CN112301069B - Enzymolysis method of genistein glycoside - Google Patents
Enzymolysis method of genistein glycoside Download PDFInfo
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- CN112301069B CN112301069B CN202011267397.2A CN202011267397A CN112301069B CN 112301069 B CN112301069 B CN 112301069B CN 202011267397 A CN202011267397 A CN 202011267397A CN 112301069 B CN112301069 B CN 112301069B
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- genistein
- glycoside
- enzymatic hydrolysis
- genistein glycoside
- enzymolysis
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- ZCOLJUOHXJRHDI-CMWLGVBASA-N genistein 7-O-beta-D-glucoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(O)=C2C(=O)C(C=3C=CC(O)=CC=3)=COC2=C1 ZCOLJUOHXJRHDI-CMWLGVBASA-N 0.000 title claims abstract description 67
- 238000000034 method Methods 0.000 title claims abstract description 41
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 23
- 239000000284 extract Substances 0.000 claims abstract description 19
- 238000006243 chemical reaction Methods 0.000 claims abstract description 16
- 239000007962 solid dispersion Substances 0.000 claims abstract description 11
- 239000002904 solvent Substances 0.000 claims abstract description 11
- 102000004190 Enzymes Human genes 0.000 claims abstract description 9
- 108090000790 Enzymes Proteins 0.000 claims abstract description 9
- 239000012429 reaction media Substances 0.000 claims abstract description 7
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims description 19
- 230000007071 enzymatic hydrolysis Effects 0.000 claims description 17
- ZCOLJUOHXJRHDI-FZHKGVQDSA-N Genistein 7-O-glucoside Natural products O([C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1)c1cc(O)c2C(=O)C(c3ccc(O)cc3)=COc2c1 ZCOLJUOHXJRHDI-FZHKGVQDSA-N 0.000 claims description 16
- CJPNHKPXZYYCME-UHFFFAOYSA-N Genistin Natural products OCC1OC(Oc2ccc(O)c3OC(=CC(=O)c23)c4ccc(O)cc4)C(O)C(O)C1O CJPNHKPXZYYCME-UHFFFAOYSA-N 0.000 claims description 16
- YCUNGEJJOMKCGZ-UHFFFAOYSA-N Pallidiflorin Natural products C1=CC(OC)=CC=C1C1=COC2=CC=CC(O)=C2C1=O YCUNGEJJOMKCGZ-UHFFFAOYSA-N 0.000 claims description 16
- 238000000108 ultra-filtration Methods 0.000 claims description 16
- 239000002245 particle Substances 0.000 claims description 14
- FRAUJUKWSKMNJY-UHFFFAOYSA-N 5-hydroxy-3-(4-hydroxyphenyl)-7-(6-malonyl-beta-D-glucopyranosyloxy)-4H-1-benzopyran-4-one Natural products OC1C(O)C(O)C(COC(=O)CC(O)=O)OC1OC1=CC(O)=C2C(=O)C(C=3C=CC(O)=CC=3)=COC2=C1 FRAUJUKWSKMNJY-UHFFFAOYSA-N 0.000 claims description 9
- FRAUJUKWSKMNJY-RSEYPYQYSA-N malonylgenistin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](COC(=O)CC(O)=O)O[C@H]1OC1=CC(O)=C2C(=O)C(C=3C=CC(O)=CC=3)=COC2=C1 FRAUJUKWSKMNJY-RSEYPYQYSA-N 0.000 claims description 9
- 102000006995 beta-Glucosidase Human genes 0.000 claims description 7
- 108010047754 beta-Glucosidase Proteins 0.000 claims description 7
- 239000005543 nano-size silicon particle Substances 0.000 claims description 7
- 235000012239 silicon dioxide Nutrition 0.000 claims description 7
- 239000012528 membrane Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000011148 porous material Substances 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 2
- 230000008569 process Effects 0.000 claims description 2
- 150000002170 ethers Chemical class 0.000 claims 1
- TZBJGXHYKVUXJN-UHFFFAOYSA-N genistein Natural products C1=CC(O)=CC=C1C1=COC2=CC(O)=CC(O)=C2C1=O TZBJGXHYKVUXJN-UHFFFAOYSA-N 0.000 description 30
- 235000006539 genistein Nutrition 0.000 description 28
- 229940045109 genistein Drugs 0.000 description 28
- 238000000605 extraction Methods 0.000 description 26
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- 229930182470 glycoside Natural products 0.000 description 11
- 239000000843 powder Substances 0.000 description 11
- 239000002994 raw material Substances 0.000 description 10
- -1 genistein glycosides Chemical class 0.000 description 9
- 244000144972 livestock Species 0.000 description 7
- 239000002798 polar solvent Substances 0.000 description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
- 150000002338 glycosides Chemical group 0.000 description 5
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- HXDOZKJGKXYMEW-UHFFFAOYSA-N 4-ethylphenol Chemical compound CCC1=CC=C(O)C=C1 HXDOZKJGKXYMEW-UHFFFAOYSA-N 0.000 description 4
- 230000001476 alcoholic effect Effects 0.000 description 4
- 230000007062 hydrolysis Effects 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 244000144977 poultry Species 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 150000002515 isoflavone derivatives Chemical class 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 244000285094 Flemingia macrophylla Species 0.000 description 2
- 244000018633 Prunus armeniaca Species 0.000 description 2
- 235000009827 Prunus armeniaca Nutrition 0.000 description 2
- 239000003674 animal food additive Substances 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 229930014626 natural product Natural products 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 238000005220 pharmaceutical analysis Methods 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000002390 rotary evaporation Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- OUJDQONJYHNTDX-UHFFFAOYSA-N 3-hydroxy-21-methyldocosa-4(E),15(E)-dien-1-yne Natural products OCC1OC(Oc2ccc(cc2)C3=COc4cc(OC5OC(CO)C(O)C(O)C5O)cc(O)c4C3=O)C(O)C(O)C1O OUJDQONJYHNTDX-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
- PLMKQQMDOMTZGG-UHFFFAOYSA-N Astrantiagenin E-methylester Natural products CC12CCC(O)C(C)(CO)C1CCC1(C)C2CC=C2C3CC(C)(C)CCC3(C(=O)OC)CCC21C PLMKQQMDOMTZGG-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 1
- 235000010523 Cicer arietinum Nutrition 0.000 description 1
- 244000045195 Cicer arietinum Species 0.000 description 1
- 241001022083 Flemingia Species 0.000 description 1
- 241000237858 Gastropoda Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical group OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 244000086363 Pterocarpus indicus Species 0.000 description 1
- 235000009984 Pterocarpus indicus Nutrition 0.000 description 1
- 241000219784 Sophora Species 0.000 description 1
- 235000015724 Trifolium pratense Nutrition 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000001430 anti-depressive effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000010420 art technique Methods 0.000 description 1
- 210000000748 cardiovascular system Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- PFOARMALXZGCHY-UHFFFAOYSA-N homoegonol Natural products C1=C(OC)C(OC)=CC=C1C1=CC2=CC(CCCO)=CC(OC)=C2O1 PFOARMALXZGCHY-UHFFFAOYSA-N 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000009057 passive transport Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 235000013526 red clover Nutrition 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
Classifications
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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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
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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
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/10—Process efficiency
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses an enzymolysis method of genistein glycoside, which comprises the steps of contacting solid dispersion containing genistein glycoside with enzyme in a reaction medium to carry out enzymolysis reaction of glycosidic bond rupture; the solid dispersion is obtained by dispersing the genistein glycoside or the extract containing the genistein glycoside in mesoporous nano silica particles by a solvent method. The enzymolysis method has higher conversion rate and shorter enzymolysis time.
Description
Technical Field
The invention belongs to the technical field of natural products, and particularly relates to an enzymolysis method of genistein glycoside.
Background
Genistein has the chemical name of 4',5, 7-trihydroxy isoflavone, genistein or genistein, and is one of isoflavone compounds with highest biological activity. The genistein molecular structure has 1 isoflavone ring and 1 phenol ring, and has 3 hydroxyl groups at the 5, 7-position of the isoflavone ring and the 4' -position of the phenol ring.
Genistein exists in the plant body in its aglycone and glycoside form under natural conditions. After entering the human and animal body, genistein glycoside is catalyzed by glycosidases of the pancreas and small intestine to produce related aglycones, which are further converted to p-ethylphenol by intestinal fluid bacteria. Finally, p-ethylphenol is absorbed by the intestinal epithelium via a form of passive transport. The metabolized genistein is excreted by urine. Genistein has antioxidant, antiaging, osteoporosis preventing, cardiovascular system health protecting, immunity enhancing, and antidepressant effects. Therefore, genistein is widely used in the fields of pharmacy, health care and the like at present, and is gradually applied to the field of livestock production.
Chinese patent application CN109170264a discloses a livestock feed additive, livestock feed or medicament. The livestock and poultry feed additive or livestock and poultry feed or drug contains genistein. The amount of genistein is as follows: 20-60mg of genistein or a drug injected according to 10-30mg/kg of body weight or 5-15mg/kg of body weight is added into each kg of livestock and poultry feed, so that the muscle endurance can be further improved, and the meat quality of livestock and poultry can be improved.
As a source of genistein, most still come from natural product extraction and are obtained by further purification. According to literature reports, genistein exists in different parts of plants such as sophora fruit, soybean, chickpea, red clover, rosewood leaf, philippine flemingia root and the like. Extracts of all plants include not only genistein but also various genistein glycosides, for example, genistein-4 ' -glucoside, genistein-7-glucoside (i.e., genistin), genistein-4 ', 7-diglucoside, genistein-4 ' -diglucoside, genistein-7-diglucoside, and the like.
Because of the large quantity of genistein glycosides, the physicochemical properties of genistein glycosides have large differences, it is difficult to obtain better extraction rate by using a single solvent or method during extraction, and to obtain higher purity during purification.
He Fengyan et al (journal of pharmaceutical analysis, 40 (1), P177) have shown that when using moghania macrophylla as an extraction raw material, the extracted genistin is mostly genistin and malonyl genistin, except genistein; both classes of genistins facilitate the overall conversion to genistein in the subsequent hydrolytic conversion step. The study used aqueous methanol and sonication to obtain genistein and its glycosides. However, the extraction yield and purity of the related substances are not high.
On the other hand, since genistein has the highest biological activity, it is generally required to convert genistein glycoside into genistein by hydrolysis, which makes it difficult to obtain a high conversion rate due to solubility of glycoside itself and complexity of operation steps. The hydrolysis methods that are more commonly used include acid hydrolysis, alkali hydrolysis and enzymatic hydrolysis. However, the hydrolysis degree of the former two methods is not easy to control, and the stability of the product is poor; the enzymatic hydrolysis condition is mild, but the cost is high;
jinxin et al (pharmaceutical journal, 47 (4), P522) used a nano silica solid dispersion technique to increase the efficiency of preparing genistein by snail enzymolysis of genistin. However, this technique only investigated the case of snailase, and no other hydrolases were studied; moreover, the enzymatic hydrolysis time of this technique is longer than most prior art techniques.
Therefore, in view of the above-mentioned drawbacks of the prior art, there is still a need to find an enzymatic hydrolysis method of genistein glycoside with higher conversion rate and shorter enzymatic hydrolysis time.
Disclosure of Invention
The invention aims to provide an enzymolysis method of genistein glycoside. Compared with the prior art, the enzymolysis method has the advantages of higher conversion rate and shorter enzymolysis time.
In order to solve the technical problems, the invention adopts the following technical scheme: a method for enzymolysis of genistein glycoside is characterized in that solid dispersion containing genistein glycoside is contacted with enzyme in a reaction medium to carry out enzymolysis reaction of glycosidic bond rupture.
The enzymatic hydrolysis method according to the invention, wherein the genistein glycoside comprises genistin and malonyl genistin.
The enzymatic hydrolysis method according to the invention, wherein the enzyme is selected from the group consisting of beta-glucosidase.
Preferably, the beta-glucosidase is selected from the group of apricot-derived beta-glucosidase.
The enzymatic hydrolysis process according to the invention, wherein the reaction medium is selected from the group consisting of water having a ph=5.0-7.0.
Preferably, the reaction medium is selected from water having a ph=5.5-6.5.
In a specific embodiment, the reaction medium is selected from water having a ph=6.0.
The enzymolysis method according to the invention, wherein the solid dispersion is obtained by dispersing the genistein glycoside or an extract containing the genistin glycoside in mesoporous nano silica particles by a solvent method.
The enzymolysis method according to the invention, wherein the enzyme is added in an amount of 0.02-0.5mg/mL.
Preferably, the enzyme is added in an amount of 0.05-0.2mg/mL.
In a specific embodiment, the enzyme is added in an amount of 0.1mg/mL.
The enzymolysis method provided by the invention, wherein the temperature of the enzymolysis reaction is 30-60 ℃; the time is 0.5-4h.
Preferably, the temperature of the enzymolysis reaction is 40-50 ℃; the time is 1-3h.
In a specific embodiment, the temperature of the enzymatic hydrolysis reaction is 45 ℃; the time was 2h.
The enzymolysis method according to the invention further comprises filtering and ultrafiltration operations.
The enzymolysis method of the invention, wherein the ultrafiltration operation is as follows: ultrafiltration was performed using a PES ultrafiltration membrane of 5-20kDa at an operating pressure of 0.05-0.25 MPa.
Preferably, the ultrafiltration operation is: ultrafiltration was performed using a PES ultrafiltration membrane of 8-15kDa at an operating pressure of 0.1-0.2 MPa.
In a specific embodiment, the ultrafiltration operation is: ultrafiltration was performed using a 10kDa PES ultrafiltration membrane at an operating pressure of 0.15 MPa.
The enzymolysis method according to the invention further comprises the operation of extraction with anhydrous diethyl ether and drying.
The enzymolysis method of the invention, wherein the weight ratio of the genistein glycoside or the extract containing the genistin glycoside to the mesoporous nano silica particles is 1:0.5-2.5.
Preferably, the weight ratio of the genistein glycoside or the extract containing the genistein glycoside to the mesoporous nano silica particles is 1:1-2.
In a specific embodiment, the weight ratio of the genistein glycoside or the extract containing the genistin glycoside to the mesoporous nano silica particles is 1:1.5.
the enzymolysis method of the invention, wherein the average particle size of the mesoporous nano silicon dioxide particles is 60-150nm; specific surface area greater than 400m 2 /g; pore volume greater than 0.3cm 3 /g。
Preferably, the average particle size of the mesoporous nano silicon dioxide particles is 80-120nm; specific surface area greater than 450m 2 /g; pore volume greater than 0.4cm 3 /g。
In a specific embodiment, the mesoporous nano silica particles have an average particle size of 90nm; specific surface area of more than 500m 2 /g; pore volume greater than 0.5cm 3 /g。
The enzymolysis method of the invention, wherein the outer surface of the mesoporous nano silicon dioxide particles is modified with water-soluble groups.
Preferably, the water-soluble group is selected from the group consisting of hydroxyl, amino, carboxyl, sulfonic acid, and phosphoric acid groups.
In a specific embodiment, the water-soluble group is selected from hydroxyl groups.
In one embodiment, the genistein glycoside-containing extract is selected from the group consisting of genistin glycoside-containing extract of Flemingiae macrophylla; the method is characterized by comprising the following steps: extracting the raw materials by using radix philippines macrophyllae as the raw materials, using a polar solvent as the extraction solvent and using ultrasonic-negative pressure cavitation cooperative operation; after centrifugation or filtration, the filtrate is dried.
Advantageously, the root of Flemingiae Philippinensis is selected from powders sieved between 100 and 300 mesh. Preferably, the root of Flemingiae Philippinensis is selected from powder sieved by 200-300 mesh sieve. In a specific embodiment, the moghania macrophylla is selected from a 280 mesh powder.
Advantageously, the polar solvent is selected from 50-90 v% by volume of aqueous alcoholic solvent. Preferably, the polar solvent is selected from 60-80v% aqueous solutions of alcoholic solvents. In a specific embodiment, the polar solvent is selected from 70v% aqueous alcoholic solvents.
Advantageously, the feed-to-liquid ratio of the extraction raw material to the polar solvent is 1:30-60 (g/g). Preferably, the feed liquid ratio of the extraction raw material to the polar solvent is 1:40-50 (g/g). In a specific embodiment, the ratio of the extractive raw material to the polar solvent is 1:45 (g/g).
Advantageously, the operating power of the ultrasonic operation is 200-500W; the working frequency is 25-45kHz. Preferably, the working power of the ultrasonic operation is 300-400W; the working frequency is 28-40kHz. In a specific embodiment, the operating power of the ultrasonic operation is 350W; the operating frequency was 33kHz.
Advantageously, the pressure of the negative pressure cavitation operation is between-0.04 and-0.12 MPa. Preferably, the negative pressure cavitation operation has a pressure of-0.06 to-0.10 MPa. In a specific embodiment, the negative pressure cavitation operation is at a pressure of-0.08 MPa.
Advantageously, the extraction temperature of the co-operation is 20-60 ℃. Preferably, the extraction temperature of the co-operation is 30-50 ℃. In a specific embodiment, the extraction temperature of the co-operation is 40 ℃.
Advantageously, the extraction time of the co-operation is 20-60min. Preferably, the extraction time of the synergistic operation is 30-50min. In a specific embodiment, the extraction time for the co-operation is 40min.
In the present invention, the alcoholic solvent is selected from methanol or ethanol; preferably methanol.
In another embodiment, the genistein glycoside is selected from a powder of genistin and/or malonyl genistin.
Without wishing to be bound by any theory, the enzymatic hydrolysis method is used not only with higher conversion rates, but also with shorter enzymatic hydrolysis times.
Detailed Description
The invention is further described below in conjunction with the detailed description.
It should be understood that the description of the specific embodiments is merely illustrative of the principles and spirit of the invention, and not in limitation thereof. Further, it should be understood that various changes, substitutions, omissions, modifications, or adaptations to the present invention may be made by those skilled in the art after having read the present disclosure, and such equivalent embodiments are within the scope of the present invention as defined in the appended claims.
In the present invention, all parts are parts by weight unless otherwise indicated.
Flemingiae Philippinensis root extract containing genistein glycoside
Pulverizing the pre-dried radix Flemingiae Philippinensis, sieving with 280 mesh sieve, and collecting 10g of sieved powder as extraction raw material. Placing the extracted raw material in an extraction tank subjected to negative pressure cavitation, wherein 70v% of methanol water solution is used as an extraction solvent; the feed liquid ratio of the extraction raw materials to the extraction solvent is 1:45 (g/g). The extraction tank and the circulating pipeline of the negative pressure cavitation are arranged in the water bath of the ultrasonic generator. The working power of the ultrasonic operation is 350W; the operating frequency was 33kHz. The pressure of negative pressure cavitation operation is-0.08 MPa. The extraction temperature is 40 ℃; the extraction time is 40min. Then, the extract was centrifuged, and the supernatant was collected. Removing methanol in the supernatant by rotary evaporation; and vacuum drying the rest liquid at 50deg.C until the quality is constant to obtain extract powder containing genistein and its glycoside.
The extraction yield and purity of the genistein and its glycosides from the obtained extract powder were determined according to the method of He Fengyan et al (journal of pharmaceutical analysis, 40 (1), P177). Wherein the extraction rate is the ratio of the weight of genistin, malonyl genistin or genistein in the extract powder to the weight of the extraction raw material; purity is the ratio of the sum of the effective content of genistin, malonyl genistin or genistein to the weight of the extract powder. The results are shown in Table 1.
TABLE 1
| Genistin (mill) | Malonyl genistin (mill) | Genistein (mill) | Purity (%) |
| 1.82 | 1.06 | 0.53 | 2.47 |
Example 1
Dissolving the radix Flemingiae Philippinensis extract with the composition shown in Table 1 in methanol, and adding solid dispersion medium; after uniform dispersion, methanol was removed by rotary evaporation to obtain a solid dispersion. The radix Flemingiae Philippinensis extract and solid componentThe weight ratio of the bulk medium is 1:1.5. the solid dispersion medium is selected from mesoporous nano silicon dioxide particles; the average particle diameter is 90nm; specific surface area of more than 500m 2 /g; pore volume greater than 0.5cm 3 /g; the outer surface is modified with hydroxyl groups (from Shanghai Sofos Biomedicine technologies Co., ltd.).
The beta-glucosidase used for the enzymolysis is selected from beta-glucosidase derived from apricot tree (from Zhongkeruitai (Beijing) biosciences). 100mg of the solid dispersion was added to 10mL of pure water, followed by addition of 0.1mg/mL of β -glucosidase, and ph=6.0 was adjusted. Enzymolysis is carried out for 2h at 45 ℃. Filtration was performed using a 0.45 micron membrane and ultrafiltration was performed using a 10kDa PES ultrafiltration membrane at an operating pressure of 0.15 MPa. The filtrate was transferred to a separatory funnel, extracted 2 times with anhydrous diethyl ether, and the organic phases were combined. The organic phase was removed by concentrating under reduced pressure, and the concentrated liquid was dried under vacuum to give a solid powder.
Comparative example 1
Other conditions were the same as in example 1 except that mesoporous nanosilica particles were replaced with nanosilica of the same average particle diameter, the outer surface of which was modified with hydroxyl groups (derived from Shanghai Sofos Biotechnology Co., ltd.).
The sum m1 of the residual weights of genistin and malonyl genistin in the solid powders obtained in example 1 and comparative example 1 was determined according to the method described above, and then the conversion was calculated, i.e. the weight (m 0-m 1)/m 0 x 100% of genistin and malonyl genistin in the extract of mozzarella macrophylla.
The results show that the conversion of example 1 is 98.6%; the conversion of comparative example 1 was 87.5%.
Without wishing to be bound by any theory, the use of the specific enzymatic hydrolysis method of the invention not only results in higher conversion rates, but also results in shorter enzymatic hydrolysis times.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention are included in the protection scope of the present invention.
Claims (6)
1. An enzymolysis method of genistein glycoside is characterized in that solid dispersion containing genistein glycoside is contacted with enzyme in a reaction medium to carry out enzymolysis reaction of glycosidic bond rupture;
the genistein glycoside comprises genistin and malonyl genistin; the enzyme is selected from beta-glucosidase;
the solid dispersion is obtained by dispersing the genistein glycoside or the extract containing the genistein glycoside in mesoporous nano silicon dioxide particles by a solvent method; the weight ratio of the genistein glycoside or the extract containing the genistein glycoside to the mesoporous nano silica particles is 1:0.5-2.5;
the average particle size of the mesoporous nano silicon dioxide particles is 60-150nm; specific surface area greater than 400m 2 /g; pore volume greater than 0.3cm 3 /g; the outer surface of the mesoporous nano silicon dioxide particles is modified with water-soluble groups;
wherein the enzyme is added in an amount of 0.02-0.5mg/mL.
2. An enzymatic hydrolysis process according to claim 1, wherein the reaction medium is selected from water having a ph=5.0-7.0.
3. The enzymatic hydrolysis method according to claim 1, wherein the temperature of the enzymatic hydrolysis reaction is 30-60 ℃; the time is 0.5-4h.
4. The enzymatic hydrolysis method of claim 1, further comprising filtration and ultrafiltration operations.
5. The enzymatic hydrolysis method of claim 4, wherein the ultrafiltration is performed as follows: ultrafiltration was performed using a PES ultrafiltration membrane of 5-20kDa at an operating pressure of 0.05-0.25 MPa.
6. The enzymatic hydrolysis method according to claim 1, further comprising the step of extracting with dehydrated ether and drying.
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