CN109265339B - Polygonum capitatum active component, extraction method and application - Google Patents
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Abstract
The invention belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to an active component of polygonum capitatum, an extraction method and application, wherein the extracted active component comprises gallic acid, quercetin and quercetin, and the extraction process comprises the following steps: mixing polygonum capitatum with ethanol according to a material-liquid ratio of 1:25g/mL, performing reflux extraction at 80 ℃ for 1.5 hours to obtain a first extracting solution and filter residue, performing reflux extraction on the filter residue under the same conditions for one time, combining the two extracting solutions, performing suction filtration, and performing reduced pressure concentration to obtain a polygonum capitatum ethanol extract; dissolving in water, sequentially extracting with petroleum ether, chloroform, ethyl acetate and n-butanol, and concentrating under reduced pressure to obtain ethyl acetate phase extract and n-butanol phase extract; separating by chromatography to obtain gallic acid, quercetin and quercetin. The gallic acid, the quercetin and the quercetin separated by the method can be applied to research on bacterial infection and treatment of diabetes.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to an active component of polygonum capitatum, an extraction method and application.
Background
Polygonum capitatum (scientific name: Polygonum capitatum Buch. -ham. ex D. Don) is a perennial herb of Polygonum of Polygonaceae. Stem creeping, fasciculation, node rooting, internode shorter than leaf, glandular or nearly unhairing, leaf oval or ellipse, apical tip, wedge-shaped base, two-sided unhairing glandular, leaf sheath cylinder, membranous, loose, apical truncated, and peripherical hair. Inflorescence head-shaped, single generation or paired generation, apical generation; inflorescence peduncle has glandular hair; the bract is long-oval and membranous; the flower stalk is extremely short; the quilt is light red, has an elliptical shape, is long oval, black brown and slightly glossy, and is wrapped in the existing quilt; blooming in 6-9 months and bearing fruit in 8-10 months. Is distributed in Jiangxi, Hunan, Hubei, Sichuan, Guizhou, Guangdong, Guangxi, Yunnan and Tibet in China. North India, Nepal, Xijin, Dane, Burma and Vietnam are also distributed; the mountain slopes and the valley wetlands grow frequently in pieces, and the elevation is 600 + 3500 meters. The plant can be used as a medicine by whole herb, is recorded in Guangxi Chinese medicinal material, Yunnan Chinese herbal medicine and Wenshan Chinese herbal medicine, has the effects of clearing heat, promoting urination, treating stranguria and stopping dysentery, and can be used for treating cystitis, pyelonephritis and dysentery.
The extraction of the active substance of the polygonum capitatum has been the key point for researching the pharmacological properties of the polygonum capitatum, and the invention researches the extraction method of the active component of the polygonum capitatum in order to enrich the research information of the polygonum capitatum.
Disclosure of Invention
The gallic acid, the quercetin and the quercetin which are separated by the invention can be used for inhibiting escherichia coli and staphylococcus aureus, and the quercetin have the function of inhibiting alpha-glucosidase and can be applied to the treatment of diabetes.
The invention provides an active component of polygonum capitatum, which comprises gallic acid, quercetin and quercetin, and the active component is extracted from polygonum capitatum.
An application of herba Polygoni Capitati active components comprises application of gallic acid, quercetin and quercetin in inhibiting Escherichia coli and Staphylococcus aureus; application of quercetin and quercetin in inhibiting alpha-glucosidase in active components.
A method for extracting the active component of the polygonum capitatum comprises the following steps:
s1, mixing polygonum capitatum with ethanol with a volume fraction of 79% in a material-liquid ratio of 1:25g/mL, performing reflux extraction at 80 ℃ for 1.5 hours to obtain a first extracting solution and filter residue, performing reflux extraction on the filter residue once under the same conditions, combining the two extracting solutions, performing suction filtration, and performing reduced pressure concentration to obtain a polygonum capitatum ethanol extract;
s2, dissolving the ethanol extract of polygonum capitatum in S1 in water, sequentially extracting with petroleum ether, chloroform, ethyl acetate and n-butanol, and concentrating the extraction phases under reduced pressure to respectively obtain an ethyl acetate phase extract and an n-butanol phase extract, wherein the n-butanol phase extract contains active components quercetin and quercetin;
s3, passing the ethyl acetate phase extract of S2 through MCI reversed phase column, and gradient eluting with methanol-water solution to obtain gallic acid, quercetin and quercetin, respectively.
Preferably, in S3, the methanol-water solution has a volume concentration of 20% to 100%, and the gallic acid is obtained when the methanol-water solution has a volume concentration of 20%, the quercetin is obtained when the methanol-water solution has a volume concentration of 60%, and the quercetin is obtained when the methanol-water solution has a volume concentration of 80%.
Compared with the prior art, the invention has the beneficial effects that:
the gallic acid, the quercetin and the quercetin separated by the method can be used for inhibiting escherichia coli and staphylococcus aureus, can be applied to the research of bacterial infection, and can be applied to the treatment of diabetes, wherein the quercetin and the quercetin have the function of inhibiting alpha-glucosidase.
Drawings
FIG. 1 is a high performance liquid chromatogram of component A in example 1 of the present invention;
FIG. 2 is a high performance liquid chromatogram of component C in example 1 of the present invention;
FIG. 3 is a high performance liquid chromatogram of component D in example 1 of the present invention;
FIG. 4 is a high performance liquid chromatogram of the mixed standard in example 1 of the present invention;
FIG. 5 is a mass spectrum of component A in example 1 of the present invention;
FIG. 6 is a mass spectrum of component C in example 1 of the present invention;
FIG. 7 is a mass spectrum of component D in example 1 of the present invention;
FIG. 8 is a graph showing the relationship between the inhibition of alpha-glucosidase by quercetin and quercetin in example 2 of the present invention.
Detailed Description
One embodiment of the present invention is described in detail below with reference to fig. 1, but it should be understood that the scope of the present invention is not limited to the embodiment.
Example 1
A method for extracting active components of Polygonum capitatum comprises the following steps:
s1, mixing 3kg of polygonum capitatum with ethanol with a volume fraction of 79% in a material-liquid ratio of 1:25g/mL, performing reflux extraction at 80 ℃ for 1.5h to obtain a first extracting solution and filter residue, performing reflux extraction on the filter residue once under the same conditions, combining the two extracting solutions, performing suction filtration, and performing reduced pressure concentration by using a rotary evaporator under the conventional use parameters to obtain 375g of polygonum capitatum ethanol extract;
s2, dissolving 365g of ethanol extract of polygonum capitatum in S1 in water, shaking up, sequentially extracting with petroleum ether, chloroform, ethyl acetate and n-butanol, combining the extracts, and performing reduced pressure concentration on each extract phase by using a conventional rotary evaporator to obtain 5.636g of petroleum ether phase extract, 33.209g of chloroform phase extract, 30.8g of ethyl acetate phase extract, 31.912g of n-butanol phase extract and 227.560g of water phase extract, wherein the n-butanol phase extract contains active components of quercetin and quercetin;
s3, 27.05g of the ethyl acetate phase extract in the S2 is subjected to MCI reverse phase column chromatography, and gradient elution is carried out by using methanol-water solutions with volume concentrations of 20%, 40%, 60%, 80% and 100% respectively, so that 8.265g of a component A (elution concentration of 20%), 4.535g of a component B (elution concentration of 40%), 5.963g of a component C (elution concentration of 60%), 1.072g of a component D (elution concentration of 80%) and 2.551g of a component E (elution concentration of 100%) are obtained respectively.
In order to verify the specific chemical structure of each component, the ethyl acetate phase extract is analyzed by high performance liquid chromatography, standard substances of gallic acid, quercetin, protocatechuic acid and rutin are purchased respectively according to the chromatogram results, the standard substances are dissolved by a methanol solution and the volume is determined to be 5mL, a mixed standard substance solution with the mass concentration of 0.5mg/mL is prepared, each component obtained by separation is dissolved by the methanol solution and the volume is determined to be 5mL respectively, solutions with the mass concentrations of 0.67mg/mL, 0.53mg/mL and 0.66mg/mL are prepared, and each solution passes through a high performance liquid chromatography column.
Chromatographic conditions are as follows: a chromatographic column C18 (4.6X 150mm, 5um) is adopted, the volume flow is 0.7ml/min, the detection wavelength is 254nm, the column temperature is 35 ℃, and the sample injection amount is 10 uL.
Chromatographic mobile phase conditions: 0-6.5 min, 13% acetonitrile- (0.1% formic acid) water-18% acetonitrile- (0.1% formic acid) water; 6.5-15 min: 18% acetonitrile- (0.1% formic acid) water-41.5% acetonitrile- (0.1% formic acid) water; 15-25.5 min: 41.5% acetonitrile- (0.1% formic acid) water-41.5% acetonitrile- (0.1% formic acid) water; 25.5-32 min: 41.5% acetonitrile- (0.1% formic acid) water-80% acetonitrile- (0.1% formic acid) water; 32-40 min: 80% acetonitrile- (0.1% formic acid) water to 100% acetonitrile- (0.1% formic acid) water.
Mass spectrum conditions: the electrospray ion source adopts a positive ion mode, the scanning range is 100-1000Da, the positive ion mode voltage is 4000V, the atomizing gas is nitrogen, the flow rate is 60L/h, and the desolventizing temperature is 400 ℃.
The results are shown in FIGS. 1-4, and it can be seen from a comparison of FIGS. 1-4 that compound component A may be gallic acid, compound component C may be quercetin, and compound component D may be quercetin.
Further performing mass spectrum identification on the component A, C, D, the result is shown in FIGS. 5-7, and comparing with the mass spectrum obtained from gallic acid, quercetin and quercetin, it is known that component A is gallic acid, has a relative molecular weight of 170.12, and may be represented as 171.12[ M + H ] in the peak detection of mass spectrum]+、193.1[M+Na]+(ii) a Component C is quercetin with relative molecular weight of 448.37, and may be represented by 449.1[ M + H ] in mass spectrum peak detection peak]+、471.1[M+Na]+、487.1[M+K]+(ii) a Component D is quercetin with a relative molecular weight of 302, which may be represented by 303.1[ M + H ] in the peak of mass spectrum peak detection]+、325.1[M+Na]+。
Example 2
In order to explore the application of each component obtained by separation, experiments and verifications prove that the carnivorous acid, the quercitrin and the quercetin have the inhibition effect on escherichia coli and staphylococcus aureus, and the quercitrin and the quercetin have the inhibition effect on alpha-glucosidase. FIG. 8 is a graph showing the relationship between inhibition of alpha-glucosidase by quercetin and quercetin.
It should be noted that the steps and methods adopted in the claims of the present invention are the same as those of the above-mentioned embodiments, and for the sake of avoiding redundancy, the present invention describes the preferred embodiments, but those skilled in the art can make other changes and modifications to these embodiments once they learn the basic inventive concept. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (1)
1. A method for extracting gallic acid, quercetin and quercetin from polygonum capitatum is characterized by comprising the following steps:
s1, mixing polygonum capitatum with ethanol with a volume fraction of 79% in a material-liquid ratio of 1:25g/mL, performing reflux extraction at 80 ℃ for 1.5 hours to obtain a first extracting solution and filter residue, performing reflux extraction on the filter residue once under the same conditions, combining the two extracting solutions, performing suction filtration, and performing reduced pressure concentration to obtain a polygonum capitatum ethanol extract;
s2, dissolving the ethanol extract of polygonum capitatum in S1 in water, sequentially extracting with petroleum ether, chloroform, ethyl acetate and n-butanol, and concentrating the extraction phases under reduced pressure to respectively obtain an ethyl acetate phase extract and an n-butanol phase extract;
s3, passing the ethyl acetate phase extract of S2 through an MCI reverse phase column, and performing gradient elution by using a methanol-water solution to respectively obtain gallic acid, quercetin and quercetin, wherein the concentration of the methanol-water solution is 20% by volume to obtain the gallic acid, the concentration of the methanol-water solution is 60% by volume to obtain the quercetin, and the concentration of the methanol-water solution is 80% by volume to obtain the quercetin.
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| CN109771483B (en) * | 2019-03-18 | 2021-11-02 | 泉州师范学院 | Method for extracting high-activity alpha-glucosidase inhibitor from polygonum hydropiper |
| CN112098563B (en) * | 2020-10-14 | 2022-07-15 | 中国中医科学院望京医院(中国中医科学院骨伤科研究所) | Detection method for chemical components of kidney-tonifying and blood-activating formula |
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