CN110564711A - gastrodin serving as alpha-glucosidase inhibitor and application thereof - Google Patents
gastrodin serving as alpha-glucosidase inhibitor and application thereof Download PDFInfo
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- CN110564711A CN110564711A CN201910894613.7A CN201910894613A CN110564711A CN 110564711 A CN110564711 A CN 110564711A CN 201910894613 A CN201910894613 A CN 201910894613A CN 110564711 A CN110564711 A CN 110564711A
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- gastrodin
- extract
- alpha
- suspension
- trichoderma viride
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- PUQSUZTXKPLAPR-UJPOAAIJSA-N Gastrodin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C(CO)C=C1 PUQSUZTXKPLAPR-UJPOAAIJSA-N 0.000 title claims abstract description 101
- PUQSUZTXKPLAPR-KSSYENDESA-N 4-(beta-D-Glucopyranosyloxy) benzyl alcohol Natural products O([C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1)c1ccc(CO)cc1 PUQSUZTXKPLAPR-KSSYENDESA-N 0.000 title claims abstract description 71
- 229930193974 gastrodin Natural products 0.000 title claims abstract description 71
- PUQSUZTXKPLAPR-NZEXEKPDSA-N helicidol Natural products O([C@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](CO)O1)c1ccc(CO)cc1 PUQSUZTXKPLAPR-NZEXEKPDSA-N 0.000 title claims abstract description 71
- 229940077274 Alpha glucosidase inhibitor Drugs 0.000 title claims abstract description 26
- 239000003888 alpha glucosidase inhibitor Substances 0.000 title claims abstract description 26
- 239000000463 material Substances 0.000 claims abstract description 55
- 239000000284 extract Substances 0.000 claims abstract description 53
- 241000223261 Trichoderma viride Species 0.000 claims abstract description 43
- 238000002360 preparation method Methods 0.000 claims abstract description 27
- 238000000855 fermentation Methods 0.000 claims abstract description 23
- 230000004151 fermentation Effects 0.000 claims abstract description 23
- 239000007787 solid Substances 0.000 claims abstract description 23
- 239000004480 active ingredient Substances 0.000 claims abstract description 21
- 235000013305 food Nutrition 0.000 claims abstract description 11
- 210000004369 blood Anatomy 0.000 claims abstract description 10
- 239000008280 blood Substances 0.000 claims abstract description 10
- 230000000291 postprandial effect Effects 0.000 claims abstract description 6
- 235000005911 diet Nutrition 0.000 claims abstract description 5
- 230000037213 diet Effects 0.000 claims abstract description 5
- 239000000725 suspension Substances 0.000 claims description 45
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 41
- 244000061456 Solanum tuberosum Species 0.000 claims description 41
- 239000001963 growth medium Substances 0.000 claims description 39
- 241000305491 Gastrodia elata Species 0.000 claims description 33
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 22
- 230000001954 sterilising effect Effects 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 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 10
- 238000001035 drying Methods 0.000 claims description 10
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 10
- 239000008103 glucose Substances 0.000 claims description 10
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- 238000004659 sterilization and disinfection Methods 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 6
- 235000012015 potatoes Nutrition 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 5
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 claims description 5
- 230000003213 activating effect Effects 0.000 claims description 5
- 239000008272 agar Substances 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 5
- 239000003708 ampul Substances 0.000 claims description 5
- 230000001580 bacterial effect Effects 0.000 claims description 5
- 238000009835 boiling Methods 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 5
- 238000005520 cutting process Methods 0.000 claims description 5
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- 238000000227 grinding Methods 0.000 claims description 5
- 238000009630 liquid culture Methods 0.000 claims description 5
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
- 238000005507 spraying Methods 0.000 claims description 5
- 238000010025 steaming Methods 0.000 claims description 5
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 claims description 5
- 229960003495 thiamine Drugs 0.000 claims description 5
- 235000019157 thiamine Nutrition 0.000 claims description 5
- 239000011721 thiamine Substances 0.000 claims description 5
- 230000001502 supplementing effect Effects 0.000 claims description 3
- 239000002775 capsule Substances 0.000 claims description 2
- 239000008187 granular material Substances 0.000 claims description 2
- 239000003826 tablet Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 3
- 239000002609 medium Substances 0.000 claims 1
- 108010028144 alpha-Glucosidases Proteins 0.000 abstract description 25
- 102100024295 Maltase-glucoamylase Human genes 0.000 abstract description 24
- 230000000694 effects Effects 0.000 abstract description 19
- 238000000605 extraction Methods 0.000 abstract description 17
- 239000000178 monomer Substances 0.000 abstract description 2
- 230000005764 inhibitory process Effects 0.000 description 21
- 239000003814 drug Substances 0.000 description 7
- 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 description 6
- 229960002632 acarbose Drugs 0.000 description 6
- 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 description 6
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 230000002401 inhibitory effect Effects 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 230000009471 action Effects 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
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- 238000010992 reflux Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241000305492 Gastrodia Species 0.000 description 3
- 229940074391 gallic acid Drugs 0.000 description 3
- 235000004515 gallic acid Nutrition 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 235000010575 Pueraria lobata Nutrition 0.000 description 2
- 244000046146 Pueraria lobata Species 0.000 description 2
- 102000019197 Superoxide Dismutase Human genes 0.000 description 2
- 108010012715 Superoxide dismutase Proteins 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
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- 230000002441 reversible effect Effects 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 206010010904 Convulsion Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000222336 Ganoderma Species 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 1
- 241000233855 Orchidaceae Species 0.000 description 1
- 208000013738 Sleep Initiation and Maintenance disease Diseases 0.000 description 1
- 230000003178 anti-diabetic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 230000036461 convulsion Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 208000002173 dizziness Diseases 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000556 factor analysis Methods 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 208000034783 hypoesthesia Diseases 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 206010022437 insomnia Diseases 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 229940118019 malondialdehyde Drugs 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
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- 230000003071 parasitic effect Effects 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000008986 qingzhen Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/0102—Alpha-glucosidase (3.2.1.20)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention discloses a gastrodin extract used as an alpha-glucosidase inhibitor and application thereof in special diet food. The gastrodin extract is used as one of active ingredients or the only active ingredient for preparing the alpha-glucosidase inhibitor; the gastrodin is prepared by performing solid fermentation treatment on the crushed radix puerariae medicinal material by using trichoderma viride, and then extracting, so that the extraction yield of the gastrodin can be greatly improved, and a gastrodin extract with high content, good quality and low cost is provided for the preparation of a gastrodin monomer. The experimental result of the gastrodin on the activity of the alpha-glucosidase provides reliable experimental and theoretical basis for the application of the gastrodin in the field of reducing postprandial blood sugar level, and also provides a new way for high-value utilization of gastrodin resources in special diet food.
Description
Technical Field
the invention relates to the technical field of functional food factor analysis and extraction, in particular to application of a gastrodin extract as an alpha-glucosidase inhibitor in blood sugar-reducing food or health-care food and a preparation method thereof.
background
Rhizoma Gastrodiae is a perennial herb parasitic plant of Gastrodia of Orchidaceae, has a thick rhizome, is used for treating dizziness, numbness of limbs, infantile convulsion, etc., is a rare Chinese medicine, and can be used together with QINGZHEN Ganoderma for treating headache and insomnia. The main medicinal component of the tuber of the tall gastrodia tuber is gastrodin which is extracted from the tuber of the tall gastrodia tuber. Gastrodin can remarkably reduce the activity of HUVECs extracellular LDH under high-sugar stimulation, and reduce the damage of high-sugar to vascular endothelial cells; the gastrodine can obviously reduce the blood sugar of a diabetic mouse, reduce the concentration of triglyceride, total cholesterol and malondialdehyde in the blood serum of the mouse, improve the activity of superoxide dismutase (SOD) and show obvious antidiabetic effect.
The traditional extraction method mainly comprises a decocting method, an immersion method, a reflux extraction method and the like. The decoction method is convenient and easy to implement, but the decoction liquid contains more impurities, is easy to mildew and rot, and some thermolabile or volatile components are easy to damage, volatilize and lose in the decoction process. The dipping method is simple, but the operation time is long, and the effective components are not easy to be completely leached. The reflux extraction method comprises extracting medicinal materials with volatile organic solvent such as ethanol, heating the extractive solution for distillation, distilling off volatile solvent, condensing, and repeatedly refluxing to the extractor for extraction until the effective components are completely extracted under reflux.
The traditional extraction method has the defects of complex extraction process, large solvent requirement, high cost, low extraction rate and the like. The continuous low-temperature high-pressure crushing and extracting technology is characterized in that under the condition of low temperature or normal temperature, a liquid sample is subjected to the sum of strong shearing force, impact and turbulence action when passing through a narrow gap at a high speed under the action of high pressure, cavitation effect generated by pressure instant release and other action effects, so that cells are instantaneously expanded and crushed, and the dissolution of effective components in the cells is promoted. At present, the prior art does not disclose a method for extracting gastrodin from gastrodia elata through continuous low-temperature high-pressure crushing. The inventor researches and optimizes the gastrodin extraction and preparation process for a long time, and surprisingly discovers that solid fermentation treatment is carried out on the crushed radix puerariae medicinal material by trichoderma viride, and then extraction is carried out, so that the extraction yield of gastrodin can be greatly improved, and a gastrodin extract with high content, good quality and low cost is provided for preparation of gastrodin monomers, thereby completing the invention.
Disclosure of Invention
The invention aims to provide a gastrodin extract as an alpha-glucosidase inhibitor and application thereof in special diet food, wherein solid fermentation treatment is carried out on crushed kudzu root medicinal materials by trichoderma viride, then extraction is carried out, alpha-glucosidase inhibitory activity, inhibition kinetics and thermodynamics experiments are carried out on the crushed kudzu root medicinal materials, an inhibition action mechanism is disclosed, experimental and theoretical bases are provided for development and utilization of the gastrodia elata, and an economic and safe alpha-glucosidase inhibitor from natural marine sources is obtained at the same time.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problem is as follows:
A gastrodine extract used as alpha-glucosidase inhibitor and its application in special diet food are provided, wherein the gastrodine extract is used as one of active ingredients or the only active ingredient for preparing alpha-glucosidase inhibitor.
a gastrodine extract is used as alpha-glucosidase inhibitor, and is used as one of active ingredients or the only active ingredient for reducing postprandial blood glucose level.
The preparation process of the gastrodin extract comprises the step of performing solid fermentation on a gastrodia elata medicinal material by using trichoderma viride, and comprises the following steps of:
(1) carrying out dry heat sterilization treatment on the dry gastrodia elata medicinal material;
(2) Crushing the sterilized gastrodia elata medicinal material, adding water and uniformly stirring to obtain a wet material;
(3) Adding the comprehensive potato culture medium and the trichoderma viride suspension, and uniformly mixing;
(4) performing solid fermentation for 4-8 days to obtain fermented material;
(5) Drying and crushing the fermented material, extracting with ethanol, and concentrating the extract to obtain gastrodin extract.
the preparation process of gastrodine extract preferably comprises the following steps:
(1) placing dry rhizoma Gastrodiae in a sterilizing pot, and dry steaming at 90-125 deg.C for 60-120min for dry heat sterilization;
(2) Crushing the sterilized gastrodia elata into 40-80-mesh coarse powder, spraying water 2-2.5 times the weight of the gastrodia elata, and uniformly stirring to form a wet material;
(3) Adding the comprehensive potato culture medium and the trichoderma viride suspension, and uniformly mixing;
(4) Performing solid fermentation at 20-30 deg.C and 80-95% relative humidity for 5-6 days to obtain fermented material;
(5) Drying and grinding the fermented material, reflux-extracting with ethanol-water solution or methanol-water solution for 3 times, and concentrating the extractive solution to obtain gastrodin extract; the weight percentage content of the ethanol or the methanol in the ethanol-water solution or the methanol-water solution is more than or equal to 92 percent.
The preparation method of the comprehensive potato culture medium per 1000mL comprises the following steps: taking 150-250g of peeled potatoes, cutting into small pieces, adding 1000mL of water, boiling for 15-25min, filtering out potato pieces, adding 10-30g of glucose, 15-30g of agar, 15-5 g of KH2PO 42, 5-10mg of thiamine, 1-3g of MgSO 4.7H2O 1, dissolving, supplementing water to 1000mL, and sterilizing at 121 ℃ for 10-20 min;
The preparation of the trichoderma viride suspension per 100mL comprises the following steps:
(1) recovering and expanding culture of strains: sucking 0.5mL of liquid culture medium by using a sterile pipette, dripping into an ampoule containing a trichoderma viride freeze-dried strain, slightly oscillating to dissolve the freeze-dried strain into a suspension state, sucking 100 mu L of thallus suspension, transplanting the thallus suspension onto a slant culture medium, and culturing and activating at a corresponding temperature of 25-32 ℃;
(2) Inoculating activated strain into 100mL of comprehensive potato culture medium at 25-32 deg.C and rotation speed of 105and (5) carrying out amplification culture for 2-4d by using a constant-temperature shaking culture box at r/min to obtain the trichoderma viride bacterial suspension.
the adding proportion of the comprehensive potato culture medium and the trichoderma viride suspension is as follows: adding 100mL of comprehensive potato culture medium and 5mL of trichoderma viride suspension into every 100g of dry gastrodia elata medicinal material; the solid fermentation is carried out at 26-28 deg.C and 92-94% relative humidity. The gastrodine can be used for preparing foods or health products for lowering blood sugar such as liquid preparation, powder, granule, tablet, capsule, oral liquid, etc
The invention has the following beneficial effects:
(1) The method for extracting gastrodin by the preparation method limits various critical conditions, and can obviously improve the extraction rate of gastrodin and the gastrodin content in a gastrodin extract.
(2) The alpha-glucosidase inhibitor disclosed by the invention is a list of gastrodin which is newly added in 2018 and is not only a food but also a drug, the gastrodin is rich in resources, and the gastrodin preparation process is simple and is easy to industrialize.
(3) the dynamic and thermodynamic experimental data of the gastrodin for inhibiting the activity of the alpha-glucosidase provide reliable theoretical basis for the application of the gastrodin in the development field of hypoglycemic foods and medicines, and also provide a new way for high-value utilization of gastrodin resources and research of safe, economic and marine alpha-glucosidase inhibitors.
(4) the gastrodin serving as an inhibitor can better inhibit the activity of alpha-glucosidase, the inhibition rate can reach 90.3 percent at most, and the inhibition rate is 16.4 percent higher than that of acarbose which is a commercially available medicine.
Drawings
FIG. 1 shows the inhibition effect of gastrodin extract on alpha-glucosidase enzyme activity;
FIG. 2 is the determination of the inhibition mechanism of gastrodin extract on alpha-glucosidase;
FIG. 3 shows the measurement of the inhibition type and inhibition constant of gastrodin extract on alpha-glucosidase. Detailed Description
the present invention is further described in detail with reference to specific examples, so that those skilled in the art can implement the invention with reference to the description.
example 1
A gastrodine extract is used as alpha-glucosidase inhibitor, and the gastrodine extract is used as one of active ingredients or the only active ingredient for preparing the alpha-glucosidase inhibitor.
a gastrodine extract is used as alpha-glucosidase inhibitor, and is used as one of active ingredients or the only active ingredient for reducing postprandial blood glucose level.
the preparation process of the gastrodin extract comprises the step of performing solid fermentation on a gastrodia elata medicinal material by using trichoderma viride, and comprises the following steps of:
(1) Carrying out dry heat sterilization treatment on the dry gastrodia elata medicinal material;
(2) Crushing the sterilized gastrodia elata medicinal material, adding water and uniformly stirring to obtain a wet material;
(3) adding the comprehensive potato culture medium and the trichoderma viride suspension, and uniformly mixing;
(4) Performing solid fermentation for 4 days to obtain a fermented material;
(5) Drying and crushing the fermented material, extracting with ethanol, and concentrating the extract to obtain gastrodin extract.
the preparation process of gastrodine extract preferably comprises the following steps:
(1) placing dry rhizoma Gastrodiae in a sterilizing pot, and dry steaming at 90 deg.C for 60min for dry heat sterilization;
(2) crushing the sterilized gastrodia elata into 40-mesh coarse powder, spraying water 2 times the weight of the gastrodia elata, and uniformly stirring to form a wet material;
(3) Adding the comprehensive potato culture medium and the trichoderma viride suspension, and uniformly mixing;
(4) Performing solid fermentation at 20 deg.C and 80% relative humidity for 5 days to obtain fermented material;
(5) drying and grinding the fermented material, reflux-extracting with ethanol-water solution or methanol-water solution for 3 times, and concentrating the extractive solution to obtain gastrodin extract; the weight percentage content of the ethanol or the methanol in the ethanol-water solution or the methanol-water solution is more than or equal to 92 percent.
the preparation method of the comprehensive potato culture medium per 1000mL comprises the following steps: taking 150g of peeled potatoes, cutting into small pieces, adding 1000mL of water, boiling for 15min, then filtering out potato pieces, adding 10g of glucose, 15g of agar, KH2PO 42 g, 5mg of thiamine and 4.7H 2O 1g of MgSO, dissolving, then supplementing water to 1000mL, and sterilizing for 10min at 121 ℃;
The preparation of the trichoderma viride suspension per 100mL comprises the following steps:
(1) recovering and expanding culture of strains: sucking 0.5mL of liquid culture medium by using a sterile pipette, dripping into an ampoule containing a trichoderma viride freeze-dried strain, slightly oscillating to dissolve the freeze-dried strain into a suspension state, sucking 100 mu L of thallus suspension, transplanting the thallus suspension onto a slant culture medium, and culturing and activating at a corresponding temperature of 25-32 ℃;
(2) Inoculating activated strain into 100mL of comprehensive potato culture medium at 25 deg.C and rotation speed of 105and (5) carrying out amplification culture for 2d by using an r/min constant-temperature oscillation culture box to obtain the trichoderma viride bacterial suspension.
the adding proportion of the comprehensive potato culture medium and the trichoderma viride suspension is as follows: adding 100mL of comprehensive potato culture medium and 5mL of trichoderma viride suspension into every 100g of dry gastrodia elata medicinal material; the solid fermentation was carried out at 26 ℃ and 92% relative humidity. The experiment was repeated three times, and the average value of the results was obtained to obtain a gastrodin extraction rate of 0.72mg/g (in terms of gallic acid equivalent).
example 2
A gastrodine extract is used as alpha-glucosidase inhibitor, and the gastrodine extract is used as one of active ingredients or the only active ingredient for preparing the alpha-glucosidase inhibitor.
A gastrodine extract is used as alpha-glucosidase inhibitor, and is used as one of active ingredients or the only active ingredient for reducing postprandial blood glucose level.
The preparation process of the gastrodin extract comprises the step of performing solid fermentation on a gastrodia elata medicinal material by using trichoderma viride, and comprises the following steps of:
(1) carrying out dry heat sterilization treatment on the dry gastrodia elata medicinal material;
(2) Crushing the sterilized gastrodia elata medicinal material, adding water and uniformly stirring to obtain a wet material;
(3) Adding the comprehensive potato culture medium and the trichoderma viride suspension, and uniformly mixing;
(4) Performing solid fermentation for 6 days to obtain a fermented material;
(5) drying and crushing the fermented material, extracting with ethanol, and concentrating the extract to obtain gastrodin extract.
The preparation process of gastrodine extract preferably comprises the following steps:
(1) Placing dry rhizoma Gastrodiae in a sterilizing pot, and dry steaming at 100 deg.C for 80min for dry heat sterilization;
(2) crushing the sterilized gastrodia elata into 60-mesh coarse powder, spraying water 2.5 times the weight of the gastrodia elata, and uniformly stirring to form a wet material;
(3) Adding the comprehensive potato culture medium and the trichoderma viride suspension, and uniformly mixing;
(4) Performing solid fermentation at 30 deg.C and 95% relative humidity for 6 days to obtain fermented material;
(5) Drying and grinding the fermented material, reflux-extracting with ethanol-water solution or methanol-water solution for 3 times, and concentrating the extractive solution to obtain gastrodin extract; the weight percentage content of the ethanol or the methanol in the ethanol-water solution or the methanol-water solution is more than or equal to 92 percent.
the preparation method of the comprehensive potato culture medium per 1000mL comprises the following steps: taking 150g of peeled potatoes, cutting into small pieces, adding 1000mL of water, boiling for 15min, then filtering out potato pieces, adding 20g of glucose, 20g of agar, KH2PO 43 g, 7mg of thiamine and 7.7H 2O 2g of MgSO4 & 7H2, adding water to 1000mL after dissolving, and sterilizing for 15min at 121 ℃;
the preparation of the trichoderma viride suspension per 100mL comprises the following steps:
(1) recovering and expanding culture of strains: sucking 0.5mL of liquid culture medium by using a sterile pipette, dripping into an ampoule containing a trichoderma viride freeze-dried strain, slightly oscillating to dissolve the freeze-dried strain into a suspension state, sucking 100 mu L of thallus suspension, transplanting the thallus suspension onto a slant culture medium, and culturing and activating at a corresponding temperature of 25-32 ℃;
(2) Inoculating activated strain into 100mL of comprehensive potato culture medium at 30 deg.C and rotation speed of 105and (5) carrying out amplification culture for 2-4d by using a constant-temperature shaking culture box at r/min to obtain the trichoderma viride bacterial suspension.
the adding proportion of the comprehensive potato culture medium and the trichoderma viride suspension is as follows: adding 100mL of comprehensive potato culture medium and 5mL of trichoderma viride suspension into every 100g of dry gastrodia elata medicinal material; the solid fermentation is carried out under the conditions of 27 ℃ and 93% relative humidity; the experiment was repeated three times, and the average value of the results was obtained to obtain a gastrodin extraction rate of 0.69mg/g (in terms of gallic acid equivalent).
example 3
a gastrodine extract is used as alpha-glucosidase inhibitor, and the gastrodine extract is used as one of active ingredients or the only active ingredient for preparing the alpha-glucosidase inhibitor.
a gastrodine extract is used as alpha-glucosidase inhibitor, and is used as one of active ingredients or the only active ingredient for reducing postprandial blood glucose level.
The preparation process of the gastrodin extract comprises the step of performing solid fermentation on a gastrodia elata medicinal material by using trichoderma viride, and comprises the following steps of:
(1) carrying out dry heat sterilization treatment on the dry gastrodia elata medicinal material;
(2) crushing the sterilized gastrodia elata medicinal material, adding water and uniformly stirring to obtain a wet material;
(3) adding the comprehensive potato culture medium and the trichoderma viride suspension, and uniformly mixing;
(4) Performing solid fermentation for 8 days to obtain a fermented material;
(5) Drying and crushing the fermented material, extracting with ethanol, and concentrating the extract to obtain gastrodin extract.
The preparation process of gastrodine extract preferably comprises the following steps:
(1) placing dry rhizoma Gastrodiae in a sterilizing pot, and dry steaming at 125 deg.C for 120min for dry heat sterilization;
(2) Crushing the sterilized gastrodia elata into 80-mesh coarse powder, spraying water 2.5 times the weight of the gastrodia elata, and uniformly stirring to form a wet material;
(3) Adding the comprehensive potato culture medium and the trichoderma viride suspension, and uniformly mixing;
(4) performing solid fermentation at 30 deg.C and 95% relative humidity for 6 days to obtain fermented material;
(5) Drying and grinding the fermented material, reflux-extracting with ethanol-water solution or methanol-water solution for 3 times, and concentrating the extractive solution to obtain gastrodin extract; the weight percentage content of the ethanol or the methanol in the ethanol-water solution or the methanol-water solution is more than or equal to 92 percent.
the preparation method of the comprehensive potato culture medium per 1000mL comprises the following steps: taking 250g of peeled potatoes, cutting the peeled potatoes into small pieces, adding 1000mL of water, boiling for 25min, then filtering the potato pieces, adding 30g of glucose, 30g of agar, KH2PO 45 g, 10mg of thiamine and 10mg of MgSO4 & 7H2O 3g, adding water to 1000mL after dissolving, and sterilizing for 20min at 121 ℃;
The preparation of the trichoderma viride suspension per 100mL comprises the following steps:
(1) recovering and expanding culture of strains: sucking 0.5mL of liquid culture medium by using a sterile pipette, dripping into an ampoule containing a trichoderma viride freeze-dried strain, slightly oscillating to dissolve the freeze-dried strain into a suspension state, sucking 100 mu L of thallus suspension, transplanting the thallus suspension onto a slant culture medium, and culturing and activating at a corresponding temperature of 32 ℃;
(2) Inoculating activated strain into 100mL of comprehensive potato culture medium at 32 deg.C and rotation speed of 105and (5) carrying out amplification culture for 4d by using a constant-temperature shaking culture box at r/min to obtain the trichoderma viride bacterial suspension.
The adding proportion of the comprehensive potato culture medium and the trichoderma viride suspension is as follows: adding 100mL of comprehensive potato culture medium and 5mL of trichoderma viride suspension into every 100g of dry gastrodia elata medicinal material; the solid fermentation is carried out under the conditions of 28 ℃ and 94% relative humidity; the experiment was repeated three times, and the average value of the results was obtained to obtain a gastrodin extraction rate of 0.72mg/g (in terms of gallic acid equivalent).
In order to verify the effect of gastrodin in inhibiting the activity of alpha-glucosidase, a commercially available drug acarbose is used as a positive reference, pNPG is used as a model substrate for testing the inhibition of the activity of alpha-glucosidase by gastrodin with different concentrations, as specifically shown in example 11. Further reveals the inhibition mechanism of gastrodin on alpha-glucosidase, and carries out experimental examinations by respectively utilizing the enzymatic reaction kinetics and the thermodynamic fluorescence method of the interaction of small molecules and proteins, specifically experimental examples 1-3. Test example 1
the determination result is shown in figure 1, the gastrodine concentration is in the range of 0.0025mg/mL to 0.0175mg/mL, the relative activity of the alpha-glucosidase is reduced along with the increase of the gastrodine concentration and is dose-dependent, when the gastrodine concentration is in the highest point of 0.0225mg/mL, the relative activity of the alpha-glucosidase is reduced to 9.3%, when the gastrodine concentration is compared with the positive control acarbose of 0.0225mg/mL, the relative activity of the alpha-glucosidase is reduced to 26.1%, the half inhibition concentrations IC50 of the gastrodine and acarbose to the alpha-glucosidase in the experimental range are respectively 0.0065mg/mL and 0.012mg/mL, and the aspect of the experimental data of the enzyme inhibition can show that the gastrodine is stronger in inhibiting the activity of the alpha-glucosidase than the acarbose of the drug on the market, is a natural source alpha-glucoronidase inhibitor with very large potential.
test example 2 mechanism of inhibition of gastrodin on α -glucosidase, fixing the concentration of substrate pNPG, changing the enzyme amount and the concentration of gastrodia elata sample in the system, and determining the relationship between the enzyme amount and the enzyme activity. As can be seen from fig. 2, the inhibition rates of gastrodin at different concentrations and α -glucosidase at different concentrations are in a linear relationship and are crossed at the origin, and the inhibition rate gradually decreases with the increase of the gastrodin concentration, indicating that the inhibition of gastrodin on α -glucosidase is reversible.
Test example 3
taking pNPG (0.5, 1.0, 2.0 and 4.0 x 10 < -5 > mol/L) with different concentrations as substrates, adding gastrodin solutions with different concentrations under the condition that the concentration of alpha-glucosidase is not changed, measuring the change of light absorption value in unit time at the wavelength of 405nm to obtain the enzymatic reaction rate (V) of the alpha-glucosidase, plotting 1/V to 1/[ S ], obtaining a Lineweaver-Burk double reciprocal diagram of different gastrodin concentrations without or after adding, judging the inhibition type between the two, and as can be seen from figure 3, the Vmax value is gradually reduced along with the increase of Km value, the two values are changed simultaneously, all the straight lines are intersected in the second quadrant, which indicates that the gastrodin can be combined with not only free enzyme but also enzyme-substrate complex, and the inhibition type of the gastrodin to the alpha-glucosidase belongs to a mixed type, the inhibition constants Ki and Kis were 0.0036mg/mL and 0.075mg/mL, respectively.
and (4) analyzing and concluding:
the gastrodin is slightly stronger than the commercial medicine acarbose in the aspect of inhibiting the activity of alpha-glucosidase, is a natural source alpha-glucosaccharase inhibitor with very large potential, the inhibition of the activity of the gastrodin on the alpha-glucosidase belongs to reversible mixed inhibition, the inhibition constant Ki < Kis, which shows that the binding capacity of the gastrodin and the alpha-glucosidase is stronger than that of a substrate pNPG, and the gastrodin is also proved to be a better alpha-glucosidase inhibitor from this aspect, and the data of the slope and the intercept of an inset part show good linear relation, which shows that the gastrodin only has one or one class of binding sites on the alpha-glucosidase.
the above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.
Claims (8)
1. a gastrodine extract used as an alpha-glucosidase inhibitor and application thereof in special diet food is characterized in that: the gastrodin extract is used as one of the active ingredients or the only active ingredient for preparing the alpha-glucosidase inhibitor.
2. the use of a gastrodine extract as an alpha-glucosidase inhibitor according to claim 1, characterized in that: the gastrodine extract is used as one or only one of the active ingredients for reducing postprandial blood glucose level.
3. The gastrodin extract as an alpha-glucosidase inhibitor according to claim 1, characterized in that: the preparation process of the gastrodin extract comprises the step of performing solid fermentation on a gastrodia elata medicinal material by using trichoderma viride.
4. the process for preparing gastrodine extract as claimed in claim 3, comprising the steps of:
(1) carrying out dry heat sterilization treatment on the dry gastrodia elata medicinal material;
(2) crushing the sterilized gastrodia elata medicinal material, adding water and uniformly stirring to obtain a wet material;
(3) Adding the comprehensive potato culture medium and the trichoderma viride suspension, and uniformly mixing;
(4) Performing solid fermentation for 4-8 days to obtain fermented material;
(5) Drying and crushing the fermented material, extracting with ethanol or methanol, and concentrating the extractive solution to obtain gastrodin extract.
5. the process for preparing gastrodine extract as claimed in claim 4, comprising the steps of:
(1) placing dry rhizoma Gastrodiae in a sterilizing pot, and dry steaming at 90-125 deg.C for 60-120min for dry heat sterilization;
(2) crushing the sterilized gastrodia elata into 40-80-mesh coarse powder, spraying water 2-2.5 times the weight of the gastrodia elata, and uniformly stirring to form a wet material;
(3) Adding the comprehensive potato culture medium and the trichoderma viride suspension, and uniformly mixing;
(4) Performing solid fermentation at 20-30 deg.C and 80-95% relative humidity for 5-6 days to obtain fermented material;
(5) Drying and grinding the fermented material, reflux-extracting with ethanol-water solution or methanol-water solution for 3 times, and concentrating the extractive solution to obtain gastrodin extract; the weight percentage content of the ethanol in the ethanol-water solution or the methanol-water solution is more than or equal to 92 percent.
6. the process for preparing gastrodine extract as claimed in claim 5, wherein:
the preparation method of the comprehensive potato culture medium per 1000mL comprises the following steps: taking 150-250g of peeled potatoes, cutting into small pieces, adding 1000mL of water, boiling for 15-25min, filtering out potato pieces, adding 10-30g of glucose, 15-30g of agar, 15-5 g of KH2PO 42, 5-10mg of thiamine, 1-3g of MgSO 4.7H2O 1, dissolving, supplementing water to 1000mL, and sterilizing at 121 ℃ for 10-20 min;
the preparation of the trichoderma viride suspension per 100mL comprises the following steps:
(1) recovering and expanding culture of strains: sucking 0.5mL of liquid culture medium by using a sterile pipette, dripping into an ampoule containing a trichoderma viride freeze-dried strain, slightly oscillating to dissolve the freeze-dried strain into a suspension state, sucking 100 mu L of thallus suspension, transplanting the thallus suspension onto a slant culture medium, and culturing and activating at a corresponding temperature of 25-32 ℃;
(2) inoculating activated strain into 100mL of comprehensive potato culture medium at 25-32 deg.C and rotation speed of 105And (5) carrying out amplification culture for 2-4d by using a constant-temperature shaking culture box at r/min to obtain the trichoderma viride bacterial suspension.
7. the method of any one of claims 1-6, wherein the integrated potato medium and the trichoderma viride suspension are added in a ratio of: adding 100mL of comprehensive potato culture medium and 5mL of trichoderma viride suspension into every 100g of dry gastrodia elata medicinal material; the solid fermentation is carried out at 26-28 deg.C and 92-94% relative humidity.
8. the gastrodin disclosed in claims 1-7 is used for preparing foods or health-care products for reducing blood sugar, such as liquid preparations, powder, granules, tablets, capsules, oral liquids and the like.
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| JPH11290023A (en) * | 1998-04-13 | 1999-10-26 | Nagaoka Jitsugyo Kk | Processed gostrodiae tuber food |
| KR20140129523A (en) * | 2013-04-30 | 2014-11-07 | 영농조합법인 이도 | Fermentation substance of gastrodia elata blume and fermentation method thereof |
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