CN118027236A - Method for flash-type efficient extraction of Cordyceps militaris polysaccharide with high hypoglycemic activity and application thereof - Google Patents
Method for flash-type efficient extraction of Cordyceps militaris polysaccharide with high hypoglycemic activity and application thereof Download PDFInfo
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- CN118027236A CN118027236A CN202410158285.5A CN202410158285A CN118027236A CN 118027236 A CN118027236 A CN 118027236A CN 202410158285 A CN202410158285 A CN 202410158285A CN 118027236 A CN118027236 A CN 118027236A
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- polysaccharide
- cordyceps militaris
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Links
- 150000004676 glycans Chemical class 0.000 title claims abstract description 146
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 144
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 144
- 241001264174 Cordyceps militaris Species 0.000 title claims abstract description 130
- 238000000034 method Methods 0.000 title claims abstract description 61
- 238000000605 extraction Methods 0.000 title claims abstract description 42
- 230000002218 hypoglycaemic effect Effects 0.000 title claims abstract description 16
- 102100024295 Maltase-glucoamylase Human genes 0.000 claims abstract description 41
- 108010028144 alpha-Glucosidases Proteins 0.000 claims abstract description 41
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- 239000000843 powder Substances 0.000 claims description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 19
- 239000006228 supernatant Substances 0.000 claims description 16
- 238000004108 freeze drying Methods 0.000 claims description 13
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 10
- MZVQCMJNVPIDEA-UHFFFAOYSA-N [CH2]CN(CC)CC Chemical group [CH2]CN(CC)CC MZVQCMJNVPIDEA-UHFFFAOYSA-N 0.000 claims description 10
- 239000003153 chemical reaction reagent Substances 0.000 claims description 10
- 239000008103 glucose Substances 0.000 claims description 10
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 9
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- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 6
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 6
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 6
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- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 claims description 5
- 238000002835 absorbance Methods 0.000 claims description 4
- 235000019441 ethanol Nutrition 0.000 claims description 4
- 229940097043 glucuronic acid Drugs 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
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- AEMOLEFTQBMNLQ-YMDCURPLSA-N D-galactopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-YMDCURPLSA-N 0.000 claims description 3
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 claims description 3
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 claims description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 229930182830 galactose Natural products 0.000 claims description 3
- 238000010298 pulverizing process Methods 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid group Chemical group S(O)(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 2
- 230000010030 glucose lowering effect Effects 0.000 claims 1
- YOBAEOGBNPPUQV-UHFFFAOYSA-N iron;trihydrate Chemical compound O.O.O.[Fe].[Fe] YOBAEOGBNPPUQV-UHFFFAOYSA-N 0.000 claims 1
- 238000011068 loading method Methods 0.000 claims 1
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- XUFXOAAUWZOOIT-SXARVLRPSA-N (2R,3R,4R,5S,6R)-5-[[(2R,3R,4R,5S,6R)-5-[[(2R,3R,4S,5S,6R)-3,4-dihydroxy-6-methyl-5-[[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl)-1-cyclohex-2-enyl]amino]-2-oxanyl]oxy]-3,4-dihydroxy-6-(hydroxymethyl)-2-oxanyl]oxy]-6-(hydroxymethyl)oxane-2,3,4-triol Chemical compound O([C@H]1O[C@H](CO)[C@H]([C@@H]([C@H]1O)O)O[C@H]1O[C@@H]([C@H]([C@H](O)[C@H]1O)N[C@@H]1[C@@H]([C@@H](O)[C@H](O)C(CO)=C1)O)C)[C@@H]1[C@@H](CO)O[C@@H](O)[C@H](O)[C@H]1O XUFXOAAUWZOOIT-SXARVLRPSA-N 0.000 abstract description 5
- 229960002632 acarbose Drugs 0.000 abstract description 5
- XUFXOAAUWZOOIT-UHFFFAOYSA-N acarviostatin I01 Natural products OC1C(O)C(NC2C(C(O)C(O)C(CO)=C2)O)C(C)OC1OC(C(C1O)O)C(CO)OC1OC1C(CO)OC(O)C(O)C1O XUFXOAAUWZOOIT-UHFFFAOYSA-N 0.000 abstract description 5
- 238000005516 engineering process Methods 0.000 abstract description 5
- 238000000874 microwave-assisted extraction Methods 0.000 abstract description 5
- 229930014626 natural product Natural products 0.000 abstract description 2
- 241001248610 Ophiocordyceps sinensis Species 0.000 abstract 1
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- 239000000243 solution Substances 0.000 description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 7
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- 230000002401 inhibitory effect Effects 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
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- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
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- 229940077274 Alpha glucosidase inhibitor Drugs 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
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- 239000003814 drug Substances 0.000 description 2
- 229940126904 hypoglycaemic agent Drugs 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 150000004804 polysaccharides Polymers 0.000 description 2
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
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- 238000012360 testing method Methods 0.000 description 2
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 2
- CHHHXKFHOYLYRE-UHFFFAOYSA-M 2,4-Hexadienoic acid, potassium salt (1:1), (2E,4E)- Chemical compound [K+].CC=CC=CC([O-])=O CHHHXKFHOYLYRE-UHFFFAOYSA-M 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 101500000959 Bacillus anthracis Protective antigen PA-20 Proteins 0.000 description 1
- 229940123208 Biguanide Drugs 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- KQLDDLUWUFBQHP-UHFFFAOYSA-N Cordycepin Natural products C1=NC=2C(N)=NC=NC=2N1C1OCC(CO)C1O KQLDDLUWUFBQHP-UHFFFAOYSA-N 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 description 1
- 239000004386 Erythritol Substances 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
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- 229920003081 Povidone K 30 Polymers 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
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- 230000009286 beneficial effect Effects 0.000 description 1
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- UBXYXCRCOKCZIT-UHFFFAOYSA-N biphenyl-3-ol Chemical group OC1=CC=CC(C=2C=CC=CC=2)=C1 UBXYXCRCOKCZIT-UHFFFAOYSA-N 0.000 description 1
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- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- OFEZSBMBBKLLBJ-BAJZRUMYSA-N cordycepin Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)C[C@H]1O OFEZSBMBBKLLBJ-BAJZRUMYSA-N 0.000 description 1
- OFEZSBMBBKLLBJ-UHFFFAOYSA-N cordycepine Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(CO)CC1O OFEZSBMBBKLLBJ-UHFFFAOYSA-N 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
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- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 description 1
- 229940009714 erythritol Drugs 0.000 description 1
- 235000019414 erythritol Nutrition 0.000 description 1
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- 238000002474 experimental method Methods 0.000 description 1
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- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 230000004190 glucose uptake Effects 0.000 description 1
- 229940127208 glucose-lowering drug Drugs 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
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- 239000012528 membrane Substances 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000013365 molecular weight analysis method Methods 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 229940069338 potassium sorbate Drugs 0.000 description 1
- 235000010241 potassium sorbate Nutrition 0.000 description 1
- 239000004302 potassium sorbate Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 150000003214 pyranose derivatives Chemical group 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000004544 sputter deposition Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L sulfate group Chemical group S(=O)(=O)([O-])[O-] QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- -1 sulfuric acid radical Chemical class 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 238000004879 turbidimetry Methods 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Diabetes (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Polymers & Plastics (AREA)
- Materials Engineering (AREA)
- Animal Behavior & Ethology (AREA)
- Biochemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Obesity (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Hematology (AREA)
- Sustainable Development (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Endocrinology (AREA)
- Emergency Medicine (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention belongs to the field of natural product preparation and application, and in particular relates to a method for efficiently preparing cordyceps militaris polysaccharide with high hypoglycemic activity by flash extraction and application thereof. The flash extraction technology provided by the invention can be used for efficiently preparing the Cordyceps militaris polysaccharide, and has the advantages of high polysaccharide yield (18.3+/-1.82%), high inhibition rate (83.2+/-7.64%) to alpha-glucosidase, low energy consumption, extremely short extraction time (only 1 min), and remarkably superior to methods of hot water extraction, enzyme-assisted extraction, microwave-assisted extraction and the like. The purified polysaccharide of Cordyceps militaris extracted and purified by the technology has strong hypoglycemic activity, and the inhibition rate of alpha-glucosidase reaches 99.4+/-1.25% at the concentration of 5mg/mL, which is obviously higher than that of acarbose with the same concentration. The purified polysaccharide of the silkworm chrysalis cordyceps sinensis, which is extracted and purified by the technical scheme of the invention, can be used for preparing products with blood sugar reducing functions, such as oral liquid, capsules, tablets and the like.
Description
Technical Field
The invention belongs to the field of natural product preparation, and particularly relates to a method for efficiently preparing cordyceps militaris polysaccharide with high hypoglycemic activity by flash extraction and application thereof.
Background
Cordyceps militaris (Cordyceps militaris), abbreviated as Cordyceps militaris, belongs to Cordyceps fungus of Clavipitaceae, is native in China, is approved by national Ministry of health as a new resource food in 2009, and is a high-value dual-purpose fungus for medicine and food. Cordyceps militaris is rich in polysaccharide, cordycepin, oxalic acid and other bioactive substances. The polysaccharide is an important active ingredient of the silkworm cordyceps, has wide pharmacological effects of enhancing organism immunity, resisting tumors, reducing blood sugar and the like (Food Bioscience,2022, 45:101503), and has high application value in the fields of biological medicines, foods and the like.
The Cordyceps militaris has the advantages of low polysaccharide content, low extraction rate and high extraction difficulty, and is a bottleneck problem for restricting industrial development. At present, extraction methods of Cordyceps militaris polysaccharide mainly comprise hot water extraction method (food science, 2017,38 (14): 91-96; CN106749742A; CN11649624A), ultrasonic auxiliary method (food research and development, 2023,44 (11): 159-165; CN107501428A), microwave auxiliary method (Jiangsu agricultural journal, 2009,25 (5): 1143-1150) and the like. However, these methods have disadvantages of long time, high energy consumption, low extraction rate, and the like.
The flash extraction Technology utilizes a high-speed shearing head to form high-strength shearing force, has extremely high wall breaking efficiency, utilizes strong stirring to quickly permeate a solvent, quickly diffuses active ingredients, and has the advantages of short extraction time, low temperature and no radiation risk (Trends in Food Science & Technology,2021, 112:581-591). At present, the preparation of Cordyceps militaris polysaccharide by using flash extraction technology has not been reported yet.
Metabolic diseases, including diabetes, remain one of the important factors that jeopardize public health. The Chinese guidelines for prevention and treatment of type 2 diabetes report that: the incidence rate of type 2 diabetes in China is 10.4%, and in addition, the incidence rate of the diabetes is higher than 20% above 60 years old, and the incidence rate of undiagnosed patients accounts for 63% of the total number. Although chemical hypoglycemic agents such as sulfonylureas and biguanides play an important role in regulating blood sugar and reducing complications, the problems of large adverse reaction, resistance and the like existing in long-term administration cannot be solved all the time. Compared with chemical hypoglycemic agents, natural hypoglycemic products are gradually accepted by people due to the characteristics of high safety, suitability for long-term administration and the like. Inhibition of alpha-glucosidase and thus reduction of glucose uptake is the most important mechanism for polysaccharide glucose reduction, and the oligosaccharide glucose-lowering drugs acarbose and analogues thereof that have been marketed are classical alpha-glucosidase inhibitors. Zhu Zhenyuan et al (modern food technology, 2014,30 (12): 55-60) found that Cordyceps militaris polysaccharide prepared by hot water extraction method can inhibit activity of alpha-glucosidase, but did not examine whether different extraction parameters and methods have influence on activity.
The processing voltage is an important technical parameter for flash extraction, and people often pay attention to the influence of the voltage on polysaccharide yield (food industry, 2020,41 (11): 42-45; food research and development, 2023,44 (9): 164-170), and neglect the influence of the voltage on polysaccharide biological activity. The invention provides a method for efficiently preparing cordyceps militaris polysaccharide with high hypoglycemic activity by flash extraction and application thereof by exploring the influence of different extraction voltages on the cordyceps militaris polysaccharide yield and hypoglycemic activity.
Disclosure of Invention
The invention aims to provide a method for efficiently preparing cordyceps militaris polysaccharide with high hypoglycemic activity by utilizing flash extraction. Compared with the traditional hot water extraction method, enzyme-assisted extraction method, microwave-assisted extraction method and ultrasonic-assisted extraction method, the cordyceps militaris polysaccharide prepared by the flash extraction technology provided by the invention has the advantages of high yield, low energy consumption and short extraction time; the purified Cordyceps militaris polysaccharide prepared by the technology has extremely high alpha-glucosidase inhibitory activity and shows excellent blood glucose reducing function.
According to a first aspect of the invention, the invention provides a method for efficiently preparing Cordyceps militaris polysaccharide by flash extraction, which takes Cordyceps militaris degreasing powder as a raw material, and is soaked in water, then placed in a flash extractor and treated under a certain voltage; centrifuging, concentrating supernatant, precipitating with ethanol, and lyophilizing to obtain Cordyceps militaris polysaccharide.
Preferably, the degreasing powder of the silkworm chrysalis cordyceps is degreased by petroleum ether, and comprises the following specific steps of: drying fresh Cordyceps militaris at 60deg.C to constant weight, pulverizing, and sieving with 40 mesh sieve to obtain Cordyceps militaris powder; adding petroleum ether, degreasing for 12h under dark condition, and vacuum filtering to obtain the defatted Cordyceps militaris powder.
Preferably, the certain treatment voltage is 70 to 150V, more preferably 110V; more preferably, a flash extractor model JHBE-20A from Siam Telecan Biotechnology Inc. is used, and the process voltage is set to 110V.
Preferably, the method for efficiently preparing the cordyceps militaris polysaccharide by utilizing flash extraction comprises the following specific steps of: soaking defatted Cordyceps militaris powder in water at a feed liquid ratio of 1:40g/mL for 2 hr, placing in a flash extractor (JHBE-20A, sieve biological technology Co., ltd.) and treating at 110V for 1min; centrifuging after the completion of the process, collecting supernatant, concentrating the supernatant to 1/4 of the original volume to obtain concentrated solution, adding 3 times of absolute ethyl alcohol of the volume of the concentrated solution, precipitating at 4 ℃ for 12 hours, and freeze-drying the precipitate to obtain Cordyceps militaris polysaccharide; under the condition, the polysaccharide yield of the Cordyceps militaris is 18.3+/-1.82%, the inhibition rate of the Cordyceps militaris to the alpha-glucosidase is 83.2+/-7.64% at 5mg/mL, and the polysaccharide is remarkably superior to the conventional hot water extraction method (polysaccharide yield: 10.7+/-0.26%, the inhibition rate of the Cordyceps militaris to the alpha-glucosidase at 5 mg/mL: 31.5+/-4.81%), the enzyme auxiliary extraction method (polysaccharide yield: 10.7+/-0.93%, the inhibition rate of the Cordyceps militaris to the alpha-glucosidase at 5 mg/mL: 35.4+/-2.95%), the microwave auxiliary extraction method (polysaccharide yield: 14.4+/-2.03%, the inhibition rate of the Cordyceps militaris to the alpha-glucosidase at 5 mg/mL: 48.6+/-5.11%) and the ultrasonic auxiliary extraction method (polysaccharide yield: 13.6+/-1.53%, and the inhibition rate of the Cordyceps militaris to the alpha-glucosidase at 5 mg/mL: 50.7+/-4.69%).
The preparation method of the cordyceps militaris polysaccharide further comprises the steps of purifying the prepared cordyceps militaris polysaccharide, deproteinizing by using a Sevag reagent, dialyzing in distilled water after removing the residual Sevag reagent, obtaining deproteinized polysaccharide after freeze drying of dialysate, and purifying by using a Diethylaminoethyl (DEAE) cellulose chromatographic column to obtain purified cordyceps militaris polysaccharide; the specific method comprises the following steps: deproteinizing 3 times with Sevag reagent (chloroform: n-butanol=4:1, volume ratio); removing denatured protein and residual Sevag reagent by centrifugation, and dialyzing in distilled water for 3 times (dialysis bag with molecular weight cutoff of 7000Da, and changing distilled water once at intervals of 12 h); freeze drying the dialyzate to obtain deproteinized polysaccharide. 100mg of deproteinized polysaccharide was weighed and dissolved in 20mL of deionized water, and loaded onto a Diethylaminoethyl (DEAE) cellulose column (2.6 cm. Times.60 cm); slowly eluting with 0.5mol/L NaCl solution at a flow rate of 0.5mL/min and 2mL per tube; and (3) measuring the absorbance of each tube of eluent by adopting a phenol-sulfuric acid method, merging the eluents after the polysaccharide is completely eluted, concentrating, dialyzing for 3 times (the method is the same as above), and freeze-drying to obtain the purified polysaccharide of the Cordyceps militaris.
According to a second aspect of the invention, the invention provides a purified polysaccharide of Cordyceps militaris, wherein the total sugar content of the purified polysaccharide of Cordyceps militaris is 83.5+/-1.46%, the protein content is 7.26+/-2.05%, the uronic acid content is 4.16+/-0.68%, and the sulfate group content is 3.71+/-0.69%; the purified polysaccharide of the Cordyceps militaris comprises arabinose, galactose, glucose, xylose, galacturonic acid and glucuronic acid according to the molar ratio of 31.6:21.6:274:23.6:1.00:3.56; the number average molecular weight (Mn) of the purified polysaccharide of the Cordyceps militaris is 1.85kDa, and the weight average molecular weight (Mw) is 5.09kDa.
According to a third aspect of the invention, the invention provides the application of the purified polysaccharide of the Cordyceps militaris in preparing a hypoglycemic functional product; can be used for preparing oral liquid, capsule, tablet, etc.
The invention has the following beneficial effects:
① The flash extraction technology provided by the invention has the remarkable advantages of shortening the extraction time, reducing the energy consumption and protecting the polysaccharide structure, can greatly improve the yield and the hypoglycemic activity of the Cordyceps militaris polysaccharide, and is remarkably superior to the prior art such as a hot water extraction method.
② The purified polysaccharide of the Cordyceps militaris provided by the invention has extremely high inhibition rate on alpha-glucosidase, is obviously higher than acarbose with the same concentration at the concentration of 5mg/mL, has excellent blood glucose reducing function, and can be used for preparing products of blood glucose reducing related diseases.
Drawings
FIG. 1. Influence of different treatment voltages on Cordyceps militaris polysaccharide yield (statistical differences in different superscript lowercase letters represent);
FIG. 2. Effects of different treatment voltages on inhibition of α -glucosidase activity by Cordyceps militaris polysaccharide (statistical differences in different upper-case lower-case representations);
FIG. 3A monosaccharide composition chromatogram of purified polysaccharide of Cordyceps militaris (peak in standard: 1-fucose; 2-rhamnose; 3-arabinose; 4-galactose; 5-glucose; 6-xylose; 7-mannose; 8-fructose; 9-galacturonic acid; 10-glucuronic acid);
FIG. 4 shows a molecular weight distribution diagram of purified polysaccharide of Cordyceps militaris;
FIG. 5 is an infrared spectrogram of purified polysaccharide of Cordyceps militaris;
FIG. 6 is a scanning electron microscope image of purified polysaccharide of Cordyceps militaris;
FIG. 7 shows the inhibition of alpha-glucosidase by purified Cordyceps militaris polysaccharide (different uppercase letters represent statistically different groups; different uppercase letters represent statistically different groups at the same concentration).
Detailed Description
Cordyceps militaris is provided by su zhou Wu Jianggu and silkworm industry professional co-worker; the flash extractor is provided by the Siam Tekang Biotechnology Co., ltd, and the model number is JHBE-20A; the molecular weight is measured by a high performance liquid chromatograph of Waters, and the model is 1525; the monosaccharide composition is measured by an ion chromatograph with the model of ICS-5000; infrared spectra were determined by a PerkinElmer fourier transform infrared microscope system, model Spotlight200i; scanning Electron Microscope (SEM) is determined by HITACHI ultra-high resolution field emission scanning electron microscope, model number Regulus8100; all experiments were three-fold, data expressed as mean ± SD, statistical analysis of data using t-test or ANOVA analysis, P <0.05 considered statistical differences.
EXAMPLE 1 flash extraction and processing Voltage screening of Cordyceps militaris polysaccharide
Drying fresh Cordyceps militaris at 60deg.C to constant weight, pulverizing, and sieving with 40 mesh sieve to obtain Cordyceps militaris powder; adding petroleum ether, degreasing for 12h under dark condition, and vacuum filtering to obtain the defatted Cordyceps militaris powder. Soaking the defatted Cordyceps militaris powder in water for 2h at a feed liquid ratio of 1:40g/mL, and then placing the soaked Cordyceps militaris powder in a flash extractor for 1min under a certain voltage (70-150V). Centrifuging after the completion of the process, collecting supernatant, concentrating the supernatant to 1/4 of the original volume, adding 3 times of absolute ethyl alcohol, precipitating at 4deg.C for 12 hr, and freeze drying the precipitate to obtain Cordyceps militaris polysaccharide.
Polysaccharide yield (%) =m/m×100, wherein: m is the mass (g) of the polysaccharide of the silkworm chrysalis cordyceps after freeze drying; m is the mass (g) of the defatted Cordyceps militaris powder. The polysaccharide content is determined by adopting a phenol-sulfuric acid method, and a glucose standard equation is as follows: y=9.541x+0.0075, r 2 =0.9997.
The Cordyceps militaris polysaccharide extracted under different voltages is respectively dissolved in distilled water to prepare 5mg/mL solution for determining the inhibition activity of alpha-glucosidase. The specific measurement method is as follows:
Accurately transferring 100 μl of Cordyceps militaris polysaccharide solution, mixing with 300 μl of 0.24U/mL α -glucosidase solution, and adding 600 μl Phosphate Buffer (PBS) buffer (pH=6.8); after shaking uniformly, incubating for 15min at 37 ℃; adding 2.74mg/mL of 4-nitrophenyl-beta-D-galactopyranoside (PNPG) solution, fully mixing, and continuously incubating at 37 ℃ for 20min; finally, 4mL of sodium carbonate was added to terminate the reaction, and the absorbance was measured at 400 nm. The α -glucosidase inhibition rate was calculated as follows: α -glucosidase inhibition rate (%) = [1- (a Sample of -A Background )/(A Negative of -A Blank space ) ] ×100; table 1 shows an experimental reaction system for the inhibitory activity of alpha-glucosidase.
TABLE 1 alpha-glucosidase inhibitory activity experimental reaction system
1.1 Influence of different treatment voltages on Cordyceps militaris polysaccharide yield
The fixed feed-liquid ratio is 1:40g/mL, the flash extraction time is 1min, the influence of different treatment voltages (70-150V) on the polysaccharide yield of the Cordyceps militaris is examined, and the result is shown in figure 1.
As can be seen from FIG. 1, in the range of 70-110V, the polysaccharide yield increases with increasing voltage value, and when the voltage value reaches 110V, the polysaccharide yield is 18.3+ -1.82%, which is significantly higher than the polysaccharide yields at 70V and 90V (P < 0.01); when the voltage value reaches 130V, the polysaccharide yield is highest and is 20.4+/-2.35 percent, and although the polysaccharide yield is higher than 110V, the polysaccharide yield is not statistically different (P is more than 0.05); when the voltage value was increased to 150V, the polysaccharide yield was significantly reduced (P < 0.05).
1.2 Influence of different treatment voltages on inhibition of alpha-glucosidase activity by Cordyceps militaris polysaccharide
The fixed feed-liquid ratio is 1:40g/mL, the flash extraction time is 1min, the concentration of Cordyceps militaris polysaccharide is 5mg/mL, the influence of different treatment voltages (70-150V) on the activity of inhibiting alpha-glucosidase by Cordyceps militaris polysaccharide is examined, and the result is shown in figure 2.
As can be seen from fig. 2, in the range of 70-110V, the α -glucosidase inhibition rate increases significantly with increasing voltage value (P < 0.01), and when the voltage value reaches 110V, the inhibition rate reaches 83.2±7.64%, which is significantly higher than the inhibition rate at 70V and 90V (P < 0.01); when the voltage value increases to 130V, the inhibition rate starts to drop significantly (P < 0.01); when the voltage value continued to rise to 150V, the inhibition rate was reduced to 53.5±6.69%.
From the comprehensive aspects of extraction effect, alpha-glucosidase inhibition activity, energy consumption and the like, the treatment voltage is preferably about 110V, and the Cordyceps militaris polysaccharide yield is 18.3+/-1.82% and the inhibition rate to the alpha-glucosidase is 83.2+/-7.64%.
Comparative example
Based on the embodiment 1, in order to further prove that the flash extraction technology provided by the invention can efficiently prepare the silkworm chrysalis cordyceps polysaccharide with high hypoglycemic activity, the invention implements the following comparative test:
Scheme a: hot water extraction method
Soaking the defatted Cordyceps militaris powder in water at a feed liquid ratio of 1:40g/mL for 2h, and extracting at 80deg.C for 2h; cooling, centrifuging to obtain supernatant, concentrating the supernatant to 1/4 of the original volume, adding 3 times of absolute ethanol, precipitating at 4deg.C for 12 hr, and lyophilizing the precipitate to obtain Cordyceps militaris polysaccharide; the procedure of example 1 is followed to calculate the polysaccharide yield and to determine the inhibition activity of the alpha-glucosidase.
The results show that: the yield of the Cordyceps militaris polysaccharide obtained by adopting a hot water extraction method is 7.52+/-1.63 percent (which is 2.4 times lower than the technical scheme of the invention); the inhibition rate of the alpha-glucosidase is 31.5+/-4.81 percent (which is 2.6 times lower than the technical proposal of the invention) at 5 mg/mL.
Scheme B: enzyme-assisted extraction method
Soaking the defatted Cordyceps militaris powder in water at a liquid-to-liquid ratio of 1:40g/mL for 2h, then regulating the pH value to about 5.0, adding cellulase (enzyme activity is 10U/mg) accounting for 2.0% of the mass of the defatted Cordyceps militaris powder, placing in a shaking table, incubating at about 50 ℃ for 2h, and placing the enzymolysis liquid in a boiling water bath for enzyme deactivation for 15min after the completion of the incubation; cooling, centrifuging to obtain supernatant, concentrating the supernatant to 1/4 of the original volume, adding 3 times of absolute ethanol, precipitating at 4deg.C for 12 hr, and lyophilizing the precipitate to obtain Cordyceps militaris polysaccharide; the procedure of example 1 is followed to calculate the polysaccharide yield and to determine the inhibition activity of the alpha-glucosidase.
The results show that: the yield of the Cordyceps militaris polysaccharide obtained by adopting the enzyme-assisted extraction method is 10.7+/-0.93 percent (which is 1.7 times lower than the technical scheme of the invention); the inhibition rate of the alpha-glucosidase is 35.4+/-2.95 percent (which is 2.4 times lower than the technical proposal of the invention) at 5 mg/mL.
Scheme C: microwave-assisted extraction method
Soaking the defatted Cordyceps militaris powder in water at a liquid-to-liquid ratio of 1:40g/mL for 2h, and transferring to a microwave-ultrasonic combined extraction instrument (Beijing Xiangnian scientific development Co., ltd., model XH-300B); starting a microwave mode, and extracting for 8min under the conditions of 500W power and 70 ℃; centrifuging after the completion of the process, collecting supernatant, concentrating the supernatant to 1/4 of the original volume, adding 3 times of absolute ethyl alcohol, precipitating at 4 ℃ for 12 hours, and freeze-drying the precipitate to obtain Cordyceps militaris polysaccharide; the procedure of example 1 is followed to calculate the polysaccharide yield and to determine the inhibition activity of the alpha-glucosidase.
The results show that: the yield of the Cordyceps militaris polysaccharide obtained by adopting the microwave-assisted extraction method is 14.4+/-2.03 percent (which is 1.3 times lower than the technical scheme of the invention); the inhibition rate of the alpha-glucosidase is 48.6+/-5.11 percent (which is 1.7 times lower than the technical proposal of the invention) at 5 mg/mL.
Scheme D: ultrasonic assisted extraction method
Soaking the defatted Cordyceps militaris powder in water at a liquid-to-liquid ratio of 1:40g/mL for 2h, and transferring to a microwave-ultrasonic combined extraction instrument (Beijing Xiangnian scientific development Co., ltd., model XH-300B); turning on ultrasonic mode, and extracting at 60deg.C under 300W power for 30min; centrifuging after the completion of the process, collecting supernatant, concentrating the supernatant to 1/4 of the original volume, adding 3 times of absolute ethyl alcohol, precipitating at 4 ℃ for 12 hours, and freeze-drying the precipitate to obtain Cordyceps militaris polysaccharide; the procedure of example 1 is followed to calculate the polysaccharide yield and to determine the inhibition activity of the alpha-glucosidase.
The results show that: the yield of the Cordyceps militaris polysaccharide obtained by adopting the ultrasonic-assisted extraction method is 13.6+/-1.53 percent (which is 1.3 times lower than the technical scheme of the invention); the inhibition rate of the alpha-glucosidase is 50.7+/-4.69 percent (which is 1.6 times lower than the technical proposal of the invention) at 5 mg/mL.
In conclusion, the Cordyceps militaris polysaccharide obtained by the flash extraction technology provided by the invention has the advantages of higher yield than the traditional hot water extraction method, enzyme-assisted extraction method, microwave-assisted extraction method and ultrasonic-assisted extraction method, low specific energy consumption and extremely high efficiency (only 1min is needed for extraction); the inhibition rate of alpha-glucosidase was also higher than that of the above method, indicating that: the flash treatment of 110V not only can improve the polysaccharide yield of the Cordyceps militaris, but also can optimize the polysaccharide structure and improve the hypoglycemic activity.
EXAMPLE 2 purification of Cordyceps militaris polysaccharide
Deproteinizing 3 times with Sevag reagent (chloroform: n-butanol=4:1, volume ratio); removing denatured protein and residual Sevag reagent by centrifugation, and dialyzing in distilled water for 3 times (dialysis bag with molecular weight cutoff of 7000Da, and changing distilled water once at intervals of 12 h); freeze drying the dialyzate to obtain deproteinized polysaccharide. 100mg of deproteinized polysaccharide was weighed and dissolved in 20mL of deionized water, and loaded onto a Diethylaminoethyl (DEAE) cellulose column (2.6 cm. Times.60 cm); slowly eluting with 0.5mol/L NaCl solution at a flow rate of 0.5mL/min and 2mL per tube; and (3) measuring the absorbance of each tube of eluent by adopting a phenol-sulfuric acid method, merging the eluents after the polysaccharide is completely eluted, concentrating, dialyzing for 3 times (the method is the same as above), and freeze-drying to obtain the purified polysaccharide of the Cordyceps militaris.
EXAMPLE 3 analysis and characterization of the fractions of purified polysaccharide of Cordyceps militaris
The total sugar content in the purified polysaccharide of the Cordyceps militaris is 83.5+/-1.46% measured by a phenol-sulfuric acid method; the content of the protein is 7.26+/-2.05% measured by a Coomassie brilliant blue method; the content of uronic acid is 4.16+/-0.68% measured by a m-hydroxybiphenyl method; the content of sulfuric acid radical is 3.71+/-0.69% measured by a barium chloride gelatin turbidimetry method.
Monosaccharide composition analysis: 5mg of purified polysaccharide of Cordyceps militaris is weighed into a 5mL test tube with a plug scale, 1mL of 2mol/L trifluoroacetic acid is added, after 2h of hydrolysis in an oven at 121 ℃, distilled water is fixed to 50mL, and after filtration by a microporous filter membrane with 0.45 micrometer, the sample is injected for analysis, and the sample injection volume is 20 mu L. Instrument and chromatographic conditions: the U.S. Daian ICS-5000 ion chromatograph, equipped with a pulsed amperometric detector, column was CarboPac PA20 (6.5 μm,3 mm. Times.150 mm). Gradient elution is adopted, and the mobile phase is as follows: 98% water+2% 250mM NaOH (0-21 min); 93% water +2%250mM NaOH+5%1M NaAc (21.1-30 min); 20% water +80%250mM NaOH (30.1-50 min). The flow rate was 0.5mL/min.
Molecular weight analysis: preparing purified polysaccharide of Cordyceps militaris into 2mg/mL solution, filtering the sample with 0.45 μm microporous membrane before sampling, and sampling with volume of 20 μl. The analytical instrument was a Waters1525 high performance liquid chromatograph, a 2414 differential refractive optical detector and an Empower 3 workstation, and the column was Ultrahydrogel TM Linear (2 μm,7.8 mm. Times.300 mm). Dextran with different molecular weights is used as standard sugar, a series of standard sugar solutions with different molecular weights are prepared, and a third-order calibration curve for measuring the molecular weight of the polysaccharide is established according to the retention time and the molecular weight value. The chromatographic conditions were mobile phase: 0.1M NaNO 3; flow rate: 0.9mL/min; column temperature: 45 ℃.
And (3) infrared spectrum analysis: mixing 2mg of dried Cordyceps militaris purified polysaccharide with 100mg of dried potassium bromide, tabletting, and measuring in an infrared spectrometer with scanning range of 4000cm -1～400cm-1.
Scanning electron microscope analysis: and (3) taking the dried purified polysaccharide of the Cordyceps militaris, adhering the purified polysaccharide to a stage of double-sided conductive adhesive by using a cotton swab, and blowing off non-adhered polysaccharide powder by using an ear washing ball. And (3) placing the sample in an ion sputtering instrument for foil spraying treatment, sequentially placing the treated sample under a scanning electron microscope, and observing the appearance of the sample after the instrument is set stable.
As shown in fig. 3, the purified polysaccharide of Cordyceps militaris was determined to be composed of arabinose, galactose, glucose, xylose, galacturonic acid, glucuronic acid in a molar ratio of 31.6:21.6:274:23.6:1.00:3.56 by comparison with standard sugar.
As shown in FIG. 4, the purified polysaccharide of Cordyceps militaris has a number average molecular weight (Mn) of 1.85kDa and a weight average molecular weight (Mw) of 5.09kDa.
As shown in FIG. 5, in the infrared spectrogram of purified polysaccharide of Cordyceps militaris, a strong absorption peak near 3422cm -1 is observed, which is caused by the stretching vibration of-OH; 2931cm -1 is methyl CH asymmetric stretching vibration; 1645cm -1 is the stretching vibration of carbonyl CO; 1404cm -1 is CO stretching vibration; the absorption peak near 1063cm -1 is the skeleton vibration of C-O-C glycosidic bond; 863cm -1 is a characteristic peak of an alpha-type glucoside bond; 598cm -1 is the absorption peak of the pyranose ring. Infrared spectrum analysis further proves that the purified polysaccharide of the Cordyceps militaris accords with the structural characteristics of the polysaccharide.
As shown in FIG. 6, the purified polysaccharide of Cordyceps militaris has a rough surface and irregular holes at magnification of x 1k, and particles with different sizes are scattered.
EXAMPLE 4 evaluation of purified polysaccharide of Cordyceps militaris for inhibiting the activity of alpha-glucosidase
In order to examine the influence of purification on the hypoglycemic activity of cordyceps militaris polysaccharide, the invention further evaluates the inhibition effect of cordyceps militaris polysaccharide on alpha-glucosidase, and uses acarbose (alpha-glucosidase inhibitor on the market) with the same concentration as a positive control, and the method is the same as in example 1.
As shown in FIG. 7, in the concentration range of 1-5 mg/mL, the inhibition effect of purified polysaccharide of Cordyceps militaris on alpha-glucosidase is obviously improved along with the increase of concentration (P < 0.01), and when the concentration reaches 5mg/mL, the inhibition rate of the purified polysaccharide of Cordyceps militaris on alpha-glucosidase reaches 99.4+/-1.25%, which is obviously higher than that of acarbose (92.5+/-2.11% and P < 0.05) with the same concentration; also significantly higher than the inhibition of alpha-glucosidase at 110V treatment voltage (83.2±7.64%, P <0.01, see example 1), indicating: the purification can further improve the hypoglycemic activity of the Cordyceps militaris polysaccharide; the half inhibition concentration of the purified Cordyceps militaris polysaccharide extracted by the technical scheme of the invention to the alpha-glucosidase is 1.48+/-0.21 mg/mL, which is 2.85 times lower than 4.22mg/mL reported in the literature (modern food technology, 2014,30 (12): 50-60), which shows that: the purified Cordyceps militaris polysaccharide extracted and purified by adopting the technical scheme of the invention has extremely high alpha-glucosidase inhibition effect and has excellent blood glucose reduction function.
EXAMPLE 5 preparation of purified polysaccharide oral liquid of Cordyceps militaris
Adding 2.0g of purified polysaccharide of Cordyceps militaris into 100mL of purified water, stirring at room temperature until the purified polysaccharide is dissolved, adding 0.5g of erythritol and 0.2g of potassium sorbate, stirring uniformly, canning, sterilizing instantly, bottling, and sealing to obtain oral liquid of purified polysaccharide of Cordyceps militaris.
EXAMPLE 6 preparation of purified polysaccharide capsule of Cordyceps militaris
Taking 5.0g of purified polysaccharide of the silkworm chrysalis cordyceps through a 40-mesh sieve, spraying 90% ethanol according to the proportion of 1:1, uniformly mixing, adding 10% starch to prepare a soft material, granulating through a 20-mesh sieve, drying in a 60-DEG C oven for 1h, sieving through a 20-mesh sieve, granulating, and filling in a No. 3 empty capsule under the environment of the relative humidity of less than 65%, thus obtaining the purified polysaccharide capsule of the silkworm chrysalis cordyceps.
EXAMPLE 7 preparation of purified polysaccharide tablet of Cordyceps militaris
Mixing 5.0g of purified polysaccharide of Cordyceps militaris with 300mg of polyvinylpyrrolidone and 4.0mg of calcium hydrophosphate, grinding, and sieving with 100 mesh sieve; adding 95% ethanol solution of 5% povidone K30 while stirring to prepare soft material, and wet granulating (sieving with 16 mesh sieve); drying at 60deg.C, sieving, granulating, adding 1% magnesium stearate and 2% silica gel micropowder, mixing, and tabletting to obtain Cordyceps militaris purified polysaccharide tablet.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the scope of the present invention.
Claims (9)
1. A method for preparing Cordyceps militaris polysaccharide with high hypoglycemic activity by flash extraction comprises soaking Cordyceps militaris defatted powder in water, placing in flash extractor, and treating under certain voltage; centrifuging, concentrating supernatant, precipitating with ethanol, and lyophilizing to obtain Cordyceps militaris polysaccharide.
2. The method of claim 1, wherein the certain process voltage is 70-150V.
3. The method of claim 1, wherein the defatted Cordyceps militaris powder is obtained by degreasing with petroleum ether.
4. The method of claim 1 or 2, wherein the certain treatment voltage is 110V, and a flash extractor of the type JHBE-20A of the sienna tecan biotechnology company.
5. The method according to claim 1 or 2, comprising the specific steps of:
Drying fresh Cordyceps militaris to constant weight, pulverizing, and sieving to obtain Cordyceps militaris powder; adding petroleum ether, degreasing in dark place, and vacuum filtering to obtain defatted Cordyceps militaris powder;
Soaking the defatted Cordyceps militaris powder in water, and then placing in a flash extractor for treatment under 110V; centrifuging after the completion of the process, collecting supernatant, concentrating the supernatant to obtain concentrated solution, adding absolute ethyl alcohol for precipitation, and freeze-drying the precipitate to obtain Cordyceps militaris polysaccharide;
the yield of the Cordyceps militaris polysaccharide is 18.3+/-1.82%, and the inhibition rate of the Cordyceps militaris polysaccharide to alpha-glucosidase is 83.2+/-7.64% when the concentration is 5 mg/mL.
6. The method of claim 1, further comprising purifying the prepared Cordyceps militaris polysaccharide, deproteinizing with Sevag reagent, removing residual Sevag reagent, dialyzing in distilled water, lyophilizing the dialysate to obtain deproteinized polysaccharide, and purifying with Diethylaminoethyl (DEAE) cellulose chromatographic column to obtain Cordyceps militaris purified polysaccharide.
7. The method according to claim 6, wherein the purification is carried out by the following steps: deproteinizing with Sevag reagent, removing residual Sevag reagent, and dialyzing in distilled water; freeze-drying the dialyzate to obtain deproteinized polysaccharide; dissolving deproteinized polysaccharide in deionized water, and loading to Diethylaminoethyl (DEAE) cellulose chromatographic column; slowly eluting with NaCl solution, measuring absorbance of each tube of eluent by phenol-sulfuric acid method, mixing eluates after polysaccharide is completely eluted, concentrating, dialyzing, and freeze-drying to obtain purified polysaccharide of Cordyceps militaris;
The purified polysaccharide of the silkworm chrysalis cordyceps can meet one or more of the following conditions: 1) The total sugar content was 83.5.+ -. 1.46%; 2) The protein content is 7.26 plus or minus 2.05 percent; 3) The uronic acid content is 4.16+/-0.68%; 4) The content of sulfuric acid groups is 3.71+/-0.69%; 5) The purified polysaccharide of the Cordyceps militaris comprises arabinose, galactose, glucose, xylose, galacturonic acid and glucuronic acid according to the molar ratio of 31.6:21.6:274:23.6:1.00:3.56; 6) The number average molecular weight (Mn) of the purified polysaccharide of the Cordyceps militaris is 1.85kDa; 7) The weight average molecular weight (Mw) was 5.09kDa.
8. Use of purified polysaccharide of Cordyceps militaris obtained by the method of claim 7 for preparing products with blood glucose lowering effect.
9. The use according to claim 8, wherein the purified polysaccharide of Cordyceps militaris is used for preparing oral liquid, capsule or tablet.
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