 rpm for 10min, add two times volume of methanol to precipitate prot...
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