CN114426478A - High-content gallic acid and preparation method thereof - Google Patents
High-content gallic acid and preparation method thereof Download PDFInfo
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- CN114426478A CN114426478A CN202210106100.7A CN202210106100A CN114426478A CN 114426478 A CN114426478 A CN 114426478A CN 202210106100 A CN202210106100 A CN 202210106100A CN 114426478 A CN114426478 A CN 114426478A
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- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 title claims abstract description 355
- 229940074391 gallic acid Drugs 0.000 title claims abstract description 173
- 235000004515 gallic acid Nutrition 0.000 title claims abstract description 173
- 238000002360 preparation method Methods 0.000 title claims abstract description 48
- 238000000034 method Methods 0.000 claims abstract description 15
- 239000002994 raw material Substances 0.000 claims abstract description 11
- 238000004440 column chromatography Methods 0.000 claims abstract description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 38
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 24
- 239000000843 powder Substances 0.000 claims description 22
- 239000013078 crystal Substances 0.000 claims description 18
- 238000001035 drying Methods 0.000 claims description 13
- 238000002425 crystallisation Methods 0.000 claims description 11
- 230000008025 crystallization Effects 0.000 claims description 11
- 238000001291 vacuum drying Methods 0.000 claims description 11
- 238000010828 elution Methods 0.000 claims description 10
- -1 hydroxypropyl Chemical group 0.000 claims description 10
- 239000002798 polar solvent Substances 0.000 claims description 10
- 238000002137 ultrasound extraction Methods 0.000 claims description 10
- 229920005654 Sephadex Polymers 0.000 claims description 9
- 239000003480 eluent Substances 0.000 claims description 9
- 108010038851 tannase Proteins 0.000 claims description 9
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 8
- 239000000945 filler Substances 0.000 claims description 8
- 238000010829 isocratic elution Methods 0.000 claims description 8
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 claims description 8
- 230000004580 weight loss Effects 0.000 claims description 8
- 238000001816 cooling Methods 0.000 claims description 7
- 239000002245 particle Substances 0.000 claims description 7
- 238000010298 pulverizing process Methods 0.000 claims description 7
- 238000007873 sieving Methods 0.000 claims description 7
- 238000001914 filtration Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 238000002481 ethanol extraction Methods 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims 1
- 238000000605 extraction Methods 0.000 abstract description 9
- 238000000746 purification Methods 0.000 abstract description 7
- 238000000926 separation method Methods 0.000 abstract description 5
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- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 8
- 239000007864 aqueous solution Substances 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 7
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- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 4
- 238000005160 1H NMR spectroscopy Methods 0.000 description 4
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- 241000196881 Castanopsis Species 0.000 description 1
- 241000499335 Castanopsis chinensis Species 0.000 description 1
- 241000237970 Conus <genus> Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
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- 238000012356 Product development Methods 0.000 description 1
- 240000003152 Rhus chinensis Species 0.000 description 1
- 235000014220 Rhus chinensis Nutrition 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
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- 230000003078 antioxidant effect Effects 0.000 description 1
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- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
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- 150000007524 organic acids Chemical class 0.000 description 1
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- 235000009566 rice Nutrition 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
- C07C51/43—Separation; Purification; Stabilisation; Use of additives by change of the physical state, e.g. crystallisation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
- C07C51/47—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
- C07C51/48—Separation; Purification; Stabilisation; Use of additives by liquid-liquid treatment
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C65/00—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C65/01—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups
- C07C65/03—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups monocyclic and having all hydroxy or O-metal groups bound to the ring
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a high-content gallic acid and a preparation method thereof, belonging to the technical field of extraction, separation and purification of traditional Chinese medicine components. The preparation method of the high-content gallic acid comprises the following steps: step 1: preparing a gallic acid extracting solution; step 2: preparing an extract rich in gallic acid; and step 3: preparing high-content gallic acid. The invention also discloses a high-content gallic acid prepared by the preparation method. The invention firstly takes the coniferous tree as the raw material, and obtains the high-content gallic acid through simple and convenient processes such as extraction, enzyme hydrolysis, column chromatography and the like, thereby providing a new supply raw material for the preparation of the gallic acid and opening up a new utilization path for conifer resources.
Description
Technical Field
The invention relates to high-content gallic acid and a preparation method thereof, belonging to the technical field of extraction, separation and purification of traditional Chinese medicine components.
Background
Cones (Castanopsis chinensis) also named as Guilin tannin extract, Castanopsis mollissima and rice cones, are trees of Conus of Fagaceae, up to 20 m, with a diameter at breast height of 0.6 m, longitudinally split bark, exfoliated, without hair on branches and leaves. The wood is widely distributed in provinces such as Guangxi province, Guangdong province and Yunnan province, the wood is brownish yellow, sometimes the core material is dirty and dark, the wood ray is very narrow, the material is lighter, the structure is slightly coarse, the texture is straight, and the wood belongs to the category of yellow cones.
At present, no relevant reports are found in researches on chemical components, pharmacological activity, product development and the like of cones, and the early stage of qualitative pre-experimental detection shows that the leaves of the cones contain polyphenol components, so that the health-care and beauty-care cones have a high prospect of being developed into health-care and beauty-care products with an antioxidant function. Gallic Acid (GA for short), its chemical name is 3,4, 5-trihydroxybenzoic Acid, and its molecular formula is C7H6O5It is also known as gallic acid and gallic acid, and is an organic acid, commonly found in gallnut, sumac, tea and other plants, and widely used in chemical, pharmaceutical, food and electronic industries, etc.
In the prior art, the preparation method of the gallic acid mainly uses organic solvent or aqueous solution to add acid for extraction, and the separation and purification adopts macroporous adsorption resin purification and anion-cation resin decoloration or activated carbon decoloration and the like to be combined, and has the defects that: the yield and purity are not high. In addition, no report about the extraction and purification process of gallic acid in cone or the extraction and purification process after enzymatic hydrolysis exists in the prior art, and no report about the separation and purification of gallic acid by adopting a high-purity ethanol system elution system when the gallic acid is purified by gel column chromatography is reported.
In view of the above, there is a need to provide a method for preparing high content gallic acid from cone, which provides a new source of gallic acid and overcomes the shortcomings of the prior art.
Disclosure of Invention
One of the purposes of the invention is to provide a preparation method of high-content gallic acid.
The technical scheme for solving the technical problems is as follows: a preparation method of high-content gallic acid comprises the following steps:
step 1: preparation of Gallic acid extract
Taking folium Coni Cone powder, adding polar solvent, and performing ultrasonic extraction to obtain gallic acid extractive solution;
and 2, step: preparation of gallic acid-rich extract
Performing enzymolysis on the gallic acid extract obtained in the step 1, and concentrating to obtain an extract rich in gallic acid;
and step 3: preparation of high content of gallic acid
Extracting the gallic acid-rich extract obtained in the step 2 by using ethanol to obtain an extract liquor; passing the extract through gel column chromatography, performing gradient elution, collecting gallic acid-containing component, concentrating until crystals are separated out, cooling for crystallization, vacuum filtering to collect white crystals, and drying to obtain high-content gallic acid.
The principle of the preparation method of the high-content gallic acid is as follows:
in step 1 of the present invention, gallic acid and gallic acid group-containing compounds in the cone leaf powder are extracted.
In step 2 of the invention, the gallic acid group-containing compound is subjected to enzymolysis to generate gallic acid and mother nucleus.
In step 3 of the invention, the impurities are separated and the gallic acid is enriched by utilizing the principle of a molecular sieve and separating the impurities through column chromatography in an ethanol solvent system.
In conclusion, the preparation method of the invention has the advantages that the content of the obtained gallic acid is high (more than or equal to 95 percent), is improved by 4 percent compared with the content of the gallic acid in the prior art, and the yield reaches more than 90 percent.
The preparation method of the high-content gallic acid has the beneficial effects that:
1. the invention firstly takes the coniferous tree as the raw material, and obtains the high-content gallic acid through simple and convenient processes such as extraction, enzyme hydrolysis, column chromatography and the like, thereby providing a new supply raw material for the preparation of the gallic acid and opening up a new utilization path for conifer resources.
2. By adopting the preparation method, the content of the obtained gallic acid is high (more than or equal to 95 percent), is improved by 4 percent compared with the content of the gallic acid in the prior art, and the yield reaches more than 90 percent.
3. The preparation method disclosed by the invention is simple, easy to operate, low in cost, wide in market prospect and suitable for large-scale popularization and application.
On the basis of the technical scheme, the invention can be further improved as follows.
Further, the cone leaf powder in the step 1 is obtained by drying, crushing and sieving fresh cone leaves with a 40-60-mesh sieve.
The adoption of the further beneficial effects is as follows: proper pulverization degree is beneficial to the extraction of the gallic acid and the gallic acid group-containing compound.
Further, in the step 1, the polar solvent is any one of water, an ethanol aqueous solution with a mass percentage of 10% and a methanol aqueous solution with a mass percentage of 10%; the mass ratio of the cone leaf powder to the polar solvent is 1 (10-30).
The adoption of the further beneficial effects is as follows: the above all are polar solvents, can extract gallic acid and compounds containing gallic acid group in folium Coni, and can reduce the use of organic solvent.
Further, in the step 1, the frequency of ultrasonic extraction is 30KHz-40KHz, the room temperature is 16 ℃ to 30 ℃, the frequency is 2 to 3 times, and each time is 30min-45 min.
The adoption of the further beneficial effects is as follows: the parameters are adopted to facilitate the extraction of the gallic acid and the compound containing the gallic acid group in the cone leaves.
Further, in the step 2, the enzymolysis is to add tannase with the weight of 0.20 per mill to 1.0 per mill of the weight of the cone leaf raw material into the gallic acid extracting solution obtained in the step 1, and carry out enzymolysis for 60min to 90 min.
The adoption of the further beneficial effects is as follows: by adopting the parameters, the gallic acid extract obtained in the step 1 can be subjected to effective enzymolysis. The tannase can hydrolyze gallic acid groups in the gallic acid group-containing compound to generate gallic acid, and can effectively increase the amount of gallic acid in the gallic acid extract.
Further, in the step 2, the concentration refers to vacuum concentration at the temperature of 60-70 ℃ and the vacuum degree of-0.04 MPa to-0.06 MPa.
The adoption of the further beneficial effects is as follows: the color of the concentrated extract is not too dark to affect the color of the final product.
Further, in step 3, the ethanol extraction specifically refers to: adding ethanol with the mass percent of 95% which is 1-2 times of the mass of the extract rich in gallic acid obtained in the step 2, extracting for 2-3 times, and combining the extract liquor.
The adoption of the further beneficial effects is as follows: the gallic acid is fully dissolved out and the large polar substances are removed, and meanwhile, the extract can be directly used as the column layer solution of the next step without concentration.
Further, in the step 3, the gel column chromatography adopts a hydroxypropyl dextran gel chromatographic column Sephadex LH-20 with the specification of 6cm multiplied by 80cm, and the grain diameter of the filler is 27-163 mu m.
The adoption of the further beneficial effects is as follows: the gallic acid has small molecular weight, adopts Sephadex LH-20 column of hydroxypropyl dextran gel chromatography, and the first effluent is separated from other substances and the gallic acid is enriched, and is easy for large-scale production.
The hydroxypropyl Sephadex LH-20 column was obtained from GE, USA.
Further, in step 3, the gradient elution is specifically: performing isocratic elution with 95% ethanol water solution of 3-5 times column volume as eluent, and washing with 3 times column volume of 100% methanol at an elution rate of 2mL/min per 0.1L.
The adoption of the further beneficial effects is as follows: the isocratic elution ensures that the gallic acid with small molecular weight flows out firstly to obtain effective enrichment, can improve the separation efficiency of target compounds and simultaneously remove impurities.
Further, in the step 3, the temperature reduction crystallization refers to standing for 12 hours at room temperature of 16-30 ℃.
The adoption of the further beneficial effects is as follows: the crystallization speed is proper in the temperature range, the crystallization yield is high, and the product purity is high.
Further, in the step 3, the drying means that the drying is carried out at the temperature of 60-70 ℃ and the vacuum degree of-0.04-0.06 MPa, and the drying is carried out in vacuum until the weight loss is less than or equal to 1 percent.
The adoption of the further beneficial effects is as follows: the product is light in color while the dryness of the product is ensured.
Further, in the step 3, the mass percentage of the gallic acid in the high-content gallic acid is more than or equal to 95%.
The adoption of the further beneficial effects is as follows: in the prior art, the mass percentage of the gallic acid is less than or equal to 91 percent. By adopting the method, the mass percentage of the gallic acid is more than or equal to 95 percent. Therefore, the invention can obviously improve the content of the gallic acid.
The second purpose of the invention is to provide a high-content gallic acid.
The technical scheme for solving the technical problems is as follows: a high content gallic acid is obtained by the above preparation method.
The beneficial effects of the high-content gallic acid of the invention are as follows:
the high-content gallic acid is prepared by the method, the preparation method is simple, the prepared gallic acid content is high, and the method is suitable for industrial production.
Drawings
FIG. 1 shows the high gallic acid content obtained in example 1 of the present invention1H NMR(500MHz,CD3OD) spectrum;
FIG. 2 shows the high gallic acid content obtained in example 1 of the present invention13C NMR(125MHz,CD3OD) spectrum.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
Example 1
The preparation method of high-content gallic acid of this embodiment includes the following steps:
step 1: preparation of Gallic acid extract
Drying fresh folium Cone, pulverizing, and sieving with 40-60 mesh sieve to obtain folium Cone powder. Taking 0.5kg of folium pyramidal powder, adding 10% ethanol water solution by mass percent, wherein the mass ratio of the folium pyramidal powder to the polar solvent is 1:30, and carrying out ultrasonic extraction, wherein the frequency of the ultrasonic extraction is 30KHz-40KHz, the temperature is 16-30 ℃ and the frequency is 2-3 times, and each time is 30min-45min, thus obtaining the gallic acid extract.
And 2, step: preparation of gallic acid-rich extract
Adding tannase with weight of 1.0 per mill of the weight of the cone leaf raw material into the gallic acid extracting solution obtained in the step 1, performing enzymolysis for 60min, and performing vacuum concentration at the temperature of 60 ℃ and the vacuum degree of-0.04 MPa to-0.06 MPa to obtain the extract rich in gallic acid.
And step 3: preparation of high content of gallic acid
Adding ethanol with the mass percent of 95% which is 1-2 times of the mass of the extract rich in gallic acid obtained in the step 2, extracting for 2-3 times, and combining the extract liquor; passing the extract through Sephadex LH-20 column of hydroxypropyl Sephadex with specification of 6cm × 80cm, filler particle diameter of 27 μm-163 μm, performing isocratic elution by using 95% ethanol aqueous solution with volume concentration of 3-5 times column volume as eluent, finally cleaning with 100% methanol with 3 times column volume, one fraction per 0.1L, eluting at 2mL/min, collecting gallic acid-containing component, concentrating until crystal is precipitated, standing at 16 deg.C for 12h at room temperature for cooling crystallization, vacuum-filtering to collect white crystal, vacuum-drying at 60 deg.C under vacuum degree of-0.04 MPa to-0.06 MPa, and vacuum-drying until weight loss is less than or equal to 1%, to obtain 21.5g high-content gallic acid.
And (3) structural identification:
carrying out MS treatment on the obtained crystals,1H-NMR and13C-NMR and other spectral data are identified as shown in figures 1 and 2, and the results are compared with the values reported in the literature.
LCIT-TOF-MS: m/z 169.0228(M-H), calculated value C7H5O5Molecular weight 169.0215.
1H-NMR(500MHz,CD3OD)δ:7.09(2H,s,H-2,6)。
13C-NMR(125MHz,CD3OD)δ:121.9(C-1),110.3(2C,C-2,6),146.3(2C,C-3,5),139.6(C-4),170.4(C-7)。
The MS mentioned above,1H-NMR and13the C-NMR data are basically consistent with the reports in the literature, so the gallic acid is identified, and the structure is as follows:
and (4) purity checking: HPLC analysis, column: cosmosil 5C18 AR II, specification 4.6mm x 250mm, filler particle size 5 μm; column temperature: 35 ℃; the mobile phase uses acetonitrile as mobile phase A, 50mM H3PO4The solution is a mobile phase B, and the volume ratio of the mobile phase A to the mobile phase B is (4-30) to (96-70) within 0-39 min; 39-54 min, wherein the volume ratio of the mobile phase A to the mobile phase B is 75: 25; flow rate: 0.8 mL/min; detection wavelength: 272 nm.
The purity of the gallic acid obtained in this example was checked, and the result showed that the gallic acid content was 98.8%.
Example 2
The preparation method of high-content gallic acid of this embodiment includes the following steps:
step 1: preparation of Gallic acid extract
Drying fresh folium Cone, pulverizing, and sieving with 40-60 mesh sieve to obtain folium Cone powder. Taking 0.5kg of folium Cone powder, adding water at a mass ratio of the folium Cone powder to the water of 1:10, and performing ultrasonic extraction at 30KHz frequency at room temperature of 30 deg.C for 2 times each for 30min-45min to obtain gallic acid extractive solution.
Step 2: preparation of gallic acid-rich extract
Adding tannase with weight of 0.20 per mill of the weight of the cone leaf raw material into the gallic acid extracting solution obtained in the step 1, performing enzymolysis for 60min, and performing vacuum concentration at the temperature of 60-70 ℃ and the vacuum degree of-0.04 MPa to-0.06 MPa to obtain the extract rich in gallic acid.
And step 3: preparation of high content of gallic acid
Adding ethanol with the mass percent of 95% which is 1-2 times of the mass of the extract rich in gallic acid obtained in the step 2, extracting for 2-3 times, and combining the extract liquor; passing the extract through Sephadex LH-20 column of hydroxypropyl Sephadex with specification of 6cm × 80cm, filler particle diameter of 27 μm-163 μm, performing isocratic elution by using 95% ethanol aqueous solution with volume concentration of 3-5 times column volume as eluent, finally cleaning with 100% methanol with 3 times column volume, one fraction per 0.1L, eluting at 2mL/min, collecting gallic acid-containing component, concentrating until crystal is precipitated, standing at 16 deg.C for 6h at room temperature for cooling crystallization, vacuum-filtering to collect white crystal, vacuum-drying at 70 deg.C under vacuum degree of-0.04 MPa to-0.06 MPa, and vacuum-drying until weight loss is less than or equal to 1%, to obtain 16.7g high-content gallic acid.
The structure was identified as in example 1.
The purity of the gallic acid obtained in this example was checked, and the gallic acid content was 97.6%.
Example 3
The preparation method of high-content gallic acid of this embodiment includes the following steps:
step 1: preparation of Gallic acid extract
Drying fresh folium Cone, pulverizing, and sieving with 40-60 mesh sieve to obtain folium Cone powder. Taking 0.5kg of cone leaf powder, adding 10% ethanol water solution by mass, performing ultrasonic extraction at 30KHz at room temperature of 30 ℃ for 2 times, and performing 30-45 min each time to obtain gallic acid extract, wherein the mass ratio of the cone leaf powder to the ethanol water solution is 1: 20.
Step 2: preparation of gallic acid-rich extract
Adding tannase with weight of 0.5 per mill of the weight of the cone leaf raw material into the gallic acid extracting solution obtained in the step 1, carrying out enzymolysis for 75min, and then carrying out vacuum concentration at the temperature of 60 ℃ and the vacuum degree of-0.04 MPa to-0.06 MPa to obtain the extract rich in gallic acid.
And step 3: preparation of high content of gallic acid
Adding ethanol with the mass percentage of 95 percent which is 1 time of the mass of the extract rich in gallic acid obtained in the step 2, extracting for 2 times, and combining the extract liquor; passing the extract through a Sephadex LH-20 column of hydroxypropyl Sephadex with the specification of 6cm multiplied by 80cm and the filler particle size of 27 to 163 mu m, performing isocratic elution by using 95 percent ethanol aqueous solution with the volume concentration of 3 times of the column volume as an eluent, finally cleaning by using 100 percent methanol with the column volume of 3 times, wherein each 0.1L of the eluent is one flow part, the elution speed is 2mL/min, collecting the components containing the gallic acid, concentrating until crystals are separated out, standing at the room temperature of 16 to 30 ℃ for 12h for cooling crystallization, performing suction filtration to collect white crystals, performing vacuum filtration at the temperature of 68 ℃ and the vacuum degree of-0.04 to-0.06 MPa, and performing vacuum drying until the weight loss is less than or equal to 1 percent to obtain 18.2g of high-content gallic acid.
The structure was identified as in example 1.
The purity of the gallic acid obtained in this example was checked, and the result showed that the gallic acid content was 98.8%.
Example 4
The preparation method of high-content gallic acid of this embodiment includes the following steps:
step 1: preparation of Gallic acid extract
Drying fresh folium Cone, pulverizing, and sieving with 40-60 mesh sieve to obtain folium Cone powder. Taking 0.5kg of cone leaf powder, adding 10% ethanol water solution by mass percent, carrying out ultrasonic extraction at the frequency of 30KHz-40KHz and at the room temperature of 28 ℃ for 3 times, wherein the time is 30min-45min each time, and obtaining the gallic acid extract, wherein the mass ratio of the cone leaf powder to the polar solvent is 1: 30.
Step 2: preparation of gallic acid-rich extract
Adding tannase with weight of 1.0 per mill of the weight of the cone leaf raw material into the gallic acid extracting solution obtained in the step 1, performing enzymolysis for 90min, and performing vacuum concentration at the temperature of 68 ℃ and the vacuum degree of-0.04 MPa to-0.06 MPa to obtain the extract rich in gallic acid.
And step 3: preparation of high content of gallic acid
Adding ethanol with the mass percentage of 95 percent which is 2 times of the mass of the extract rich in gallic acid obtained in the step 2, extracting for 3 times, and combining the extract liquor; passing the extract through a Sephadex LH-20 column of hydroxypropyl Sephadex with the specification of 6cm multiplied by 80cm and the filler particle size of 27 to 163 mu m, performing isocratic elution by using 95 percent ethanol aqueous solution with the volume concentration of 5 times of the column volume as an eluent, finally cleaning by using 100 percent methanol with the volume of 3 times of the column volume, wherein each 0.1L of the eluent has the elution speed of 2mL/min, collecting the components containing the gallic acid, concentrating until crystals are separated out, standing at the room temperature of 16 to 30 ℃ for 12h for cooling crystallization, performing suction filtration to collect white crystals, performing vacuum filtration at the vacuum degree of between-0.04 MPa and-0.06 MPa at the temperature of 62 ℃, and performing vacuum drying until the weight loss is less than or equal to 1 percent to obtain 12.5g of high-content gallic acid.
The structure was identified as in example 1.
The purity of the gallic acid obtained in this example was checked, and the gallic acid content was 86.4%.
Example 5
The preparation method of high-content gallic acid of this embodiment includes the following steps:
step 1: preparation of Gallic acid extract
Drying fresh folium Cone, pulverizing, and sieving with 40-60 mesh sieve to obtain folium Cone powder. Taking 0.5kg of folium Cone powder, adding 10% ethanol water solution by mass, wherein the mass ratio of the folium Cone powder to the polar solvent is 1:30, and performing ultrasonic extraction at the frequency of 30KHz-40KHz at room temperature of 16 ℃ for 3 times, each time for 45min to obtain gallic acid extract.
Step 2: preparation of gallic acid-rich extract
Adding tannase with weight of 1.0 per mill of the weight of the cone leaf raw material into the gallic acid extracting solution obtained in the step 1, performing enzymolysis for 90min, and performing vacuum concentration at the temperature of 65 ℃ and the vacuum degree of-0.04 MPa to-0.06 MPa to obtain the extract rich in gallic acid.
And step 3: preparation of high content of gallic acid
Adding ethanol with the mass percent of 95% which is 1-2 times of the mass of the extract rich in gallic acid obtained in the step 2, extracting for 2-3 times, and combining the extract liquor; passing the extract through Sephadex LH-20 column of hydroxypropyl Sephadex with specification of 6cm × 80cm, filler particle diameter of 27 μm-163 μm, performing isocratic elution by using 95% ethanol aqueous solution with volume concentration of 3-5 times column volume as eluent, finally cleaning with 100% methanol with 3 times column volume, one fraction per 0.1L, eluting at 2mL/min, collecting gallic acid-containing component, concentrating until crystal is precipitated, standing at 22 deg.C for 12h at room temperature for cooling crystallization, performing suction filtration to collect white crystal, vacuum drying at 67 deg.C under vacuum degree of-0.04 MPa to-0.06 MPa, and vacuum drying until weight loss is less than or equal to 1% to obtain 12.7g high-content gallic acid.
The structure was identified as in example 1.
The purity of the gallic acid obtained in this example was checked, and the gallic acid content was 83.5%.
Comparative example 1
The comparative example is different from example 1 in that no tannase is added in step 2, and specifically comprises the following steps:
step 1: the same as in example 1.
And 2, step: preparation of gallic acid-rich extract
And (3) carrying out vacuum concentration on the gallic acid extract obtained in the step (1) at the temperature of 60 ℃ and the vacuum degree of-0.04 MPa to-0.06 MPa to obtain the extract rich in gallic acid.
And step 3: 6.2g of gallic acid was obtained in the same manner as in example 1.
The structure was identified as in example 1.
The purity of the gallic acid obtained in this example was checked, and the gallic acid content was 96.9%. The yield of the gallic acid obtained by the comparative example is far less than that of the gallic acid obtained by the example 1, and the purity of the gallic acid is also less than that of the gallic acid obtained by the example 1.
Comparative example 2
This comparative example differs from example 1 in that step 3 does not use a hydroxypropyl sephadex column, but uses a polystyrene type macroporous adsorbent resin (D101). Specifically, the method comprises the following steps:
step 1: the same as in example 1.
Step 2: the same as in example 1.
And step 3: preparation of gallic acid
Adding ethanol with the mass percent of 95% which is 1-2 times of the mass of the extract rich in gallic acid obtained in the step 2, extracting for 2-3 times, and combining the extract liquor; concentrating the extract, adsorbing with polystyrene macroporous adsorbent resin (D101), eluting with 3 times column volume of deionized water, then eluting with 4 times column volume of ethanol and water at a volume ratio of 30:70, one fraction per 0.1L, at an elution speed of 2mL/min, collecting gallic acid-containing component, concentrating until crystal is precipitated, adding 100g of activated carbon to decolorize for 2h, filtering, concentrating until crystal is precipitated, standing at 16 deg.C for 12h to cool and crystallize, filtering to collect white crystal, vacuum drying at 60 deg.C under a vacuum degree of-0.04 MPa to-0.06 MPa, and vacuum drying until weight loss is less than or equal to 1% to obtain 20.1g of gallic acid.
The structure was identified as in example 1.
The purity of the gallic acid obtained in this example was checked, and the gallic acid content was 90.5%. The purity of the gallic acid obtained by the comparative example is far less than that of the gallic acid obtained by the example 1, and the yield of the gallic acid is also less than that of the gallic acid obtained by the example 1.
As can be seen from examples 1-5 and comparative examples 1-2, the content of the obtained gallic acid (more than or equal to 95%) is increased by 4% compared with the content of the gallic acid in the prior art by adopting the preparation method of the invention, and the yield reaches more than 90%.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A preparation method of high-content gallic acid is characterized by comprising the following steps:
step 1: preparation of Gallic acid extract
Taking folium Coni Cone powder, adding polar solvent, and performing ultrasonic extraction to obtain gallic acid extractive solution;
step 2: preparation of gallic acid-rich extract
Performing enzymolysis on the gallic acid extract obtained in the step 1, and concentrating to obtain an extract rich in gallic acid;
and step 3: preparation of high content of gallic acid
Extracting the gallic acid-rich extract obtained in the step 2 by using ethanol to obtain an extract liquor; passing the extract through gel column chromatography, performing gradient elution, collecting gallic acid-containing component, concentrating until crystals are separated out, cooling for crystallization, vacuum filtering to collect white crystals, and drying to obtain high-content gallic acid.
2. The method for preparing gallic acid in high content according to claim 1, wherein in step 1, the folium pyramidal powder is prepared by drying fresh folium pyramidal, pulverizing, and sieving with 40-60 mesh sieve; the polar solvent is any one of water, 10% by mass of ethanol water solution and 10% by mass of methanol water solution; the mass ratio of the cone leaf powder to the polar solvent is 1 (10-30).
3. The method for preparing high-content gallic acid according to claim 1, wherein in step 1, the frequency of ultrasonic extraction is 30KHz-40KHz, and the temperature is 16-30 deg.C, and the frequency is 2-3 times, each time for 30min-45 min.
4. The method for preparing gallic acid with high content according to claim 1, wherein in step 2, the enzymolysis is performed by adding tannase with weight of 0.20-1.0 ‰ of cone leaf raw material into gallic acid extract obtained in step 1, and performing enzymolysis for 60-90 min.
5. The method for preparing high-content gallic acid according to claim 1, wherein in step 2, the concentration is performed under vacuum at a temperature of 60-70 ℃ and a vacuum degree of-0.04 MPa to-0.06 MPa.
6. The method for preparing high-content gallic acid according to claim 1, wherein in step 3, said ethanol extraction specifically comprises: adding ethanol with the mass percent of 95% which is 1-2 times of the mass of the extract rich in gallic acid obtained in the step 2, extracting for 2-3 times, and combining the extract liquor; the gel column chromatography adopts Sephadex LH-20 of hydroxypropyl dextran gel chromatographic column with specification of 6cm × 80cm, and filler particle diameter of 27 μm-163 μm.
7. The method for preparing high-content gallic acid according to claim 1, wherein in step 3, said gradient elution is specifically: performing isocratic elution with 95% ethanol water solution of 3-5 times column volume as eluent, and washing with 3 times column volume of 100% methanol at an elution rate of 2mL/min per 0.1L.
8. The method for preparing high-content gallic acid according to claim 1, wherein in step 3, said crystallization at reduced temperature is performed by standing at 16-30 ℃ for 12 h; the drying means that the vacuum degree is between-0.04 MPa and-0.06 MPa at the temperature of between 60 and 70 ℃, and the vacuum drying is carried out until the weight loss is less than or equal to 1 percent.
9. The method for preparing high-content gallic acid according to claim 1, wherein in step 3, the mass percentage of gallic acid in the high-content gallic acid is not less than 95%.
10. High content of gallic acid obtained by the production method according to any one of claims 1 to 9.
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