In the process of transferring from plants to solvents, the main influencing factor is the solubility of flavonoids in the selected solvents. At present, commonly used organic solvents are methanol, ethanol, ethyl acetate, ether, petroleum ether, acetone and so on. Among them, ethanol is a common solvent for extracting total flavonoids because of the toxicity of methanol and acetone, and ethanol is relatively safe and easy to recover. The main methods of extracting total flavonoids from white clover based on ethanol are reflux method and ultrasonic method. Among them, reflux method is the most commonly used method and the extraction efficiency is the highest. Reflux method refers to extracting the components of Trifolium Trifolium pratense with volatile organic solvent, heating the extract, evaporating the solvent, and then concentrating and reflowing to the medicinal materials, and so on until the effective components are completely extracted. Including reflux hot soaking method and reflux cold soaking method, in which the hot soaking method has a large amount of solvent and cannot be recycled, while the cold soaking method has a small amount of solvent and can be recycled during extraction, which is more suitable for the extraction of total flavonoids from white clover. Ultrasonic extraction of flavonoids is a relatively new method. Its principle is to use ultrasonic cavitation in liquid to accelerate the extraction of flavonoids from plant medicinal materials, and also to use secondary effects such as mechanical vibration, diffusion and pulverization to accelerate the release of extracted substances. The method has the advantages of simple equipment, convenient operation and short extraction time. The extraction rate of total flavonoids from white clover was 55.5%. In addition, it also includes a new extraction method based on macroporous adsorption resin. Among the extraction methods of total flavonoids from Trifolium repens, there are patents on purification method of total flavonoids from aboveground parts of Trifolium repens invented by Chen Dan 35 1 et al. and patents on extraction of total flavonoids from Trifolium repens based on supercritical carbon dioxide invented by Liu Dongfeng et al., which provide new methodological support for the extraction technology of total flavonoids from Trifolium repens.
Second, the determination of flavonoids
At present, there are many methods to determine flavonoids, mainly spectrophotometry and chromatography. Spectrophotometry is the most commonly used method to determine the content of total flavonoids in clover, including direct determination method and chromogenic direct determination method. The direct determination method determines the content at the characteristic absorption peak wavelength of flavonoids structure, which has the advantages of convenience, rapidity and accuracy for the detection of single-component samples. However, there are many interference factors for the complex components of traditional Chinese medicine, which will affect the determination results and cause great errors. Another method to determine the content of total flavonoids is the chromogenic method under alkaline conditions. There are two main methods of color development. One is the NaNO2-A 1(NO3)3-NaOH colorimetric method, that is, the solution of NaNO2, A(NO3)3 and NaOH is added in sequence in the determination process to generate a stable red compound, and the content is determined by colorimetric method at (505 5) m. This method is the most widely used method for the determination of flavonoids at present, and can effectively exclude polysaccharides and saponins. The other is aluminum chloride color development method, that is, aluminum chloride solution is added to the determination solution, and A 1+ complexs with 3- or 5- hydroxyl and 4- carbonyl or 3', 4- ortho-diphenol hydroxyl of flavonoids to develop color. Maximum absorption chromatography at 4 13mm has been widely used in qualitative and quantitative analysis of various natural products, and the commonly used chromatographic techniques are divided into gas chromatography. Because of the high boiling point and thermal instability of flavonoids, it is necessary to derivatize the samples before gas chromatography. Therefore, high performance liquid chromatography is widely used in the determination of total flavonoids in clover, and it is also recognized as the best method at present. However, HPLC has some disadvantages, such as high price, long analysis time and low separation efficiency.