| Radix polygoni multiflori is the dried root of polygonum plant Polygonum multiflorum Thunb.. It has many pharmacological actions. Recent studies showed that Radix polygoni multiflori had antihyperlipidemic and antiatherosclerotic effects. 2, 3, 5, 4′-Tetrahydroxystilbene-2-O-β-D-glycoside (stilbene glycoside, TSG) is one of the water soluble bioactive components in Radix polygoni multiflori. Many studies have documented the beneficial properties of TSG, including its strong antioxidant and free radical-scavenging. Possibly because of these properties, recent studies suggested that TSG could have preventive and therapeutic effect against some chronic diseases, such as apolexis, senile dementia (Alzheimer's disease), hyperlipemia and atherosclerosis and so on.In this present study, the antihyperlipidemic effects of active fraction and TSG from Radix polygoni multiflori has been studied and compared. The study suggested that TSG was the active constitute of Radix polygoni multiflori for its antihyperlipidemic effects. Absorption, distribution, metabolism and excretion of TSG in rat were investigated. The results of this work will contribution to its development and utilization.Qibaomeiran pill, a well-known compound preparation of traditional Chinese medicine, consists of seven herbs. Radix Polygoni Multiflori was the principal drug. In order to preferably control the quality of Qibaomeiran pill overall, a new RP-HPLC method was established for simultaneous determination of seven active components with photo diode array detection.Part one Experimental studies on antihyperlipidemic effects of constitute from Radix polygoni multifloriObjective: To investigate the antihyperlipidemic effects of active fraction (HSWAF) and active constitute stilbene glycoside (TSG) from Radix polygoni multiflori. To illuminate the therapeutical basis of Radix polygoni multiflori for its antihyperlipidemic effectsMethods: The effects of HSWAF and TSG on serum lipids and liver index were studied in normal mice. Using the concentrations of lipids, glutamic oxalacetic transaminase enzyme (GOT) and glutamate-pyruvate transaminase (GPT) in serum in the model mice with acute hyperlipidemia induced by intraperitoneal injection of Triton as markers, the influences of HSWAF and TSG were observed. The investigation was also employed in the hyperlipidemic model rats induced by feeding with the high-lipid diet accompanied by oral administration of HSWAF and TSG to the rats for 28 days, respectively. The effects of HSWAF and TSG were investigated by measurement of the concentrations of lipids, malondialdehyde (MDA), nitric oxide (NO), and the activity of superoxide dismutase (SOD) in serum in the hyperlipidemic rats. The contents of total cholesterol (TC) and triglyceride (TG) in liver as well as liver index were also determined.Results: In the normal mice, HSWAF and TSG not only increased the level of serum high density lipoprotein cholesterol (HDL-C), also decreased the level of serum low density lipoprotein cholesterol (LDL-C). HSWAF and TSG could lower serum TC and TG, and increase serum HDL-C in the hyperlipidemic mice. Administration of HSWAF (60 mg/kg) significantly reduced the liver index in hyperlipidemic rats. Administration of HSWAF (30, 60 mg/kg) could remarkably decrease the levels of serum TC, TG, LDL-C, MDA as well as the ratio of TC/HDL-C, also remarkably increase serum HDL-C, NO and SOD. Compared with HSWAF, TSG had the somewhat lower effect but no significant difference.Conclusion: HSWAF and TSG possess obvious lipid-regulating, antioxidative and protecting vascular endothelium effects. The main bioactive constitute in HSWAF was TSG. Such characteristics will be of significance to prevent and/ or treat hyperlipidemia.Part two Studies on pharmacokinetics and tissue distribution of TSG in the hyperlipidemia model rats Objective: To study the characteristics of pharmacokinetics and tissue distribution of TSG in the hyperlipidemia model rats induced by different factor.Methods: 1. Normal rats, the hyperlipidemic model rats induced by feeding with the high-lipid diet and the hyperlipidemic model rats induced by intraperitoneal injection of Triton, all of them were orally administrated TSG at a dose of 60 mg/kg, and blood samples were obtained from fossa orbitalis vein according to the specific schedule and collected in heparinized centrifuge tube, respectively. Plasma was obtained by centrifugation at approximately 3 000 r/min for 10 min. Polydatin was taked as the internal standard. Three-time volume of methanol was added and the content of tube was mixed to precipitate the plasma protein, and then centrifuged. The supernatant was evaporated to dryness under a stream of nitrogen in the thermostatically controlled water-bath maintained at 50℃. Thereafter, quantitative volume of methanol was added to it and vortexed for 45 s and then centrifuged at 12000r/min. The supernatant (20μl) was injected into the HPLC system. The C18 column (250×4.6 mm, 5μm) was used as the stationary phase with the mobile phase consisting of acetonitrile-methanol-0.1 % glacial acetic acid (12∶10∶78). The flow rate was 1 ml/min. The UV detector was set at 320 nm. The peak area ratio of TSG and Polydatin were used for predicting unknown concentrations from the regression equation. 2. Normal rats, the hyperlipidemic model rats induced by feeding with the high-lipid diet and the hyperlipidemic model rats induced by intraperitoneal injection of Triton were randomly assigned to three groups. After oral administration of 60 mg/kg water solution of TSG , Heart, liver, spleen, lung, kidney, brain, stomach and small intestine samples were obtained at 5, 15, and 45 min, respectively. Tissue samples were homogenized in saline solution (1∶2 w/v). Three-time volume of methanol was added to the tissue homogenate to precipitate the protein, and then centrifuged. The supernatant (20μl) was injected into the HPLC system. The C18 column (250×4.6 mm, 5μm) was used as the stationary phase with the mobile phase consisting of acetonitrile-methanol-0.1 % glacial acetic acid (15∶18∶67). The flow rate was 1 ml/min. The UV detector was set at 320 nm. The peak area of TSG was recorded to determine the content of TSG in each sample.Results: The calibration curve in plasma was linear over the range of 0.13~81.00μg/ml and the RSD values of intra-day and inter-day were less than 3 %. The recoveries of TSG in three different concentrations (high, middle and low) were 99.7 %,98.9 % and 98.3 %, respectively. Compared with the normal rats, in the hyperlipidemic model rats induced by Triton, the Tmax was lower, t1/2 was highly significant shorter (p<0.01), Cmax, Ke, AUC0-t and AUC0-∞were all highly significant increased (p<0.01); in the hyperlipidemic model rats induced by high-lipid diet, only AUC0-t and AUC0-∞had highly significant increase (p<0.05). Compared with the normal rats, the relative bioavailability of TSG in Triton model rats and high-lipid diet model rats were 188 % and 155 %, respectively. 2. The standard curve range were 0.65~162.00μg/ml in all tissue homogenate samples. The recoveries of TSG in three different concentrations (high, middle and low) were 100.5 %,102.3 % and 100.9 %, respectively, and the RSD values of intra-day and inter-day were less than 3 %. Compared with the normal rats, in the hyperlipidemic model rats induced by Triton, the concentration of TSG in kidney was slightly decreased; in the hyperlipidemic model rats induced by high-lipid diet, the concentration of TSG in heart was highly significant decreased, while there were obviously increased in liver, spleen and kidney.Conclusion: The characteristics of pharmacokinetics and tissue distribution of TSG in the hyperlipidemia model rats had taken place some change compared with the normal rats. The results suggested that the physiologic condition of animal must be consideration to during the investigation. Patho-model animal should be choosing under the possible conditions in order to draw the real conclusion.Part three Drug-protein binding determination of TSGObjective: To study the binding of TSG to plasma, albumin andα1-AGP and to prove the availability of cloud-point extraction (CPE) in the determination of protein binding ratio of TSG.Methods: In the present investigation, the binding of TSG to plasma, albumin andα1-AGP (2.0, 10.0 or 50μg/ml) was investigated by three different methods- ultrafiltration, equilibrium dialysis and CPE, and compare the results obtained from the three methods. The process of CPE as follow: In a test tube 1.0 ml of a 5 % (w/v) aqueous solution of Triton X-114 is added to 0.2 ml of sample. The phase separation was observed at 35℃. The extraction mixture was kept at this temperature for 10 min. After centrifugation at 3 000 r/min for 5 min the upper dilute aqueous phase (850μl) with the free fraction of TSG was removed from the surfactant-rich lower phase (350μl). The quantitive surfactant-rich lower phase was transferred into a tube for determining the concentration of bound drug.Results: The results to plasma obtained by CPE were in good agreement to these observed by ultrafiltration and equilibrium. The binding ratios(%) of TSG to plasma in three different concentrations (0.05, 0.25 and 1.25mg/ml) obtained by equilibrium, ultrafiltration and CPE were 55.8±4.1, 53.4±4.4, 51.9±5.0; 79.6±3.3, 74.2±3.3, 72.5±2.6; 89.8±1.8, 85.0±3.6, 87.4±1.3, respectively. The results obtained by ultrafiltration and equilibrium showed that binding albumin was constant (about 60 %) within concentration range studied, while the binding toα1-AGP decreased with increasing drug concentration, but the results obtained by CPE was very different form these.Conclusion: CPE was a highly sensitive and selective method for the measurement to plasma protein binding of TSG. Both albumin andα1-AGP were important for TSG protein binding. It is possible that some other proteins could contribution to the binding of TSG to plasma.Part four Studies on the absorption kinetics of TSG in ratsObjective: To develop a high-performance liquid chromatography coupled with diode array detection (HPLC/PDA) method for simultaneous determination of TSG and phenolsulfonphthalein in the circulation solution and to study the absorption kinetics of TSG in stomach and intestine in rats with the in situ perfusion. Methods: The experiments were performed using the in situ perfusion method in rats. The concentrations of TSG and phenolsulfonphthalein were determined by HPLC/PDA with C18 column (250 mm×4.6 mm, 5μm) as an analytical column and a mixture of acetonitrile-methanol-0.2% phosphoric acid (v/v) (35∶15∶50) as a mobile phase at 1.0 ml/min of the flow rate. The column compartment was kept at the temperature of 30℃. The detection wavelenghs were chosen at 320 nm and 430 nm to record chromatograms of TSG and phenolsulfonphthalein, respectively.Results: Calibration curves were generated over a concentration range of 3.5~140μg/ml for TSG and 1~40μg/ml for phenolsulfonphthalein, respectively. Intra- and inter-days variations were less than 3.1 %; Recoverys of TSG and phenolsulfonphthalein were among 99.48 % to 102.5 %. The hourly absorption percentages of TSG (2.5, 5 and 10 mg/ml) in stomach were 72.7 %, 67.7 % and 56.6 %, respectively. The absorption rate constants of TSG (30, 60 and 120μg/ml) in intestine were 0.047 7, 0.051 4 and 0.056 3, respectively and no significant difference (P>0.05) among them.Conclusion: The measurement of TSG and phenolsulfonphthalein in the circulation solution was achieved by HPLC/PDA method. The method was sensitive, accurate, and simple. The results indicated that TSG was poor absorbed at intestine but well absorbed at stomach in rats. So TSG could be prepared as floating sustained-release tablet to prolong the retention time at stomach to improve bioavailability.Part five Metabolism of TSG in vivo and in vitroObjective: To study the metabolites and metabolic pathway of TSG in vivo and in vitro.Methods: Culture solution of intestinal bacteria produced from rat feces and hepatic microsomal enzyme were used to research the metabolism of TSG in vitro. After taking orally TSG, blood, bile, urine, feces, contents of stomach and contents of intestine were pretreated and analyzed by HPLC/PDA. Metabolites of TSG were purified by HPLC and identified by 1H-NMR, 13C-NMR and MS. Results: Only TSG was present in both gastric content and culture solution of intestinal bacteria. TSG and its metabolites were not detected in the urine samples. In addition to free TSG, metabolite M2 was detected in the intestine content. TSG and metabolite M1 (TSG-glucuronide) were detected in the plasma sample. In the bile sample, slight TSG, metabolite M1 and considerable metabolite M2 were all detected. In the feces, free TSG was the major form of TSG. In the liver microsome incubation mixture, metabolite M2 was high in proportion. Metabolite M2 was C3-OH TSG-glucuronide identified by 1H-NMR, 13C-NMR and MS, which different from metabolite M1.Conclusion: The results of this work demonstrated that TSG is metabolized to TSG-glucuronide mainly in rat liver, and be more easily excreted through bile. In the intestinal tract, TSG-glucuronides were hydrolyzed to TSG by bacteria or enzymes, and then out of body with defecation.Part six Studies on the excretion of TSG in ratsObjective: To develop a HPLC method to assess the concentrations of TSG and its metabolite in rat bile and identify the metabolic pathway of TSG in rat.Methods: After a single oral administration at a dose of 60 mg/kg, the bile of rat was collected. After methanol (10μl) was added into 200μl bile or diluted bile, and then centrifuged at a high speed for 10 min, the supernatant was injected into the liquid chromatographic system to determine the concentrations of TSG and its metabolite. Dikma Diamonsil C18 column (250 mm×4.6 mm, 5μm) was used. The mobile phase was composed of acetonitrile and 0.1 % (v/v) acetic acid solution (0~10 min, 15∶85; 15~23 min, 25∶75; 25~30 min, 100∶0). The flow rate was 1.0 ml/min and the column compartment was kept at the temperature of 30℃, 320 nm was chosen to record chromatograms.Results: Calibration curves were generated over a concentration range of 12.0~0.8μg/ml for TSG and 110~0.9μg/ml for the metabolite, respectively. Intra- and inter- days variations were less than 7.0 %, and relative errors were ranged within±4.33 %. Extraction recoverys of TSG and metabolite were all above 99.14 %. Mean biliary accumulated excretion of TSG and metabolite for 24 h after administration were (0.084±0.04) % and (36.87±12.94) %, respectively.Conclusion: This specific, sensitive and precise method is suitable for the determination of TSG and its metabolite in rat bile. Biliary excretion should be the main excretion route of TSG in the form of its metabolite. Part seven Development of the study model for physiological disposition of drug in rats and its application on TSGObjective: To develop a simple study model to research the absorption, distribution, metabolism and excretion of drug in rat, and to make use of it on TSG.Methods: The rats were anesthetized and kept alive under anesthesia throughout the experiments (In situ gastric absorption). At first, the biliary duct was cannulated. Bile was collected for up to 20 min after the administration. Second, the pylorus was ligated, and 15 mg of TSG in 0.5 ml artificial gastric juice at 37℃was injected into the stomach through the cardia by a syringe with plastic tubing. Immediately, the cardia was also ligated. At 20 min post-administration, blood was withdrawn with heparinized syringes from the portal vein and the celiac artery, respectively. Thereafter, the whole stomach was removed and immediately placed on ice (0℃). Urine was withdrawn from the bladder. Each sample was determined by HPLC after samples were prepared by the use of combined enzymatic hydrolysis to evaluate the content of free TSG, TSG-sulfate, TSG-glucuronide and TSG-diconjugate in each sample.Results: After 15 mg of TSG was incubated in rat stomach in vitro for 20 min, (64±9.8) % of administered TSG seemed to be absorbed from the stomach, (4±2) % of the dose was stored in the gastric mucosa, and (1.1±0.5) % of the dose was excreted through bile, but none TSG was found in the urine. From the concentration of total TSG in the plasma, it could be also estimated that about 1 % of the dose was in the blood pool. Compared with the portal vein plasma, in the arteries plasma, the proportion of free TSG to total TSG was declined, while the proportion of conjugated TSG to total TSG was increased. In the bile, TSG-glucuronide was the major form of TSG.Conclusion: TSG was absorbed from the stomach in the free form and transhepatic portal vein transported into the liver. In the liver, TSG was metabolized into conjugated TSG. TSG was excreted through bile mainly in the conjugated forms.Part eight Simultaneous determination of seven components including stilbene glycoside in Qibaomeiran pillObjective: To develop a high performance liquid chromatography coupled with photo diode array detection (HPLC/PDA) method for simultaneous determination of seven active components in Qibaomeiran pill.Methods: The separation was performed by a Dikma Diamonsil C18 column using gradient acetonitrile-0.1 % glacial acetic acid as mobile phase. The detected wavelengths were set at 245, 320, 290 and 350 nm. The column temperature was 30℃. In order to extract the seven components completely, different extraction methods, solvents and extraction time were compared. The optimal method was refluxing extraction 2.0 hours with alcohol as solvent.Results: The linear rangers of rutin, stilbene glucoside, ferulic acid, psoralen, isopsoralen, emodin and physcion were 0.0215~0.536, 0.0450~1.125, 0.0052~0.131, 0.0113~0.283, 0.0137~0.342, 0.0046~0.116 and 0.0018~0.044 mg/ml, respectively. Their average recoveries were 102.1 %, 101.4 %, 100.4 %, 100.9 %, 100.0 %, 100.2 % and 99.99 %, respectively. The results indicated that the precision and reproducibility were suitable. There is notable difference among the different manufacturers.Conclution: The method was proved to be very accurate, quick and stable to the quality control for Qibaomeiran pill. |
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