Study On The Extraction And Separation Of The Active Components In The Leaves Of Diospyros Kaki

The chemical constituents of the leaves of Diospyros kaki are very complex, mainly including naphthoquinones, flavonoids, coumarins, triterpenoids, organic acids, steroids, and they have wide biological activities. To fully utilize the leaves, the processing of the leaves of Diospyros kaki was studied. The total triterpenoids were first extracted from the leaves of Diospyros kaki. Subsequently, the total flavonoids were extracted from the extracted leaves using the aqueous ethanol solution, while ursolic acid was isolated from the total triterpenoids by a relatively simpler process and the residues of the total triterpenoids were used for the isolation of other triterpene acids. The main contents of this work are as following:(1) To obtaint the optimal conditions of the extraction of the total triterpenoids, The effects of temperature, ratio of solvent volume to the leaves of diospyros kaki mass, extraction time and extraction times on the degreasing by petroleum ether (bp 60-90℃) and the extraction of the total triterpenoids by ethyl acetate were investigated, respectively.(2) The total triterpenoids obtained above were degreased by petroleum ether (bp 60-90℃) and decolorized by active carbon, subsequently. The conditions of separation and purification of ursolic acid were optimized, such as ratio of solvent volume to the total triterpenoids mass, temprature, experimental time and times. The total triterpenoids purified were recrystallized in ethanol to obtain ursolic acid. The chemical structure of ursolic acid was identified by EI-MS, ESI-MS, ¹H NMR and ¹³C NMR.and analysed by HPLC.(3) Six colorimetric methods for the determination of total flavonoids in the extracts of the leaves of Diospyros kaki were compared with two HPLC methods. A new colorimetric method, i.e. AlCl₃-(HAc-NaAc) colorimetry was proposed.(4) The extracted leaves of Diospyros kaki, which were extracted by hot petroleum ether (bp 60-90 ℃) and ethyl acetate succesively, were further extracted with aqueous ethanol solution for obtaining total flavonoids. The effects of temperature, ratio of solvent volume to the leaves of diospyros kaki mass, extraction time and extraction times on the extraction of the total flavonoids were investigated.The crude extracts containing total flavonoids were purified by extraction using ethyl acetate-water solutions at different pH.(5) A single-step preparative high-speed counter-current chromatography (HSCCC, with a lOOOmL coil column) method was developed for the separation and purification of four bioactive pentacyclic triterpene acids from the extract of the leaves of Diospyros kaki. The ratio of compositions of the solvent system composed of n-hexane-ethyl acetate-methanol-water were optimized. The effects of rotation speed of the multilayer coil column, flow rate of mobile phase and temperature on the retention of the stationary phase were investigated. In a single operation, barbinervic acid ( 3a,19a,24-trihydroxy-urs-12-en-28-oic acid) and its epimer, rotungenic acid ( 3/?,19a,24-trihydroxy-urs-12-en-28-oic acid) along with 24-hydroxy ursolic acid (3/?,24-dihydroxy-urs-12-en-28-oic acid), which was reported for the first time from the leaves of Diospyros kaki, and ursolic acid (3/?-hydroxy-urs-12-en-28-oic acid) were obtained from the extract of the leaves of Diospyros kaki. The chemical structure of the four compounds was elucidated by EI-MS, ESI-MS, !H NMR and 13C NMR.(6) In order to improve the quality control of the leaves of Diospyros kaki, derived extracts and phytomedicines, a simple, rapid and accurate high-performance liquid chromatography method was developed to simultaneously assess the three bioactive triterpene acids: barbinervic acid (BA) and its epimer, rotungenic acid (RA), along with 24-hydroxy ursolic acid (HA). This HPLC assay was performed on a reversed-phase Ci8 column with methanol and aqueous H3PO4 solution as the mobile phase and using a monitoring wavelength at 210 nm. This method was successfully applied to quantify these three bioactive triterpene acids in five different solvent extracts and in the leaves from six different locations in China.