Two flavonoid compounds commonly used in laboratories
The extraction of flavonoids is mainly based on the nature of the extracted substance and whether the impurities accompanying the extraction process are easy to remove.
Flavonoid glycosides and polar glycosides, such as hydroxyflavonoids, diflavonoids, orange ketones, chalcone, etc., can generally be used in acetone, ethyl acetate,EthanolMethanol, water or mixed solvent extraction with high polarity, among which methanol-water (1:1) or methanol is the most used. Polysaccharides can be extracted with boiling water to destroy the activity of hydrolases and avoid hydrolysis of glycosides, such as rutin extracted from locust rice. When extracting anthocyanins, a small amount of acid, such as 0.1% hydrochloric acid, can be added, but acid should not be added when extracting general flavonoid glycosides to avoid hydrolysis. The extraction of flavonoid glycosides should use solvents with low polarity, such as chloroform, ether, ethyl acetate, etc. For poly-methoxy flavonoids with low polarity, benzene can also be used for extraction.
For the obtained crude extract, the solvent can be further recovered and concentrated into an extract, which can be preliminarily refined by solvent extraction, alkali acid extraction and precipitation, macroporous adsorption resin, supercritical fluid extraction, and ultrasonic wave.
1. Solvent extraction
Using flavonoids and mixed impurities with different polarities, selecting different solvents for extraction can not only separate flavonoids from impurities, but also separate glycosides from each other or polar glycosides from non-polar glycosides. For example, the alcohol extract of plant leaves, extracted with petroleum ether, can remove chlorophyll andcaroteneIsofat-soluble pigments; after the water extract is concentrated, 3-4 times the amount of ethanol can be added to precipitate and remove water-soluble impurities such as proteins and polysaccharides.
Sometimes the solvent extraction process can also be carried out continuously by countercurrent distribution method. Commonly used solvent systems are: water-ethyl acetate, n-butanol-petroleum ether, etc.
2. Alkaline extraction and acid precipitation
Most of the flavonoid molecules contain phenolic hydroxyl groups, which are weakly acidic and easily soluble in alkaline water but poorly soluble in acidic water. Therefore, they can be extracted with alkaline aqueous solutions (such as saturated lime water, 5% sodium carbonate or dilute sodium hydroxide solutions, etc.) or alkaline dilute alcohols. After the extract is acidified with acid, flavonoids can precipitate and precipitate.
When using alkali extraction and acid precipitation for extraction and purification, it should be noted that the alkali concentration used should not be too high, so as to avoid destroying the mother nucleus of flavonoids under strong alkali conditions, especially when heating; when adding acidification, the acidity should not be too strong, so as not to generate detailed salts, resulting in the re-dissolution of the precipitated flavonoids and reducing the yield of the product; when the raw materials contain a large amount of water-soluble impurities such as pectin and mucilage, such as flowers, fruits and other raw materials, lime milk or lime water should be used instead of other alkaline aqueous solutions for extraction, so that the above-mentioned impurities containing carboxyl groups can form calcium salt precipitation without being dissolved, which is more conducive to the purification of flavonoids.
This method is simple, economical and safe, such as rutin, hesperidin,BaicalinThis method is used for the extraction of. The operation process of extracting rutin from locust flower rice is as follows: add about 6 times the amount of water to locust flower rice, bring to a boil, and slowly add lime milk to pH8~ 9 under stirring. Under this pH condition, slightly boil for 20-30min, and filter while hot; add 4 times the water to decocate once, and filter while hot. The combined filtrate is at 60-70 ° C, adjust the pH to 5 with concentrated hydrochloric acid, stir well, let stand for 24h, and filter. The sediment is washed to neutral, dried at 60 ° C to obtain crude rutin, recrystallized in water, and dried at 70-80 ° C.RutinPure.
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