CN114426478B - High-content gallic acid and preparation method thereof - Google Patents
High-content gallic acid and preparation method thereof Download PDFInfo
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- CN114426478B CN114426478B CN202210106100.7A CN202210106100A CN114426478B CN 114426478 B CN114426478 B CN 114426478B CN 202210106100 A CN202210106100 A CN 202210106100A CN 114426478 B CN114426478 B CN 114426478B
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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 333
- 235000004515 gallic acid Nutrition 0.000 title claims abstract description 162
- 229940074391 gallic acid Drugs 0.000 title claims abstract description 162
- 238000002360 preparation method Methods 0.000 title claims abstract description 46
- 239000000284 extract Substances 0.000 claims abstract description 68
- 238000000034 method Methods 0.000 claims abstract description 22
- 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 32
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- 239000000243 solution Substances 0.000 claims description 22
- 239000000843 powder Substances 0.000 claims description 21
- 239000013078 crystal Substances 0.000 claims description 18
- 238000002137 ultrasound extraction Methods 0.000 claims description 13
- 238000001035 drying Methods 0.000 claims description 11
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 11
- 238000001816 cooling Methods 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 9
- 239000002798 polar solvent Substances 0.000 claims description 9
- 238000010829 isocratic elution Methods 0.000 claims description 8
- 239000002245 particle Substances 0.000 claims description 8
- 230000004580 weight loss Effects 0.000 claims description 8
- 239000003480 eluent Substances 0.000 claims description 7
- 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 7
- 238000007873 sieving Methods 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 238000002425 crystallisation Methods 0.000 claims description 5
- 230000008025 crystallization Effects 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 5
- 239000000945 filler Substances 0.000 claims description 5
- -1 hydroxypropyl Chemical group 0.000 claims description 5
- TUSDEZXZIZRFGC-UHFFFAOYSA-N 1-O-galloyl-3,6-(R)-HHDP-beta-D-glucose Natural products OC1C(O2)COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC1C(O)C2OC(=O)C1=CC(O)=C(O)C(O)=C1 TUSDEZXZIZRFGC-UHFFFAOYSA-N 0.000 claims description 4
- 239000001263 FEMA 3042 Substances 0.000 claims description 4
- LRBQNJMCXXYXIU-PPKXGCFTSA-N Penta-digallate-beta-D-glucose Natural products OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 235000015523 tannic acid Nutrition 0.000 claims description 4
- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 claims description 4
- 229940033123 tannic acid Drugs 0.000 claims description 4
- 229920002258 tannic acid Polymers 0.000 claims description 4
- 229920005654 Sephadex Polymers 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 2
- 238000002481 ethanol extraction Methods 0.000 claims description 2
- 238000000605 extraction Methods 0.000 abstract description 8
- 238000000746 purification Methods 0.000 abstract description 6
- 238000000926 separation method Methods 0.000 abstract description 6
- 241000218631 Coniferophyta Species 0.000 abstract description 4
- 239000003814 drug Substances 0.000 abstract description 3
- 108090000790 Enzymes Proteins 0.000 abstract description 2
- 102000004190 Enzymes Human genes 0.000 abstract description 2
- 230000007062 hydrolysis Effects 0.000 abstract description 2
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 2
- 230000009286 beneficial effect Effects 0.000 description 14
- 230000000052 comparative effect Effects 0.000 description 7
- 150000001875 compounds Chemical class 0.000 description 7
- 238000010298 pulverizing process Methods 0.000 description 6
- 238000001291 vacuum drying Methods 0.000 description 6
- 239000000126 substance Substances 0.000 description 5
- 108010038851 tannase Proteins 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000011347 resin Substances 0.000 description 4
- 229920005989 resin Polymers 0.000 description 4
- 239000002023 wood Substances 0.000 description 4
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 238000012856 packing Methods 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 241001070941 Castanea Species 0.000 description 1
- 235000014036 Castanea Nutrition 0.000 description 1
- 241000499335 Castanopsis chinensis Species 0.000 description 1
- 241000237970 Conus <genus> Species 0.000 description 1
- 241000219428 Fagaceae Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 238000012356 Product development Methods 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- 244000044283 Toxicodendron succedaneum Species 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 230000003796 beauty Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 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
- 239000002904 solvent Substances 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 229920001864 tannin Polymers 0.000 description 1
- 235000018553 tannin Nutrition 0.000 description 1
- 239000001648 tannin Substances 0.000 description 1
Classifications
-
- 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
-
- 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 gallic acid extract; step 2: preparing an extract enriched in gallic acid; step 3: preparing high content gallic acid. The invention also discloses the high-content gallic acid obtained by the preparation method. The invention takes the conifer leaves as raw materials for the first time, 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 raw material for preparing the gallic acid and opening up a new utilization way for the conifer plant resources.
Description
Technical Field
The invention relates to 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.
Background
Cone (Castanopsis chinensis) is also called cortex Cinnamomi Lin Kao, tannin chestnut and rice cone, etc., and is made of plant of Conus of Fagaceae, with a diameter of 20 m, a diameter of 0.6 m, and bark with longitudinal crack, flaking, and no hair on branch and leaf. The wood is widely distributed in provinces such as Guangxi, guangdong and Yunnan, the wood is brown yellow, sometimes the core is dark in color and stain, the wood rays are very narrow, the material is light, the structure is slightly thick, the texture is straight, and the wood belongs to yellow cones.
At present, the researches on chemical components, pharmacological activity, product development and the like of the cone are not reported, and the leaves of the cone are detected to contain polyphenol components through qualitative pre-experiment in the early stage, so that the cone has higherIs developed into health care and beauty products with antioxidant function. Gallic Acid (GA) with chemical name of 3,4, 5-trihydroxybenzoic Acid and molecular formula of C 7 H 6 O 5 Also called as acid and gallic acid, is an organic acid commonly used in plants such as gallnut, lacquer tree, tea and the like, and is widely applied to the fields of chemical industry, medicine, food, electronic industry and the like.
In the prior art, the preparation method of gallic acid mainly comprises the steps of extracting by adding acid into an organic solvent or an aqueous solution, purifying by adopting macroporous adsorption resin, decoloring by anion-cation resin or decoloring by activated carbon, and the like, and has the following defects: the yield and purity were not high. In addition, no report about the extraction and purification process of gallic acid in cones or the extraction and purification process after enzymatic hydrolysis is available in the prior art, and no report about the separation and purification of gallic acid by using a high-purity ethanol system elution system during the purification of gallic acid by gel column chromatography is available.
In view of the above, it is necessary to provide a method for preparing high-content gallic acid from cones, which provides a new source for gallic acid and overcomes the shortcomings of the prior art.
Disclosure of Invention
The invention aims at providing 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
Adding polar solvent into the cone leaf powder, and performing ultrasonic extraction to obtain gallic acid extract;
step 2: preparation of gallic acid-enriched extract
Carrying out enzymolysis and concentration on the gallic acid extract obtained in the step 1 to obtain an extract rich in gallic acid;
step 3: preparation of high content gallic acid
Extracting the extract rich in gallic acid obtained in the step 2 by ethanol to obtain an extract; subjecting the extract to gel column chromatography, gradient eluting, collecting gallic acid-containing component, concentrating until crystals are separated out, cooling, crystallizing, vacuum filtering, collecting white crystals, and drying to obtain high-content gallic acid.
The preparation method of the high-content gallic acid comprises the following steps:
in step 1 of the present invention, gallic acid and gallic acid group-containing compounds in the cone-leaf powder are extracted.
In the step 2 of the invention, gallic acid group-containing compounds are subjected to enzymolysis to generate gallic acid and parent nucleus.
In the step 3 of the invention, the separation of impurities and the enrichment of gallic acid are realized by separating the impurities through column chromatography in an ethanol solvent system by utilizing a molecular sieve principle.
In conclusion, the content of the gallic acid obtained by adopting the preparation method of the invention is high (more than or equal to 95 percent), which is 4 percent higher than that of the gallic acid in the prior art, and the yield is more than 90 percent.
The preparation method of the high-content gallic acid has the beneficial effects that:
1. the invention takes the conifer leaves as raw materials for the first time, 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 raw material for preparing the gallic acid and opening up a new utilization way for the conifer plant resources.
2. The content of the gallic acid obtained by adopting the preparation method is high (more than or equal to 95 percent), which is 4 percent higher than that of the gallic acid in the prior art, and the yield is more than 90 percent.
3. The preparation method provided 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 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 method has the further beneficial effects that: the appropriate pulverizing degree is beneficial to extraction of gallic acid and compound containing gallic acid groups.
Further, in step 1, the polar solvent is any one of water, an aqueous ethanol solution with a mass percentage of 10% and an aqueous methanol 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 method has the further beneficial effects that: the above polar solvents can extract gallic acid and compounds containing gallic acid groups from folium Coni, and reduce the use of organic solvents.
Further, in the step 1, the frequency of ultrasonic extraction is 30KHz-40KHz, the room temperature is 16-30 ℃, the times are 2-3 times, and each time is 30-45 min.
The adoption of the method has the further beneficial effects that: the above parameters are adopted to facilitate extraction of gallic acid and compounds containing gallic acid groups in the cone leaves.
In step 2, the enzymolysis is that tannic acid of 0.20-1.0 per mill of the weight of the cone leaf raw material is added into the gallic acid extracting solution obtained in step 1, and the enzymolysis is carried out for 60-90 min.
The adoption of the method has the further beneficial effects that: 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 compound containing gallic acid groups 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 a temperature of 60-70 ℃ and a vacuum degree of-0.04 MPa to-0.06 MPa.
The adoption of the method has the further beneficial effects that: 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 percentage of 95% 1-2 times of the extract rich in gallic acid obtained in the step 2, extracting for 2-3 times, and mixing the extracts.
The adoption of the method has the further beneficial effects that: the gallic acid is fully dissolved out and the large polar substances are removed, and meanwhile, the extract can be directly used as a column layer solution of the next step without concentration.
In the step 3, the gel column chromatography adopts a hydroxypropyl dextran gel chromatographic column Sephadex LH-20, the specification is 6cm multiplied by 80cm, and the particle size of the filler is 27 mu m-163 mu m.
The adoption of the method has the further beneficial effects that: the gallic acid has small molecular weight, adopts the hydroxypropyl Sephadex LH-20 chromatographic column, has good separation effect with other substances and enrichment effect of the gallic acid when separating, and is easy for large-scale production.
The above-described hydroxypropyl Sephadex LH-20 was purchased from GE company of America.
Further, in step 3, the gradient elution specifically includes: and (3) performing isocratic elution by using 3-5 times of ethanol water solution with the volume concentration of 95% of the column volume as an eluent, and finally cleaning with 100% of methanol with the volume of 3 times of the column volume, wherein each 0.1L is one flow part, and the elution speed is 2mL/min.
The adoption of the method has the further beneficial effects that: the isocratic elution ensures that gallic acid with small molecular weight flows out first to obtain effective enrichment, can improve the separation efficiency of target compounds, and simultaneously removes impurities.
In the step 3, the cooling crystallization refers to standing for 12 hours at the room temperature of 16-30 ℃.
The adoption of the method has the further beneficial effects that: the crystallization speed is proper, the crystallization yield is high, and the product purity is high in the temperature range.
In the step 3, the drying is that the vacuum degree is between-0.04 MPa and-0.06 MPa at the temperature of 60 ℃ to 70 ℃ and the weight loss is less than or equal to 1 percent.
The adoption of the method has the further beneficial effects that: the product has light color while ensuring the dryness of the product.
In 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 method has the further beneficial effects that: in the prior art, the mass percentage of the gallic acid is less than or equal to 91 percent. By adopting the method of the invention, the mass percentage of the gallic acid is more than or equal to 95 percent. Therefore, the invention can obviously improve the content of gallic acid.
The second object of the present invention is to provide a high content of gallic acid.
The technical scheme for solving the technical problems is as follows: the high content gallic acid obtained by the above preparation method.
The high-content gallic acid has the beneficial effects that:
the high-content gallic acid is prepared by the method, the preparation method is simple, and the prepared gallic acid has high content and is suitable for industrial production.
Drawings
FIG. 1 shows the high content of gallic acid obtained in example 1 of the present invention 1 H NMR(500MHz,CD 3 OD) spectra;
FIG. 2 shows the high content of gallic acid obtained in example 1 of the present invention 13 C NMR(125MHz,CD 3 OD) spectra.
Detailed Description
The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.
Example 1
The preparation method of the high-content gallic acid comprises 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 an ethanol aqueous solution with the mass percentage of 10%, performing ultrasonic extraction at the frequency of 30KHz-40KHz and the room temperature of 16-30 ℃ for 2-3 times and 30-45 min each time, so as to obtain gallic acid extract.
Step 2: preparation of gallic acid-enriched extract
Adding tannase with the weight of 1.0 per mill of that of the cone leaf raw material into the gallic acid extracting solution obtained in the step 1, carrying out enzymolysis for 60min, and 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.
Step 3: preparation of high content gallic acid
Adding ethanol with the mass percentage 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 extracts; subjecting the extract to Sephadex LH-20 with specification of 6cm×80cm, packing particle diameter of 27-163 μm, performing isocratic elution with 3-5 times of column volume of 95% ethanol water solution as eluent, washing with 3 times of column volume of 100% methanol at a flow rate of 0.1L, collecting gallic acid component, concentrating until crystals are separated, standing at 16deg.C for 12 hr, cooling, crystallizing, vacuum filtering, collecting white crystals, and vacuum drying at 60deg.C under vacuum degree of-0.04 MPa to-0.06 MPa until weight loss is less than or equal to 1.5g of high-content gallic acid.
And (3) structural identification:
MS is carried out on the obtained crystal, 1 H-NMR 13 The C-NMR and other spectroscopic data are identified as shown in FIGS. 1 and 2, and the results are compared with literature report values.
LCIT-TOF-MS: m/z 169.0228 (M-H), calculated C 7 H 5 O 5 Molecular weight 169.0215.
1 H-NMR(500MHz,CD 3 OD)δ:7.09(2H,s,H-2,6)。
13 C-NMR(125MHz,CD 3 OD)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 is, 1 H-NMR 13 The C-NMR data are substantially identical to those reported in the literature, and therefore are identified as gallic acid, having the following structure:
purity inspection: HPLC analysis, column: cosmosil 5C18 AR II, specification is 4.6mm multiplied by 250mm, and the particle size of the filler is 5 mu m; column temperature: 35 ℃; mobile phase acetonitrile as mobile phase A,50mM H 3 PO 4 The solution is a mobile phase B, the volume ratio of the mobile phase A to the mobile phase B is (4-30) (96-70); 39-54 min, the volume ratio of the mobile phase A to the mobile phase B is 75:25; flow rate: 0.8mL/min; detection wavelength: 272nm.
The purity of the gallic acid obtained in this example was examined, and the result showed that the content of the gallic acid obtained was 98.8%.
Example 2
The preparation method of the high-content gallic acid comprises 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 water, wherein the mass ratio of the cone leaf powder to the water is 1:10, and performing ultrasonic extraction, wherein the frequency of ultrasonic extraction is 30KHz, the room temperature is 30 ℃, the times are 2 times, and each time is 30-45 min, so as to obtain gallic acid extract.
Step 2: preparation of gallic acid-enriched extract
Adding tannic acid 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, carrying out enzymolysis for 60min, and carrying out 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.
Step 3: preparation of high content gallic acid
Adding ethanol with the mass percentage 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 extracts; subjecting the extract to Sephadex LH-20 with specification of 6cm×80cm, packing particle diameter of 27-163 μm, performing isocratic elution with 3-5 times of column volume of 95% ethanol water solution as eluent, washing with 3 times of column volume of 100% methanol at a flow rate of 0.1L, collecting gallic acid component, concentrating until crystals are separated, standing at 16deg.C for 6 hr, cooling, crystallizing, vacuum filtering, collecting white crystals, and vacuum drying at 70deg.C under vacuum degree of-0.04 MPa to-0.06 MPa until weight loss is less than or equal to 1.7 g.
Structural identification was the same as in example 1.
The purity of the gallic acid obtained in this example was measured, and the result showed that the content of the gallic acid obtained was 97.6%.
Example 3
The preparation method of the high-content gallic acid comprises 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 an ethanol water solution with the mass percentage of 10%, wherein the mass ratio of the cone-leaf powder to the ethanol water solution is 1:20, and performing ultrasonic extraction, the frequency of the ultrasonic extraction is 30KHz, the room temperature is 30 ℃, the times are 2 times, and each time is 30-45 min, so as to obtain gallic acid extract.
Step 2: preparation of gallic acid-enriched extract
Adding tannic acid with the weight of 0.5 per mill of that of the cone leaf raw material into the gallic acid extracting solution obtained in the step 1, carrying out enzymolysis for 75min, and 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.
Step 3: preparation of high content gallic acid
Adding ethanol with the mass percentage of 95% 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 extracts; subjecting the extract to Sephadex LH-20 with specification of 6cm×80cm, and filler particle diameter of 27-163 μm, performing isocratic elution with 95% ethanol water solution with volume concentration of 3 times of column volume as eluent, washing with 100% methanol with volume of 3 times of column volume, eluting at a flow rate of 2mL/min every 0.1L, collecting gallic acid-containing component, concentrating until crystals are separated, standing at 16-30deg.C for 12 hr, cooling, crystallizing, vacuum filtering, collecting white crystals, vacuum drying at 68 deg.C under vacuum degree of-0.04 MPa to-0.06 MPa until weight loss is less than or equal to 1%, and obtaining 18.2g high-content gallic acid.
Structural identification was the same as in example 1.
The purity of the gallic acid obtained in this example was examined, and the result showed that the content of the gallic acid obtained was 98.8%.
Example 4
The preparation method of the high-content gallic acid comprises 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 ethanol water solution with the mass percentage of 10%, wherein the mass ratio of the cone-leaf powder to the polar solvent is 1:30, and performing ultrasonic extraction, the frequency of the ultrasonic extraction is 30KHz-40KHz, the room temperature is 28 ℃, the times are 3 times, and each time is 30min-45min, so as to obtain gallic acid extract.
Step 2: preparation of gallic acid-enriched extract
Adding tannase with the weight of 1.0 per mill of that of the cone leaf raw material into the gallic acid extracting solution obtained in the step 1, carrying out enzymolysis for 90min, and carrying out 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.
Step 3: preparation of high content gallic acid
Adding ethanol with the mass percentage of 95% 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 extracts; subjecting the extract to Sephadex LH-20 with specification of 6cm×80cm, and filler particle diameter of 27-163 μm, performing isocratic elution with 95% ethanol water solution with volume concentration of 5 times of column volume as eluent, washing with 100% methanol with volume of 3 times of column volume, eluting at a flow rate of 2mL/min, collecting gallic acid-containing component, concentrating until crystals are separated, standing at 16-30deg.C for 12 hr, cooling, crystallizing, vacuum filtering, collecting white crystals, vacuum drying at 62 deg.C under vacuum degree of-0.04 MPa to-0.06 MPa until weight loss is less than or equal to 1%, and obtaining 12.5g high-content gallic acid.
Structural identification was the same as in example 1.
The purity of the gallic acid obtained in this example was measured, and the result showed that the content of the gallic acid obtained was 86.4%.
Example 5
The preparation method of the high-content gallic acid comprises 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 ethanol water solution with the mass percentage of 10%, wherein the mass ratio of the cone-leaf powder to the polar solvent is 1:30, and performing ultrasonic extraction, the frequency of the ultrasonic extraction is 30KHz-40KHz, the room temperature is 16 ℃, the times are 3 times, and 45min each time, so as to obtain gallic acid extract.
Step 2: preparation of gallic acid-enriched extract
Adding tannase with the weight of 1.0 per mill of that of the cone leaf raw material into the gallic acid extracting solution obtained in the step 1, carrying out enzymolysis for 90min, and carrying out 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.
Step 3: preparation of high content gallic acid
Adding ethanol with the mass percentage 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 extracts; subjecting the extract to Sephadex LH-20 with specification of 6cm×80cm, packing particle diameter of 27-163 μm, performing isocratic elution with 3-5 times of column volume of 95% ethanol water solution as eluent, washing with 3 times of column volume of 100% methanol at a flow rate of 0.1L, collecting gallic acid component, concentrating until there is crystal precipitation, standing at 22deg.C for 12 hr, cooling, crystallizing, vacuum filtering, collecting white crystals, and vacuum drying at 67 deg.C under vacuum degree of-0.04 MPa to-0.06 MPa until weight loss is less than or equal to 1.7 g, to obtain 12.7g high-content gallic acid.
Structural identification was the same as in example 1.
The purity of the gallic acid obtained in this example was measured, and the result showed that the content of the gallic acid obtained was 83.5%.
Comparative example 1
The comparative example differs from example 1 in that no tannase is added in step 2, specifically, the following steps are included:
step 1: the same as in example 1.
Step 2: preparation of gallic acid-enriched extract
Concentrating the gallic acid extract obtained in the step 1 at 60 ℃ and a vacuum degree of-0.04 MPa to-0.06 MPa in vacuum to obtain an extract rich in gallic acid.
Step 3: 6.2g of gallic acid was obtained in the same manner as in example 1.
Structural identification was the same as in example 1.
The purity of the gallic acid obtained in this example was examined, and the result showed that the content of the gallic acid obtained was 96.9%. The yield of gallic acid obtained in this comparative example was far less than that of example 1 and the purity was also less than that of example 1.
Comparative example 2
This comparative example differs from example 1 in that step 3 does not use a hydroxypropyl dextran gel column, but rather 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.
Step 3: preparation of gallic acid
Adding ethanol with the mass percentage 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 extracts; concentrating the extract, adsorbing with polystyrene macroporous adsorption resin (D101), eluting with 3 times of column volume deionized water, eluting with 4 times of column volume ethanol and water according to a volume ratio of 30:70, wherein each 0.1L is a fraction, eluting at a rate of 2mL/min, collecting gallic acid-containing component, concentrating until crystals are separated out, adding 100g of activated carbon for decolorizing for 2h, filtering, concentrating until crystals are separated out, standing at 16 ℃ at room temperature for 12h for cooling crystallization, filtering to collect white crystals, and vacuum drying at 60 ℃ at a vacuum degree of-0.04 MPa to-0.06 MPa until the weight loss is less than or equal to 1%, thereby obtaining 20.1g of gallic acid.
Structural identification was the same as in example 1.
The purity of the gallic acid obtained in this example was measured, and the result showed that the content of the gallic acid obtained was 90.5%. The purity of gallic acid obtained in this comparative example was far lower than that in example 1, and the yield was also lower than that in example 1.
As is clear from examples 1-5 and comparative examples 1-2, the content of gallic acid obtained by the preparation method of the invention is high (more than or equal to 95%), which is 4% higher than that of gallic acid in the prior art, and the yield is more than 90%.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (6)
1. A preparation method of high-content gallic acid is characterized by comprising the following steps:
step 1: preparation of gallic acid extract
Adding polar solvent into the cone leaf powder, and performing ultrasonic extraction to obtain gallic acid extract; the polar solvent is any one of water, an ethanol aqueous solution with the mass percentage of 10 percent and a methanol aqueous solution with the mass percentage of 10 percent;
step 2: preparation of gallic acid-enriched extract
Carrying out enzymolysis and concentration on the gallic acid extract obtained in the step 1 to obtain an extract rich in gallic acid; the enzymolysis is that tannic acid of 0.20 to 1.0 per mill of the weight of the cone leaf raw material is added into the gallic acid extracting solution obtained in the step 1, and the enzymolysis is carried out for 60 to 90 minutes;
step 3: preparation of high content gallic acid
Extracting the extract rich in gallic acid obtained in the step 2 by ethanol to obtain an extract; subjecting the extract to gel column chromatography, gradient eluting, collecting gallic acid-containing component, concentrating until crystals are separated out, cooling, crystallizing, vacuum filtering, collecting white crystals, and drying to obtain high-content gallic acid; the gradient elution specifically comprises the following steps: performing isocratic elution by using 3-5 times of ethanol water solution with the volume concentration of 95% of the column volume as an eluent, and finally cleaning with 100% of methanol with the volume of 3 times of the column volume, wherein each 0.1L is a fraction, and the elution speed is 2mL/min; in the high-content gallic acid, the mass percentage of the gallic acid is more than or equal to 95 percent.
2. The method for preparing high-content gallic acid according to claim 1, wherein in step 1, the cone-leaf powder is obtained by drying, crushing and sieving fresh cone-leaves with a 40-60 mesh sieve; 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, the room temperature is 16-30 ℃, the times are 2-3 times, each time is 30-45 min.
4. The method for preparing gallic acid with high content according to claim 1, wherein in the step 2, the concentration is vacuum concentration at a temperature of 60 ℃ to 70 ℃ and a vacuum degree of-0.04 MPa to-0.06 MPa.
5. The method for preparing high-content gallic acid according to claim 1, wherein in step 3, the ethanol extraction specifically means: adding ethanol with the mass percentage 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 extracts; the gel column chromatography adopts a hydroxypropyl dextran gel chromatographic column Sephadex LH-20, the specification is 6cm multiplied by 80cm, and the particle size of the filler is 27 mu m-163 mu m.
6. The method for preparing high-content gallic acid according to claim 1, wherein in step 3, the cooling crystallization means standing at room temperature of 16 ℃ to 30 ℃ for 12 hours; the drying is carried out at 60-70 ℃ and the vacuum degree of-0.04 MPa to-0.06 MPa, and the weight loss is less than or equal to 1 percent.
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| CA2215251A1 (en) * | 1997-10-01 | 1999-04-01 | Kemestrie Inc. | Industrial preparation of high purity gallic acid |
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| CN112877373A (en) * | 2021-01-26 | 2021-06-01 | 桂林莱茵生物科技股份有限公司 | Preparation method for obtaining gallic acid with content of more than 99% |
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| CA2215251A1 (en) * | 1997-10-01 | 1999-04-01 | Kemestrie Inc. | Industrial preparation of high purity gallic acid |
| JP2005516585A (en) * | 2001-06-22 | 2005-06-09 | インディアン インスティチュート オブ テクノロジー | Preparation of gallic acid by co-culture |
| CN101864460A (en) * | 2010-05-26 | 2010-10-20 | 南京泽朗农业发展有限公司 | Method for extracting gallic acid from bistort rhizome |
| CN111217696A (en) * | 2018-11-23 | 2020-06-02 | 南京泽朗医药科技有限公司 | Method for extracting gallic acid from rhus chinensis roots |
| CN112877373A (en) * | 2021-01-26 | 2021-06-01 | 桂林莱茵生物科技股份有限公司 | Preparation method for obtaining gallic acid with content of more than 99% |
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