CN114214379A - A process for converting and extracting icariin by coupling flavonidase - Google Patents
A process for converting and extracting icariin by coupling flavonidase Download PDFInfo
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- CN114214379A CN114214379A CN202111612018.3A CN202111612018A CN114214379A CN 114214379 A CN114214379 A CN 114214379A CN 202111612018 A CN202111612018 A CN 202111612018A CN 114214379 A CN114214379 A CN 114214379A
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- flavonoid
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- 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 45
- 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 45
- 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 45
- 238000000034 method Methods 0.000 title claims abstract description 29
- 230000008878 coupling Effects 0.000 title claims abstract description 21
- 238000010168 coupling process Methods 0.000 title claims abstract description 21
- 238000005859 coupling reaction Methods 0.000 title claims abstract description 21
- 238000006243 chemical reaction Methods 0.000 claims abstract description 23
- 238000002156 mixing Methods 0.000 claims abstract description 22
- 108010059820 Polygalacturonase Proteins 0.000 claims abstract description 21
- 108010093305 exopolygalacturonase Proteins 0.000 claims abstract description 21
- 229930003935 flavonoid Natural products 0.000 claims abstract description 21
- 150000002215 flavonoids Chemical class 0.000 claims abstract description 21
- 235000017173 flavonoids Nutrition 0.000 claims abstract description 21
- 239000006228 supernatant Substances 0.000 claims abstract description 21
- 108010031186 Glycoside Hydrolases Proteins 0.000 claims abstract description 18
- 102000005744 Glycoside Hydrolases Human genes 0.000 claims abstract description 18
- 235000020696 epimedium extract Nutrition 0.000 claims abstract description 18
- 108010044879 alpha-L-rhamnosidase Proteins 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 11
- 230000002255 enzymatic effect Effects 0.000 claims abstract description 9
- 239000000413 hydrolysate Substances 0.000 claims abstract description 8
- 239000000126 substance Substances 0.000 claims abstract description 8
- 238000010438 heat treatment Methods 0.000 claims abstract description 7
- 238000001035 drying Methods 0.000 claims abstract description 6
- 230000002779 inactivation Effects 0.000 claims abstract description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 20
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 20
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- 239000007864 aqueous solution Substances 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 8
- 239000000284 extract Substances 0.000 claims description 5
- 238000009776 industrial production Methods 0.000 abstract description 2
- 108090000790 Enzymes Proteins 0.000 description 12
- 102000004190 Enzymes Human genes 0.000 description 12
- 241000893536 Epimedium Species 0.000 description 12
- YPFSXWUDSOVOGG-UHFFFAOYSA-N epimedin C Natural products COc1ccc(cc1)C2=C(OC3OC(C)C(O)C(OC(=O)C)C3OC4OC(CO)C(O)C(O)C4O)C(=O)c5c(O)cc(OC6OC(CO)C(O)C(O)C6O)c(CC=C(C)C)c5O2 YPFSXWUDSOVOGG-UHFFFAOYSA-N 0.000 description 12
- 235000018905 epimedium Nutrition 0.000 description 12
- ULZLIYVOYYQJRO-JIYCBSMMSA-N Epimedin C 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@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 ULZLIYVOYYQJRO-JIYCBSMMSA-N 0.000 description 10
- 230000000694 effects Effects 0.000 description 10
- 239000008346 aqueous phase Substances 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 9
- 239000012071 phase Substances 0.000 description 8
- 238000003756 stirring Methods 0.000 description 8
- 239000007788 liquid Substances 0.000 description 7
- 238000010828 elution Methods 0.000 description 5
- LKNITMBRWDCKMG-UHFFFAOYSA-N Epimedin B Natural products COc1ccc(cc1)C2=C(OC3OC(C)C(O)C(O)C3OC4OC(CO)C(O)C4O)C(=O)c5c(O)cc(OC6OC(CO)C(O)C(O)C6O)c(CC=C(C)C)c5O2 LKNITMBRWDCKMG-UHFFFAOYSA-N 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- SVXJDTNFJXKATR-KQSLYFRASA-N Epimedin A Natural products O([C@@H]1[C@@H](O)[C@@H](O)[C@H](C)O[C@H]1OC1=C(c2ccc(OC)cc2)Oc2c(C/C=C(\C)/C)c(O[C@H]3[C@@H](O)[C@@H](O)[C@H](O)[C@H](CO)O3)cc(O)c2C1=O)[C@H]1[C@@H](O)[C@H](O)[C@H](O)[C@@H](CO)O1 SVXJDTNFJXKATR-KQSLYFRASA-N 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- SVXJDTNFJXKATR-UHFFFAOYSA-N hexandraside A Natural products C1=CC(OC)=CC=C1C1=C(OC2C(C(O)C(O)C(C)O2)OC2C(C(O)C(O)C(CO)O2)O)C(=O)C2=C(O)C=C(OC3C(C(O)C(O)C(CO)O3)O)C(CC=C(C)C)=C2O1 SVXJDTNFJXKATR-UHFFFAOYSA-N 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- OCZZCFAOOWZSRX-LRHLXKJSSA-N Epimedin B 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@H]2[C@@H]([C@@H](O)[C@H](O)CO2)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 OCZZCFAOOWZSRX-LRHLXKJSSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 229930003944 flavone Natural products 0.000 description 2
- 235000011949 flavones Nutrition 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 241000133570 Berberidaceae Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 208000010228 Erectile Dysfunction Diseases 0.000 description 1
- 206010062575 Muscle contracture Diseases 0.000 description 1
- 206010041497 Spermatorrhoea Diseases 0.000 description 1
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 230000004097 bone metabolism Effects 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 208000006111 contracture Diseases 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 229930183477 epimedin Natural products 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 150000002212 flavone derivatives Chemical class 0.000 description 1
- 150000002213 flavones Chemical class 0.000 description 1
- 229930182486 flavonoid glycoside Natural products 0.000 description 1
- 150000007955 flavonoid glycosides Chemical class 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 201000001881 impotence Diseases 0.000 description 1
- 231100000862 numbness Toxicity 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000009759 skin aging Effects 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 230000002087 whitening effect Effects 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
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- 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
- 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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Abstract
The invention discloses a process for extracting icariin by coupling flavonoid glycosidase conversion, which comprises the following steps: s1, mixing the epimedium extract, water and pectinase uniformly, carrying out primary enzymolysis, heating for inactivation, adjusting pH, adding alpha-L-rhamnosidase uniformly, and carrying out secondary enzymolysis to obtain an enzymolysis solution; and S2, adding the enzymatic hydrolysate into the aqueous two-phase system, uniformly mixing, standing for layering, taking supernatant, centrifuging the supernatant, standing, taking supernatant, concentrating, and drying to obtain the converted substance. The method can improve the content of icariin, is simple to operate and is suitable for industrial production.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine extraction, in particular to a process for extracting icariin by coupling flavonoid glycosidase conversion.
Background
Epimedium herb (Herba Epimedii), also known as Epimedium herb, is the aerial part of various plants in Epimedium of berberidaceae, and is listed as a Chinese medicine in Shen nong Ben Cao Jing. Herba Epimedii is a traditional Chinese medicine for invigorating in China, and can be used for treating sexual impotence, spermatorrhea, tendons and bones weakness, rheumatalgia, numbness and contracture, etc. The recent research shows that the epimedium herb has quite wide pharmacological action on organisms, and has the effects of increasing the blood flow of heart and cerebral vessels, promoting the hematopoietic function, the immunologic function and the bone metabolism, resisting aging, resisting tumors and the like.
The epimedium flavonoid glycoside is the main effective component of epimedium, more than 70 kinds of epimedium flavonoids are found, and more than 80-90% of the flavonoids in the epimedium are Icariin (Icariin), epimedin A (epimidA), epimedin B (epimidB) and epimedin C (epimidc); wherein more than 50% is icariin, and the other about 40% flavone is epimedin A, B and C.
The structures of icariin, epimedin a, epimedin B and epimedin C are shown below.
The quality of the domestic epimedium extract is expressed by the icariin content. Icariin has effects of preventing depression, resisting tumor, resisting oxidation, enhancing immunity, resisting skin aging, resisting wrinkle, and whitening skin. However, the epimedium extract contains flavones such as epimedin A, B and C in addition to icariin, which reduces the quality and efficacy of the epimedium extract and also causes waste.
Other epimedium flavonoids can be converted into icariin by enzymatic conversion, but the conversion rate is lower; and in the process of enzyme conversion, there is also a problem that icariin is converted into other substances; in addition, the separation and purification process of the later-stage column elution is complex in operation.
Disclosure of Invention
Based on the technical problems in the background art, the invention provides a process for extracting icariin by coupling flavonoid glycosidase conversion.
The invention provides a process for extracting icariin by coupling flavonoid glycosidase conversion, which comprises the following steps:
s1, mixing the epimedium extract, water and pectinase uniformly, carrying out primary enzymolysis, heating for inactivation, adjusting pH, adding alpha-L-rhamnosidase uniformly, and carrying out secondary enzymolysis to obtain an enzymolysis solution;
and S2, adding the enzymatic hydrolysate into the aqueous two-phase system, uniformly mixing, standing for layering, taking supernatant, centrifuging the supernatant, standing, taking supernatant, concentrating, and drying to obtain the converted substance.
Preferably, in S1, the temperature of the first enzymolysis is 40-45 deg.C, and the time of the first enzymolysis is 3-4 h.
Preferably, in S1, the temperature of the second enzymolysis is 53-57 deg.C, and the time of the second enzymolysis is 30-60 min.
Preferably, in S1, the pH is adjusted to 6.0-6.2.
The inventor finds that the content of icariin and epimedin C can be improved by carrying out enzymolysis on the epimedium extract for a proper time by pectinase under proper conditions, and the content of icariin and epimedin C can be reduced by carrying out enzymolysis for too long time, so that the content of icariin and epimedin C is improved by carrying out enzymolysis on the epimedium extract by the pectinase, and then enzyme inactivation is carried out;
and then, selecting alpha-L-rhamnosidase for continuous enzymolysis, wherein the alpha-L-rhamnosidase can selectively carry out enzymolysis to remove rhamnosyl on the epimedin C, and selecting proper enzymolysis conditions and time to convert the epimedin C into icariin through enzymolysis, so that the phenomenon that the enzymolysis time is too long, the icariin and the rhamnosyl in the epimedin C are excessively subjected to enzymolysis is avoided, and the content of the icariin is reduced.
The method adopts a two-aqueous-phase system to purify the enzymolysis liquid, and has simple operation and short time consumption; the time consumption is much less than for the column elution process.
Preferably, in S1, the ratio of the epimedium extract to the pectinase is 1g: 400-800U.
Preferably, in S1, the ratio of the epimedium extract to the alpha-L-rhamnosidase is 1g: 300-500U.
Preferably, in S1, the weight ratio of epimedium extract to water is 1: 40-60.
Preferably, in S1, the pH is adjusted with ammonium sulfate.
Preferably, in S2, the two aqueous phase system consists of ethanol and an aqueous solution of ammonium sulfate.
Preferably, the mass fraction of ethanol is 40-45 wt%.
Preferably, the concentration of ammonium sulfate in the aqueous solution of ammonium sulfate is 0.22-0.25 g/ml.
Preferably, in S2, the weight ratio of the enzymatic hydrolysate to the aqueous two-phase system is 0.7-1: 10.
Preferably, in S2, standing for 30-60min for layering.
Has the advantages that:
according to the invention, pectinase is selected for enzymolysis at a proper temperature for a proper time to improve the icariin and epimedin C content in the epimedium extract, and then enzyme inactivation is carried out, so that the enzymolysis of the icariin by the pectinase is avoided, and the icariin content is reduced; then carrying out enzymolysis for a proper time under an adaptive condition by using alpha-L-rhamnosidase to convert epimedin C into icariin, thereby greatly improving the content of the icariin; then, the double aqueous phase system is used for purifying the enzymolysis liquid, so that the operation is simple, the consumed time is short, and the method is suitable for industrial production; and the raw materials of the aqueous two-phase system are cheap and easy to obtain, and the cost is low.
Detailed Description
The technical solution of the present invention will be described in detail below with reference to specific examples.
Example 1
A process for extracting icariin by coupling flavonoid glycosidase conversion comprises the following steps:
s1, uniformly mixing and dissolving 10g of epimedium herb extract and 400ml of water, adding 4000U of pectinase (the enzyme activity of the pectinase is 10 ten thousand U/g), stirring for 30min, uniformly mixing, adjusting the temperature to 45 ℃, preserving heat for enzymolysis for 3h for one time, heating to inactivate the pectinase, cooling to room temperature, adjusting the pH to 6.2 by using ammonium sulfate, adding 3000U of alpha-L-rhamnosidase (the enzyme activity of the alpha-L-rhamnosidase is 10 ten thousand U/g), stirring for 30min, uniformly mixing, adjusting the temperature to 57 ℃, and preserving heat for secondary enzymolysis for 30min to obtain an enzymolysis solution;
s2, adding the enzymatic hydrolysate into a double-aqueous-phase system, uniformly mixing, standing for layering for 60min, taking supernatant, centrifuging the supernatant at a speed of 3000r/min, standing, taking supernatant, concentrating the supernatant under reduced pressure, and drying in vacuum to obtain a converted substance, wherein the double-aqueous-phase system consists of 40 wt% of ethanol and 0.25g/ml of ammonium sulfate in an ammonium sulfate aqueous solution; the weight ratio of the enzymolysis liquid to the aqueous two-phase system is 0.7: 10.
Example 2
A process for extracting icariin by coupling flavonoid glycosidase conversion comprises the following steps:
s1, mixing 10g of epimedium extract with 600ml of water for dissolving, adding 8000U of pectinase (the enzyme activity of the pectinase is 10 ten thousand U/g), stirring for 30min, mixing, adjusting the temperature to 40 ℃, preserving heat for enzymolysis for 4h for the first time, heating to inactivate the pectinase, cooling to room temperature, adjusting the pH to 6.0 by using ammonium sulfate, adding 5000U of alpha-L-rhamnosidase (the enzyme activity of the alpha-L-rhamnosidase is 10 ten thousand U/g), stirring for 30min, mixing, adjusting the temperature to 53 ℃, preserving heat for secondary enzymolysis for 60min to obtain an enzymolysis solution;
s2, adding the enzymatic hydrolysate into a double-aqueous-phase system, uniformly mixing, standing for layering for 30min, taking supernatant, centrifuging the supernatant at a speed of 3000r/min, standing, taking supernatant, concentrating the supernatant under reduced pressure, and drying in vacuum to obtain a converted substance, wherein the double-aqueous-phase system consists of ethanol and an ammonium sulfate aqueous solution, the mass fraction of the ethanol is 45 wt%, and the concentration of the ammonium sulfate in the ammonium sulfate aqueous solution is 0.22 g/ml; the weight ratio of the enzymolysis liquid to the aqueous two-phase system is 0.9: 10.
Example 3
A process for extracting icariin by coupling flavonoid glycosidase conversion comprises the following steps:
s1, uniformly mixing and dissolving 10g of epimedium herb extract and 500ml of water, adding 6000U of pectinase (the enzyme activity of the pectinase is 10 ten thousand U/g), stirring for 30min, uniformly mixing, adjusting the temperature to 43 ℃, preserving heat for primary enzymolysis for 3.5h, heating to inactivate the pectinase, cooling to room temperature, adjusting the pH value to 6.1 by using ammonium sulfate, adding 4000U of alpha-L-rhamnosidase (the enzyme activity of the alpha-L-rhamnosidase is 10 ten thousand U/g), stirring for 30min, uniformly mixing, adjusting the temperature to 55 ℃, preserving heat for secondary enzymolysis for 45min to obtain an enzymolysis liquid;
s2, adding the enzymatic hydrolysate into a double-aqueous-phase system, uniformly mixing, standing for layering for 50min, taking supernatant, centrifuging the supernatant at a speed of 3000r/min, standing, taking supernatant, concentrating the supernatant under reduced pressure, and drying in vacuum to obtain a converted substance, wherein the double-aqueous-phase system consists of ethanol and an ammonium sulfate aqueous solution, the mass fraction of the ethanol is 42 wt%, and the concentration of the ammonium sulfate in the ammonium sulfate aqueous solution is 0.24 g/ml; the weight ratio of the enzymolysis liquid to the aqueous two-phase system is 1: 10.
Comparative example 1
A process for extracting icariin by coupling flavonoid glycosidase conversion comprises the following steps:
s1, uniformly mixing and dissolving 10g of epimedium herb extract and 500ml of water, adding 6000U of pectinase (the enzyme activity of the pectinase is 10 ten thousand U/g), stirring for 30min, uniformly mixing, adjusting the temperature to 43 ℃, carrying out heat preservation and enzymolysis for 3.5h, heating to inactivate the pectinase, and cooling to room temperature to obtain an enzymolysis solution;
s2, same as S2 of example 3.
Comparative example 2
A process for extracting icariin by coupling flavonoid glycosidase conversion comprises the following steps:
s1, mixing 10g of epimedium extract with 500ml of water uniformly for dissolving, then adding 4000U of alpha-L-rhamnosidase (the enzyme activity of the alpha-L-rhamnosidase is 10 ten thousand U/g), stirring for 30min, mixing uniformly, adjusting the pH value to 6.1 by using ammonium sulfate, adjusting the temperature to 55 ℃, and carrying out heat preservation and enzymolysis for 45min to obtain an enzymolysis liquid;
s2, same as S2 of example 3.
Comparative example 3
The same procedure as in example 3 was repeated for 8 hours.
Comparative example 4
The secondary enzymolysis is carried out for 2h, and the rest is the same as example 3.
The epimedium extracts in the above examples 1-3 and comparative examples 1-4 are prepared in the same batch, and the components are the same.
Detecting icariin content in herba Epimedii extract and converted substance by high performance liquid chromatography.
The conditions of the high performance liquid chromatography are as follows: gradient elution was carried out using an Agilent1200 high performance liquid chromatography apparatus with Kromasil C18 column (5 μm, 250 mm. times.4.6 mm), a sample volume of 10 μ l, a column temperature of 35 ℃, a flow rate of 1.0ml/min, a detection wavelength of 273nm, mobile phase A of acetonitrile, and mobile phase B of purified water, and the specific elution procedure is shown in Table 1.
TABLE 1 elution procedure
The results are shown in Table 2.
TABLE 2 test results
| Grouping | Icariin content% |
| Epimedium extract | 58 |
| Example 1 | 72 |
| Example 2 | 71 |
| Example 3 | 73 |
| Comparative example 1 | 60 |
| Comparative example 2 | 67 |
| Comparative example 3 | 32 |
| Comparative example 4 | 40 |
It can be seen from table 2 that the icariin content can be increased by selecting appropriate enzymes and enzymolysis conditions.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (10)
1. A process for extracting icariin by coupling flavonoid glycosidase conversion is characterized by comprising the following steps:
s1, mixing the epimedium extract, water and pectinase uniformly, carrying out primary enzymolysis, heating for inactivation, adjusting pH, adding alpha-L-rhamnosidase uniformly, and carrying out secondary enzymolysis to obtain an enzymolysis solution;
and S2, adding the enzymatic hydrolysate into the aqueous two-phase system, uniformly mixing, standing for layering, taking supernatant, centrifuging the supernatant, standing, taking supernatant, concentrating, and drying to obtain the converted substance.
2. The process for extracting icariin by coupling flavonoid glycosidase conversion according to claim 1, wherein the temperature of the first enzymolysis is 40-45 ℃ and the time of the first enzymolysis is 3-4h in S1.
3. The process for extracting icariin by coupling flavonoid glycosidase conversion according to claim 1 or 2, wherein in S1, the temperature of secondary enzymolysis is 53-57 ℃, and the time of secondary enzymolysis is 30-60 min; preferably, in S1, the pH is adjusted to 6.0-6.2.
4. The process for extracting icariin by coupling flavonoid glycosidase conversion according to any one of claims 1 to 3, wherein the ratio of epimedium extract to pectinase is 1g:400-800U in S1.
5. The process for extracting icariin by coupling flavonoid glycosidase conversion according to any one of claims 1 to 4, wherein the ratio of epimedium extract to alpha-L-rhamnosidase in S1 is 1g: 300-500U.
6. The process for extracting icariin by coupling flavoneglycosidase conversion as claimed in any one of claims 1 to 5, wherein in S1, the weight ratio of herba Epimedii extract to water is 1: 40-60.
7. The process for extracting icariin by coupling flavoneglycosidase conversion as claimed in any one of claims 1 to 6, wherein in S1, pH is adjusted with ammonium sulfate.
8. The process for extracting icariin by coupling flavonoid glycosidase conversion according to any one of claims 1 to 7, wherein in S2, the aqueous two-phase system consists of ethanol and aqueous ammonium sulfate solution.
9. The process for extracting icariin by coupling flavonoid glycosidase conversion according to claim 8, wherein the mass fraction of ethanol is 40-45 wt%; preferably, the concentration of ammonium sulfate in the aqueous solution of ammonium sulfate is 0.22-0.25 g/ml.
10. The process for extracting icariin by coupling flavonoid glycosidase conversion according to any one of claims 1 to 9, wherein the weight ratio of the enzymatic hydrolysate to the aqueous two-phase system in S2 is 0.7-1: 10; preferably, in S2, standing for 30-60min for layering.
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