CN111560409B - Preparation method of icariin - Google Patents
Preparation method of icariin Download PDFInfo
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- CN111560409B CN111560409B CN202010383284.2A CN202010383284A CN111560409B CN 111560409 B CN111560409 B CN 111560409B CN 202010383284 A CN202010383284 A CN 202010383284A CN 111560409 B CN111560409 B CN 111560409B
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- extraction
- enzymolysis
- icariine
- epimedium
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- TZJALUIVHRYQQB-XLRXWWTNSA-N icariin Chemical compound C1=CC(OC)=CC=C1C1=C(O[C@H]2[C@@H]([C@H](O)[C@@H](O)[C@H](C)O2)O)C(=O)C2=C(O)C=C(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)C(CC=C(C)C)=C2O1 TZJALUIVHRYQQB-XLRXWWTNSA-N 0.000 title claims abstract description 103
- TZJALUIVHRYQQB-UHFFFAOYSA-N icariine Natural products C1=CC(OC)=CC=C1C1=C(OC2C(C(O)C(O)C(C)O2)O)C(=O)C2=C(O)C=C(OC3C(C(O)C(O)C(CO)O3)O)C(CC=C(C)C)=C2O1 TZJALUIVHRYQQB-UHFFFAOYSA-N 0.000 title claims abstract description 103
- TZJALUIVHRYQQB-XFDQAQKOSA-N Icariin Natural products O(C)c1ccc(C2=C(O[C@H]3[C@@H](O)[C@H](O)[C@@H](O)[C@H](C)O3)C(=O)c3c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O4)c(C/C=C(\C)/C)c3O2)cc1 TZJALUIVHRYQQB-XFDQAQKOSA-N 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
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- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 claims abstract description 25
- 238000001179 sorption measurement Methods 0.000 claims abstract description 25
- 102000004190 Enzymes Human genes 0.000 claims abstract description 22
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- 229930013930 alkaloid Natural products 0.000 claims abstract description 20
- 150000003797 alkaloid derivatives Chemical class 0.000 claims abstract description 12
- 238000000194 supercritical-fluid extraction Methods 0.000 claims abstract description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 55
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 46
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- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 23
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- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical group [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 18
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- SVPXDRXYRYOSEX-UHFFFAOYSA-N bentoquatam Chemical compound O.O=[Si]=O.O=[Al]O[Al]=O SVPXDRXYRYOSEX-UHFFFAOYSA-N 0.000 claims description 12
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- HWDGVJUIHRPKFR-UHFFFAOYSA-I copper;trisodium;18-(2-carboxylatoethyl)-20-(carboxylatomethyl)-12-ethenyl-7-ethyl-3,8,13,17-tetramethyl-17,18-dihydroporphyrin-21,23-diide-2-carboxylate Chemical compound [Na+].[Na+].[Na+].[Cu+2].N1=C(C(CC([O-])=O)=C2C(C(C)C(C=C3C(=C(C=C)C(=C4)[N-]3)C)=N2)CCC([O-])=O)C(=C([O-])[O-])C(C)=C1C=C1C(CC)=C(C)C4=N1 HWDGVJUIHRPKFR-UHFFFAOYSA-I 0.000 description 7
- 229940079841 sodium copper chlorophyllin Drugs 0.000 description 7
- 235000013758 sodium copper chlorophyllin Nutrition 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 150000001414 amino alcohols Chemical class 0.000 description 6
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
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- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
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- 229940106157 cellulase Drugs 0.000 description 2
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- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- TWCMVXMQHSVIOJ-UHFFFAOYSA-N Aglycone of yadanzioside D Natural products COC(=O)C12OCC34C(CC5C(=CC(O)C(O)C5(C)C3C(O)C1O)C)OC(=O)C(OC(=O)C)C24 TWCMVXMQHSVIOJ-UHFFFAOYSA-N 0.000 description 1
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- NGMYNFJANBHLKA-SENBMHEBSA-N Icariside II Natural products O(C)c1ccc(C2=C(O[C@H]3[C@@H](O)[C@H](O)[C@@H](O)[C@H](C)O3)C(=O)c3c(O)cc(O)c(C/C=C(\C)/C)c3O2)cc1 NGMYNFJANBHLKA-SENBMHEBSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains four or more hetero rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07G—COMPOUNDS OF UNKNOWN CONSTITUTION
- C07G5/00—Alkaloids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/06—Benzopyran radicals
- C07H17/065—Benzo[b]pyrans
- C07H17/07—Benzo[b]pyran-4-ones
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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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
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Abstract
The invention relates to a preparation method of icariin. The method solves the problems of low yield, low recovery rate of active ingredients, high cost, complex process, large wastewater treatment capacity, environmental pollution, unfavorable industrial production and the like in the existing method. The method comprises the steps of supercritical extraction, enzymolysis, adsorption, analysis, ion resin purification and membrane filtration, and adopts a complex enzyme enzymolysis process to convert the higher glucoside in the epimedium into the icariine as much as possible, so as to maximally increase the content of the icariine in the raw materials, improve the extraction rate, ensure that the recovery rate of the effective components reaches more than 90 percent, and maximally develop the active components in the epimedium raw materials through the supercritical extraction, the ion resin purification and the membrane filtration to obtain chlorophyll, icariin and alkaloid, thereby greatly reducing the production cost of single products and greatly improving the production efficiency.
Description
Technical Field
The invention relates to a preparation method of icariin.
Background
Herba Epimedii is perennial herb of Epimedium of berberidaceae, and the medicinal part in pharmacopoeia is dry leaf. The traditional Chinese medicine has over 2000 medicinal history and is mainly distributed in regions of Shanxi, gansu, shanxi, henan, qinghai, hubei, sichuan and the like of China. Herba Epimedii is a traditional Chinese medicine for supplementing medicine, and is used for treating impotence, premature ejaculation, soreness of waist, leg pain, numbness of limbs, hemiplegia, neurasthenia, amnesia, tinnitus, and giddiness. The herba Epimedii contains flavone and polysaccharide, and also contains alkaloid, lignan, anthraquinone, volatile oil, stearic acid, linolenic acid, oleic acid, etc.
The Chinese pharmacopoeia prescribes that the main component of the epimedium is icariine, and the content of the icariine in the raw materials is not less than 0.5 percent, so that the icariine can effectively improve the cardiovascular system and regulate endocrine; can also obviously reduce bone loss and increase bone density of the osteoporosis rat.
In addition, the epimedium herb also contains abundant effective components such as chlorophyll, polysaccharide, alkaloid and the like. Chlorophyll is the main pigment for photosynthesis of plants, and has various purposes of hematopoiesis, vitamin provision, detoxification, disease resistance and the like. However, chlorophyll is not very stable, and light, acid, alkali, oxygen, oxidant and the like can decompose the chlorophyll, so that most of the chlorophyll is synthesized into sodium copper chlorophyllin at present, the stability and water solubility are improved, and the application range is widened. The epimedium polysaccharide can control cell division and differentiation, regulate cell growth and aging, has obvious effects in resisting tumor, resisting virus, reducing blood fat, enhancing immunity and the like, and has good development prospect. The alkaloid has various biological effects, can tonify kidney yang, strengthen tendons and bones, dispel wind-damp, and has good application prospect.
According to the reference data, icariine in the prior art is mostly obtained by directly adopting an organic reagent and a chromatographic column for separation or enzymolysis purification, the process is complex, the cost is high, the yield is low, the industrial production is not easy, and polysaccharide and alkaloid remained in mother liquor are treated as waste, so that a large amount of resources are wasted, the environment is extremely polluted, the great workload is brought to sewage treatment, and the production cost is increased.
Patent CN1225470C discloses a method for extracting icariine from Epimedium plant, which adopts aqueous organic solvent extraction, column chromatography and recrystallization to obtain 98% icariine, and the recovery rate of the effective components is about 88%. The macroporous column chromatography is adopted, then the silica gel column is adopted for separation and recrystallization, the process is complex, the period is long, the organic solvent consumption is large, the recovery rate of the effective components is low, the production cost is high, and the method is not a good choice for industrial production.
Icariine or aglycone is obtained by enzymolysis in patent CN 1268759C. Fermenting to obtain enzymolysis solution, adding ammonium sulfate and ethanol extraction enzyme, icariine, acetic acid or phosphoric acid, and reacting. The method has strong theories, weak practicality, complex products after enzymolysis, low yield and low suitability for industrial production, and high icariin content is difficult to obtain after silica gel column chromatography.
Patent CN101845067B relates to a preparation of icariin with high content. Dissolving 15-40% of herba Epimedii extract with high concentration ethanol, dissolving residue with low concentration ethanol, standing for crystallization, and recrystallizing to obtain 98% icariin. The process is simple, but does not convert the higher glycoside in the raw material into icariine, so that a great amount of raw material is wasted, and the cost is high.
Patent CN103910772B mentions a method for extracting icariin. Extracting with ethanol, acidifying, separating with macroporous resin, separating with simulated moving bed chromatography, crystallizing to obtain icariine, performing enzymolysis with tween 80 and glucosidase or cellulase, and separating and purifying with chromatographic column filled with D101 and AB-8 macroporous resin to obtain final product baohuoside I, II. Although the process provides a process route with a short period, the higher glycoside in the raw material is not converted into icariine, and a specific yield is not given. The simulated moving bed chromatographic separation is used in the purification process, and the equipment is hardly applied to industrial production, and has the advantages of complex operation, high cost and inapplicability to industrial production.
A preparation method of icariine is provided in patent CN106831913 a. Extracting the raw materials with ethanol, concentrating, extracting with ethyl acetate or n-butanol, washing with absolute methanol or absolute ethanol or 40-95% ethanol or 30-95% methanol, and crystallizing the residue to obtain icariine with concentration of 98% or more. The recovery rate of the effective components is only 89%, the effective components are low, and the higher glucoside is not converted, so that the waste of raw materials is caused.
The patent CN110343731A adopts cellulase to hydrolyze the epimedium extract, the addition amount is 0.1-1% of the mass of the raw material, and the low-glucoside icariine is obtained after hydrolysis, and the icariine is not obtained, but is directly hydrolyzed into the secondary glucoside. The conversion rate is 97%, and the content of low glucoside is 86%.
Patent CN110343731A relates to a process for obtaining icariine by an enzymolysis method. The raw material is epimedium wushanense, and more than 98 percent of icariine can be obtained by ethanol extraction, rhamnosidase enzymolysis, ethyl acetate extraction, crystallization and recrystallization. The total yield was 81.2%. Although the conversion is very high, the overall yield is low.
Disclosure of Invention
The invention aims to provide a preparation method of icariin with low cost and high recovery rate, which aims to solve the problems of low yield, low recovery rate of active ingredients, high cost, complex process, large wastewater treatment capacity, environmental pollution, adverse industrialized production and the like in the method. The invention separates and purifies icariine, simultaneously enriches polysaccharide and alkaloid to obtain a series of products with commercial value, realizes the maximization of resource utilization, reduces energy consumption, reduces sewage treatment and obviously reduces production cost.
The technical scheme of the invention is to provide a preparation method of icariin, which comprises the following steps:
crushing the epimedium raw material, extracting the epimedium raw material by adopting a carbon dioxide supercritical extraction method by taking a weak alkaline organic solvent as an entrainer to obtain an extract and extraction residues, wherein the extract is chlorophyll;
step 2, extracting and carrying out enzymolysis;
2.1, steaming the extraction slag obtained in the step 1 with alkaline water, adding active carbon, mixing uniformly, adding ethanol, stirring at normal temperature for extraction, filtering, and concentrating the filtrate;
step 2.2, adding a mixed reagent of calcium chloride and magnesium sulfate into the filtrate obtained in the step 2.1, stirring, filtering, adding rhamnosidase and xylosidase into the filtrate for enzymolysis at 45-55 ℃, inactivating enzyme, and concentrating to obtain an extract after enzymolysis;
step 3, adsorption;
adding a composite adsorbent with the mass accounting for 20-30% of the mass of the extract into the extract after enzymolysis, adsorbing icariine, heating to 55-65 ℃, stirring, filtering, and collecting precipitate and mother liquor; the composite adsorbent consists of active carbon, bentonite and chitosan components;
step 4, analyzing;
adding isopropanol into the precipitate obtained in the step 3, heating for dissolving, filtering, concentrating the filtrate, cooling for crystallization, adding acetone, heating, rinsing, and drying to obtain icariine with content of more than 98%;
adsorbing the mother solution obtained in the step 3 by cation resin, washing with water to be neutral, and eluting with ammonia alcohol; clarifying the adsorption effluent with clarifier, adding protease for enzymolysis, filtering with ultrafiltration membrane, and collecting concentrated solution to obtain herba Epimedii polysaccharide; concentrating the ammonia alcohol eluent to obtain the epimedium total alkaloids.
Further, in the step 1, the epimedium is epimedium wushanense; the organic solvent is acetone with pH of 8-8.5 and concentration of 70-90%, and the dosage of acetone is 0.5-1.5% (ml/g) of herba Epimedii raw material.
Further, the pressure of the supercritical carbon dioxide extraction method in the step 1 is 38-45MPa, the extraction temperature is 45-50 ℃, the extraction time is 3-4h, the pressure of the I-stage separation kettle is 8-10MPa, the temperature is 40-45 ℃, the pressure of the II-stage separation kettle is 6-8MPa, and the temperature is 35-40 ℃.
Further, in step 2.1, the alkaline water is ammonia water or ammonium hydroxide with the mass fraction of 5-8% in order to maximize the icariine content in the raw material; the mass of the active carbon is 0.4-0.6% of the mass of the extraction slag; concentrating the filtrate to a specific gravity of 1.02-1.03;
in the step 2.2, the mass ratio of the calcium chloride to the magnesium sulfate is 2.8:1, and the addition amount is 0.01-0.02% of the mass of the extraction slag in the step 1; concentrating the mixture to a specific gravity of 1.05-1.08 after enzyme deactivation;
the addition amount of the rhamnosidase is 7-9% of the mass of the extraction slag in the step 1, and the enzyme activity is 1600u/ml; the addition amount of the xylosidase is 0.5-1% of the mass of the extraction slag in the step 1, and the enzyme activity is 500u/ml.
Further, the addition amount of the rhamnosidase is 8% of the mass of the extraction slag in the step 1; the addition amount of the xylosidase is 0.6% of the mass of the extraction slag in the step 1, and the addition amount of the calcium chloride and the magnesium sulfate is 0.015% of the mass of the extraction slag in the step 1.
Further, in order to fully adsorb icariin, in step 3, the active carbon in the composite adsorbent accounts for 70-80%, bentonite accounts for 5-10%, and chitosan accounts for 15-20%.
Further, the composite adsorbent accounts for 75% of active carbon, 9% of bentonite and 16% of chitosan.
Further, in the step 4, the concentration of the isopropanol is 60-80%, and the dosage is 8-10 times of the precipitation quality obtained in the step 3.
Further, in order to improve the recovery rate of the active ingredients, the consumption of the protease in the step 5 is 0.2-0.5% of the quality of the extraction slag in the step 1, the enzymolysis temperature is 40-50 ℃, the enzymolysis time is 1.5-2.5h, the enzymolysis pH is 6-7, and the molecular weight cut-off of the ultrafiltration membrane is 20-50KD; the ammonia alcohol eluent is 50% ethanol with the mass concentration of 5% of ammonia water, and the dosage is 3BV.
The beneficial effects of the invention are as follows:
1. the invention adopts the complex enzyme enzymolysis technology to convert the higher glucoside in the epimedium into the icariine as much as possible, thereby maximally increasing the content of the icariine in the raw materials, reducing the cost, improving the extraction rate and enabling the recovery rate of the active ingredients to reach more than 90 percent. And by adding the mixed reagent of calcium chloride and magnesium sulfate, the enzymolysis effect is greatly improved, the use amount of enzyme is reduced, a good foundation is laid for the subsequent separation and purification, and the yield is further improved.
2. The invention abandons the traditional process of macroporous adsorption resin, and forms a new way, adopts the composite adsorbent to adsorb icariine, has good selectivity, can well separate water-soluble components, reduces the use of organic solvents and acid-base treatment, reduces environmental pollution, ensures that the whole process is simple and easy to operate, avoids dead adsorption of the icariine in column separation, and improves the yield. Meanwhile, the water-soluble components are adsorbed by adopting cationic resin, so that the epimedium polysaccharide and the alkaloid can be well separated, and the recovery rate of the effective components is improved.
3. The method adopts supercritical carbon dioxide extraction technology to separate chlorophyll from the epimedium raw material, has mild conditions and avoids the degradation of chlorophyll. Not only can obtain additional chlorophyll products, but also provides a good basis for separation and purification and identification of icariin.
4. The invention adopts the combination of a plurality of technologies of supercritical extraction, enzymolysis, adsorption, analysis, ion resin purification and membrane filtration to develop active ingredients in the epimedium raw material to the maximum extent, and except for icariine, chlorophyll, epimedium polysaccharide and alkaloid are obtained, thereby greatly reducing the production cost of single products and greatly improving the production efficiency.
Drawings
FIG. 1 is a process flow diagram of the method of the present invention;
FIG. 2 is an HPLC chromatogram of Epimedium wushanense material;
FIG. 3 is a graph showing the effect of enzymolysis without adding a mixed reagent of calcium chloride and magnesium sulfate;
FIG. 4 is a graph showing the effect of enzymolysis by adding a mixed reagent of calcium chloride and magnesium sulfate;
FIG. 5 effect of temperature on enzyme activity;
FIG. 6 is a HPLC chart of the mother liquor after adsorption by the composite adsorbent;
FIG. 7 is a HPLC chart of icariin in example I of the present invention;
FIG. 8 is a HPLC chart of icariin in the second embodiment of the present invention;
FIG. 9 is a HPLC chart of icariin in the third embodiment of the present invention;
FIG. 10 is a HPLC chart of icariin in the fourth embodiment of the present invention;
FIG. 11 is a HPLC chart of icariin in fifth embodiment of the present invention;
FIG. 12 is a HPLC chart of icariin in example six of the present invention.
Detailed Description
The invention is further described below with reference to the drawings and specific embodiments.
Referring to fig. 1, the preparation method of the invention mainly comprises the processes of supercritical carbon dioxide extraction, enzymolysis, adsorption, resolution and preparation of polysaccharide and alkaloid, wherein in the process of supercritical carbon dioxide extraction, a weakly alkaline organic solvent is used as an entrainer, and the separated extract is chlorophyll; calcium chloride and magnesium sulfate are added in the extraction and enzymolysis processes, so that the content of icariin in the raw materials is increased to the maximum extent, the cost for preparing the icariin is reduced, and the extraction yield is improved. Experiments prove that the specific comparison between the figure 3 and the figure 4 shows that the figure 3 shows the enzymolysis effect of the mixed reagent without adding calcium chloride and magnesium sulfate, the icariine content after enzymolysis is 14.52 percent, and the figure 4 shows the enzymolysis effect of the mixed reagent with adding calcium chloride and magnesium sulfate, the icariine content after enzymolysis is 27.92 percent, and the addition of the mixed reagent with calcium chloride and magnesium sulfate can greatly improve the enzymolysis effect, reduce the use amount of enzyme and lay a good foundation for subsequent separation and purification. And examined the influence of temperature on the enzyme activity, as shown in FIG. 5, it can be seen that the enzyme activity is high at 45-55℃and thus the following examples conducted the enzymatic hydrolysis process in this temperature range. Icariine is adsorbed by the composite adsorbent in the adsorption and analysis process, as shown in fig. 6, the icariine in the mother solution basically has no content after the adsorption by the composite adsorbent, and the adsorption is complete without loss. The method reduces the use of organic solvents and acid-base treatment, reduces environmental pollution, ensures that the whole process is simple and easy to operate, and finally obtains the icariin and the alkaloid except the icariin by using simple operation, thereby greatly reducing the production cost of single products and greatly improving the production efficiency.
Example 1
The embodiment is realized by the following processes:
100Kg of epimedium wushanense raw material is taken and crushed into 80 meshes, and the HPLC spectrogram is shown in figure 2, wherein the icariine content is 0.3 percent. The method comprises the steps of taking acetone with the mass of 1% of the raw material, the pH value of 8 and the concentration of 80% as an entrainer, performing supercritical carbon dioxide extraction, wherein the extraction pressure is 38MPa, the extraction temperature is 45 ℃, the extraction time is 3h, the pressure of a first-stage separation kettle is 8MPa, the temperature is 45 ℃, the pressure of a second-stage separation kettle is 6MPa, the temperature is 40 ℃, and chlorophyll and extraction residues are obtained after separation. Chlorophyll can be used for preparing sodium copper chlorophyllin through a conventional process.
Decocting the obtained extraction residue with 5% ammonia water for 1h, cooling at normal temperature, adding 0.5% active carbon, mixing, extracting with 5% ethanol for 2 times at normal temperature under stirring, filtering, mixing the filtrates, concentrating the filtrate to specific gravity of 1.02, adding 0.02% calcium chloride and magnesium sulfate mixed reagent (wherein the mass ratio of calcium chloride to magnesium sulfate is 2.8:1), stirring for 30min, filtering, adding 7% rhamnosidase and 0.5% xylosidase, performing enzymolysis at 50deg.C for 3h, inactivating enzyme, concentrating to specific gravity of 1.05, and improving icariine content by 4.7 times compared with original icariine content of 0.3%; then adding a composite adsorbent (wherein the active carbon accounts for 70%, the bentonite accounts for 10% and the chitosan accounts for 20%) accounting for 20% of the mass of the extract, stirring for 1h at 55 ℃, filtering, and respectively collecting precipitate and mother liquor.
The precipitate was dissolved by heating with 8 times of 80% isopropyl alcohol, filtered, concentrated, cooled to crystallize, rinsed with hot acetone, and dried to obtain 1.58Kg of solid, which was detected by HPLC as shown in FIG. 7, wherein icariine content was 98.3%. The yield is 1.58%, the content of icariine in the reduced raw material is 1.55%, and the recovery rate of the effective components is 90.6% compared with the content of the icariine in the raw material after enzymolysis of 1.71%.
The mother liquor is adsorbed by 001 x 7 cation resin (adsorption amount is 12g crude drug/ml resin), washed to be neutral by water, and then eluted by 3BV of amino alcohol (50% ethanol with mass concentration of 5% of ammonia water). Clarifying the adsorption effluent by a clarifier, adding protease 45 ℃ (pH 6-7) accounting for 0.2% of the mass of the extraction residue, performing enzymolysis for 2 hours, filtering by an ultrafiltration membrane with a molecular weight cut-off of 20KD, and collecting a concentrated solution part to obtain 8.35Kg of epimedium polysaccharide with a content of 82.6%. Concentrating the ammonia alcohol eluent to obtain 0.82Kg of epimedium total alkaloids with the content of 67.3 percent.
Example two
The embodiment is realized by the following processes:
100Kg of epimedium wushanense raw material (icariine content is 0.3%) is taken, crushed into 80 meshes, acetone with the mass of 0.5% of the raw material mass, pH value of 8.5 and concentration of 70% is taken as entrainer, supercritical carbon dioxide extraction is adopted, the extraction pressure is 45MPa, the extraction temperature is 50 ℃, the extraction time is 4 hours, the pressure of a first-stage separation kettle is 10MPa, the temperature is 40 ℃, the pressure of a second-stage separation kettle is 8MPa, and the temperature is 35 ℃, so that chlorophyll and extraction residues are obtained after separation. Chlorophyll can be used for preparing sodium copper chlorophyllin through a conventional process.
Steaming the obtained extraction slag for 1h by adopting 8% ammonia water, cooling at normal temperature, adding active carbon with the mass of 0.4% of the mass of the extraction slag, uniformly mixing, adopting 40% ethanol with the mass of 5 times of the mass of the extraction slag, stirring and extracting for 2 times at normal temperature, 1.5h each time, filtering, combining the two filtrates, concentrating the combined filtrate to have the specific gravity of 1.03, adding a mixed reagent with the mass of 0.01% calcium chloride and magnesium sulfate (wherein the mass ratio of the calcium chloride to the magnesium sulfate is 2.8:1), stirring for 30min, filtering, adding rhamnosidase with the mass of 9% of the extraction slag and xylosidase with the mass of 1% into the filtrate, carrying out enzymolysis for 3h at 45 ℃, inactivating enzyme, concentrating to have the specific gravity of 1.06, and obtaining the reduced icariine content of 1.96% after enzymolysis, and improving the icariine content by 5.5 times compared with the original icariine content in the raw material; then adding a composite adsorbent (wherein the active carbon accounts for 80%, the bentonite accounts for 5% and the chitosan accounts for 15%) accounting for 30% of the mass of the extract, stirring for 1h at 65 ℃, filtering, and respectively collecting precipitate and mother liquor.
The precipitate was dissolved by heating with 80% isopropyl alcohol 10 times the mass thereof, filtered, concentrated, cooled for crystallization, rinsed with hot acetone and dried to obtain 1.82Kg of solid, which was detected by HPLC as shown in FIG. 8, wherein icariine content was 98.2%. The yield is 1.82%, the content of icariine in the reduced raw material is 1.79%, and compared with the content of icariine in the raw material after enzymolysis, the recovery rate of the effective components is 91.3%.
The mother liquor is adsorbed by 001 x 7 cation resin (adsorption amount is 12g crude drug/ml resin), washed to be neutral by water, and then eluted by 3BV of amino alcohol (50% ethanol with mass concentration of 5% of ammonia water). Clarifying the adsorption effluent by a clarifier, adding protease 40 ℃ (pH 6-7) accounting for 0.5% of the mass of the extraction residue, performing enzymolysis for 2.5 hours, filtering by an ultrafiltration membrane with a molecular weight cutoff of 50KD, and collecting a concentrated solution part to obtain 7.95Kg of epimedium polysaccharide with a content of 85.2%. Concentrating the ammonia alcohol eluent to obtain 0.84Kg of epimedium total alkaloids with the content of 66.0 percent.
Example III
100Kg of epimedium wushanense raw material (icariine content is 0.3%) is taken, crushed into 80 meshes, acetone with the mass of 1.5% of the raw material mass, pH value of 8 and concentration of 90% is taken as entrainer, supercritical carbon dioxide extraction is adopted, the extraction pressure is 40MPa, the extraction temperature is 48 ℃, the extraction time is 4 hours, the pressure of a first-stage separation kettle is 9MPa, the temperature is 42 ℃, the pressure of a second-stage separation kettle is 7MPa, the temperature is 38 ℃, and chlorophyll and extraction residues are obtained after separation. Chlorophyll can be used for preparing sodium copper chlorophyllin through a conventional process.
Steaming the obtained extraction slag for 1h by adopting ammonium hydroxide with the mass fraction of 6%, cooling at normal temperature, adding active carbon with the mass of 0.6% of the extraction slag, uniformly mixing, adopting 40% ethanol with the mass of 5 times of the mass of the extraction slag, stirring and extracting for 2 times at normal temperature, 1.5h each time, filtering, combining the two filtrates, concentrating the combined filtrate to have the specific gravity of 1.02, adding a mixed reagent (the mass ratio of 2.8:1) of calcium chloride and magnesium sulfate with the mass of 0.012% of the extraction slag, stirring for 30min, filtering, adding rhamnosidase with the mass of 8% of the extraction slag and xylosidase with the mass of 0.9% of the extraction slag, carrying out enzymolysis for 3h at 55 ℃, inactivating enzyme, and concentrating to have the specific gravity of 1.08 to obtain an extract after enzymolysis; the icariine content in the folded raw material after enzymolysis is 1.88%, which is improved by 5.3 times compared with the original icariine content in the raw material of 0.3%; then adding a composite adsorbent (wherein the active carbon accounts for 72%, the bentonite accounts for 8% and the chitosan accounts for 20%) accounting for 25% of the mass of the extract, stirring for 1h at 60 ℃, filtering, and respectively collecting the precipitate and the mother liquor.
The precipitate was dissolved by heating with 80% isopropyl alcohol 9 times the mass thereof, filtered, concentrated, cooled for crystallization, rinsed with hot acetone and dried to obtain 1.75Kg of solid, which was detected by HPLC as shown in FIG. 9, wherein icariine content was 98.5%. The yield is 1.75%, the content of icariine in the reduced raw material is 1.72%, and compared with the content of icariine in the raw material after enzymolysis, the recovery rate of the effective components is 91.5%.
The mother liquor is adsorbed by 001 x 7 cation resin (adsorption amount is 12g crude drug/ml resin), washed to be neutral by water, and then eluted by 3BV of amino alcohol (50% ethanol with mass concentration of 5% of ammonia water). Clarifying the adsorption effluent by a clarifier, adding protease 50 ℃ (pH 6-7) with the mass of 0.3% of the extract residue for enzymolysis for 2 hours, filtering by an ultrafiltration membrane with the molecular weight cut-off of 30KD, and collecting the concentrated solution to obtain 8.34Kg of epimedium polysaccharide with the content of 80.9%. Concentrating the ammonia alcohol eluent to obtain 0.80Kg of epimedium total alkaloids with the content of 68.3 percent.
Example IV
100Kg of epimedium wushanense raw material (icariine content is 0.3%) is taken, crushed into 80 meshes, acetone with the mass of 1% of the raw material mass, pH=8 and concentration of 80% is taken as entrainer, supercritical carbon dioxide extraction is adopted, the extraction pressure is 40MPa, the extraction temperature is 46 ℃, the extraction time is 3 hours, the pressure of a grade I separation kettle is 9MPa, the temperature is 43 ℃, the pressure of a grade II separation kettle is 7MPa, and the temperature is 36 ℃, so that chlorophyll and extraction residues are obtained after separation. Chlorophyll can be used for preparing sodium copper chlorophyllin through a conventional process.
Steaming the obtained extraction slag for 1h by adopting 8% ammonium hydroxide, cooling at normal temperature, adding active carbon with the mass of 0.5% of the extraction slag, uniformly mixing, adopting 40% ethanol with the mass of 5 times of the extraction slag, stirring and extracting for 2 times at normal temperature, 1.5h each time, filtering, combining the two filtrates, concentrating the combined filtrate to have the specific gravity of 1.02, adding a mixed reagent (the mass ratio of 2.8:1) with the mass of 0.015% calcium chloride and magnesium sulfate of the extraction slag, stirring for 30min, filtering, adding rhamnosidase with the mass of 8% of the extraction slag and xylosidase with the mass of 0.6% into the filtrate, carrying out enzymolysis for 3h at 50 ℃, inactivating enzyme, concentrating to have the specific gravity of 1.05, and obtaining an extract after enzymolysis, wherein the icariin content in the folded raw material is 1.94%, and is improved by 5.5 times compared with the original icariin content in the raw material; then adding 25% of the liquid volume of the composite adsorbent (wherein, the active carbon accounts for 75%, the bentonite accounts for 9% and the chitosan accounts for 16%), stirring for 1h at 60 ℃, filtering, and collecting the precipitate and the mother liquor respectively. The precipitate was dissolved by heating with 80% isopropyl alcohol in an amount 10 times the mass thereof, filtered, concentrated, cooled for crystallization, rinsed with hot acetone and dried to obtain 1.83Kg of solid, which was detected by HPLC as shown in FIG. 10, wherein icariine content was 98.7%. The yield is 1.8,3%, the content of icariine in the reduced raw material is 1.83%, and compared with the content of icariine in the raw material after enzymolysis of 1.94%, the recovery rate of the effective components is 93.3%.
The mother liquor is adsorbed by 001 x 7 cation resin (adsorption amount is 12g crude drug/ml resin), washed to be neutral by water, and then eluted by 3BV of amino alcohol (50% ethanol with mass concentration of 5% of ammonia water). Clarifying the adsorption effluent by a clarifier, adding protease 45 ℃ (pH 6-7) accounting for 0.4% of the mass of the extraction residue, performing enzymolysis for 2 hours, filtering by an ultrafiltration membrane with a molecular weight cut-off of 40KD, and collecting a concentrated solution part to obtain 8.04Kg of epimedium polysaccharide with a content of 85.2%. Concentrating the ammonia alcohol eluent to obtain 0.82Kg of epimedium total alkaloids with the content of 70.2 percent.
Example five
100Kg of epimedium wushanense raw material (icariine content is 0.3%) is taken, crushed into 80 meshes, acetone with the mass of 1% of the raw material mass, pH=8 and concentration of 80% is taken as entrainer, supercritical carbon dioxide extraction is adopted, the extraction pressure is 42MPa, the extraction temperature is 45 ℃, the extraction time is 3 hours, the pressure of a first-stage separation kettle is 10MPa, the temperature is 40 ℃, the pressure of a second-stage separation kettle is 8MPa, and the temperature is 35 ℃, so that chlorophyll and extraction residues are obtained after separation. Chlorophyll can be used for preparing sodium copper chlorophyllin through a conventional process.
Steaming the obtained extraction slag for 1h by adopting 7% ammonia water, cooling at normal temperature, adding 0.5% active carbon with the mass of the extraction slag, mixing uniformly, adopting 40% ethanol with the mass of 5 times of the mass of the extraction slag, stirring and extracting for 2 times at normal temperature, 1.5h each time, filtering, combining the two filtrates, concentrating the combined filtrate to a specific gravity of 1.02, adding a mixed reagent with 0.018% calcium chloride and magnesium sulfate with the mass of 0.018% calcium chloride and magnesium sulfate (wherein the mass ratio of the calcium chloride to the magnesium sulfate is 2.8:1), stirring for 30min, filtering, adding rhamnosidase with the mass of 9% of the extraction slag and xylosidase with the mass of 0.7% into the filtrate, carrying out enzymolysis for 3h at 50 ℃, inactivating enzyme, concentrating to a specific gravity of 1.05, obtaining an extract after enzymolysis, and improving the icariin content in the folded raw material by 5.3 times compared with the original icariin content in the raw material; then adding 28% of the liquid volume of the composite adsorbent (wherein the active carbon accounts for 76%, the bentonite accounts for 6% and the chitosan accounts for 18%), stirring for 1h at 60 ℃, filtering, and collecting the precipitate and the mother liquor respectively.
The precipitate was dissolved by heating with 80% isopropyl alcohol in an amount 10 times the mass thereof, filtered, concentrated, cooled for crystallization, rinsed with hot acetone and dried to obtain 1.77Kg of solid, which was detected by HPLC as shown in FIG. 11, wherein icariine content was 98.3%. The yield is 1.77%, the icariine content in the reduced raw material is 1.57%, and the recovery rate of the effective components is 92.1% compared with the icariine content of the raw material after enzymolysis of 1.89%.
The mother liquor is adsorbed by 001 x 7 cation resin (adsorption amount is 12g crude drug/ml resin), washed to be neutral by water, and then eluted by 3BV of amino alcohol (50% ethanol with mass concentration of 5% of ammonia water). Clarifying the adsorption effluent by a clarifier, adding protease 50 ℃ (pH 6-7) with the mass of 0.5% of the extraction residue, performing enzymolysis for 1.5h, filtering by an ultrafiltration membrane with the molecular weight cut-off of 20KD, and collecting the concentrated solution to obtain 8.29Kg of epimedium polysaccharide with the content of 80.4%. Concentrating the ammonia alcohol eluent to obtain 0.80Kg of epimedium total alkaloids with the content of 68.0 percent.
Example six
100Kg of epimedium wushanense raw material (icariine content is 0.3%) is taken, crushed into 80 meshes, acetone with the mass of 1% of the raw material mass, pH=8 and concentration of 80% is taken as entrainer, supercritical carbon dioxide extraction is adopted, the extraction pressure is 39MPa, the extraction temperature is 49 ℃, the extraction time is 4 hours, the pressure of a grade I separation kettle is 8MPa, the temperature is 43 ℃, the pressure of a grade II separation kettle is 7MPa, the temperature is 39 ℃, and chlorophyll and extraction residues are obtained after separation. Chlorophyll can be used for preparing sodium copper chlorophyllin through a conventional process.
Steaming the obtained extraction slag for 1h by adopting ammonium hydroxide with the mass fraction of 5%, cooling at normal temperature, adding active carbon with the mass of 0.5% of the extraction slag, uniformly mixing, adopting 40% ethanol with the mass of 5 times of the mass of the extraction slag, stirring and extracting for 2 times at normal temperature, 1.5h each time, filtering, combining the two filtrates, concentrating the combined filtrate to the specific gravity of 1.02, adding a mixed reagent (the mass ratio of 2.8:1) of calcium chloride and magnesium sulfate with the mass of 0.01% of the extraction slag, stirring for 30min, filtering, adding rhamnosidase with the mass of 7% of the raw materials and xylosidase with the mass of 1% of the raw materials, carrying out enzymolysis for 3h at 50 ℃, inactivating enzymes, concentrating to the specific gravity of 1.05, obtaining an extract after enzymolysis, and reducing the icariine content of the raw materials by 1.79%, and improving the icariine content by 5 times compared with the original icariine content of the raw materials; then adding a composite adsorbent (wherein the active carbon accounts for 79%, the bentonite accounts for 7% and the chitosan accounts for 14%) with the mass of 28% of the extract, stirring for 1h at 60 ℃, filtering, and respectively collecting the precipitate and the mother liquor.
The precipitate was dissolved by heating with 8 times of 80% isopropyl alcohol, filtered, concentrated, cooled to crystallize, rinsed with hot acetone, and dried to obtain 1.65Kg of solid, which was detected by HPLC as shown in FIG. 12, wherein icariine content was 98.5%. The yield is 1.65%, the content of icariine in the reduced raw material is 1.63%, and compared with the content of icariine in the raw material after enzymolysis, the recovery rate of the effective components is 91.1%.
The mother liquor is adsorbed by 001 x 7 cation resin (adsorption amount is 12g crude drug/ml resin), washed to be neutral by water, and then eluted by 3BV of amino alcohol (50% ethanol with mass concentration of 5% of ammonia water). Clarifying the adsorption effluent by a clarifier, adding protease 45 ℃ (pH 6-7) with the mass of 0.2% of the extraction residue, performing enzymolysis for 2 hours, filtering by an ultrafiltration membrane with the molecular weight cut-off of 50KD, and collecting the concentrated solution to obtain 8.03Kg of epimedium polysaccharide with the content of 82.4%. Concentrating the ammonia alcohol eluent to obtain 0.83Kg of epimedium total alkaloids with the content of 68.6 percent.
Claims (8)
1. The preparation method of icariin is characterized by comprising the following steps:
step 1, supercritical extraction;
crushing the epimedium raw material, extracting the epimedium raw material by adopting a carbon dioxide supercritical extraction method by taking a weak alkaline organic solvent as an entrainer to obtain an extract and extraction residues, wherein the extract is chlorophyll;
step 2, extracting and carrying out enzymolysis;
2.1, steaming the extraction slag obtained in the step 1 with alkaline water, adding active carbon, mixing uniformly, adding ethanol, stirring at normal temperature for extraction, filtering, and concentrating the filtrate;
step 2.2, adding a mixed reagent of calcium chloride and magnesium sulfate into the filtrate obtained in the step 2.1, stirring, filtering, adding rhamnosidase and xylosidase into the filtrate for enzymolysis at 45-55 ℃, inactivating enzyme, and concentrating to obtain an extract after enzymolysis; the mass ratio of the calcium chloride to the magnesium sulfate is 2.8:1, the adding amount is 0.01-0.02% of the mass of the extraction slag in the step 1, and the concentration proportion is 1.05-1.08 after enzyme deactivation; the addition amount of the rhamnosidase is 7-9% of the mass of the extraction slag in the step 1, and the enzyme activity is 1600U/ml; the addition amount of the xylosidase is 0.5-1% of the mass of the extraction slag in the step 1, and the enzyme activity is 500U/ml;
step 3, adsorption;
adding a composite adsorbent with the mass accounting for 20-30% of the mass of the extract into the extract after enzymolysis, adsorbing icariine, heating to 55-65 ℃, stirring, filtering, and collecting precipitate and mother liquor; the composite adsorbent consists of active carbon, bentonite and chitosan components; the active carbon in the composite adsorbent accounts for 70-80%, the bentonite accounts for 5-10%, and the chitosan accounts for 15-20%;
step 4, analyzing;
adding isopropanol into the precipitate obtained in the step 3, heating for dissolving, filtering, concentrating the filtrate, cooling for crystallization, rinsing with hot acetone, and drying to obtain icariine with content of more than 98%;
step 5, preparing polysaccharide and alkaloid;
adsorbing the mother solution obtained in the step 3 by cation resin, washing with water to be neutral, and eluting with ammonia alcohol; clarifying the adsorption effluent with clarifier, adding protease for enzymolysis, filtering with ultrafiltration membrane, and collecting concentrated solution to obtain herba Epimedii polysaccharide; concentrating the ammonia alcohol eluent to obtain the epimedium total alkaloids.
2. The method for preparing icariin according to claim 1, wherein:
the epimedium in the step 1 is epimedium wushanense; the organic solvent is acetone with pH of 8-8.5 and concentration of 70-90%, and the dosage of acetone is 0.5-1.5% ml/g of herba Epimedii raw material.
3. The method for preparing icariin according to claim 2, wherein: the supercritical carbon dioxide extraction method in the step 1 has the pressure of 38-45MPa, the extraction temperature of 45-50 ℃, the extraction time of 3-4h, the pressure of a first-stage separation kettle of 8-10MPa, the temperature of 40-45 ℃, the pressure of a second-stage separation kettle of 6-8MPa and the temperature of 35-40 ℃.
4. The method for preparing icariin according to claim 3, wherein: in the step 2.1, the alkaline water is ammonia water or ammonium hydroxide with the mass fraction of 5-8%; the mass of the active carbon is 0.4-0.6% of the mass of the extraction slag; concentrating the filtrate to a specific gravity of 1.02-1.03.
5. The method for preparing icariin according to claim 4, wherein: the addition amount of the rhamnosidase is 8% of the mass of the extraction slag in the step 1; the addition amount of the xylosidase is 0.6% of the mass of the extraction slag in the step 1; the addition amount of the calcium chloride and the magnesium sulfate is 0.015 percent of the mass of the extraction slag in the step 1.
6. The method for preparing icariin according to claim 5, wherein: the composite adsorbent comprises 75% of active carbon, 9% of bentonite and 16% of chitosan.
7. The method for preparing icariin according to claim 5, wherein: in the step 4, the concentration of the isopropanol is 60-80%, and the dosage is 8-10 times of the precipitation quality obtained in the step 3.
8. The method for preparing icariin according to claim 5, wherein: the consumption of the protease in the step 5 is 0.2-0.5% of the mass of the extraction slag in the step 1, the enzymolysis temperature is 40-50 ℃, the enzymolysis time is 1.5-2.5h, the enzymolysis pH is 6-7, and the interception molecular weight of the ultrafiltration membrane is 20-50KD; the ammonia alcohol eluent is 50% ethanol with the mass concentration of 5% of ammonia water, and the dosage is 3BV.
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Application publication date: 20200821 Assignee: SHAANXI JIAHE PHARMACEUTICAL CO.,LTD. Assignor: SHAANXI JIAHE PHYTOCHEM Co.,Ltd. Contract record no.: X2023980049450 Denomination of invention: A preparation method of icariin Granted publication date: 20230428 License type: Common License Record date: 20231201 |