CN113662983A - Method for extracting pueraria isoflavone - Google Patents
Method for extracting pueraria isoflavone Download PDFInfo
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- CN113662983A CN113662983A CN202110959848.7A CN202110959848A CN113662983A CN 113662983 A CN113662983 A CN 113662983A CN 202110959848 A CN202110959848 A CN 202110959848A CN 113662983 A CN113662983 A CN 113662983A
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- isoflavone
- urea
- radix puerariae
- choline chloride
- solution
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- GOMNOOKGLZYEJT-UHFFFAOYSA-N isoflavone Chemical compound C=1OC2=CC=CC=C2C(=O)C=1C1=CC=CC=C1 GOMNOOKGLZYEJT-UHFFFAOYSA-N 0.000 title claims abstract description 91
- CJWQYWQDLBZGPD-UHFFFAOYSA-N isoflavone Natural products C1=C(OC)C(OC)=CC(OC)=C1C1=COC2=C(C=CC(C)(C)O3)C3=C(OC)C=C2C1=O CJWQYWQDLBZGPD-UHFFFAOYSA-N 0.000 title claims abstract description 91
- 235000008696 isoflavones Nutrition 0.000 title claims abstract description 91
- 241000219780 Pueraria Species 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 19
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 107
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 57
- 239000001763 2-hydroxyethyl(trimethyl)azanium Substances 0.000 claims abstract description 34
- 235000019743 Choline chloride Nutrition 0.000 claims abstract description 34
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims abstract description 34
- 239000004202 carbamide Substances 0.000 claims abstract description 34
- 229960003178 choline chloride Drugs 0.000 claims abstract description 34
- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical compound [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 claims abstract description 34
- 238000000605 extraction Methods 0.000 claims abstract description 34
- 239000000287 crude extract Substances 0.000 claims abstract description 24
- 239000000843 powder Substances 0.000 claims abstract description 17
- 230000005496 eutectics Effects 0.000 claims abstract description 14
- 238000001291 vacuum drying Methods 0.000 claims abstract description 14
- 239000002904 solvent Substances 0.000 claims abstract description 10
- 238000002360 preparation method Methods 0.000 claims abstract description 3
- 238000001914 filtration Methods 0.000 claims description 20
- 239000000203 mixture Substances 0.000 claims description 20
- 239000012153 distilled water Substances 0.000 claims description 17
- 238000001816 cooling Methods 0.000 claims description 14
- 238000010438 heat treatment Methods 0.000 claims description 13
- 238000005406 washing Methods 0.000 claims description 12
- 239000000284 extract Substances 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 8
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 claims description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 239000013078 crystal Substances 0.000 claims description 6
- 239000008367 deionised water Substances 0.000 claims description 6
- 229910021641 deionized water Inorganic materials 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
- 239000000706 filtrate Substances 0.000 claims description 6
- 239000012535 impurity Substances 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 3
- 238000001704 evaporation Methods 0.000 claims 1
- 238000001035 drying Methods 0.000 abstract description 17
- 235000010575 Pueraria lobata Nutrition 0.000 abstract description 16
- 241000219781 Pueraria montana var. lobata Species 0.000 abstract description 16
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 abstract description 12
- -1 flavonoid compounds Chemical class 0.000 abstract description 8
- 238000005303 weighing Methods 0.000 abstract description 8
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 abstract description 7
- 229930003935 flavonoid Natural products 0.000 abstract description 7
- 235000017173 flavonoids Nutrition 0.000 abstract description 7
- 238000002156 mixing Methods 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 5
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 abstract description 5
- 241000196324 Embryophyta Species 0.000 abstract description 4
- 238000004090 dissolution Methods 0.000 abstract description 4
- 210000002421 cell wall Anatomy 0.000 abstract description 3
- 229910052739 hydrogen Inorganic materials 0.000 abstract description 3
- 239000001257 hydrogen Substances 0.000 abstract description 3
- 239000002994 raw material Substances 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 54
- 235000019441 ethanol Nutrition 0.000 description 31
- JMGZEFIQIZZSBH-UHFFFAOYSA-N Bioquercetin Natural products CC1OC(OCC(O)C2OC(OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5)C(O)C2O)C(O)C(O)C1O JMGZEFIQIZZSBH-UHFFFAOYSA-N 0.000 description 12
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 12
- IVTMALDHFAHOGL-UHFFFAOYSA-N eriodictyol 7-O-rutinoside Natural products OC1C(O)C(O)C(C)OC1OCC1C(O)C(O)C(O)C(OC=2C=C3C(C(C(O)=C(O3)C=3C=C(O)C(O)=CC=3)=O)=C(O)C=2)O1 IVTMALDHFAHOGL-UHFFFAOYSA-N 0.000 description 12
- FDRQPMVGJOQVTL-UHFFFAOYSA-N quercetin rutinoside Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 FDRQPMVGJOQVTL-UHFFFAOYSA-N 0.000 description 12
- IKGXIBQEEMLURG-BKUODXTLSA-N rutin Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@@H]1OC[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 IKGXIBQEEMLURG-BKUODXTLSA-N 0.000 description 12
- ALABRVAAKCSLSC-UHFFFAOYSA-N rutin Natural products CC1OC(OCC2OC(O)C(O)C(O)C2O)C(O)C(O)C1OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5 ALABRVAAKCSLSC-UHFFFAOYSA-N 0.000 description 12
- 235000005493 rutin Nutrition 0.000 description 12
- 229960004555 rutoside Drugs 0.000 description 12
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 9
- 239000003814 drug Substances 0.000 description 6
- 238000000227 grinding Methods 0.000 description 5
- 238000007789 sealing Methods 0.000 description 5
- 238000007873 sieving Methods 0.000 description 5
- 238000003860 storage Methods 0.000 description 5
- 239000002244 precipitate Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 4
- 229960005070 ascorbic acid Drugs 0.000 description 3
- 235000010323 ascorbic acid Nutrition 0.000 description 3
- 239000011668 ascorbic acid Substances 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 229960004889 salicylic acid Drugs 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 1
- 230000002292 Radical scavenging effect Effects 0.000 description 1
- 238000003916 acid precipitation Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 239000012267 brine Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 1
- 235000018927 edible plant Nutrition 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 150000002515 isoflavone derivatives Chemical class 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
- A61K36/488—Pueraria (kudzu)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
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- Animal Behavior & Ethology (AREA)
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- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
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Abstract
The invention provides a method for extracting pueraria isoflavone. The extraction method comprises the following steps: preparation of choline chloride/urea: mixing 139.6g of choline chloride and 120.1-240.2g of urea until a transparent solution is formed, then drying in a vacuum drying oven at 60 ℃ for 36h to obtain a deep eutectic solvent choline chloride/urea, extracting in a constant-temperature water bath to obtain a crude extract of pueraria isoflavone, and then separating and purifying to obtain the pueraria isoflavone. The invention aims to provide a method for extracting pueraria isoflavone, which utilizes the characteristics that a deep eutectic solvent, namely choline chloride/urea, has the effect of dissolving and destroying plant cell walls and forms hydrogen bonds with flavonoid compounds to promote the dissolution of the flavonoid compounds, and simultaneously ethanol is added to improve the extraction efficiency of the pueraria isoflavone; weighing the powder of the isoflavone of the kudzuvine root, wherein the final extraction rate is 21.48-27.23mg/g (calculated according to the raw material of the kudzuvine root), and determining the hydroxyl radical clearance rate, DPPH clearance rate and ABTS clearance rate of the isoflavone of the kudzuvine root, and the result shows that the isoflavone of the kudzuvine root is consistent with VC activity.
Description
Technical Field
The invention relates to the technical field, in particular to a method for extracting pueraria isoflavone, belonging to the field of food processing.
Background
The kudzu root is the dry root of Pueraria lobata (Willd) which is a leguminous plant, belongs to a medicinal and edible plant, contains rich functional isoflavone compounds, has the effects of improving blood circulation, reducing blood pressure, reducing blood sugar, preventing cancer, enhancing body immunity and the like, and has wide prospects in the fields of food industry, daily use, health care, medicine and the like, so that the research on the extraction process of the kudzu root isoflavone has important significance.
The most common extraction method at present is an organic solvent extraction method, which is simple and convenient to operate and has low requirements on instruments and equipment, but has the defects of long extraction time, low efficiency and high organic solvent consumption.
Summary of the invention
In the method, the deep eutectic solvent choline chloride/urea is utilized to have the characteristics of dissolving and destroying the plant cell walls, forming hydrogen bonds with flavonoid compounds to promote the dissolution of the flavonoid compounds and the like, and simultaneously, ethanol is added to improve the extraction efficiency of the pueraria isoflavone.
The invention can achieve the purpose by the following scheme, including the following steps, but not limited to:
(1) preparation of choline chloride/urea: 139.6g of choline chloride and 120.1-240.2g of urea are mixed (i.e. the molar ratio is controlled to be 1: 2-4), magnetically stirred in a water bath kettle at 80 ℃ for 2-3h until a transparent solution is formed, then dried in a vacuum drying oven at 60 ℃ for 36h to obtain the deep eutectic solvent choline chloride/urea, and the deep eutectic solvent choline chloride/urea is preserved in a dryer for standby.
(2) Preparing an extraction solution with distilled water, wherein the extraction solution comprises 20-40% choline chloride/urea and 20-40% ethanol, adding into 60-mesh radix Puerariae powder, stirring, extracting in a constant temperature water bath to obtain a crude radix Puerariae isoflavone extract, and separating and purifying to obtain radix Puerariae isoflavone.
(3) The material-liquid ratio of the radix puerariae powder to the extracting solution is 1: 10-25.
(4) The extraction process is carried out at 40-60 deg.C for 30-60 min.
(5) After extraction, removing pueraria residue by high-speed centrifugation to obtain a crude extract, wherein the content of pueraria isoflavone in the crude extract is 1.19-1.54mg/mL, and the extraction rate is 3.58-4.62%.
(6) The separation and purification uses AB-8 type macroporous resin as a filler, and the purification is realized through the separation and purification of a chromatographic column. The separation and purification process comprises the following steps: loading on column by wet method, loading the crude extract, eluting with ethanol solution, collecting eluate, concentrating the eluate by rotary evaporation, adjusting pH, cooling to separate out precipitate, filtering to obtain crude radix Puerariae isoflavone precipitate, dissolving the precipitate with lime water, adjusting pH, cooling to separate out precipitate, filtering, and freeze drying to obtain refined radix Puerariae isoflavone.
(7) The AB-8 resin was loaded into a chromatography column (15mm by 500mm) with a column volume of 50mL by wet packing.
(8) The loading volume of the crude extract is 15 mL.
(9) The eluate was collected at 70% ethanol concentration.
(10) The pH of the concentrated eluate and the redissolved solution was adjusted to pH 3.0 with brine.
(11) Weighing the pueraria isoflavone powder, wherein the extraction rate is 21.48-27.23mg/g (calculated according to the pueraria raw material).
(12) And (3) determining the hydroxyl free radical clearance rate, DPPH clearance rate and ABTS clearance rate of the pueraria isoflavone.
Has the advantages that:
the method is characterized in that a deep eutectic solvent with low volatility, non-flammability and explosiveness, good thermal stability, adjustable viscosity, density and conductivity and other excellent characteristics is adopted to replace the traditional solvent, the deep eutectic solvent choline chloride/urea has the dissolution and destruction effects on plant cell walls, and forms hydrogen bonds with flavonoid compounds to promote the dissolution of the flavonoid compounds, and the high-quality flavonoid is prepared by a recrystallization method of alkali extraction and acid precipitation; weighing the powder of the isoflavone of the kudzuvine root, wherein the final extraction rate is 21.48-27.23mg/g (calculated according to the raw material of the kudzuvine root), and determining the hydroxyl radical clearance rate, DPPH clearance rate and ABTS clearance rate of the isoflavone of the kudzuvine root, and the result shows that the isoflavone of the kudzuvine root is consistent with VC activity.
The first embodiment is as follows:
(1) and (3) preparing a deep eutectic solution. Drying choline chloride and urea with the purity of 99% in a vacuum drying oven at the temperature of 60 ℃ for 24 hours, and then carrying out vacuum drying according to the weight ratio of 1:2, 1: 3,1: 4, filling the mixture into a 50mL round-bottom flask, plugging a stopper, and stirring and heating the mixture for about 2 to 3 hours in a water bath kettle at the temperature of 100 ℃ and at the rotating speed of 300rpm until a uniform and transparent solution is formed. Cooling to room temperature, drying in a vacuum drying oven at 60 deg.C for 36h, transferring to a dryer, and sealing for storage. Before use, choline chloride/urea, ethanol and distilled water are mixed according to a certain proportion to obtain an extracting solution with the concentration of the choline chloride/urea being 20 percent and the concentration of the ethanol being 40 percent.
(2) And (4) pretreating the kudzuvine root. Drying the purchased radix Puerariae in the sun, drying for 12h, slicing, grinding with a traditional Chinese medicine grinder, sieving with a 60-mesh sieve, bagging the fine powder in a room temperature dryer, and storing.
(3) Extracting radix Puerariae isoflavone. Weighing fine powder according to a material-liquid ratio of 1 g: 20mL of the extract was added and stirred well. Then extracting for 1h in a water bath at the constant temperature of 60 ℃. Centrifuging the mixture at 12000g for 10min to obtain radix Puerariae isoflavone extractive solution. Then using a rotary evaporator to evaporate and remove ethanol in the pueraria isoflavone extracting solution under the conditions of vacuum and heating temperature of 50 ℃, and then adding distilled water to adjust the volume after concentration to the original volume, thus obtaining the crude extracting solution.
(4) Separating and purifying rutin. 15mL of the crude extract was slowly added to the column, followed by elution of other impurities with 600mL of deionized water, followed by 70% ethanol solution, and collection of the ethanol eluate. The eluate was then concentrated to 10mL using a rotary evaporator and the pH was adjusted to 3.0 with dilute hydrochloric acid. Placing the concentrated solution in 4 deg.C environment, standing for 24 hr to separate out radix Puerariae isoflavone crystal, filtering, and washing with cold water to obtain crude radix Puerariae isoflavone. Dissolving crude radix Puerariae isoflavone in lime water under heating, filtering with 0.22um membrane, adjusting pH of the filtrate to 3.0 with saline water, cooling, standing, recrystallizing to precipitate, filtering, washing with cold water, and lyophilizing to obtain refined radix Puerariae isoflavone.
| Molar ratio of | 1：2 | 1：3 | 1：4 |
| Content of radix Puerariae isoflavone (mg/mL) in the crude extractive solution | 1.27 | 1.35 | 1.19 |
| Extraction ratio (%) of isoflavone in the crude extract | 3.82 | 4.06 | 3.58 |
| Extraction rate (mg/g) of refined rutin | 22.95 | 24.27 | 21.48 |
Note that: the ratio of the content of the pueraria isoflavone in the crude extract to the extraction rate of the isoflavone in the crude extract is fixed. Example two:
(1) and (3) preparing a deep eutectic solution. After choline chloride and urea with the purity of 99% are dried in a vacuum drying oven at 60 ℃ for 24 hours, the mixture is mixed with ethylene glycol according to the molar ratio of 1:2, the mixture is placed in a 50mL round-bottom flask, a plug is plugged, and the mixture is stirred and heated in a water bath kettle at 100 ℃ at the rotating speed of 300rpm for about 2-3 hours until a uniform and transparent solution is formed. Cooling to room temperature, drying in a vacuum drying oven at 60 deg.C for 36h, transferring to a dryer, and sealing for storage. Mixing choline chloride/urea, ethanol and distilled water according to a certain proportion before use to obtain an extracting solution: 1) choline chloride/urea concentration of 30% and ethanol concentration of 30%; 2) the choline chloride/urea concentration was 40% and the ethanol concentration was 20%.
(2) And (4) pretreating the kudzuvine root. Drying the purchased radix Puerariae in the sun, drying for 12h, slicing, grinding with a traditional Chinese medicine grinder, sieving with a 60-mesh sieve, bagging the fine powder in a room temperature dryer, and storing.
(3) Extracting radix Puerariae isoflavone. Weighing fine powder according to a material-liquid ratio of 1 g: 20mL of the extract was added and stirred well. Then extracting for 1h in a water bath at the constant temperature of 60 ℃. Centrifuging the mixture at 12000g for 10min to obtain radix Puerariae isoflavone extractive solution. Then using a rotary evaporator to evaporate and remove ethanol in the pueraria isoflavone extracting solution under the conditions of vacuum and heating temperature of 50 ℃, and then adding distilled water to adjust the volume after concentration to the original volume, thus obtaining the crude extracting solution.
(4) Separating and purifying rutin. 15mL of the crude extract was slowly added to the column, followed by elution of other impurities with 600mL of deionized water, followed by 70% ethanol solution, and collection of the ethanol eluate. The eluate was then concentrated to 10mL using a rotary evaporator and the pH was adjusted to 3.0 with dilute hydrochloric acid. Placing the concentrated solution in 4 deg.C environment, standing for 24 hr to separate out radix Puerariae isoflavone crystal, filtering, and washing with cold water to obtain crude radix Puerariae isoflavone. Dissolving crude radix Puerariae isoflavone in lime water under heating, filtering with 0.22um membrane, adjusting pH of the filtrate to 3.0 with saline water, cooling, standing, recrystallizing to precipitate, filtering, washing with cold water, and lyophilizing to obtain refined radix Puerariae isoflavone.
| DES concentration%) | 10 | 20 | 30 | 40 |
| Content of radix Puerariae isoflavone (mg/mL) in the crude extractive solution | 1.21 | 1.35 | 1.31 | 1.21 |
| Extraction ratio (%) of isoflavone in the crude extract | 3.64 | 4.06 | 3.94 | 3.62 |
| Extraction rate (mg/g) of refined rutin | 21.85 | 24.47 | 23.24 | 21.72 |
Note that: the ratio of the content of the pueraria isoflavone in the crude extract to the extraction rate of the isoflavone in the crude extract is fixed. Example three:
(1) and (3) preparing a deep eutectic solution. After choline chloride and urea with the purity of 99% are dried in a vacuum drying oven at 60 ℃ for 24 hours, the mixture is mixed with ethylene glycol according to the molar ratio of 1:2, the mixture is placed in a 50mL round-bottom flask, a plug is plugged, and the mixture is stirred and heated in a water bath kettle at 100 ℃ at the rotating speed of 300rpm for about 2-3 hours until a uniform and transparent solution is formed. Cooling to room temperature, drying in a vacuum drying oven at 60 deg.C for 36h, transferring to a dryer, and sealing for storage. The choline chloride/urea, ethanol and distilled water are mixed according to a certain proportion before use to obtain the extract with the concentration of the choline chloride/urea being 30 percent and the concentration of the ethanol being 30 percent.
(2) And (4) pretreating the kudzuvine root. Drying the purchased radix Puerariae in the sun, drying for 12h, slicing, grinding with a traditional Chinese medicine grinder, sieving with a 60-mesh sieve, bagging the fine powder in a room temperature dryer, and storing.
(3) Extracting radix Puerariae isoflavone. Weighing fine powder according to a material-liquid ratio of 1 g: 10, 20 and 25mL of the extract is added and stirred uniformly. Then extracting for 1h in a water bath at the constant temperature of 60 ℃. Centrifuging the mixture at 12000g for 10min to obtain radix Puerariae isoflavone extractive solution. Then using a rotary evaporator to evaporate and remove ethanol in the pueraria isoflavone extracting solution under the conditions of vacuum and heating temperature of 50 ℃, and then adding distilled water to adjust the volume after concentration to the original volume, thus obtaining the crude extracting solution.
(4) Separating and purifying rutin. 15mL of the crude extract was slowly added to the column, followed by elution of other impurities with 600mL of deionized water, followed by 70% ethanol solution, and collection of the ethanol eluate. The eluate was then concentrated to 10mL using a rotary evaporator and the pH was adjusted to 3.0 with dilute hydrochloric acid. Placing the concentrated solution in 4 deg.C environment, standing for 24 hr to separate out radix Puerariae isoflavone crystal, filtering, and washing with cold water to obtain crude radix Puerariae isoflavone. Dissolving crude radix Puerariae isoflavone in lime water under heating, filtering with 0.22um membrane, adjusting pH of the filtrate to 3.0 with saline water, cooling, standing, recrystallizing to precipitate, filtering, washing with cold water, and lyophilizing to obtain refined radix Puerariae isoflavone.
| Ratio of material to liquid | 10 | 20 | 25 |
| Content of radix Puerariae isoflavone (mg/mL) in the crude extractive solution | 1.22 | 1.35 | 1.44 |
| Extraction ratio (%) of isoflavone in the crude extract | 3.67 | 4.06 | 4.32 |
| Extraction rate (mg/g) of refined rutin | 22.01 | 24.47 | 25.81 |
Example four:
(1) and (3) preparing a deep eutectic solution. After choline chloride and urea with the purity of 99% are dried in a vacuum drying oven at 60 ℃ for 24 hours, the mixture is mixed with ethylene glycol according to the molar ratio of 1:2, the mixture is placed in a 50mL round-bottom flask, a plug is plugged, and the mixture is stirred and heated in a water bath kettle at 100 ℃ at the rotating speed of 300rpm for about 2-3 hours until a uniform and transparent solution is formed. Cooling to room temperature, drying in a vacuum drying oven at 60 deg.C for 36h, transferring to a dryer, and sealing for storage. The choline chloride/urea, ethanol and distilled water are mixed according to a certain proportion before use to obtain the extract with the concentration of the choline chloride/urea being 30 percent and the concentration of the ethanol being 30 percent.
(2) And (4) pretreating the kudzuvine root. Drying the purchased radix Puerariae in the sun, drying for 12h, slicing, grinding with a traditional Chinese medicine grinder, sieving with a 60-mesh sieve, bagging the fine powder in a room temperature dryer, and storing.
(3) Extracting radix Puerariae isoflavone. Weighing fine powder according to a material-liquid ratio of 1 g: 10, 20 and 25mL of the extract is added and stirred uniformly. Then extracting in 40, 50, 60 deg.C water bath for 1 h. Centrifuging the mixture at 12000g for 10min to obtain radix Puerariae isoflavone extractive solution. Then using a rotary evaporator to evaporate and remove ethanol in the pueraria isoflavone extracting solution under the conditions of vacuum and heating temperature of 50 ℃, and then adding distilled water to adjust the volume after concentration to the original volume, thus obtaining the crude extracting solution.
(4) Separating and purifying rutin. 15mL of the crude extract was slowly added to the column, followed by elution of other impurities with 600mL of deionized water, followed by 70% ethanol solution, and collection of the ethanol eluate. The eluate was then concentrated to 10mL using a rotary evaporator and the pH was adjusted to 3.0 with dilute hydrochloric acid. Placing the concentrated solution in 4 deg.C environment, standing for 24 hr to separate out radix Puerariae isoflavone crystal, filtering, and washing with cold water to obtain crude radix Puerariae isoflavone. Dissolving crude radix Puerariae isoflavone in lime water under heating, filtering with 0.22um membrane, adjusting pH of the filtrate to 3.0 with saline water, cooling, standing, recrystallizing to precipitate, filtering, washing with cold water, and lyophilizing to obtain refined radix Puerariae isoflavone.
| Temperature (. degree.C.) | 60 | 70 | 80 |
| Content of radix Puerariae isoflavone (mg/mL) in the crude extractive solution | 1.51 | 1.54 | 1.36 |
| Extraction ratio (%) of isoflavone in the crude extract | 4.53 | 4.62 | 4.09 |
| Extraction rate (mg/g) of refined rutin | 26.72 | 27.22 | 23.72 |
Note that: the ratio of the content of the pueraria isoflavone in the crude extract to the extraction rate of the isoflavone in the crude extract is fixed. Example five:
(1) and (3) preparing a deep eutectic solution. After choline chloride and urea with the purity of 99% are dried in a vacuum drying oven at 60 ℃ for 24 hours, the mixture is mixed with ethylene glycol according to the molar ratio of 1:2, the mixture is placed in a 50mL round-bottom flask, a plug is plugged, and the mixture is stirred and heated in a water bath kettle at 100 ℃ at the rotating speed of 300rpm for about 2-3 hours until a uniform and transparent solution is formed. Cooling to room temperature, drying in a vacuum drying oven at 60 deg.C for 36h, transferring to a dryer, and sealing for storage. The choline chloride/urea, ethanol and distilled water are mixed according to a certain proportion before use to obtain the extract with the concentration of the choline chloride/urea being 30 percent and the concentration of the ethanol being 30 percent.
(2) And (4) pretreating the kudzuvine root. Drying the purchased radix Puerariae in the sun, drying for 12h, slicing, grinding with a traditional Chinese medicine grinder, sieving with a 60-mesh sieve, bagging the fine powder in a room temperature dryer, and storing.
(3) Extracting radix Puerariae isoflavone. Weighing fine powder according to a material-liquid ratio of 1 g: 10, 20 and 25mL of the extract is added and stirred uniformly. Then extracting in a water bath at 50 deg.C for 30min and 60 min. Centrifuging the mixture at 12000g for 10min to obtain radix Puerariae isoflavone extractive solution. Then using a rotary evaporator to evaporate and remove ethanol in the pueraria isoflavone extracting solution under the conditions of vacuum and heating temperature of 50 ℃, and then adding distilled water to adjust the volume after concentration to the original volume, thus obtaining the crude extracting solution.
(4) Separating and purifying rutin. 15mL of the crude extract was slowly added to the column, followed by elution of other impurities with 600mL of deionized water, followed by 70% ethanol solution, and collection of the ethanol eluate. The eluate was then concentrated to 10mL using a rotary evaporator and the pH was adjusted to 3.0 with dilute hydrochloric acid. Placing the concentrated solution in 4 deg.C environment, standing for 24 hr to separate out radix Puerariae isoflavone crystal, filtering, and washing with cold water to obtain crude radix Puerariae isoflavone. Dissolving crude radix Puerariae isoflavone in lime water under heating, filtering with 0.22um membrane, adjusting pH of the filtrate to 3.0 with saline water, cooling, standing, recrystallizing to precipitate, filtering, washing with cold water, and lyophilizing to obtain refined radix Puerariae isoflavone.
| Extraction time (min) | 30 | 60 |
| Content of radix Puerariae isoflavone (mg/mL) in the crude extractive solution | 1.40 | 1.54 |
| Extraction ratio (%) of isoflavone in the crude extract | 4.20 | 4.62 |
| Extraction rate (mg/g) of refined rutin | 24.54 | 27.23 |
Note that: the ratio of the content of the pueraria isoflavone in the crude extract to the extraction rate of the isoflavone in the crude extract is fixed. And (3) determining the hydroxyl free radical clearance rate, DPPH clearance rate and ABTS clearance rate of the pueraria isoflavone.
| Hydroxyl radical scavenging ratio (%) | DPPH clearance (%) | ABTS clearance (%) | |
| Refining rutin | 25.3 | 60.1 | 62.7 |
The hydroxyl radical clearance (%) measurement method was modified as follows: preparing 8mmol/L FeSO4Solution, 20mmol/L H2O2And 3mmol/L salicylic acid. Taking 0.335mL of fermentation broth, 0.1mL of FeSO4, 0.335mL of salicylic acid and 0.08mL of H2O2Mixing, and water bathing at 37 deg.C for 30 min. Cooling, supplementing to volume of 1mL, centrifuging at 8000rpm for 10min, measuring absorbance of supernatant at 510nm wavelength, and respectively replacing sample and H with distilled water2O2The solution served as blank and control. Ascorbic acid (Vc) was used as a positive control group, and the hydroxyl radical clearance rate of 0.5mmol/L Vc was 27.3%.
The DPPH clearance (%) determination method was modified as follows: adding 2mL of newly prepared 0.2mmol/L DPPH solution into 2mL of rutin solution with different concentrations, uniformly mixing, carrying out dark water bath at 37 ℃ for 30min, and then measuring the light absorption value at the wavelength of 517 nm. The sample was replaced with absolute ethanol as a blank control. Ascorbic acid (Vc) was used as a positive control group. The DPPH radical clearance rate of 0.5mmol/L Vc is 61.8%.
The ABTS clearance (%) determination method was modified as follows: 1mL of ABTS solution (7.4mmol/L) and an equal volume of 2.6mmol/L K2S2O4Mixing, and standing in dark for 16h to obtain ABTS + stock solution; before use, ABTS + is diluted properly by absolute ethyl alcohol solution, and the absorbance range of the mixed solution at 734nm is 0.7 +/-0.02, so that ABTS + working solution is obtained; evenly mixing 1.980mL of ABTS + with 20 mu L of sample, reacting for 10min at 30 ℃, and measuring the light absorption value at 734 nm; the distilled water and the ethanol are uniformly mixed to form the absorbentThe light value is a sample control; replacing the light absorption value of the sample with distilled water as a blank control; ascorbic acid (Vc) was used as a positive control group. ABTS clearance of 0.5mmol/L Vc is 59.7%.
Claims (6)
1. A method for extracting pueraria isoflavone is characterized by comprising the following steps: the method comprises the following steps:
(1) preparation of choline chloride/urea: 139.6g of choline chloride and 120.1-240.2g of urea are mixed (i.e. the molar ratio is controlled to be 1: 2-4), magnetically stirred in a water bath kettle at 80 ℃ for 2-3h until a transparent solution is formed, then dried in a vacuum drying oven at 60 ℃ for 36h to obtain the deep eutectic solvent choline chloride/urea, and the deep eutectic solvent choline chloride/urea is preserved in a dryer for standby.
(2) Preparing an extraction solution with distilled water, wherein the extraction solution comprises 20-40% choline chloride/urea and 20-40% ethanol, adding into 60-mesh radix Puerariae powder, stirring, extracting in a constant temperature water bath to obtain a crude radix Puerariae isoflavone extract, and separating and purifying to obtain radix Puerariae isoflavone.
2. The method for extracting pueraria isoflavone according to claim 1, wherein the steps of: 139.6g of choline chloride was mixed with 120.1 to 240.2g of urea (i.e., the molar ratio was controlled at 1:2 to 4).
3. The method for extracting pueraria isoflavone according to claim 1, wherein the steps of: preparing an extraction solution with distilled water, comprising 20-40% choline chloride/urea and 20-40% ethanol.
4. The method for extracting pueraria isoflavone according to claim 1, wherein the steps of: the material-liquid ratio of the radix puerariae powder to the extracting solution is 1: 10-25.
5. The method for extracting pueraria isoflavone according to claim 1, wherein the steps of: the extraction process is carried out at 40-60 deg.C for 30-60 min. Centrifuging the mixture at 12000g for 10min to obtain radix Puerariae isoflavone extractive solution. Then evaporating ethanol in the radix Puerariae isoflavone extractive solution with rotary evaporator under vacuum and heating temperature of 50 deg.C, and adding distilled water to adjust the volume of the concentrated solution to original volume to obtain crude extractive solution.
6. The method for extracting pueraria isoflavone according to claim 1, wherein the steps of: 15mL of the crude extract was slowly added to the column, followed by elution of other impurities with 600mL of deionized water, followed by 70% ethanol solution, and collection of the ethanol eluate. The eluate was then concentrated to 10mL using a rotary evaporator and the pH was adjusted to 3.0 with dilute hydrochloric acid. Placing the concentrated solution in 4 deg.C environment, standing for 24 hr to separate out radix Puerariae isoflavone crystal, filtering, and washing with cold water to obtain crude radix Puerariae isoflavone. Dissolving crude radix Puerariae isoflavone in lime water under heating, filtering with 0.22um membrane, adjusting pH of the filtrate to 3.0 with saline water, cooling, standing, recrystallizing to precipitate, filtering, washing with cold water, and lyophilizing to obtain refined radix Puerariae isoflavone.
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