| Objective:Rhodiola, Ganoderma lucidum, Grape seed and American ginseng were clinical experience prescription with effect of anti-anoxia and anti-exercise-induced fatigue, the purpose of this project was to make this prescription to be chewable tablets with good test which was more convenient for patients to take, by resaching for the preparation technology and Quality standard. Using resveratrol as a model drug, to investigate the effect of Rhodiola, Ganoderma lucidum and Grape seed on the glucuronidation of resveratrol.Methods:(1) According to the physical and chemical properties of the active ingredient in each herb used in this prescription, chose eligible extraction method, and optimized the extraction process by orthogonal experiment.(2) According to the taste, appearance and hardness request of chewable tablets, the appropriate excipients of fillers, corrigents, wetting agents and lubricants were screened by the single factor test.(3) TLC qualitative distinction was used to inspect the Rhodiola, Grape seed and American ginseng in the prescription, the assay method of salidroside in Rhodiola was established through methodology investigation.(4) Developing an Ultra Performance Liquid Chromatography (UPLC) spectrometry method for the analyzing both trans-resveratrol and trans-resveratrol-3-O-glucuronide. To investigate the effects of each herb, used in this prescriptionl, on the glucuronidation of resveratrol which was assessed with liver microsomes, the everted gut sac model and the in vivo experiment.Results:(1) Extraction process of Rhodiola was determined:with10times of80%ethanol by triple, each time for2hour.Extraction process of Ganoderma lucidum was to extract with water decoction for4times, each time20-time water for2hour. Extraction procss of Grape seed was determined:with10times of50%ethanol by triple for4.Oh (the first time for2.Oh, the last two times for1.Oh each time.(2) The chewable tablet was prepared:Rhodiola, Ganoderma lucidum, Grape seed were mingled with the1.5times β-Cyclodextrin and American ginseng,1.5times mannito,3%Steviosides,0.5%peppermint flavor,1.5%orange flavor were mixed in evenly.95%ethanol and1%magnesium stearatewas were used as wetting agent and lubricant respectively. After granulation,sieving and tabletting came the end item.(3) Quality specification of the chewable tablets was established. TLC qualitative distinction could inspect the active agents in Rhodiola, Grape seed and American ginseng. The methodology consideration result indicated that:the precision, the stability, the reproduction quality and application of sample recovery rate can meet the requirement.(4) It was found that all the three selected compounds could produce a dose-dependent inhibition on the glucuronidation of resveratrol. The IC50of each compound was:Grape seed (0.001%), Rhodiola (0.030%). Ganoderma lucidum (0.006%), Grape seed was the best in the inhibition on formation of the glucuronidation of resveratrol in liver microsomes model. However the opposite results was observed using the everted gut sac model, the three selected compounds had no effect on the absorption and glucuronidation of resveratrol, no significant difference in overall was seen between the groups. The results of the experiment in vivo was demonstrated that there was no significant difference of the AUC of trans-resveratrol and rans-resveratrol-3-O-glucuronide when gavage trans-resveratrol alone compared with the three selected compounds combined with the trans-resveratrol.Conclusion:Extraction process of each ingredients in the prescription was reasonable, the data was reliable; The chewable tablets had good test and was more convenient for patients to take. The chewable tablets conformed to regulations of the tablet item in2010edition of Chinese Pharmacopoeia. The quality standard of the preparation was established and the determination method was stable and reliable.The reason for that different results were observed in liver microsomes model and the everted gut sac model, maybe that liver microsomes model was deficient in consistency comparing with the metabolization in vivo, even though this method could eliminate the interference factor of vivo and obseved the metabolization of drug more directly, while the everted gut sac had healthy tissue and mucous membrane, was more close to situation in vivo. The results of the experiment in vivo was consistent with the everted gut sac model.Consequently, the conclusion was the three selected compounds had no effect on the absorption and glucuronidation of resveratrol. |
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