Studies On The Preparation Technology Of Total Flavonoids And Determination Of Major Components From Litsea Coreana L.

Laoying tea is leaf of Litsea Coreana Leve which belongs to Neolitsea Lauraceae department. It has been reported that the extract of Litsea Coreana L. exerts various pharmacological effects including antioxygen, antihyperlipidemic, antihypertensive, anti-inflammatory anti-hyperlipidemic and immunomodulation. It contains many chemical compositions, such as flavonoid, saponin, organic acid, coumarin and tannin etal. In it, content of flavonoids is high, and flavonoid is the effective component of Litsea Coreana L. In the pre-study, five effective flavonoids have been separated from the plant. Ttheir structures are identified as catechin, quercetin-3-O-β-D-galactoside, quercetin-3-O-β-D-glucoside, kaempferol-3-O-β-D-galactose and kaempferol-3-O-β- D-glucoside.With deepening research on pharmacological effect of Litsea Coreana total flavonoid(TFLC), the preparation and determination of TFLC should be given further exploration and research. This paper is aimed to research the preparation technology of TFLC; use a RP-HPLC method for determination the content of five major flavonoids in Litsea coreana L.; and compare extracs from different preparation technologies.The main contents were divided into two sections as follows:1. Studies on the preparation technology and separation of total flavonoids from Litsea Coreana L.With the flavonoids content as a evaluation index, orthogonal design L9(33) is applied to optimize the water extraction technique for total flavonoids of Litsea Coreana L. The best extraction technology is that 12BV water, 1 hour of reflux, and 3 times of extraction. Macroporous resin is a kind of non-ionic polymers ,which have been widely used in traditional Chinese medicine, especially the purification of flavonoids.In this paper, through investing the adsorption capacity and desorption rate, 4 kinds of resins are compared for isolation and purification of TFLC. The D101 macroporous resin (non-polar type) is the suitable medium to prepare TFLC. And the conditions for preparation of TFLC are studied. The technology conditions are as follow : the concentration of the extraction sample is 1~1.5mg·ml-1, the maximum adsorbing capacity for the total flavonoids of Litsea Coreana L. is 7.14 mg·g-1 with the adsorption velocity 1.5BV·h-1, 70% ethanol is used to elute the total flavonoids, the eluting volume is 3BV and the eluting velocity is 3BV·h-1. On this condition to adsorption and elution, the D101 resin could be used 5 times repeatedly. After adsorption on macroporous resin and desorption by ethanol, the content of total flavonoids in Litsea Coreana L. is 48.59%, and the recovery rate is > 90 %. In this paper, four flavonoids are separated from TFLC by the normal-phase and reversed-phase silica gel column chromatography. Compared with the known compounds, they are identified as: quercetin-3-O-β-D-galactoside, quercetin-3-O-β-D-glucoside, kaempferol-3-O-β-D-galactoside, and kaempferol-3-O-β-D-glucoside.2. Studies on determination of the major components from TFLCHPLC method is used to determine the contents of catechin, quercetin-3-O-β-D-galactoside, quercetin-3-O-β-D-glucoside, kaempferol-3-O-β-D-galactoside, kaempferol-3-O-β-D-glucoside in TFLC. Chromatography conditions are as follows: Hypersil ODS reversed-phase chromatographic column (4.6 mm×200 mm, 5μm); acetonitrile: 3 % aqueous acetic acid(18:82) as the mobile phase; flow rate is 1ml·min- 1; detection wavelength is set at 280 nm and 355 nm. This HPLC analysis method is simple, accurate, sensitive, with good separation of flavonoids, so it is suitable for content determination of TFLC. On this basis, to use the HPLC method , the components and contents of flavonoids in different preparation products are compared. The result shows that major components of two different preparation technique (ethanol extract and water extract) are both flavonoids. Catechin, quercetin-3-O-β-D-galactoside, quercetin-3-O-β-D-glucoside, kaempferol-3-O-β-D-galactose and kaempferol-3-O-β-D- glucoside are with the high levels in TFLC. Catechin contents of water extract and ethanol extract have a significant difference, and the other four flavonoid glycosides content is basically the same.