Studies On Extraction, Separation And Purification Of The Active Constituents From The Leaves Of Cajanus Cajan (L.) Millsp

In this study, analysis methods were established to determine contents of flavonoids, polysaccharides and vitexin from the leaves of pigeonpea(Cajanus cajan (L.) Millsp). As results, the harvest period and the medicinal parts were determined. And then, according to the contents of the three constituents, the process of water extraction was studied and the better extraction process was gotten. After that, the water-soluble extract was purified by alcohol precipitations, the chromatography and crystallizations and three extracts,50% flavonoids,70% polysaccharides and 60% vitexin were obtained. At last, the vitexin extract of 60% was purified to the content over 97% by recrystallizations and the chromatography.Ⅰ. Establishments of analysis methods:To identify the crude pigeonpea leaves, a silica gel thin-layer chromatography (TLC) method was established. The mobile phase was benzene-ethyl acetate-acetone-methanoic acid-water(3:4:2:0.7:0.3), and the detection wavelength was 254nm.A method was established to determine the content of flavonoids, using vitexin as the standard, at 276.50nm. After aluminium chloride color reaction, the content of the analytes were determined by UV-visible spectrometer.A high-performance liquid chromatography(HPLC) method was established for determining the content of vitexin. The analytes were separated on a Kromasil KR100-5 C18 (250×4.6mm) column, using methanol-0.05% acetic acid(23:73, volume ratio) as the mobile phase in the first 50 minutes, and using methanol-0.05% acetic acid(90:10) as the mobile phase in the last 15 minutes, flow rate 1mL/min, column temperature 32℃and detection wavelength 269nm.For determining the contents of the polysaccharides, a UV-visible spectrometer analysis method was established, using glucose as the standard. After the phenol-sulfur ic acid color reaction, at 483.50nm, the contents of the analytes were determined.At last, the contents of the pigeonpea leaves in different harvest periods and the different parts of the crude materials, leaves, tender stems and old stems, were determined. As results, in different harvest periods, there were significant differences on the contents of flavonoids, polysaccharides and vitexin. The contents of leaves, tender stems and old stems were so different that the leaves and tender stems could be the better medicinal parts.Ⅱ. Study of the extraction process:The most important factors of extraction, the solvents, the ratios of the solvents to crude pigeonpea leaves, temperature, extraction degree and time, were studied. The better extraction process was obtained as follows:Extraction solvent waterWeight ratio of water 10:1Extraction temperature 100℃Extraction degree 3timesExtraction time 1.5hⅢ. Purification of the water-soluble extract:By two degree alcohol precipitations, the water-soluble extract of the pigeonpea leaves was purified. After that, a polysaccharide extract with an average content of 73.54% was obtained. The leachate was concentrated and then was separated on macroporous adsorption resin column. As a result, a flavonoid extract was gotten with an average content of 51.37%. And then, the flavonoid extract was extracted by ethyl acetate and after methanol recrystallization, a vitexin extract with a content of 61.26% was gotten. At the same time, an unknow compound in the the vitexin extract was found with a content of 37.98%, the structure of which was identified to be Orientin. At last, the vitexin extract of over 60% was purified by a silica gel adsorption resin column and methanol recrystallization and then a vitexin extract was gotten with a content of 97.40%.