| Rhizoma Smilacis Glabrae(RSG), the dried root of Smilax glabra Roxb.(Family Liliaceae), first recorded in the appendix of Shennong’s classic of Materia Medica is a well known Chinese herbal medicine with the homology of medicine and food. It has long been used in folk medicine to treat syphilis, leptospirosis, dermatitis, brucellosis, acute bacterial dysentery, acute and chronic nephritis and metal poisoning. However, for RSG, a drug with a long history of over one thousand years, there are still no generally accepted quality standards. Few researches have been reported about the advanced extraction techniques,pharmacodynamic material basis and metabolic processes in vivo of RSG. In this study, the advanced sample pretreatment technique and the multidimensional chromatography combined with mass spectrometry were used in the extraction, sepeartion, identification and preparation of RSG, which contribution to establishment of the generally accepted quality standard in vitro and illumination of the metabolic process in vivo of RSG. First of all, optimum extraction parameters of advanced extraction method ASE were investigated using mono-factor analysis, and high performance liquid chromatagraph coupled with diode array, electrospray ionization ion trap tandem mass spectrometry(HPLC-DAD-ESI-ITMSn) method was established to separate and analyze the component of effective components from RSG. Then, two different ploarities of compounds groups in RSG alcohol extractive separated by macroporous resin together with the RSG polysaccharide group were evaluated the pharmacological effects through the experimental hiperuricemia mice model and the model of lead poisoning of male wistar rats. The active ingredients were further preparated by high-speed counter current chromatography combined with preparative HPLC. Finally, the flavonoids prototypes and metabolites were detected in plasma and urine after oral administration of RSG to speculate the possible metabolic pathway of RSG in vivo. In all, the investigation on the material basis and metabolic characteristics to clarify the pharmacological ingredients and mechanisms is of great significance for further development and utilization of RSG. The detailed contents were presented as follows:1. Optimal extraction and qualitative fingerprint analysis of Rhizoma Smilacis Glabrae by accelerated solvent extraction and high-performance liquid chromatography coupled with tandem mass spectrometryFirstly, the operational parameters of ASE including extraction solvent, extractiontemperature, static extraction time, solid-to-liquid ratio and extraction cycles were optimized, and the results showed that effective extraction can be carried out at 40℃ with4 minutes for one static and 0.5/10(g/ml) of solid-to-liquid ratio using a 20% ethanol as extraction solvent for all the active compounds studied. Meanwhile, compared to traditional extraction methods, such as reflux extraction and ultrasonic extraction under the optimal conditions, in which extraction contents for the 6 active flavonoids were similar, the ASE technique has the advantage of saving time and energy and better reproducibility. Eight active compounds were identified in RSG extraction by using HPLC-DAD-MSn, and further confirmed coupled with UPLC-TOF-MS, of which retention orders were contradictory among the literatures. Therefore, the qualitative fingerprint for RSG established is the foundation for the quality control and action mechanism of this herb.2. Material foundation study of medicinal effectiveness for decreasing the concentration of uric acid in serum on Kunming mice and lead-excreting function on saturnism wistar rats and the preparation of effective compoundsRSG has long been used in clinical to treat hyperuricaemia and gout, as well as in the elimination of heavy metal with the effects of heat-clearing, detoxication, dampness dispelling and collaterals dredging as a compound Chinese herb or medicine preparation.In this chapter, firstly, the RSG extraction were separated into two groups with small polarity and middle polarity by using macroporous resin, as well as RSG polysaccharide group using the method of alcohol sedimentation.Then, the hyperuricemia mice model was established to screen the effective compound group with the alloporinol as positive control drug. The results showed that the middle polarity group exhibited hypoglycemic effect on hyperuricemia model animals, and there was no significant difference between the group and positive group(p>0.05). The results of determination of activeties of xanthine oxidase(XOD) in the liver of each group of mice also showed the values of XOD in the middle polarity group were lower than that of blank group as well as in the positive group. it could be speculated that its mechanisms of lowing serum urate might be associated with inhibition of XO activity just like alloporinol.The model of lead poisoning of male wistar rats was also established to screen the effective compound group of RSG with the dimercaptosuccinic acid(DMSA) as positive control drug. The content of lead in the blood, urine, heart, liver, spleen, kieney and femurof lead-exposured rats were determined by non flame atom absorptance before and after administration for 21 days. The results showed that, the lead concentration of blood and contents in liver, spleen and kidney of rats in all groups were decreased compared with that before treatment, and there was significant difference between before and after treatment(p<0.01). Besides, lead contents in heat of rats in group with small polarity, in femur of rats in two groups of two polarities, and in brain of rats in group with middle polarity were all decreased in addition to the positive control group. Comparison of every two groups that of before and after treatment were significant different(p<0.05).What’s more, based on the fact that the effective compounds groups of RSG was found, analytical high speed counter chromatography(HSCCC) combined with preparative HPLC were employed to prepare effective monomers of RSG. Four monomers were larg-scale obtained, which were neoastilbin, astilbin, neoisoastilbin and isoastilbin. In all,the technique platform to preparation of active monomers from RSG was established based on efficacy tests on the hyperuricemia models and lead-exposured models by using multidimensional column, HSCCC and preparative HPLC.3. Quality control of multi-compounds for RSG by quantitative fingerprint analysis coupled with pattern recognitionIn china, although abundant RSG resources can be obtained from several provinces,there were obvious differences of chemical components among the RSG samples from various origins, which may affect the clinical efficacy seriously for lacking of quality control of multi-compounds so far. In this chapter, based on the developed ASE and HPLC methods, chromatography fingerprints were established for the discrimination of sixteen batches of Rhizoma Smilacis Glabrae samples and further quality control. Firstly,Hierarchical cluster analysis(HCA) was performed to classify 16 samples based on the similarities of their chemical properties, where RSG samples were clustered to two groups,labeled as ‘Unqualified’ and ‘Qualified’. Next, the 12 RSG samples clustered in“Qualified” group were analyzed by similarity measurement to examine the similarity of the samples, which results indicated that the twelve qualified samples shared similar chromatographic patterns. Principal component analysis(PCA) was then utilized to provide an overview of the capacity to distinguish RSG based on HPLC chromatographic data. According to the loading plot, quite a few discriminating components were obtained,based on which six main flavonoids whose reference substances are available, namely toxifolin, neoastilbin, astilbin, neoisoastilbin, isoastilbin, engeletin, were selected for thequantitative determination of RSG. It was found that although 12 qualified samples shared similar fingerprint properties from a chemical viewpoint, content of each analyte varied significantly among the different samples. Therefore, the quantitative analysis of multi-compounds is very important to the quality control of Chinese herb medicinethe(HB). Meanwhile, chromatographic profile of HB combined with pattern recognition methods could comprehensively evaluate the quality of HB.Above all, with modern techniques and pharmacodynamics screening models experiments, the platform was reliable, efficient and comprehensive for the study of mainly active flavonoids from RSG containing extraction, separation, identification,quality control and metabolic analysis. So the study is meaningful for the investigation of pharmacodynamic material basis and the mechanism of action of RSG. |
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