Study On Microwave-assisted Two-phase Extraction Of The Effective Constituents From Sophorae Tonkinensis Radix.

Sophorae tonkinesis Radix, the dried roots and rhizomes of Sophora tonkinensis Gapnep, also known as sophorae subprostrata radix, herb of sweetclover-like milkvetch, vietnamese sophora root, have the effect of detoxification and relieving pain. Modern research shows that, the extract of sophorae tonkinensis Radix not only has the activity of anti-inflammatory, anti-bacterial, anti-tumor, but it can also regulate the immune system. The main active ingredients of sophorae tonkinensis Radix are alkaloids and polysaccharides, which are often extracted by the methods of reflux and soxhlet extraction. However, these traditional methods are inefficient, reagent-consuming and harmful to the environment. Microwave-assisted extraction is a rapid, efficient, and environmentalfriendly technique with fewer reagents and less time due to advantages of the thermal and the non-thermal effects between microwave and media. Aqueous two-phase extraction(ATPE), the stable, transparent, non-incompatible two phases of inorganic salts, water mixed with organic reagents, could be adjusted according to the nature and scale of the extract to obtain the high extraction efficiency while ensuring its activity. In this study, the microwave-assisted aqueous two-phase extraction was used to select and separate the active ingredients of sophorae tonkinensis Radix, investigating the effects of different types of salts to the aqueous two-phase, phasing diagram fitting the data and correlating with phase balance data, and then screening out the best extraction system of sophorae tonkinensis Radix, optimizing extraction conditions, at last, analysising and quantificating the active ingredients with the HPLC method, the main contents are as follows:1、At the temperature of 20、40、60、80 ℃, measuring and plotting the phase diagrams of Et OH with sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium phosphate, using Merchuk empirical equation to fit the Binodal-line data; Aqueous two-phase liquid-liquid equilibrium data using gravimetric analysis, calculated by Matlab, using Othmer-Tobias and Bancroft empirical equations to calculate the accuracy. The yield of total alkaloids and polysaccharides as the standards, selecting the most suitable system for aqueous two-phase extraction.2、The methods to determination of alkaloids and monosaccharide of sophorae tonkinensis Radix polysaccharide were developed by reversed HPLC. Acetonitrile-m Ethanol-0.1% ammonium hydroxide as mobile phase, detection wavelength was 220 nm, the column temperature was 30℃. The result shows that the alkaloids from radix sophorae tonkinesis contains matrine, oxymatrine, sophocarpine, sophoridine and oxysophocarpine; their linear range were 0.002~0.2 mg/m L, recoveries were between 97.14~102.7 %. Detectting the monosaccharide derivatives at the condition of acetonitrile-50 mmo L phosphate buffered saline as the mobile phase, detection wavelength was 245 nm, the column temperature was 30℃, their linear range were 0.001~0.5 mg/m L, recoveries were between 97.19~102.7 %. The result shows that the polysaccharides from radix sophorae tonkinesis mainly contain mannose, rhamnose, glucuronic acid, galactose, glucose, arabinose.3、Using Et OH-Na2HPO4 aqueous two-phase system as extraction solvent, microwaveassisted aqueous two-phase extraction of the total alkaloids from radix sophorae tonkinesis, and examing their contents through acid dye colorimetry. To discuss the effects of microwave-assisted aqueous two-phase extraction to the contents of total alkaloids from radix sophorae tonkinesis, a response surface methodology was established, based on the single factor(selected from four significant factors) optimization experiment. The optimum extraction conditions were: the ratio of aqueous two-phase was 2.43, ratio of solvent to material was 74.36:1, the granularity was 89.31, the extraction temperature was 80 ℃, the prediction extraction yield was 29.13 mg/g. The experimental extraction yield was 29.41 mg/g, which was very close to the prediction extraction yield, indicating that the model was adequate for the extraction process. Using ethanol- Na2HPO4 aqueous two-phase system, the alkaloids from sophorae tonkinensis Radix could be enrichment to top phase, one step can be completed extraction and purification process, Analysising by HPLC method established, the mainly contents of matrine, oxymatrine, and less sophocarpine, oxysophocarpine, sophoridine.4、Using Et OH-Na2HPO4 aqueous two-phase system as extraction solvent, microwaveassisted aqueous two-phase extraction of the polysaccharides from radix sophorae tonkinesis, and examing their contents through phenol-sulfuric acid method. To discuss the effects of microwave-assisted aqueous two-phase extraction to the polysaccharides, a response surface methodology was established, based on the single factor(selected from four significant factors) optimization experiment. The optimum extraction conditions were: the percentage of salt was 42.14 %, the percentage of Ethanol was 15.48 %, the extraction temperaturewas 91.53 ℃, the extraction time was 6.44 min, the prediction extraction quality was 108.74 mg/g. The experimental extraction quality was 109.3 mg/g, which was very close to the prediction extraction quality, indicating that the model was adequate for the extraction process. Using Infrared spectroscopy, Gel permeation chromatography and the established HPLC monosaccharide component detection method, Analyzed the polysaccharide distribution to aqueous two-phase structure, molecular weight and monosaccharide Composition. The qualitative and quantitative of monosaccharides in the two phases from radix sophorae tonkinesis polysaccharide can be analysised by the established HPLC method and analysis distribution of the polysaccharide mechanism.