Study On The Chemical Constituents From The Stems Of Ilex Pubescens And Its Fingerprints Analysis

The traditional chinese medicine, Maodongqing, dry roots of of Ilex pubescens Hook. et Arn. of Aquifoliaceae, has been common used in some provinces in southern China. It has the funtion of dissipating heat and detoxifying, activating the blood and invigorating the pulse, detumescence. It is a kind drug for treating on colds, asthma, tonsillitis, stroke, paralysis, burns, bone trauma, bleeding, and carbuncle swollen. Terpenes, coumarins, sterols, tannins, sugars and amino acids and other types of constituents have been found in Maodongqing, triterpene saponins compounds is the main active component.Now, with the development and application of Ilex pubescens, more and more medicinal herbs in demand and the wild medicinal resources have been severely damaged. The problems such as mixed roots, quality varies greatly has arisen. Ilex pubescens study focused on roots based on separation of main chemical components and pharmacological action of activity, while studies on stems have not been reported. To ensure the drug safe, effective, standard medicine production and rational use of resources and development, the study from the viewpoint of the exploration of new resources, based on the study on the chemical constituents of the stems. Focusing on the quality of evaluation. On the basis of the studies on chemical constituents. Establish the chromatographic fingerprint of Ilex pubescens. Looking forward to determine whether the stems can replace root to be used and to improve its development and utilization.Study includes the chemical constituents of the stems of Ilex pubescens, the HPTLC chromatographic fingerprint analysis of Ilex pubescens and its related species and HPTLC-ELSD chromatographic fingerprint analysis of Ilex pubescens. The results are as follows:1. Five compounds were isolated and purified form the petroleum ether and the ethyl acetate extract of the stems. Though use the via silica gel and Sephadex LH-20column chromatography as well as recrystallization Their structures were determined on the basis of spectral analysis and physicochemical properties. Four of them were triterpenoid saponins compounds as IlexgeninA, IlexsaponinA,, IlexsaponinB1, IlexsaponinB2. One of them was steroid:β-Sitosterol. All of the five compounds were firstly isolated from the stems of Ilex pubescens.2. To develop the High-performance thin-layer chromatographic (HPTLC) fingerprint of triterpenoid from Ilex pubescens and compare fingerprints similarity with its related species, such as Ilex rotunda and Ilex asprella. The separation was performed on pre-coated HPTLC GF254silica-gel plate stationary phase with trichloromethane-ethyl acetate-methanol-water-formic acid(3:22:5:2:0.2)as solvent system,10%sulfuric acid alcoholic solution as color development reagent. The common pattern of HPTLC fingerprints were obtained through ’Chromafinger2005’ solution software, and authentication and quality assessment were analyzed by similarity and Principle Commonent Analysis. The common pattern of the pileus of Ilex pubescens consists of13characteristic peaks. There are some difference between different parts of Ilex pubescens and there are also notable difference between Ilex pubescens and its related species. The different parts of Ilex pubescens can be distinguished by comparison with the HPTLC chromatograph. The other relatives can be easily differentiated from Ilex pubescens.3. To develop the High performance liquid chromatography-Evaporative light scattering detector (HPLC-ELSD) fingerprint of the Roots and Stems fingerprinting of Ilex pubescens and compare fingerprints similarity with commercial herbs and its related species. The conditions of experment were confirmed and the validation of the method was also tested. The chromatography conditions were defined as Luna-C18(250mm×4.6mm,5μm), temperature of column was30℃, mobile phase was A:acetonitrile containing10%propan-2-ol, B:0.4%formic acid, gradient elution, flow speed was1.0mL/min, injection volume:20μL. The ELSD conditions were as follows:the temperature of drift tube was40℃, the gas pressure was2.0Bar, the value of gain was2. The HPLC-ELSD fingerprints of the roots and stems were established, and the similarity commercial herbs were compared. The total15peaks were selected as the characteristic peaks of the roots of Ilex pubescens and17peaks were selected as the characteristic peaks of its stems. Furthermore, four peaks in HPLC-ELSD chromatogram were assigned as IlexgeninA, IlexsaponinA1, IlexsaponinB1, llexsaponinB2. The characteristics of the roots and stems of Ilex pubescens was obtained.The method can be used for the quality control of Ilex pubescens.This paper study on chemical constituents in stems of Ilex pubescens, enriched the study of its chemical constituents. The chromatographic fingerprint include the HPTLC chromatographic fingerprint analysis and HPLC-ELSD fingerprint analysis. Focus on the comparison of Ilex pubescens difference between stems and roots, which was useful for the quality control of Ilex pubescens.Vie also compare fingerprints similarity with commercial herbs and its related species. The research provide a reference for the expanded use of medicinal plants and the further study of quality standards of Ilex pubescens as well.