Glycogen, aliasAglycone, YesGlycosideIn compounds like, sugarcondensationThe non sugar portion of.Glycosides can be alcohols such as methanol, ethanolsterol , such glycosides are calledOxyglycoside；Can bemercaptanasEthanethiol, such glycosides are calledGlucosinolate；Can becarboxylic acidFor example, fatty acids, such glycosides are called sugar esters;Can beamine , such glycosides are calledNitrogen glycoside；It can also be that the carbon atom of aglycone is directly connected with sugar, calledCarboside。
In terms of chemical structure,saponinIt is composed of aglycone skeleton and glycosylGlycosidic bondGlycosidic Linkage.The saponins in natural plants are mainlyDammaraneType, andClelou typeandOleanolic acid typeLess.[1]
Synthesis of daidzein
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The synthetic methods of daidzein have been reported in many literatures, and the methods are basically the same.Deoxybenzoin reaction is generally adopted.There are two main catalytic systems for the synthesis of deoxybenzoin from phenylacetylated polyphenols: BFthree/EttwoO catalytic system Bass process and ZnCltwo/POClthreeCatalytic system.BFthreewrongStrong acid and strong baseIt has high activity and can be used with EttwoO forms solvent complex, which is conducive to the reaction.
Using p-hydroxyphenylacetic acid and resorcinol as raw materials, 2,4-dihydroxyphenyl-4 '- hydroxybenzyl ketone was synthesized by Friedel Crafts reaction in the presence of boron trifluoride, and then daidzein was synthesized with N, N-dimethylformamide ring by Bass process.If the reactant isPhloroglucinolIt has strong nucleophilic and reductive properties, so the acylation conditions must be quite mild, and ZnCl is often usedtwo/POClthreeThe catalytic system has low yield.Isoflavones can be easily cyclized by Bass process, which is characterized by BFthree/EttwoO and deoxybenzophenol hydroxyl complex passivates the aromatic ring, thus making DMF MeSOtwoCl selectively formylates its methylene, and then dehydrates and cyclizes to produce isoflavones.The yield can be increased.The synthetic methods of daidzein have been reported in many literatures, and the methods are basically the same.[1]
Preparation and identification
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principle
1. Use rutin to extract because it is easily soluble in alkali solution but easy to precipitate in acid solution.
2. Flavone glycosides can be hydrolyzed by acid to obtain aglycones and sugars, and can be identified by TLC, PC and acetylate preparation.
3. Different ketone compounds have specific absorption of UV light, which can be used for qualitative and quantitative determination.[2]
step
1. Extraction of rutin
Weigh 120g of Huaihua rice (extracted separately in three times), grind it, put it into a 500ml beaker, add ten times of boiling water and a proper amount of borax, stir it, heat it on the asbestos screen until slightly boiling, use saturated lime water (use a spoon to take a proper amount of quicklime into the beaker, add distilled water to dissolve it to obtain suspended liquid, and filter it to obtain clear filtrate, which is saturated lime water), adjust the pH value of the solution between 8-9,And keep stirring.After 30 minutes of slight boiling, filter and collect the filtrate.Collect the filtrate again by the same method and concentrate it to about 300ml. When the temperature of the filtrate drops to 60~70 ℃, acidify it with concentrated hydrochloric acid to PH4~5. Put it in the refrigerator for 12h at room temperature to make the precipitation complete.Filter by suction, wash the precipitate with distilled water until it is neutral, and obtain crude rutin.
2. Refining of rutin
After weighing the above precipitation, add 30-40 times of hot water to dissolve it, filter it while it is hot, let it cool to precipitate crystallization, filter it, and naturally dry the crystallization (or dry it under infrared light) to obtain high-quality rutin.
Then take refined rutin water and recrystallize it with MeOH (1:10~15) to obtain refined rutin.
3. Identification of rutin
Rutin hydrolysis method: take 1g of refined rutin, add 80mL of 0.5% dilute sulfuric acid, heat and reflux for hydrolysis for 1h, cool, filter, and wash the precipitation with distilled water to neutral.Add the precipitate into 95% ethanol, heat it to make it completely dissolved, and filter it while hot.The filtrate is added with equal amount of distilled water to recrystallize, and filtered to obtain quercetin and rutin hydrolysate.It can be seen from the references that the yield of quercetin produced by rutin hydrolysis in aqueous solution is higher than that in methanol solution, and the production cost is lower;The yield of hydrochloric acid or sulfuric acid is almost unchanged;When the acid concentration is 0.5% and the heating time is 1h, the hydrolysis is complete, and the yield is 91.0%.[2]
Extraction, separation and identification
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principle
Rhubarb, a plant of Polygonaceae, is bitter in taste and cold in nature. It has the effects of purging heat, purging intestines, cooling blood and detoxification, and removing blood stasis and meridians.Its main effective ingredients are emodinAloe emodinEmodin methyl ether, etcAnthraquinonesMost of them are combined anthraquinones, and a few are free anthraquinones.The combined anthraquinone compounds are weakly acidic and soluble in water, ethanolsodium bicarbonateSolution, but the solubility in organic solvents is very small.Free anthraquinone is easily soluble in chloroform, ether and other organic solvents, but insoluble in water.Among them, rhein has a carboxyl group and is the most acidic;Emodin has β - phenolic hydroxyl, the second acid;Aloe emodin is connected with hydroxymethyl group, the third acid;Emodin methyl etherAnd chrysophanol are the weakest.According to the acidity difference of the above compounds, gradient extraction separation can be carried out with solutions with different alkalinity.[2]
step
（1） Acid hydrolysis
Weigh 10g of rhubarb powder with a balance, place it in a 500ml beaker, and add 20% HtwoSOfour100ml of water solution, heated on a straight fire for 1h, filtered with a Buchner funnel, washed with water and dried at about 70 ℃.Turn off the power supply when the solution is boiling, and turn it on again when it is not boiling. Be careful not to burn the solution dry, and turn the rhubarb powder from yellowish brown to black.
（2） Extraction of total hydroxyanthraquinone aglycone
After drying the filter cake, put it into Soxhlet extractor, add 150ml of ether, reflux and extract for 2h to obtain ether extract.The ether extract has rheinAloe emodinEmodin, emodin methyl ether and chrysophanol.The thin layer plate is a silica gel CMC Na plate, and the developing agent is petroleum ether (boiling range 60~90 ℃) - ethyl acetate (7:3). It is developed nearly horizontally or vertically, and four spots can be seen under visible light,The yellow spot with Rf ≈ 0.9 is the mixture of chrysophanol and emodin methyl ether, which cannot be separated under this condition. The other three spots are emodin (orange spot), aloe emodin (yellow spot) and rhein (yellow spot) according to Rf value.
（3） PH gradient extraction separation
1. Separation and purification of rhein: add 5% of the above ether extractsodium bicarbonateThe water layer is purple red after solution shaking extraction.Separate the water layer and extract it repeatedly for several times until no red color appears (about 60ml in total, extracted in 3~4 times).Combine the aqueous extract and acidize it to about pH3 with hydrochloric acid to obtain yellow precipitate.Filter, wash and precipitate several times with water, and then wash with a small amount of cold acetone to remove colored impurities.After drying, crystallize with glacial acetic acid or pyridine for 2-3 times to obtain yellow needle like crystals.It was identified as rhein by melting point determination, paper chromatography or thin layer chromatography, and compared with the standard substance.
2. Isolation and purification of emodin:sodium bicarbonateThe ether layer extracted from the solution is shaken with 5% sodium carbonate solution for several times (about 120ml in total, extracted in 3~4 times), and the water layer is red. Combine the water layer extraction solution, add hydrochloric acid to the acid (about pH 6) to obtain yellow precipitate, filter, wash with water to precipitate, wash with cold acetone, crystallize in glacial acetic acid or pyridine for several times, and obtain orange large needle shaped crystal.It was identified as emodin by melting point determination, paper chromatography or thin layer chromatography, and compared with the standard substance.
3、Aloe emodinSeparation and purification of: the ether layer extracted by sodium carbonate solution is shaken by 0.25% sodium hydroxide solution for several times (about 100ml, extracted in 3~4 times), and the water layer is red. Combine the water layer extraction solution, add hydrochloric acid to acidity (about pH 6), and get orange precipitate.After filtration, wash and precipitate with water, and dry it in glacial acetic acid orethyl acetateMedium crystallization several times to obtain orange long needle shaped crystals.It was identified as aloe emodin by melting point determination, paper chromatography or thin layer chromatography.
4. Chrysophanol andEmodin methyl etherSeparation and purification of:Aloe emodin5% for separated ether solutionsodium hydroxideShake the solution for several times until it is colorless.Combine the dark red aqueous solution and add hydrochloric acid to make it acidic to obtain yellow precipitate.After filtration, water washing and drying, a mixture of chrysophanol and emodin methyl ether is obtained. The mixture is dissolved in ethyl acetate (silica gel thin-layer plate can be used, and petroleum ether: ethyl acetate (15:1) can be used as developing agent to develop. Two spots can be seen, and chrysophanol is the one with higher Rf value,Emodin methyl ether) is separated by silica gel column chromatography. The eluent is the mixture of petroleum ether (boiling range 60~90 ℃) - ethyl acetate (15:1). The compound eluted first is chrysophanol, and the compound eluted later is emodin methyl ether. Then it is purified by ethyl acetate crystallization. Both are pure by melting point determination and thin layer chromatography.[2]
