Study On Separation And Analysis Of Effective Constituents From Some Medicinal Plants With RP-HPLC

Traditional Chinese medicine coming from the nature is the intelligence gathering of human beings and has been used widely in clinic as a special drug. Chinese patent medicines account for one thirds of all drugs listed in the catalogue of basic medicines of the state promulgate in 2009. Limited by the level of technology in history, many bioactive components and the corresponding mechanism of action of the Chinese patent medicines are not clearly understood. Thus it is hard to make sure the security, the effectiveness and the quality control of these drugs. Over the past decade, our government promoted the researches of the traditional Chinese medicine with modern technologies to improve the quality standard and the clinic curative effect of them. On the other hand, the state carries out major science and technology projects and promotes the development of new traditional and herbal drugs for the aim of bioactive components basically known and the corresponding mechanism of action basically clear. So it poses a rigorous challenge for the study of traditional Chinese medicine and natural pharmaceutical chemistry. At the same time, it also affords a chance to open up a new field for biochemical engineering discipline.It is the heart of the matter to find out the active components from traditional Chinese medicines or medicinal plants and reveal the bioactive components and the corresponding mechanism of action in the field of the research of Chinese medicine. The key point is to build up the suitable method for separation and analysis. In other words, it is the emphasis of the modern investigation of traditional Chinese medicines or medicinal plants to separating the bioactive components and identifying the corresponding compounds. Preparative high performance liquid chromatography (HPLC), with high separation effect and a lot of separation modes, has been powerful tool for separating complex systems including many compounds. The separation effect of preparative HPLC is decided by operation conditions of chromatography. At present, the operation conditions for the preparative HPLC are mainly selected by operators according their experiences, which is blindfold. So it is necessary to study the theoretical model of separation and the elution rules of preparative HPLC intensively and to develop the new method for optimizing the operation conditions of chromatography.In this paper, we selected Viburnum dilatatum Thunb. and Caulis Spatholobi as subjects investigated at first and separated compounds with traditional methods. To analyze compounds extracted from Viburnum dilatatum Thunb. with methanol, a new compound,2-(β-glucopyranosyloxy)-benzyl 3-((3-glucopyranosyloxy)-benzoate, was found out. According to the anti-tumor experiment in vitro, we screened out bioactive components and analyzed the structures of the corresponding compounds with HPLC-MSn. The results showed that the bioactive components mainly contained nine flavones and three isoflavones:7-hydroxy-3',4'-dimethoxy-isoflavone, 7-hydroxy-6,2',4'-trimethoxy-isoflavone and 3'-hydroxy-7,4'-dimethoxy-isoflavone were reported at first time in this plant.Basing on the analysis of flavones from Caulis Spatholobi with analytical reverse-phase HPLC (RP-HPLC) and according to the retention models of chromatography, we predicted the retention time and the peak width of the same substance in preparative RP-HPLC with the computer programs wrote with matlab soft. The optimized operation conditions would be predicted after setting the separation index. Compared with traditional method for deciding the operation conditions of chromatography, the method developed in this paper was with advantages of flexible, convenient, high effective and economical.On the base of the study of the new method for prediction of separation conditions of preparative RP-HPLC, SID, a new integration method, was developed again, which could be used to separate and identify phenolic acids from plants and determine parameter of pKa of compounds in complex system at the same time. The SID method included processes of phenolic acids judgement, pKa prediction, conditions of preparative RP-HPLC optimization, sample purification, substance identification and pKa determination. In this paper, we selected phenolic acids from Danshen as subject investigated and determined the pKa values of danshensu, caffeic acid, lithospermic acid, rosmarinci acid, salvianic acid B and salvianic acid A. The unknown compound prepared with preparative RP-HPLC was identified with HPLC-MS and 'H NMR. The result showed it was salvianic acid A. The method could improved the separation effect of phenolic acids and test the pKa values of them without getting pure samples of phenolic acids. So the SID method was a promising method.