CN112321450A - Preparation method of hydroxyl-alpha-sanshool monomer - Google Patents
Preparation method of hydroxyl-alpha-sanshool monomer Download PDFInfo
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- CN112321450A CN112321450A CN202011146305.5A CN202011146305A CN112321450A CN 112321450 A CN112321450 A CN 112321450A CN 202011146305 A CN202011146305 A CN 202011146305A CN 112321450 A CN112321450 A CN 112321450A
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Abstract
The invention provides a preparation method of a hydroxyl-alpha-sanshool monomer, belonging to the technical field of natural medicine extraction and separation. The preparation method comprises the following steps: (1) leaching with lower alcohol to obtain fructus Zanthoxyli extract total extract; (2) extracting with ethyl acetate, and enriching the mixture of hydroxy sanshool by column chromatography; (3) removing impurities by adopting a freezing crystallization method, and enriching hydroxy-alpha-sanshool; (4) and separating and purifying the hydroxy-alpha-sanshool. The preparation method of the hydroxyl-alpha-sanshool monomer is simple and convenient to operate and has low technical requirements on operators; the preparation process has mild conditions, thereby ensuring the stability of the hydroxy-alpha-sanshool and being very safe; meanwhile, the hydroxyl-alpha-sanshool monomer prepared by the preparation method has high purity, and the purity of the prepared hydroxyl-alpha-sanshool monomer can reach more than 99 percent and can be used as a hydroxyl-alpha-sanshool monomer standard product. The preparation method has low cost, is suitable for large-scale industrial popularization, and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of natural medicine extraction and separation, and particularly relates to a preparation method of a hydroxyl-alpha-sanshool monomer.
Background
Zanthoxylum bungeanum L, Rutaceae Zanthoxylum, has a very long history as traditional Chinese condiment and Chinese medicinal material, and its fruit has strong fragrance and pungent taste, and has the function of reducing the smell of mutton and fishy smell, and is one of eight major condiments in China. The spicy flavor is the most main flavor characteristic of the pepper and is the most important index for evaluating the quality of the pepper. In addition, the pepper is also a common Chinese medicine in China, has the effects of warming middle-jiao to relieve pain, resisting bacteria and killing insects, can be used for treating diseases such as vomit, diarrhea, ascariasis and the like, and also has the effects of analgesia, anti-inflammation, bacteriostasis, anti-aging and the like.
In the application of the zanthoxylum, the flavor is still the most important, and the unsaturated fatty amide compounds in the zanthoxylum are the main sources of the pungent taste of the zanthoxylum. The substances mainly comprise more than thirty species such as alpha-sanshool, beta-sanshool, gamma-sanshool, hydroxyl-alpha-sanshool, hydroxyl-beta-sanshool, hydroxyl-gamma-sanshool, hydroxyl-epsilon-sanshool and the like. Wherein the hydroxy-alpha-sanshool (1) and the hydroxy-beta-sanshool (2) shown in the following formulas have the highest content, and are specifically contained active ingredients in Zanthoxylum plants. Wherein, the hydroxyl-alpha-sanshool has strong tingling and numbing feelings and is the main active compound of the mechanism of action of generating numbing taste of the pepper.
Since the structures of the hydroxy sanshool compounds in the pepper are very similar and are mostly cis-trans isomers, the high-purity sanshool compound is obtained by extracting, separating and purifyingThe difficulty of the monomer is extremely high. Patent CN 103099163a only obtained a mixture of hydroxy sanshool. CN 105481711a discloses a preparation method of hydroxy- β -sanshool monomer, but does not disclose how to prepare hydroxy- α -sanshool monomer. CN106977416A discloses a method for preparing hydroxy-alpha-sanshool by ultraviolet irradiation and n-hexane extraction, but the method needs ultraviolet irradiation, the hydroxy-alpha-sanshool is very unstable, and the change is easy to occur in illumination or room temperature placement. Other methods for extracting fructus Zanthoxyli with pungent taste are provided, such as supercritical CO extraction2Only mixed hydroxy sanshool can be obtained by methods such as extraction, TLC thin layer and the like. The only monomers that can be used as standards are also hydroxy-beta-sanshool monomers. The preparation of the hydroxyl-alpha-sanshool monomer with high purity and high yield is still blank, so that the standard substance for detecting the spicy substance of the zanthoxylum is single, and the research and the application of the zanthoxylum are limited to a great extent.
Disclosure of Invention
In order to solve the above problems, the present invention provides a highly efficient method for preparing a high-purity hydroxy- α -sanshool monomer that can be used as a standard.
The invention provides a preparation method of a hydroxyl-alpha-sanshool monomer, which comprises the following steps:
(1) preparing a pepper extract total extract: leaching fructus Zanthoxyli with lower alcohol as extraction solvent to obtain leaching solution, and concentrating the leaching solution under reduced pressure to obtain fructus Zanthoxyli extract total extract;
(2) enrichment of mixture of hydroxy sanshools: diluting the total extract of the pepper extract obtained in the step (1) with water, extracting with ethyl acetate, collecting ethyl acetate extract, concentrating under reduced pressure to obtain sanshool mixture extract, performing column chromatography on the sanshool mixture extract, collecting eluent, and concentrating to obtain hydroxyl sanshool mixture extract;
(3) enrichment of hydroxy-alpha-sanshool: dissolving the hydroxy sanshool mixture extract obtained in the step (2) in an organic solvent, freezing and crystallizing, and filtering and collecting a hydroxy sanshool mixture mother solution;
(4) separation and purification of hydroxy-alpha-sanshool: and (3) subjecting the hydroxyl sanshool mixture mother liquor obtained in the step (3) to RP-18 reverse phase silica gel column chromatography, eluting with an acetone aqueous solution to obtain a hydroxyl-alpha-sanshool monomer acetone aqueous solution, adding NaCl until no precipitate is separated out, filtering, concentrating the filtrate under reduced pressure, subjecting the filtrate to non-polar polystyrene type macroporous adsorption resin column chromatography to obtain a hydroxyl-alpha-sanshool solution, concentrating under reduced pressure, and drying to obtain the hydroxyl-alpha-sanshool compound.
Further, the air conditioner is provided with a fan,
in the step (1), the lower alcohol is 70-100% lower alcohol aqueous solution by volume fraction;
and/or in the step (1), the feed-liquid ratio of the pepper to the extraction solvent is 1: 5-1: 10;
and/or, in the step (1), the leaching is cold leaching extraction;
and/or in the step (1), the temperature of the reduced pressure concentration is 40-50 ℃;
and/or in the step (1), the volume of a concentrated solution obtained by the reduced pressure concentration is 1/5-1/4 of the leaching solution.
Further, the air conditioner is provided with a fan,
in the step (1), the lower alcohol is 85-100% lower alcohol aqueous solution by volume fraction;
and/or in the step (1), the feed-liquid ratio of the pepper to the extraction solvent is 1: 8-1: 10;
and/or, in the step (1), the pepper is dried pepper powder;
and/or in the step (1), the extraction time of the leaching is 48 hours;
and/or in the step (1), the extraction times of the leaching are 2-3 times;
preferably, the first and second electrodes are formed of a metal,
in the step (1), the pepper is dried pepper peel and crushed into 20-40 meshes of powder.
Further, in the step (1), the lower alcohol is one or a mixture of more than one of methanol and ethanol.
Further, the air conditioner is provided with a fan,
in the step (2), diluting the total extract of the pepper extract obtained in the step (1) with water to a volume fraction of a lower alcohol solution of 10-20%;
and/or in the step (2), the volume ratio of the ethyl acetate to the water is 1: 0.8-1;
and/or in the step (2), the temperature of the reduced pressure concentration is 40-50 ℃;
and/or in the step (2), performing column chromatography by using 100-200 mesh silica gel;
and/or, in the step (2), in the column chromatography, the elution is gradient elution;
and/or, in the step (2), in the column chromatography, the eluent to be eluted is a mixed solution of petroleum ether and ethyl acetate;
preferably, the first and second electrodes are formed of a metal,
in the step (2), the extraction is carried out for 3 times;
and/or, in the step (2), during gradient elution, gradient elution is carried out according to the volume ratio of petroleum ether to ethyl acetate of 4:1, 2:1, 1:1 and 0: 1.
Further, in the step (3), the organic solvent is one or a mixture of more than one of petroleum ether, acetone, diethyl ether and ethyl acetate.
Further, in the step (3), the organic solvent is a mixed solution of petroleum ether and acetone or ethyl acetate;
preferably, in the step (3), in the mixed solution of petroleum ether and acetone, the volume ratio of petroleum ether to acetone is 1:1 to 2.
Further, the air conditioner is provided with a fan,
in the step (4), the acetone aqueous solution is acetone and AgNO3A mixed solution of aqueous solutions;
and/or in the step (4), the temperature of decompression concentration after filtration is 40 ℃;
preferably, the acetone and AgNO3The volume ratio of the aqueous solution was 45: 55.
Further, the air conditioner is provided with a fan,
the AgNO3The concentration of the aqueous solution is 1.5-2%.
Further, in the step (4), the model of the nonpolar polystyrene type macroporous resin is D101, AB-8 and HP 20.
Compared with the prior art, the invention has the advantages and effects that:
the method can obtain the high-purity hydroxy-alpha-sanshool monomer from the pepper peel extract through four steps of extraction, mixture enrichment, target substance enrichment, separation and purification. The whole preparation process has mild conditions, the highest temperature is not more than 50 ℃, the stability of the hydroxy-alpha-sanshool is ensured, the yield is high, the operation is simple and convenient, the technical requirement on operators is low, and the method is suitable for industrial popularization.
Secondly, the preparation of the hydroxyl-alpha-sanshool monomer provided by the invention adopts a low-temperature freezing crystallization method, so that the content of the hydroxyl-alpha-sanshool in a hydroxyl sanshool mixture is greatly improved, and simultaneously, the hydroxyl-alpha-sanshool is separated and purified by adopting a method of combining Ag ion reverse phase column chromatography with macroporous resin column chromatography, so that the problem that cis-trans isomers such as the hydroxyl-alpha-sanshool, the hydroxyl-beta-sanshool, the hydroxyl-gamma-sanshool and the like are difficult to separate is solved, the process is simple to operate and short in time consumption, the defect of yield reduction caused by the change in the preparation process due to the instability of the hydroxyl-alpha-sanshool is avoided, the purity of the obtained product is high, and the purity of the prepared hydroxyl-alpha-sanshool monomer can reach more than 98%. Fills the blank that the hydroxyl-alpha-sanshool is taken as a reference sample in the prior zanthoxylum tingling degree detection and zanthoxylum quality detection.
The equipment used by the method mainly comprises a chromatographic column during column chromatography and a rotary evaporator during reduced pressure concentration, and the chromatographic column and the rotary evaporator are low in price and low in production cost.
In conclusion, the preparation method of the hydroxy-alpha-sanshool monomer is simple and convenient to operate and has low technical requirements on operators; the preparation process has mild conditions, thereby ensuring the stability of the hydroxy-alpha-sanshool and being very safe; meanwhile, the hydroxyl-alpha-sanshool monomer prepared by the preparation method has high purity, and the purity of the prepared hydroxyl-alpha-sanshool monomer can reach more than 99 percent and can be used as a hydroxyl-alpha-sanshool monomer standard product. The preparation method has low cost, is suitable for large-scale industrial popularization, and has good application prospect.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
Fig. 1 is a high performance liquid chromatography analysis diagram of a hydroxy- α -sanshool monomer prepared in example 5 of the present invention, wherein the peak at t ═ 11.60min is hydroxy- α -sanshool.
Detailed Description
The raw materials and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products.
The final product of the example, hydroxy- α -sanshool monomer, was purity checked using High Performance Liquid Chromatography (HPLC). The detection chromatographic conditions are as follows: the mobile phase composition is acetonitrile-water 45:55(v/v), chromatographic column model YMC-Pack C18 chromatographic column (4.6mm × 250mm, 5 μm); the flow rate is 1.0 mL/min; column temperature: room temperature; the detection wavelength is 266 nm.
Example 1 preparation of hydroxy-alpha-sanshool monomer of the invention
The preparation method comprises the following steps:
(1) preparation of general extract of pepper extract
Extracting 2kg of dried pricklyash peel powder with 95% ethanol water for 48h at a material-liquid ratio of 1:10(kg/L) for 3 times, mixing the extractive solutions, removing residues, concentrating the extractive solution at 50 deg.C under reduced pressure to obtain concentrated solution with volume of 1/5 of the extractive solution, and collecting the total extract.
(2) Enrichment of mixture of hydroxy sanshools
Diluting the total extract of the pepper extract obtained in the step (1) with water until the volume fraction of ethanol is 20%, and extracting with ethyl acetate, wherein the volume ratio of ethyl acetate to water is 1L:1L, repeating the extraction process for 3 times, mixing the ethyl acetate solutions obtained by extraction, and concentrating under reduced pressure at 50 deg.C to obtain 70g sanshool mixture extract. Performing column chromatography on the sanshool mixture extract by using 100-200-mesh silica gel, performing gradient elution by using petroleum ether and ethyl acetate according to the proportion sequence of 4:1, 2:1, 1:1 and 0:1(v/v), collecting eluent, and concentrating to obtain 10g of hydroxyl sanshool mixture.
(3) Enrichment of hydroxy-alpha-sanshool
30mL of petroleum ether-acetone (1:1v/v) mixed solution is added into each gram of the mixture of the hydroxy sanshools to dissolve the mixture of the hydroxy sanshools, the content of the hydroxy-alpha-sanshools in the solution is 64.1 percent through HPLC detection, the solution is placed in a refrigerator to be frozen for 18 hours at low temperature (-18 ℃), the solid is removed through filtration, and the content of the hydroxy-alpha-sanshools in the mother solution is 84.1 percent through HPLC detection. The content of the hydroxy-alpha-sanshool in the hydroxy sanshool mixture is improved.
(4) Separation and purification of hydroxy-alpha-sanshool
Performing RP-18 reverse phase silica gel column chromatography on the hydroxyl sanshool mixture mother liquor obtained in the step (3) under the condition of keeping out of the sun, and performing AgNO chromatography by using acetone of 2 percent3Eluting with water solution 45:55(v/v) to obtain water solution of hydroxy-alpha-sanshool monomer acetone; then adding NaCl until no precipitate is separated out, and filtering to obtain the product containing no Ag+The obtained hydroxy-alpha-sanshool monomer acetone aqueous solution is subjected to vacuum concentration at the temperature of 40 ℃, then is subjected to column chromatography by using a nonpolar polystyrene type macroporous adsorption resin to obtain a hydroxy-alpha-sanshool solution, and is subjected to vacuum concentration and drying to obtain the hydroxy-alpha-sanshool monomer with the purity of 98.5 percent (29.2g, the yield is 1.46 percent).
Example 2 preparation of hydroxy-alpha-sanshool monomer of the invention
The preparation method comprises the following steps:
(1) preparation of general extract of pepper extract
2kg of dried pepper powder is taken, and extracted by 85% ethanol water solution for 48h, wherein the material-liquid ratio is 1:8(kg/L), extracting for 3 times, mixing leaching liquor, removing residue, concentrating the leaching liquor at 50 deg.C under reduced pressure to obtain concentrated solution, wherein the volume of the concentrated solution is 1/5 of the leaching liquor, and obtaining fructus Zanthoxyli extract total extract.
(2) Enrichment of mixture of hydroxy sanshools
Diluting the total extract of the pepper extract obtained in the step (1) with water until the volume fraction of ethanol is 20%, extracting with ethyl acetate, wherein the volume ratio of ethyl acetate to water is 1L:1L, repeating the extraction process for 3 times, combining the ethyl acetate solutions obtained by extraction, and concentrating under reduced pressure at 50 ℃ to obtain 65g of sanshool mixture extract. Performing column chromatography on the sanshool mixture extract by using 100-200-mesh silica gel, performing gradient elution by using petroleum ether and ethyl acetate according to the proportion sequence of 4:1, 2:1, 1:1 and 0:1(v/v), collecting eluent, and concentrating to obtain 8.9g of hydroxyl sanshool mixture.
(3) Enrichment of hydroxy-alpha-sanshool
30mL of petroleum ether-acetone (1:1v/v) mixed solution is added into each gram of the mixture of the hydroxy sanshools to dissolve the mixture of the hydroxy sanshools, the content of the hydroxy-alpha-sanshools in the solution is 62.9 percent through HPLC detection, the solution is placed in a refrigerator to be frozen for 24 hours at low temperature (-18 ℃), the solid is removed through filtration, and the content of the hydroxy-alpha-sanshools in the mother solution is 79.7 percent through HPLC detection. The content of the hydroxy-alpha-sanshool in the hydroxy sanshool mixture is improved.
(4) Separation and purification of hydroxy-alpha-sanshool
Performing RP-18 reverse phase silica gel column chromatography on the hydroxyl sanshool mixture mother liquor obtained in the step (3) under the condition of keeping out of the sun, and performing AgNO chromatography by using acetone of which the ratio is 1.5 percent3Eluting with water solution 45:55(v/v) to obtain water solution of hydroxy-alpha-sanshool monomer acetone; then adding NaCl until no precipitate is separated out, and filtering to obtain the product containing no Ag+The obtained hydroxy-alpha-sanshool monomer acetone aqueous solution is subjected to vacuum concentration at the temperature of 40 ℃, then is subjected to column chromatography by using a nonpolar polystyrene type macroporous adsorption resin to obtain a hydroxy-alpha-sanshool solution, and is subjected to vacuum concentration and drying to obtain the hydroxy-alpha-sanshool monomer with the purity of 98.9% (24.2g, the yield is 1.21%).
Example 3 preparation of hydroxy-alpha-sanshool monomer of the invention
The preparation method comprises the following steps:
(1) preparation of general extract of pepper extract
2kg of dried pepper powder is taken and extracted by 90 percent methanol water solution for 48 hours, and the material-liquid ratio is 1:10(kg/L), extracting for 3 times, mixing leaching solutions, removing residue, concentrating the leaching solution at 50 deg.C under reduced pressure to obtain concentrated solution, wherein the volume of the concentrated solution is 1/5 of the leaching solution, and obtaining fructus Zanthoxyli extract total extract.
(2) Enrichment of mixture of hydroxy sanshools
Diluting the total extract of the pepper extract obtained in the step (1) with water until the volume fraction of methanol is 20%, extracting with ethyl acetate, wherein the volume ratio of ethyl acetate to water is 1L:1L, repeating the extraction process for 3 times, combining the ethyl acetate solutions obtained by extraction, and concentrating under reduced pressure at 50 ℃ to obtain 58g of sanshool mixture extract. Performing column chromatography on the sanshool mixture extract by using 100-200-mesh silica gel, performing gradient elution by using petroleum ether and ethyl acetate according to the proportion sequence of 4:1, 2:1, 1:1 and 0:1(v/v), collecting eluent, and concentrating to obtain 8.1g of hydroxyl sanshool mixture.
(3) Enrichment of hydroxy-alpha-sanshool
Adding petroleum ether-acetone (1:1v/v) mixed solution into 30mL of the mixture per gram of the mixture of the hydroxy sanshools to dissolve the mixture of the hydroxy sanshools, detecting by HPLC to find that the content of the hydroxy-alpha-sanshools in the solution is 59.0%, placing the solution in a refrigerator for freezing at low temperature (-18 ℃) for 36h, filtering to remove solids, and detecting by HPLC to find that the content of the hydroxy-alpha-sanshools in mother liquor after crystallization is 78.2%. The content of the hydroxyl-alpha-sanshool in the hydroxyl sanshool mixture solution is improved.
(4) Separation and purification of hydroxy-alpha-sanshool
Performing RP-18 reverse phase silica gel column chromatography on the hydroxyl sanshool mixture mother liquor obtained in the step (3) under the condition of keeping out of the sun, and performing AgNO chromatography by using acetone of which the ratio is 1.5 percent3Eluting with water solution 45:55(v/v) to obtain water solution of hydroxy-alpha-sanshool monomer acetone; then adding NaCl until no precipitate is separated out, and filtering to obtain the product containing no Ag+The obtained hydroxy-alpha-sanshool monomer acetone aqueous solution is subjected to vacuum concentration at the temperature of 40 ℃, then is subjected to column chromatography by using non-polar polystyrene type macroporous adsorption resin to obtain a hydroxy-alpha-sanshool solution, and is subjected to vacuum concentration and drying to obtain the hydroxy-alpha-sanshool monomer with the purity of 98.6 percent (30.6g, the yield is 1.53 percent).
Example 4 preparation of hydroxy-alpha-sanshool monomer of the invention
The preparation method comprises the following steps:
(1) preparation of general extract of pepper extract
Extracting 2kg of dried fructus Zanthoxyli powder with anhydrous methanol at a material-to-liquid ratio of 1:8(kg/L) for 48h for 3 times, mixing extractive solutions, removing residue, concentrating the extractive solution at 50 deg.C under reduced pressure to obtain concentrated solution with volume of 1/5 of the extractive solution, and obtaining fructus Zanthoxyli extract total extract.
(2) Enrichment of mixture of hydroxy sanshools
Diluting the total extract of the pepper extract obtained in the step (1) with water until the volume fraction of methanol is 20%, and extracting with ethyl acetate, wherein the volume ratio of ethyl acetate to water is 1L:1L, repeating the extraction process for 3 times, mixing ethyl acetate solutions obtained by extraction, and concentrating under reduced pressure at 50 deg.C to obtain sanshool mixture extract 65 g. Performing column chromatography on the sanshool mixture extract by using 100-200-mesh silica gel, performing gradient elution by using petroleum ether and ethyl acetate according to the proportion sequence of 4:1, 2:1, 1:1 and 0:1(v/v), collecting eluent, and concentrating to obtain 9.3g of hydroxyl sanshool mixture.
(3) Enrichment of hydroxy-alpha-sanshool
Adding 30mL of petroleum ether-acetone (1:1v/v) mixed solution into each gram of the mixture of the hydroxy sanshools to dissolve the mixture of the hydroxy sanshools, detecting by HPLC to find that the content of the hydroxy-alpha-sanshools in the solution is 65.9 percent, placing the solution in a refrigerator to be frozen for 20 hours at low temperature (-18 ℃), filtering to remove solids, and detecting by HPLC to find that the content of the hydroxy-alpha-sanshools in the mother liquor after crystallization is 80.6 percent. The content of the hydroxyl-alpha-sanshool in the hydroxyl sanshool mixture solution is improved.
(4) Separation and purification of hydroxy-alpha-sanshool
Performing RP-18 reverse phase silica gel column chromatography on the hydroxyl sanshool mixture mother liquor obtained in the step (3) under the condition of keeping out of the sun, and performing AgNO chromatography by using acetone of which the ratio is 1.5 percent3Eluting with water solution 45:55(v/v) to obtain water solution of hydroxy-alpha-sanshool monomer acetone; then adding NaCl until no precipitate is separated out, and filtering to obtain the product containing no Ag+The hydroxyl-alpha-sanshool monomer acetone aqueous solution is decompressed and concentrated at the temperature of 40 ℃, and then the hydroxyl-alpha-sanshool is obtained by the chromatography of a nonpolar polystyrene type macroporous absorption resin columnThe resulting solution was concentrated under reduced pressure and dried to obtain a hydroxy- α -sanshool monomer (26.4g, yield 1.32%) having a purity of 99.1%.
Example 5 preparation of hydroxy-alpha-sanshool monomer of the invention
The preparation method comprises the following steps:
(1) preparation of general extract of pepper extract
Taking 10kg of dried pepper powder, leaching for 48h by using 95% methanol aqueous solution by volume fraction, wherein the material-liquid ratio is 1:8(kg/L), extracting for 3 times, combining leaching liquor, removing dregs of a decoction, concentrating the leaching liquor at 50 ℃ under reduced pressure to obtain concentrated solution, wherein the volume of the concentrated solution is 1/4 of the leaching liquor, and obtaining the total extract of the pepper extract.
(2) Enrichment of mixture of hydroxy sanshools
Diluting the total extract of the pepper extract obtained in the step (1) with water until the volume fraction of methanol is 10%, extracting with ethyl acetate, wherein the volume ratio of ethyl acetate to water is 1L:1L, repeating the extraction process for 3 times, combining the ethyl acetate solutions obtained by extraction, and concentrating under reduced pressure at 50 ℃ to obtain 150g of sanshool mixture extract. Performing column chromatography on the sanshool mixture extract by using 100-200-mesh silica gel, performing gradient elution by using petroleum ether and ethyl acetate according to the proportion sequence of 4:1, 2:1, 1:1 and 0:1(v/v), collecting eluent, and concentrating to obtain 28.5g of hydroxyl sanshool mixture.
(3) Enrichment of hydroxy-alpha-sanshool
Adding 30mL of petroleum ether-acetone (1:2v/v) mixed solution into each gram of the mixture of the hydroxy sanshools to dissolve the mixture of the hydroxy sanshools, detecting by HPLC to find that the content of the hydroxy-alpha-sanshools in the solution is 66.6 percent, placing the solution in a refrigerator to be frozen for 18 hours at low temperature (-18 ℃), filtering to remove solids, and detecting by HPLC to find that the content of the hydroxy-alpha-sanshools in mother liquor after crystallization is 84.4 percent. The content of the hydroxyl-alpha-sanshool in the hydroxyl sanshool mixture solution is improved.
(4) Separation and purification of hydroxy-alpha-sanshool
Performing RP-18 reverse phase silica gel column chromatography on the hydroxyl sanshool mixture mother liquor obtained in the step (3) under the condition of keeping out of the sun, and performing AgNO chromatography by using acetone of 2 percent3Eluting with aqueous solution 45:55(v/v) to obtain hydroxyl-alpha-a sanshool monomer aqueous acetone solution; then adding NaCl until no precipitate is separated out, and filtering to obtain the product containing no Ag+The hydroxy-alpha-sanshool monomer acetone aqueous solution is decompressed and concentrated at the temperature of 40 ℃, then the hydroxy-alpha-sanshool solution is obtained by the chromatography of a nonpolar polystyrene type macroporous absorption resin column, and the hydroxy-alpha-sanshool monomer (148g, the yield is 1.48%) with the purity of 98.7 percent is obtained by decompression, concentration and drying.
Fig. 1 is a high performance liquid chromatography analysis diagram of the hydroxy- α -sanshool monomer prepared in this example, in which the peak at t ═ 11.60min is hydroxy- α -sanshool.
In conclusion, the preparation method of the hydroxy-alpha-sanshool monomer is simple and convenient to operate and has low technical requirements on operators; the preparation process has mild conditions, thereby ensuring the stability of the hydroxy-alpha-sanshool and being very safe; meanwhile, the hydroxyl-alpha-sanshool monomer prepared by the preparation method has high purity, and the purity of the prepared hydroxyl-alpha-sanshool monomer can reach more than 99 percent and can be used as a hydroxyl-alpha-sanshool monomer standard product. The preparation method has low cost, is suitable for large-scale industrial popularization, and has good application prospect.
Claims (10)
1. A preparation method of a hydroxyl-alpha-sanshool monomer is characterized by comprising the following steps: it comprises the following steps:
(1) preparing a pepper extract total extract: leaching fructus Zanthoxyli with lower alcohol as extraction solvent to obtain leaching solution, and concentrating the leaching solution under reduced pressure to obtain fructus Zanthoxyli extract total extract;
(2) enrichment of mixture of hydroxy sanshools: diluting the total extract of the pepper extract obtained in the step (1) with water, extracting with ethyl acetate, collecting ethyl acetate extract, concentrating under reduced pressure to obtain sanshool mixture extract, performing column chromatography on the sanshool mixture extract, collecting eluent, and concentrating to obtain hydroxyl sanshool mixture extract;
(3) enrichment of hydroxy-alpha-sanshool: dissolving the hydroxy sanshool mixture extract obtained in the step (2) in an organic solvent, freezing and crystallizing, and filtering and collecting a hydroxy sanshool mixture mother solution;
(4) separation and purification of hydroxy-alpha-sanshool: and (3) subjecting the hydroxyl sanshool mixture mother liquor obtained in the step (3) to RP-18 reverse phase silica gel column chromatography, eluting with an acetone aqueous solution to obtain a hydroxyl-alpha-sanshool monomer acetone aqueous solution, adding NaCl until no precipitate is separated out, filtering, concentrating the filtrate under reduced pressure, subjecting the filtrate to non-polar polystyrene type macroporous adsorption resin column chromatography to obtain a hydroxyl-alpha-sanshool solution, concentrating under reduced pressure, and drying to obtain the hydroxyl-alpha-sanshool compound.
2. The method of claim 1, wherein:
in the step (1), the lower alcohol is 70-100% lower alcohol aqueous solution by volume fraction;
and/or in the step (1), the feed-liquid ratio of the pepper to the extraction solvent is 1: 5-1: 10;
and/or, in the step (1), the leaching is cold leaching extraction;
and/or in the step (1), the temperature of the reduced pressure concentration is 40-50 ℃;
and/or in the step (1), the volume of a concentrated solution obtained by the reduced pressure concentration is 1/5-1/4 of the leaching solution.
3. The method of claim 1, wherein:
in the step (1), the lower alcohol is 85-100% lower alcohol aqueous solution by volume fraction;
and/or in the step (1), the feed-liquid ratio of the pepper to the extraction solvent is 1: 8-1: 10;
and/or, in the step (1), the pepper is dried pepper powder;
and/or in the step (1), the extraction time of the leaching is 48 hours;
and/or in the step (1), the extraction times of the leaching are 2-3 times;
preferably, the first and second electrodes are formed of a metal,
in the step (1), the pepper is dried pepper peel and crushed into 20-40 meshes of powder.
4. The method of claim 1, wherein: in the step (1), the lower alcohol is one or a mixture of more than one of methanol and ethanol.
5. The method of claim 1, wherein:
in the step (2), diluting the total extract of the pepper extract obtained in the step (1) with water to a volume fraction of a lower alcohol solution of 10-20%;
and/or in the step (2), the volume ratio of the ethyl acetate to the water is 1: 0.8-1;
and/or in the step (2), the temperature of the reduced pressure concentration is 40-50 ℃;
and/or in the step (2), performing column chromatography by using 100-200 mesh silica gel;
and/or, in the step (2), in the column chromatography, the elution is gradient elution;
and/or, in the step (2), in the column chromatography, the eluent to be eluted is a mixed solution of petroleum ether and ethyl acetate;
preferably, the first and second electrodes are formed of a metal,
in the step (2), the extraction is carried out for 3 times;
and/or, in the step (2), during gradient elution, gradient elution is carried out according to the volume ratio of petroleum ether to ethyl acetate of 4:1, 2:1, 1:1 and 0: 1.
6. The method of claim 1, wherein: in the step (3), the organic solvent is one or a mixture of more than one of petroleum ether, acetone, diethyl ether and ethyl acetate.
7. The method of claim 6, wherein: in the step (3), the organic solvent is a mixed solution of petroleum ether and acetone or ethyl acetate;
preferably, in the step (3), in the mixed solution of petroleum ether and acetone, the volume ratio of petroleum ether to acetone is 1:1 to 2.
8. The method of claim 1, wherein:
in the step (4), the acetone aqueous solution is acetone and AgNO3A mixed solution of aqueous solutions;
and/or in the step (4), the temperature of decompression concentration after filtration is 40 ℃;
preferably, the acetone and AgNO3The volume ratio of the aqueous solution was 45: 55.
9. The method of claim 8, wherein:
the AgNO3The concentration of the aqueous solution is 1.5-2%.
10. The method of claim 1, wherein: in the step (4), the types of the nonpolar polystyrene macroporous resin are D101, AB-8 and HP 20.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113816870A (en) * | 2021-08-25 | 2021-12-21 | 沈阳药科大学 | Method for rapidly extracting different sanshool monomers |
| CN115385817A (en) * | 2022-07-08 | 2022-11-25 | 四川大学 | Method for improving photoprotection stability of sanshool by utilizing polyphenol material |
| CN116036225A (en) * | 2023-02-10 | 2023-05-02 | 西南交通大学 | A natural external pharmaceutical composition for treating arthritis |
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| CN105237430A (en) * | 2015-10-27 | 2016-01-13 | 峨眉山万佛绿色食品有限公司 | Method for extracting numb-taste substance from Zanthoxylum armatum DC. |
| CN106977416A (en) * | 2017-04-25 | 2017-07-25 | 四川大学 | A kind of method of quick preparation high-purity hydroxyl α sanshools |
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| CN105237430A (en) * | 2015-10-27 | 2016-01-13 | 峨眉山万佛绿色食品有限公司 | Method for extracting numb-taste substance from Zanthoxylum armatum DC. |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113816870A (en) * | 2021-08-25 | 2021-12-21 | 沈阳药科大学 | Method for rapidly extracting different sanshool monomers |
| CN113816870B (en) * | 2021-08-25 | 2023-10-31 | 沈阳药科大学 | Method for rapidly extracting different sanshool monomers |
| CN115385817A (en) * | 2022-07-08 | 2022-11-25 | 四川大学 | Method for improving photoprotection stability of sanshool by utilizing polyphenol material |
| CN115385817B (en) * | 2022-07-08 | 2023-10-20 | 四川大学 | Method for improving photoprotection stability of sanshool by using polyphenol material |
| CN116036225A (en) * | 2023-02-10 | 2023-05-02 | 西南交通大学 | A natural external pharmaceutical composition for treating arthritis |
| CN116036225B (en) * | 2023-02-10 | 2023-11-24 | 西南交通大学 | A natural external pharmaceutical composition for treating arthritis |
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