| This research is part of the National Basic Research Development Program of China (National973Program, No.2007CB512605)-The essential research focus on Traditional Chinese Medicine (TCM) properties is based on the "three-element theory of Chinese Medicine". The central hypothesis is that TCM properties are a result of the combined effect of chemical components, bioactivity, and biological status of the organism. The organism, which has been considered to be the most important of the three elements, is a carrier of the expression of drugs’bioactivity. In the three-element theory of TCM, the status of an organism determines how drugs are absorbed and metabolized, thus leading to different biological effects. Curculigo orchioides Gaertn., a traditional Chinese medicinal herb, is one of our candidates. In our previous study, we found that orcinol glucoside, a component from the water extraction of Curculigo orchioides Gaertn. rootstalk, could be absorbed in plasma. Experiments on the absorption and distribution of orcinol glucoside in rat tissues, found that the highest amounts of the compound concentrated in the liver and kidney. Additionally, the absorption of orcinol glucoside is significantly different depending on the status of the organism. However, more research is needed to understand the absorption mechanism of orcinol glucoside.The first experimental objective, based on our previous study, was to isolate orcinol glucoside from Curculigo orchioides Gaertn. in sufficient quantities, and determine its purity using High Performance Liquid Chromatography (HPLC). Next, the mechanism of absorption of orcinol glucoside was investigated by using non-everted intestinal sacs as an in vitro model, in order to explain why the metabolism of orcinol glucoside is significantly different in different biological status.This dissertation is presented in two parts.In part1, the various chemical constituents and pharmacological effects of Curculigo orchioides Gaertn. were reviewed. Moreover, experimental methods, models, and current research on the intestinal absorption of drugs were investigated and discussed.In part2, phytochemical isolation, purity confirmation and small intestinal absorption studies of orcinol glucoside from Curculigo orchioides Gaertn. were performed.1. Based on our previous study of Curculigo orchioides Gaertn., we found that good extraction efficiency for orcinol glucoside was achieved by using80%ethanol in water as an extraction solvent. Through our isolation method,500milligrams of orcinol glucoside were isolated from Curculigo orchioides Gaertn. By HPLC determination, the purity of orcinol glucoside isolated was≥97%.2. The intestinal absorption study of orcinol glucoside(1):The detection and quantification of orcinol glucoside by HPLC was investigated as a method for monitoring the presence of the compound in rats’intestine. We simplified the method of processing samples, and finalized the HPLC methodology to be used in the absorption studies. The HPLC methodology includes Linear, Recovery Rate, Precision, Stability and Specificity results. Our conclusion was that this methodology is accurate, reliable and stable, which means it could be applicable to studying the intestinal absorption of orcinol glucoside. Our HPLC studies will be the basis for the following experiment.(2):To study the transport properties of orcinal glucoside, absorption studies were performed using the non-everted intestinal sac method. Studies of different sections of the rats’intestines showed that there was no significant difference among duodenum, jejunum and ileum (p>0.05) in absorptivities, but the apparent absorption coefficient of ileum ranked the highest. Efflux transporter P-glycoprotein (P-gp) and influx transporter organic anion transporting polypeptide (Oatp) were both studied. The results revealed that the efflux transporter P-glycoprotein (P-gp), and the influx transporter family of organic anion transporting polypeptides (Oatp) are involved in the absorption of orcinol glucoside across the rats’ intestinal membrane. It was found that the P-gp inhibitor, verapamil hydrochloride, significantly increased the absorption of orcinol glucoside from apical to basal side of the intestinal membrane, while the Oatp inhibitor, indometacin, decreased absorption. Additionally, different pH values were studied. Our conclusion, based on these varying pH studies, was that the absorption of orcinol glucoside showed no significant difference in buffer solutions with different pH values.One conclusion is that the bioavailability of orcinol glucoside is mechanistically determined by the active transporters of cell membranes-P-gp and the Oatp’s. This conclusion corresponds with our previous findings-the level of P-glycoprotein in the small intestine of normal conditioned rats is significantly higher than that of the Yang-deficiency status group, while the distribution of orcinol glucoside in liver and kidney of Yang-deficiency rats is significantly higher than that of the normal conditioned group. Because orcinol glucoside is a substrate of P-glycoprotein, the absorption of orcinol glucoside is affected by the drug efflux of P-gp. When the level of P-glycoprotein is lower in Yang-deficiency rats, than in the normal status group, the drug efflux effect of P-glycoprotein is correspondingly slower, leading to a higher transportation rate of orcinol glucoside from apical to basal side across the rats’ intestinal membrane, and a higher distribution of the glucoside in liver and kidney tissues. However, further research on the level and changes of Oatp’s in different biological status is still needed.Until now, no references about the mechanism of gastric and intestinal absorption of Curculigo orchioides Gaertn. are available. This dissertation focuses on the intestinal absorption of orcinol glucoside, the content of which is high in Curculigo orchioides Gaertn. In our previous study we were able to show that orcinol glucoside is absorbed in blood. Now, by adopting the non-everted gut sac method, we can show how different factors affect the intestinal absorption of orcinol glucoside. Our studies have given some experimental basis for elucidating the differences of absorption of orcinol glucoside in different biological status. |
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