Radix Angelicae Dahuricae Radix Angelicae Dahuricae has the effects of expelling wind and removing dampness, inducing resuscitation and relieving pain, reducing swelling and expelling pus, and can be used for treating common cold, headache, stuffy nose and sores. It mainly contains coumarin and volatile oil, mostly furan coumarin, and imperatorin, angelica dahurica lactone, angelica dahurica lactone and so on can represent the curative effect.
Peucedanum praeruptorum has the effects of dispelling wind, clearing heat, lowering qi and resolving phlegm, and can be used for treating wind-heat cough and excessive phlegm. It mainly contains coumarins, a small amount of saponins, tetracyclic triterpenoids and volatile oil. More than 20 kinds of coumarins were isolated from Peucedanum praeruptorum, most of which were 7,8-dihydropyran coumarins, and a few were furan coumarins and simple coumarins. More than 10 coumarins have been isolated from Peucedanum purpurea, most of which are linear dihydropyrans or furocoumarins, and their glucose disaccharides.
Radix Angelicae Pubescentis has the effects of expelling wind, removing dampness, removing arthralgia and relieving pain, and can be used for treating arthralgia due to wind-cold and dampness, and lumbago and knee pain. Its main components are coumarins, among which osthole is the main component, and there are also many kinds of furan coumarins.
root of large-flowered skullcap
Scutellaria baicalensis Georgi contains many kinds of flavonoids, the highest content is baicalin, and its aglycone is baicalein (5,6,7- triol flavone), and baicalin is 5,6-diol, 7-O- glucuronic acid flavone glycoside. Except for the color reaction of FeCl3, it is almost insoluble in water and acidic water, but hardly soluble in MeOH. Because it is a glycoside of glucuronic acid, it is difficult to be acidified. It can be hydrolyzed by baicalein, and the generated aglycone baicalein has ortho-pyrogallol hydroxyl, which is easy to be oxidized into quinone derivatives and turn green, which is the reason why scutellaria baicalensis Georgi can turn green if it is not preserved or processed properly. After Scutellaria baicalensis Georgi turns green, the effective components are destroyed and the quality is reduced. The sodium and potassium salts of baicalin are easily soluble in water.
[Some Flavonoids in Scutellaria baicalensis Georgi] [Extraction Technology of Baicalin]
Baicalin is almost insoluble in water. It exists in the form of salt in plants, but it is soluble in water. Therefore, baicalin (in the form of baicalin salt) can be extracted from Scutellaria baicalensis coarse powder by adding water. When HCL is added to the solution to adjust the pH to 1 ~ 2, the baicalin salt is dissociated, and the generated baicalin is almost insoluble in acidic water and precipitated out. Then the precipitate was suspended in water, and 40%NaOH was added dropwise, and baicalin became sodium salt again and dissolved. At this time, it should be noted that NaOH should not be excessive, otherwise when the same amount of ethanol is added, it will appear jelly-like and difficult to filter. Continue to add HCl to dilute alcohol solution to adjust pH to 1-2, and baicalin sodium salt dissociates again, which is insoluble in dilute alcohol and precipitates out. This precipitate can be washed with water, 50% ethanol and 95% ethanol to obtain pure baicalin, and then crystallized with methanol for many times to obtain higher purity baicalin.
The meaning and existing forms of flavonoids
Classical meaning: a class of compounds with 2- phenylchromone as the basic nucleus, called flavonoids. At that time, these compounds were called flavonoids because they were yellow and had ketocarbonyl groups at the 4 th position.
Modern significance: Two benzene rings (A ring and B ring) are connected by a three-carbon chain, which is called flavonoids. The compounds included in this meaning are yellow, light yellow and white. The chemical structure includes ketone carbonyl, carbonyl-free, 2-position benzene ring and 3-position benzene ring, etc. As can be seen from the following classification. Of course, yellow flavonoids account for the vast majority.
Flavonoids are widely found in plants, and many commonly used Chinese medicines mainly contain such components. Most of them combine with sugar to form glycosides (called flavonoid glycosides), and some of them combine with glucuronic acid to form glycosides, such as baicalin in Scutellaria baicalensis Georgi. Some of them exist in free form, which is called free flavonoids or flavonoid aglycones. Free flavonoids and their glycosides may exist in the same Chinese medicine. For example, Scutellaria baicalensis Georgi contains baicalein (baicalein 5,6,7- triol flavone) and baicalin (5,6-diol, 7-o glucuronic acid flavone glycoside).
The substituents of flavonoids include hydroxyl, methoxy, methyl, methylenedioxy (-o-CH2-o-) isopentenyl, etc.
Flavonoid glycoside can be monosaccharide glycoside, disaccharide glycoside and triglycoside. Some structures are more complex, and some exist in the form of carbon glycosides, such as puerarin in Pueraria lobata and cinnamoyl derivatives obtained from Ginkgo biloba leaves, such as kaempferol -3- rhamnose -2-(6- p-hydroxy-trans-cinnamoyl)-glucoside. (Kaempferol is 3,5,7,4'-tetrahydroxyflavone), etc.
classify
According to the oxidation degree of the three-carbon chain between ring A and ring B (whether there is OH in C3 position, whether there is C=O in C4 position, whether C2 and C3 are double bonds, etc. ), whether the connecting position of B ring (No.2 or No.3) and C ring constitute a cyclic structure, flavonoids can be divided into many small categories.
Main structural types of flavonoid aglycones
Flavone dihydrochalcone
Flavonoid anthocyanin
Dihydroflavone flavan -3- ol
Dihydroflavonol flavan-3,4-diol
Isoflavone benzophenone (ketone)
Dihydroisoflavone
Oh (orange ketone)
Chalcone
Characteristics of flavonoids
form
Flavonoids are mostly crystalline solids, and a few (such as flavonoid glycosides) are amorphous powders.
colour
Generally speaking, flavones, flavonols and their glycosides are mostly grayish yellow to yellow, chalcone is yellow to orange yellow, and dihydroflavones, flavonols and isoflavones are almost colorless because there is no crossed yoke system in the molecular structure. For example, electron donor groups such as -OH and OCH3 are introduced into flavonoids and flavonol molecules, especially in the 7-position or 4'- position, resulting in ρ-π * * * yoke, which promotes electron rearrangement, prolongs the * * * yoke system and deepens the color of the compound. However, the introduction of -OH and OCH3 at other positions in the molecular structure has little effect on the color.
[Structural changes of flavonoids and flavonols]
If the double bond between C2 and C3 is hydrogenated, the cross yoke system and addition relationship are interrupted, so dihydroflavones and dihydroflavonols are almost colorless. Isoflavone yokes are rare and only slightly yellow.
The color of anthocyanins changes with different pH, generally pH8.5 is blue, and different pH may promote the reversible change of structure.
Optical rotation/rotation
Biflavonoids, dihydroflavonols, flavanols, dihydroisoflavones and their derivatives, esteramine and rotenone are optically active because they contain asymmetric carbon atoms in their molecules. Other flavonoid aglycones have no optical activity. Flavonoid glycosides are optically active because of the introduction of sugar molecules into their structures, and most of them are left-handed.
Acidity and alkalinity
Flavonoids are acidic because they contain phenolic hydroxyl groups in their molecules and can be dissolved in alkaline aqueous solutions and pyridine. Its acidity is related to the number and position of phenolic hydroxyl groups. For example, the order of phenolic hydroxyl acidity of flavonoids from strong to weak is:
7，4′-= OH & gt； 7- or 4 '-oh >；; Generally phenolic hydroxyl group >; 5- Oh
Under the influence of ρ-π * * * yoke effect, compounds with phenolic hydroxyl groups at 7- position and 4'- position can enhance acidity and dissolve in sodium bicarbonate aqueous solution. Phenolic hydroxyl groups at the 7- or 4'- position are only soluble in sodium carbonate aqueous solution, but insoluble in sodium bicarbonate aqueous solution. With ordinary phenolic hydroxyl group, it is only soluble in sodium hydroxide aqueous solution. Only the hydroxyl group of 5- phenol can form intramolecular hydrogen bond with C4=O, so the acidity is the weakest. Therefore, pH gradient method can be used to separate flavonoids.
The oxygen atom at the 1- position on the γ-pyrone ring in the molecule of flavonoids is weakly alkaline due to the unused electron pair (fully methylated polyhydroxy flavonoids are more alkaline), and can form salts with strong inorganic acids, such as concentrated sulfuric acid and hydrochloric acid, which is extremely unstable and will decompose immediately after adding water.
The salt produced by dissolving flavonoids in concentrated sulfuric acid often presents special color, which can be used to identify the types of components to be detected.
solubility
The solubility of flavonoids varies greatly with different structures.
1. Generally, flavonoid aglycone is insoluble in water, but soluble in organic solvents such as methanol, ethanol, chloroform, ether and dilute alkali solution. Among them, flavonoids, flavonols and chalcones are planar molecules, which are not easily soluble in water because of their close packing and large intermolecular attraction. However, dihydroflavones and dihydroflavonols are non-planar molecules [as shown in the figure], which are not closely arranged, and the intermolecular attraction is reduced, which is beneficial to the entry of water molecules, so their solubility in water is slightly higher.
Isoflavones are more hydrophilic than planar molecules. Anthocyanin is hydrophilic, although it also belongs to the plane structure, but it is water-soluble because it exists in the form of ions and has the universality of salt.
2. Flavonoids are mostly polyhydroxy compounds, which are generally insoluble in petroleum ether, so they can be separated from lipophilic impurities.
3. After the hydroxyl glycosylation of flavonoids, the water solubility increased and the fat solubility decreased. Generally, it is soluble in hot water, methanol, ethanol, pyridine and dilute alkali solution, but insoluble in organic solvents such as benzene, ether, chloroform and petroleum ether.
The number and binding position of sugar groups in glycoside molecules also have some influence on solubility. Generally, polysaccharide glycosides are more water-soluble than monosaccharide glycosides; 3- hydroxyglycoside is more soluble in water than the corresponding 7- hydroxyglycoside.
Extraction and separation of flavonoids
extraction method
The extraction of flavonoids mainly depends on the properties of extraction materials and impurities. Generally, glycosides and aglycones with higher polarity can be extracted with ethyl acetate, acetone, ethanol, methanol, water or some mixed solvents with higher polarity [such as methanol-water (1: 1)]. Most aglycones should be extracted with less polar solvents, such as ether, chloroform and ethyl acetate. Polymethoxyflavone aglycone can even be extracted with benzene.
Ethanol or methanol extraction
Ethanol or methanol is the most commonly used solvent for extracting flavonoids, and high concentration ethanol (such as 90% ~ 95%) is suitable for extracting aglycones. About 60% ethanol is suitable for extracting glycosides. The extraction times are generally 2 ~ 4 times, and the heating extraction method or cold soaking method can be used. For example, ginkgo flavone glycosides can be extracted by reflux with 65% ethanol.
Hot water extraction method
Hot water is limited to extracting glycosides, such as rutin in Sophora japonica. Because there are many impurities extracted from hot water, it is not often used.
Alkaline water or alkaline dilute alcohol extraction
Because most flavonoids have phenolic hydroxyl groups, they can be leached by alkaline water or alkaline dilute alcohol (such as 50% ethanol), and flavonoids can be separated out after acidification. Dilute sodium hydroxide aqueous solution has great leaching ability, but there are many impurities. If the leaching solution is quickly acidified and filtered (for example, within half an hour), the first precipitate (mostly impurities) may be pure flavonoids. [Advantages of limewater] [Disadvantages of limewater] The leaching effect of 5% sodium hydroxide dilute ethanol solution is better, but after acidification of the leaching solution, the flavonoids separated out have certain solubility in dilute ethanol, which reduces the product yield. When extracting with alkaline solvent, the concentration of alkali used should not be too high, so as not to destroy the mother nucleus of flavonoids when heating under strong alkali. When catechol has hydroxyl groups, boric acid can be added for protection.
Systematic solvent extraction is a sequential extraction with solvents from small polarity to large polarity. For example, degreasing with petroleum ether or hexane, and then extracting polymethoxyflavone or flavone containing isopentenyl and methyl with benzene. Chloroform, ether and ethyl acetate can extract most of the free flavonoids. Flavonoids, biflavones and chalcones can be extracted from acetone, ethanol, methanol and methanol-water (1: 1). Glycosides can be extracted with dilute alcohol and boiling water, and anthocyanins can be extracted with 1%HCl.
Separation method
Solvent extraction method
This method is a preliminary separation method, mainly to separate aglycone and glycoside. The common operation is to add a proper amount of water to the ethanol extract, and then extract it with petroleum ether, ether, ethyl acetate and water-saturated n-butanol. Petroleum ether can contain no flavone or only polymethoxyflavone, while ether can contain some free flavone, ethyl acetate can contain polyhydroxyflavone and flavone monosaccharide glycoside, and n-butanol can contain flavone glycoside of more than two sugars.
Polyamide adsorption method
Most flavonoids have phenolic hydroxyl groups, which can be adsorbed by polyamide and separated from components without phenolic hydroxyl groups.
Coarse powder of traditional Chinese medicine
│
↓ 70% ~ 80% ethanol extraction
Ethanol extract
│
│ Recovering ethanol under reduced pressure, and standing.
┌──────────┴──────────┐
↓ ↓
Insoluble solution
(Fat-soluble impurities such as resin. Through the polyamide column, water is used in turn.
There may be free flavonoids) eluted with 95% ethanol.
┌──────┴──────┐
↓ ↓
95% ethanol eluent water eluent
│ (hydrophilic impurities such as sugar)
Recover ethanol under reduced pressure to dryness.
general flavone
If the insoluble matter contains free flavonoids, the following methods can be adopted: ① alkali dissolution and acid precipitation. (2) cold washing with petroleum ether to remove lipophilic impurities, then dissolving with organic solvent such as benzene, removing polar impurities through silica gel column, eluting with benzene, and concentrating the obtained benzene solution under reduced pressure to obtain free flavonoids.
Lead salt method
This method used to be used in research, but it is rarely used now. Generally, an appropriate amount of neutral lead acetate and alkaline lead acetate aqueous solution are sequentially added into ethanol or methanol solution, so that components with ortho-diphenol hydroxyl groups (including flavonoids) and components with hydroxyl groups are separated from components with ortho-phenol hydroxyl groups respectively, and then lead salts are respectively precipitated and suspended in ethanol, and the components are obtained after lead removal.
Chromium borate method
Flavonoids containing catechol hydroxyl group can be complexed with boric acid, and the product is easily soluble in water, so it can be separated from flavonoids without catechol hydroxyl group.
PH gradient extraction method
PH gradient extraction method is suitable for the separation of free flavonoids with different acidity. Dissolve the mixture in an organic solvent (such as ether), and sequentially use 5%NaHCO3 (extracting 7,4 '- dihydroxyflavone), 5%Na2CO3 (extracting 7- or 4'-hydroxyflavone), 0.2%NaOH (extracting total phenolic hydroxyflavone) and 4%NaOH (extracting phenolic hydroxyflavone).
Macroporous resin method
This method can be used for the purification of total flavonoids, such as passing the aqueous solution through a macroporous resin column, washing with water first, and then eluting with ethanol with different concentrations, and the ethanol eluate with a certain concentration contains flavonoids. For example, the Ginkgo biloba extract is passed through macroporous resin, washed with water, and then eluted with 25% ethanol and 70% ethanol, and the 70% ethanol eluent contains total flavonoids of Ginkgo biloba leaves.
Column chromatography and droplet countercurrent chromatography
Include silica gel, polyamide, SephadexLH-20, C 18 filler, alumina, etc. Alumina is rarely used. Column chromatography is an effective method to separate monomers. According to the particle size of the filler, atmospheric pressure chromatography, low pressure chromatography, medium pressure chromatography and high performance (high pressure) liquid chromatography can be used.
Droplet countercurrent chromatography (DCCC) is usually used to separate polar components, such as glycosides.
Examples of traditional Chinese medicine containing flavonoids
Huaihuami
Sophora japonica L. is called Sophora japonica L. for short, and its main component is rutin, also called rutin, namely quercetin 3-O- rutin, and quercetin is 5,7,3', 4'- tetrahydroxyflavone.
The solubility of rutin is 1: 10000 in cold water, 1:200 in boiling water, 1:60 in boiling ethanol and 1:7 in boiling methanol, and it is soluble in ethanol, pyridine, formamide and so on.
Rutin molecules contain polyphenol hydroxyl groups, which are weakly acidic and easily soluble in alkaline solution, and then precipitate after acidification, so rutin can be extracted by alkali dissolution and acid precipitation.
Rutin is unstable because it contains catechol hydroxyl groups. Exposure to air can slowly oxidize to dark brown, and it is easier to oxidize and decompose under alkaline conditions. Borate can combine with the hydroxyl group of catechol to achieve the purpose of protection, so when rutin is extracted by heating in alkaline solution, a small amount of borax is often added. There are 7-OH and 4'-OH, which have strong acidity and can be used for extraction. Rutin is soluble in boiling water (1: 200) and slightly soluble in cold water (1: 10000), which can be used for extraction and refining.
The extraction method of rutin, called alkali extraction and acid precipitation for short, can be used in production. It should be noted that the alkalinity of alkali extraction must not be too strong (pH 8 ~ 9). If the pH value is too high, the structure may degrade, thus reducing the yield and even completely destroying the structure.
Another extraction method of rutin is boiling water extraction, that is, taking coarse powder of Sophora japonica, adding hot water 0/0 times of/kloc-0, boiling for 20-30 minutes, filtering, adding water to boil 1-2 times, combining decoctions for several times, cooling for several hours, separating crude rutin, filtering, recrystallizing with water once to obtain refined rutin, and recrystallizing with methanol once.