CN118307604B - Extraction method of gastrodin, prepared gastrodin composition and application - Google Patents
Extraction method of gastrodin, prepared gastrodin composition and application Download PDFInfo
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- CN118307604B CN118307604B CN202410741052.8A CN202410741052A CN118307604B CN 118307604 B CN118307604 B CN 118307604B CN 202410741052 A CN202410741052 A CN 202410741052A CN 118307604 B CN118307604 B CN 118307604B
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- Prior art keywords
- gastrodin
- silica gel
- water
- prepared
- enzymolysis
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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 145
- 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 140
- 229930193974 gastrodin Natural products 0.000 title claims abstract description 140
- 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 140
- 239000000203 mixture Substances 0.000 title claims abstract description 49
- 238000000605 extraction Methods 0.000 title claims abstract description 39
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 62
- 238000002360 preparation method Methods 0.000 claims abstract description 54
- 238000000034 method Methods 0.000 claims abstract description 40
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- KSEBMYQBYZTDHS-HWKANZROSA-N ferulic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-N 0.000 claims description 44
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
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- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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- A—HUMAN NECESSITIES
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- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- C—CHEMISTRY; METALLURGY
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- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
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- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention provides a gastrodin extraction method, a prepared gastrodin composition and application thereof, and belongs to the technical field of medicines. The method comprises the following steps: s1, adding gastrodia elata into water, and adding complex enzyme for enzymolysis to obtain an enzymolysis complex; s2, extracting the enzymolysis compound under ultrahigh pressure to obtain an extracting solution; s3, primarily purifying the extracting solution through a chromatographic column to obtain a gastrodin primary pure product; s4, purifying the crude gastrodin product by using molecular engram polymer microspheres to obtain the pure gastrodin product; s5, dissolving the pure gastrodin product in water to obtain a water phase; dissolving the benzomarie and lecithin in ethanol to obtain an alcohol phase; the alcohol phase is dripped into the water phase for hydration reaction, and the prepared gastrodin composition has good extraction rate for gastrodin, high purity of the gastrodin, less consumption, simple preparation method and low energy consumption, and the alcohol plastid containing ethanol prepared from the gastrodin and the benzohessian has good effects of preventing and treating senile dementia and parkinsonism.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a gastrodin extraction method, a prepared gastrodin composition and application.
Background
Gastrodia elata is a plant of Orchidaceae, also called red arrow, rhizoma Bolbostemmatis, dudu mail, SHENCAO, duzhi, red arrow fat, difengcao, he Ji Cao, du Ji, herba Achilleae or rhizoma Solani Tuber osi. The rhizoma Gastrodiae contains gastrodine, also called gastrodine, and has good sedative and hypnotic effects, and can be used for relieving neurasthenia, insomnia and headache. Gastrodin can be extracted from rhizoma Gastrodiae or obtained by chemical synthesis. Gastrodine used in the pharmaceutical industry is mostly synthesized chemically, the yield is high, and the Gastrodine accords with the modern pharmaceutical production law, but phenols, phosphates and bromides are used in the chemical synthesis process, which is likely to involve environmental pollution. Therefore, the extraction of gastrodin from gastrodia elata is safer and more environment-friendly. At present, a large number of reports about extraction of gastrodin from gastrodia elata exist, and solvent extraction is generally used under heating.
Chinese patent document CN101704855A discloses a preparation method of gastrodin, which comprises percolating coarse powder of rhizoma Gastrodiae with 60-90% ethanol, concentrating under reduced pressure, drying, dissolving with methanol, countercurrent extracting, collecting gastrodin section, crystallizing, and recrystallizing with methanol-ethyl acetate.
Chinese patent document CN102250164A discloses a method for purifying gastrodin, which comprises the steps of ultrasonically extracting gastrodia elata powder by using an alcohol-containing solution, concentrating until no alcohol exists, purifying the concentrated solution by using polyamide resin, adopting ethanol-water gradient elution, passing target solution through an ODS silica gel column, and eluting by using a methanol-water or acetonitrile-water mixed solvent.
Chinese patent document CN103242461A discloses a method for simultaneously extracting gastrodia polysaccharide and gastrodin, which comprises the steps of extracting gastrodia elata powder with water, precipitating with ethanol, washing precipitate with ethanol, drying under reduced pressure, crushing to obtain gastrodia elata polysaccharide, and concentrating alcohol filtrate under reduced pressure to obtain gastrodin extract.
Chinese patent document CN101531689B discloses a method for extracting gastrodin by biological enzyme method, dissolving rhizoma Gastrodiae powder in acetic acid-sodium acetate buffer solution, adding complex enzyme for enzymolysis, adding alcohol, filtering to remove residue, concentrating into extract by rotary evaporation, and drying to obtain gastrodin extract.
However, the gastrodin obtained by the above technical method has the problems of low extraction rate, low purity and the like. In addition, gastrodin is easy to dissolve in water and ethanol, difficult to dissolve in chloroform and diethyl ether, weak in penetration capacity and short in half-life, so that the bioavailability of the gastrodin preparation is generally low, and various limitations are brought to preparation development.
Disclosure of Invention
The invention aims to provide a gastrodin extraction method, a gastrodin composition prepared by the same and application thereof, and the gastrodin composition has good extraction rate for gastrodin, high purity, less consumption, simple preparation method and low energy consumption of extracted gastrodin, and an ethosome containing 20-50% of ethanol prepared from the gastrodin and the phenylmarine rope has good effects of preventing and treating senile dementia and parkinsonism and has wide application prospect.
The technical scheme of the invention is realized as follows:
The invention provides a gastrodin extraction method, which comprises the following steps:
S1, enzymolysis of gastrodia elata: cleaning rhizoma Gastrodiae, pulverizing, adding into water, and adding complex enzyme for enzymolysis to obtain enzymolysis complex; s2, ultrahigh pressure extraction: extracting the enzymolysis complex in the step S1 under ultrahigh pressure, filtering, and concentrating the filtrate to obtain an extracting solution; s3, primary purification: the extract obtained in the step S2 is subjected to primary purification through a chromatographic column to obtain a gastrodin primary pure product; s4, fine purification of a molecularly imprinted polymer: purifying the crude gastrodin product prepared in the step S3 by using molecular engram polymer microspheres to obtain the pure gastrodin product; s5, preparing a gastrodin composition: dissolving the pure gastrodin product obtained in the step S4 in water to obtain a water phase; dissolving the benzomarie and lecithin in ethanol, and uniformly mixing to obtain an alcohol phase; dropping the alcohol phase into the water phase with a syringe, carrying out hydration reaction, and filtering with a microporous filter membrane to obtain the gastrodin composition.
As a further improvement of the invention, the compound enzyme in the step S1 is cellulase and pectase, the mass ratio is 10-12:7-10, the enzymolysis temperature is 40-50 ℃ and the time is 2-4h, and the solid-liquid ratio of the gastrodia elata to the water is 1:7-10g/mL.
As a further improvement of the invention, the pressure of the ultrahigh pressure extraction in the step S2 is 100-120MPa, the extraction time is 15-30min, and the relative density of the extracting solution is 1.05-1.1.
As a further improvement of the invention, the filler added in the chromatographic column in the step S3 is ferulic acid/abietic acid crosslinked silica gel, and the preparation method is as follows:
T1. Modified silica gel: adding silica gel powder into ethanol, adding a silane coupling agent KH570, heating and stirring for reaction, filtering, washing and drying to obtain modified silica gel; t2. Preparation of ferulic acid/abietic acid crosslinked silica gel: adding ferulic acid, abietic acid and the modified silica gel prepared in the step T1 into water, uniformly dispersing by ultrasonic, adding an initiator under the protection of inert gas, heating, stirring, reacting, filtering, washing and drying to obtain the ferulic acid/abietic acid crosslinked silica gel.
As a further improvement of the invention, the temperature of the heating and stirring reaction in the step T1 is 40-50 ℃ and the time is 1-2h, and the mass ratio of the silica gel powder to the silane coupling agent KH570 is 10:1-2; in the step T2, the mass ratio of the ferulic acid to the abietic acid to the modified silica gel to the initiator is 4-6:3-5:15-20:0.1-0.2, the initiator is at least one of potassium persulfate, sodium persulfate and ammonium persulfate, the power of ultrasonic is 800-1000W, the temperature of the heating and stirring reaction is 65-75 ℃, and the time is 3-5h.
As a further improvement of the present invention, the preparation method of the molecularly imprinted polymer microsphere in the step S4 is as follows: mixing gastrodin, monomer, cross-linking agent, emulsifier and pore-forming agent, adding into acetonitrile, mixing uniformly, dripping into water, emulsifying, adding initiator under the protection of inert gas, heating, stirring, reacting, filtering the product, adding into Soxhlet extractor, extracting with mixed solvent until gastrodin is not detected, washing, and drying to obtain the molecularly imprinted polymer microsphere.
As a further improvement of the invention, the mass ratio of the gastrodin to the monomer to the crosslinking agent to the emulsifying agent to the pore-forming agent to the initiator is 1-2:15-18:0.5-1:1-2:0.3-0.6:0.1-0.2, the initiator is a mixture of azodiisobutyronitrile and ammonium persulfate, the mass ratio is 4-6:2, the crosslinking agent is ethylene glycol dimethacrylate, the emulsifying agent is at least one selected from span-20, span-40, span-60, span-80 and span-85, the pore-forming agent is cetyl trimethyl ammonium chloride, the monomer is a mixture of methyl methacrylate, 4-vinyl pyridine, acrylamide and styrene, the mass ratio is 7-10:4-6:2-4:1-3, the temperature of the heating and stirring reaction is 60-70 ℃ for 3-5 hours, and the mixed solvent is a mixed solution of ethanol and acetic acid according to the volume ratio of 8-10:1.
As a further improvement of the invention, the mass ratio of the pure gastrodin to the phenylhalicarb and the lecithin in the step S5 is 4-6:2-4:2-3, and the hydration reaction time is 20-40min.
The invention further provides a gastrodin composition prepared by the extraction method.
The invention further provides application of the gastrodin composition in preparation of medicines for preventing and treating senile dementia and parkinsonism.
The invention has the following beneficial effects:
the gastrodia elata is subjected to enzymolysis by the compound enzyme, the cell wall of the gastrodia elata plant is broken under the synergistic effect of the cellulase and the pectase, and the complex glycoprotein, the sugar complex and other structures are degraded, so that active components in the gastrodia elata are promoted to be dissolved out, and most of gastrodin is dissolved into the enzymolysis complex.
The method further promotes the extraction of the gastrodin under the action of ultrahigh pressure extraction, has short extraction time, less solvent consumption, high extraction efficiency, simple operation and low energy consumption, and greatly improves the extraction rate of the gastrodin.
The extracting solution obtained by extraction is primarily purified by a chromatographic column, the filler in the chromatographic column is ferulic acid/abietic acid crosslinked silica gel, the abietic acid is of a polycyclic structure, the ferulic acid is of a benzene ring structure, the benzene ring is connected with hydroxyl, the extracting solution has rich three-dimensional structures, macromolecular substances can be selectively identified, and meanwhile, the abietic acid and the ferulic acid are of double-bond structures and can be crosslinked and connected with the silica gel powder modified by KH570, so that the ferulic acid/abietic acid crosslinked silica gel is prepared, has a better purification effect on gastrodin compounds, and obtains purer gastrodin.
Then, the molecular engram polymer is adopted to further purify the crude gastrodin product, the molecular engram polymer microsphere prepared by the template method is convenient to store, has high mechanical strength, high selectivity to gastrodin and convenient for repeated use, and the prepared pure gastrodin product has high purity and low consumption.
The gastrodin composition prepared by the invention is an ethosome containing 20% -50% of ethanol and prepared from gastrodin and benzomaria, wherein the benzomaria is a fat-soluble drug, the gastrodin is a water-soluble drug, and the gastrodin composition is a novel liposome, has the advantages of high encapsulation efficiency, low leakage rate, small skin irritation, good percutaneous absorption effect and the like, can effectively solve the problems of low bioavailability and the like of the gastrodin, improves the release property of the gastrodin, and improves the in-vivo absorption capacity, thereby improving the drug effect.
The extraction method has good extraction rate for gastrodin, high purity of the extracted gastrodin, less consumption, simple preparation method and low energy consumption, and the ethosome containing 20-50% of ethanol prepared from the gastrodin and the phenylhaline has good effects of preventing and treating senile dementia and parkinsonism and has wide application prospect.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Cellulase, 5U/g, pectase, 2.5U/g, available from Cangzhou Xia Chengmei Biotechnology Inc.
Preparation example 1 preparation of ferulic acid/abietic acid crosslinked silica gel
The method comprises the following steps: t1. Modified silica gel: 10g of silica gel powder is added into 100mL of ethanol, 1g of silane coupling agent KH570 is added, the mixture is heated to 40 ℃, stirred and reacted for 1h, filtered, washed and dried, and the modified silica gel is prepared.
T2. Preparation of ferulic acid/abietic acid crosslinked silica gel: adding 4g of ferulic acid, 3g of abietic acid and 15g of modified silica gel prepared in the step T1 into 100mL of water, performing ultrasonic dispersion for 10min at 800W, adding 0.1g of sodium persulfate under the protection of nitrogen, heating to 65 ℃, stirring and reacting for 3h, filtering, washing and drying to obtain the ferulic acid/abietic acid crosslinked silica gel.
Preparation example 2 preparation of ferulic acid/abietic acid crosslinked silica gel
The method comprises the following steps: t1. Modified silica gel: 10g of silica gel powder is added into 100mL of ethanol, 2g of silane coupling agent KH570 is added, the mixture is heated to 50 ℃, stirred and reacted for 2 hours, filtered, washed and dried, and the modified silica gel is prepared.
T2. Preparation of ferulic acid/abietic acid crosslinked silica gel: adding 6g of ferulic acid, 5g of abietic acid and 20g of modified silica gel prepared in the step T1 into 100mL of water, performing 1000W ultrasonic dispersion for 10min, adding 0.2g of potassium persulfate under the protection of nitrogen, heating to 75 ℃, stirring and reacting for 5h, filtering, washing and drying to obtain the ferulic acid/abietic acid crosslinked silica gel.
Preparation example 3 preparation of ferulic acid/abietic acid crosslinked silica gel
The method comprises the following steps: t1. Modified silica gel: 10g of silica gel powder is added into 100mL of ethanol, 1.5g of silane coupling agent KH570 is added, the mixture is heated to 45 ℃, stirred and reacted for 1.5h, filtered, washed and dried, and the modified silica gel is prepared.
T2. Preparation of ferulic acid/abietic acid crosslinked silica gel: adding 5g of ferulic acid, 4g of abietic acid and 17g of modified silica gel prepared in the step T1 into 100mL of water, performing 900W ultrasonic dispersion for 10min, adding 0.15g of ammonium persulfate under the protection of nitrogen, heating to 70 ℃, stirring and reacting for 4h, filtering, washing and drying to obtain the ferulic acid/abietic acid crosslinked silica gel.
Comparative preparation example 1
In comparison with preparation example 3, the difference is that no ferulic acid was added in step T2.
The method comprises the following steps: t1. Modified silica gel: 10g of silica gel powder is added into 100mL of ethanol, 1.5g of silane coupling agent KH570 is added, the mixture is heated to 45 ℃, stirred and reacted for 1.5h, filtered, washed and dried, and the modified silica gel is prepared.
T2. Preparation of abietic acid crosslinked silica gel: adding 9g of abietic acid and 17g of modified silica gel prepared in the step T1 into 100mL of water, performing ultrasonic dispersion for 10min at 900W, adding 0.15g of ammonium persulfate under the protection of nitrogen, heating to 70 ℃, stirring and reacting for 4h, filtering, washing and drying to obtain the abietic acid crosslinked silica gel.
Comparative preparation example 2
In comparison with preparation example 3, except that abietic acid was not added in step T2.
The method comprises the following steps: t1. Modified silica gel: 10g of silica gel powder is added into 100mL of ethanol, 1.5g of silane coupling agent KH570 is added, the mixture is heated to 45 ℃, stirred and reacted for 1.5h, filtered, washed and dried, and the modified silica gel is prepared.
T2. Preparation of ferulic acid crosslinked silica gel: adding 9g of ferulic acid and 17g of modified silica gel prepared in the step T1 into 100mL of water, performing ultrasonic dispersion for 10min at 900W, adding 0.15g of ammonium persulfate under the protection of nitrogen, heating to 70 ℃, stirring and reacting for 4h, filtering, washing and drying to obtain the ferulic acid crosslinked silica gel.
Preparation example 4 preparation of molecularly imprinted Polymer microspheres
The method comprises the following steps: mixing 1g of gastrodin, 15g of monomer, 0.5g of crosslinking agent ethylene glycol dimethacrylate, 1g of span-20 and 0.3g of pore-forming agent cetyl trimethyl ammonium chloride, adding into 100mL of acetonitrile, stirring and mixing for 15min, dripping into 200mL of water, emulsifying for 15min at 7000r/min, adding 0.1g of initiator under the protection of nitrogen, heating to 60 ℃, stirring and reacting for 3h, filtering the product, adding into a Soxhlet extractor, extracting until gastrodin is not detected by using a mixed solvent, washing, and drying to obtain the molecularly imprinted polymer microsphere. The initiator is a mixture of azodiisobutyronitrile and ammonium persulfate, and the mass ratio is 4:2. The monomer is a mixture of methyl methacrylate, 4-vinyl pyridine, acrylamide and styrene, and the mass ratio is 7:4:2:1; the mixed solvent is a mixed solution of ethanol and acetic acid according to the volume ratio of 8:1.
Preparation example 5 preparation of molecularly imprinted Polymer microspheres
The method comprises the following steps: mixing 2g of gastrodin, 18g of monomer, 1g of crosslinking agent ethylene glycol dimethacrylate, 2g of span-60 and 0.6g of pore-forming agent cetyl trimethyl ammonium chloride, adding into 100mL of acetonitrile, stirring and mixing for 15min, dripping into 200mL of water, emulsifying for 15min at 7000r/min, adding 0.2g of initiator under the protection of nitrogen, heating to 70 ℃, stirring and reacting for 5h, filtering the product, adding into a Soxhlet extractor, extracting until the gastrodin is not detected by a mixed solvent, washing and drying to obtain the molecularly imprinted polymer microsphere; the initiator is a mixture of azodiisobutyronitrile and ammonium persulfate, and the mass ratio is 6:2; the monomer is a mixture of methyl methacrylate, 4-vinyl pyridine, acrylamide and styrene, and the mass ratio is 10:6:4:3; the mixed solvent is a mixed solution of ethanol and acetic acid according to the volume ratio of 10:1.
Preparation example 6 preparation of molecularly imprinted Polymer microspheres
The method comprises the following steps: mixing 1.5g of gastrodin, 16.5g of monomer, 0.7g of cross-linking agent ethylene glycol dimethacrylate, 1.5g of span-85 and 0.45g of pore-forming agent cetyl trimethyl ammonium chloride, adding into 100mL of acetonitrile, stirring and mixing for 15min, dropwise adding into 200mL of water, emulsifying for 15min at 7000r/min, adding 0.15g of initiator under the protection of nitrogen, heating to 65 ℃, stirring and reacting for 4h, filtering the product, adding into a Soxhlet extractor, extracting until gastrodin is not detected by using a mixed solvent, washing and drying to obtain the molecularly imprinted polymer microsphere; the initiator is a mixture of azodiisobutyronitrile and ammonium persulfate, and the mass ratio is 5:2; the monomer is a mixture of methyl methacrylate, 4-vinyl pyridine, acrylamide and styrene, and the mass ratio is 8:5:3:2; the mixed solvent is a mixed solution of ethanol and acetic acid according to the volume ratio of 9:1.
Example 1
The embodiment provides a gastrodin extraction method, which comprises the following steps:
S1, enzymolysis of gastrodia elata: cleaning 10g of gastrodia elata, crushing, adding the crushed gastrodia elata into 70mL of water, adding 1g of complex enzyme, and carrying out enzymolysis for 2 hours at 40 ℃ to obtain an enzymolysis complex; the compound enzyme is cellulase and pectase, and the mass ratio is 10:7.
S2, ultrahigh pressure extraction: extracting the enzymolysis complex in the step S1 at an ultrahigh pressure of 100MPa for 15min, filtering, and concentrating the filtrate to obtain an extracting solution with a relative density of 1.05.
S3, primary purification: the extract obtained in the step S2 is primarily purified by a chromatographic column, wherein the filler of the chromatographic column is the ferulic acid/abietic acid crosslinked silica gel prepared in the preparation example 1, and a gastrodin primary pure product is obtained; filling a column by a wet method, filling ferulic acid/abietic acid crosslinked silica gel into a stainless steel chromatographic empty column (250 mm multiplied by 5 mm), filling the column for 20min under the pressure of 45MPa, filling the column for 15min under the pressure of 22MPa, unloading the chromatographic column after the column pressure is balanced, filling the column head and a sieve plate, adding the extracting solution into the column, and using acetonitrile-0.1 wt% phosphoric acid (2:98, v/v) as a mobile phase, wherein the flow rate is 0.5mL/min, and the column temperature is 25 ℃.
S4, fine purification of a molecularly imprinted polymer: purifying the crude gastrodin product prepared in the step S3 by using the molecularly imprinted polymer microspheres prepared in the preparation example 4 to obtain the pure gastrodin product; filling a column by adopting a wet method, filling molecularly imprinted polymer microspheres into a chromatographic column, wherein the column volume is 150mL, adding a gastrodin initial pure product into the column, eluting with an ethanol-acetic acid mixed solution (95:5, v/v) to obtain a mobile phase, wherein the flow rate is 0.3mL/min, and the column temperature is 25 ℃.
S5, preparing a gastrodin composition: dissolving 4g of the pure gastrodin prepared in the step S4 in 70mL of water to obtain a water phase; 2g of benzomarie and 2g of lecithin are dissolved in 50mL of ethanol and stirred and mixed for 10min to prepare an alcohol phase; dropwise adding the alcohol phase into the water phase by using a syringe, carrying out hydration reaction for 20min, and filtering by using a microporous filter membrane with the diameter of 0.22 mu m to obtain the gastrodin composition.
Example 2
The embodiment provides a gastrodin extraction method, which comprises the following steps:
S1, enzymolysis of gastrodia elata: cleaning 10g of gastrodia elata, crushing, adding the crushed gastrodia elata into 100mL of water, adding 2g of complex enzyme, and carrying out enzymolysis for 4 hours at 50 ℃ to obtain an enzymolysis complex; the compound enzyme is cellulase and pectase, and the mass ratio is 12:10.
S2, ultrahigh pressure extraction: extracting the enzymolysis complex in the step S1 at an ultrahigh pressure of 120MPa for 30min, filtering, and concentrating the filtrate to obtain an extracting solution with a relative density of 1.1.
S3, primary purification: the extract obtained in the step S2 is primarily purified by a chromatographic column, wherein the filler of the chromatographic column is the ferulic acid/abietic acid crosslinked silica gel prepared in the preparation example 2, and a gastrodin primary pure product is obtained; filling a column by a wet method, filling ferulic acid/abietic acid crosslinked silica gel into a stainless steel chromatographic empty column (250 mm multiplied by 5 mm), filling the column for 20min under the pressure of 45MPa, filling the column for 15min under the pressure of 22MPa, unloading the chromatographic column after the column pressure is balanced, filling the column head and a sieve plate, adding the extracting solution into the column, and using acetonitrile-0.1 wt% phosphoric acid (2:98, v/v) as a mobile phase, wherein the flow rate is 0.5mL/min, and the column temperature is 25 ℃.
S4, fine purification of a molecularly imprinted polymer: purifying the crude gastrodin product prepared in the step S3 by using the molecularly imprinted polymer microspheres prepared in the preparation example 5 to obtain the pure gastrodin product; filling a column by adopting a wet method, filling molecularly imprinted polymer microspheres into a chromatographic column, wherein the column volume is 150mL, adding a gastrodin initial pure product into the column, eluting with an ethanol-acetic acid mixed solution (95:5, v/v) to obtain a mobile phase, wherein the flow rate is 0.3mL/min, and the column temperature is 25 ℃.
S5, preparing a gastrodin composition: dissolving 6g of the pure gastrodin prepared in the step S4 in 70mL of water to obtain a water phase; dissolving 4g of benzomarie and 3g of lecithin in 50mL of ethanol, and stirring and mixing for 10min to prepare an alcohol phase; dropping the alcohol phase into the water phase with a syringe, carrying out hydration reaction for 40min, and filtering with a 0.22 μm microporous filter membrane to obtain the gastrodin composition.
Example 3
The embodiment provides a gastrodin extraction method, which comprises the following steps:
S1, enzymolysis of gastrodia elata: cleaning 10g of gastrodia elata, crushing, adding the crushed gastrodia elata into 85mL of water, adding 1.5g of complex enzyme, and carrying out enzymolysis for 3 hours at 45 ℃ to obtain an enzymolysis complex; the compound enzyme is cellulase and pectase, and the mass ratio is 11:8.
S2, ultrahigh pressure extraction: extracting the enzymolysis complex in the step S1 at an ultrahigh pressure of 110MPa for 20min, filtering, and concentrating the filtrate to obtain an extracting solution with a relative density of 1.07.
S3, primary purification: the extract obtained in the step S2 is primarily purified by a chromatographic column, wherein the filler of the chromatographic column is the ferulic acid/abietic acid crosslinked silica gel prepared in the preparation example 3, and a gastrodin primary pure product is obtained; filling a column by a wet method, filling ferulic acid/abietic acid crosslinked silica gel into a stainless steel chromatographic empty column (250 mm multiplied by 5 mm), filling the column for 20min under the pressure of 45MPa, filling the column for 15min under the pressure of 22MPa, unloading the chromatographic column after the column pressure is balanced, filling the column head and a sieve plate, adding the extracting solution into the column, and using acetonitrile-0.1 wt% phosphoric acid (2:98, v/v) as a mobile phase, wherein the flow rate is 0.5mL/min, and the column temperature is 25 ℃.
S4, fine purification of a molecularly imprinted polymer: purifying the crude gastrodin product prepared in the step S3 by using the molecularly imprinted polymer microspheres prepared in the preparation example 6 to obtain the pure gastrodin product; filling a column by adopting a wet method, filling molecularly imprinted polymer microspheres into a chromatographic column, wherein the column volume is 150mL, adding a gastrodin initial pure product into the column, eluting with an ethanol-acetic acid mixed solution (95:5, v/v) to obtain a mobile phase, wherein the flow rate is 0.3mL/min, and the column temperature is 25 ℃.
S5, preparing a gastrodin composition: dissolving 5g of the pure gastrodin prepared in the step S4 in 70mL of water to obtain a water phase; dissolving 3g of benzomarie and 2.5g of lecithin in 50mL of ethanol, and stirring and mixing for 10min to prepare an alcohol phase; dropping the alcohol phase into the water phase with a syringe, carrying out hydration reaction for 30min, and filtering with a 0.22 μm microporous filter membrane to obtain the gastrodin composition.
Comparative examples 1 to 2
The difference compared with example 3 is that the ferulic acid/abietic acid crosslinked silica gel is replaced by the abietic acid crosslinked silica gel prepared in comparative preparation examples 1 to 2, respectively.
Comparative example 3
The difference compared to example 3 is that the ferulic acid/abietic acid crosslinked silica gel is replaced by silica gel powder.
Comparative example 4
The difference compared to example 3 is that the complex enzyme is a single cellulase.
Comparative example 5
The difference compared to example 3 is that the complex enzyme is a single pectase.
Comparative example 6
In comparison with example 3, the difference is that no complex enzyme was added in step S1.
The method comprises the following steps: s1, extracting gastrodia elata: 10g of gastrodia elata is cleaned, crushed, added into 85mL of water, heated and boiled for extraction for 3h, and then the water extract is obtained.
Comparative example 7
In comparison with example 3, the difference is that step S2 is not performed.
The method comprises the following steps: s1, enzymolysis of gastrodia elata: washing 10g of gastrodia elata, crushing, adding the crushed gastrodia elata into 85mL of water, adding 1.5g of complex enzyme, carrying out enzymolysis for 3 hours at 45 ℃ to obtain an enzymolysis complex, filtering, and concentrating to obtain filtrate; the compound enzyme is cellulase and pectase, and the mass ratio is 11:8.
S2, primary purification: the filtrate obtained in the step S1 is primarily purified by a chromatographic column, wherein the filler of the chromatographic column is ferulic acid/abietic acid crosslinked silica gel prepared in the preparation example 3, and a gastrodin primary pure product is obtained; filling a column by a wet method, filling ferulic acid/abietic acid crosslinked silica gel into a stainless steel chromatographic empty column (250 mm multiplied by 5 mm), filling the column for 20min under the pressure of 45MPa, filling the column for 15min under the pressure of 22MPa, unloading the chromatographic column after the column pressure is balanced, filling the column head and a sieve plate, adding the extracting solution into the column, and using acetonitrile-0.1 wt% phosphoric acid (2:98, v/v) as a mobile phase, wherein the flow rate is 0.5mL/min, and the column temperature is 25 ℃.
S3, fine purification of a molecularly imprinted polymer: purifying the crude gastrodin product prepared in the step S2 by using the molecularly imprinted polymer microspheres prepared in the preparation example 6 to obtain the pure gastrodin product; filling a column by adopting a wet method, filling molecularly imprinted polymer microspheres into a chromatographic column, wherein the column volume is 150mL, adding a gastrodin initial pure product into the column, eluting with an ethanol-acetic acid mixed solution (95:5, v/v) to obtain a mobile phase, wherein the flow rate is 0.3mL/min, and the column temperature is 25 ℃.
S4, preparing a gastrodin composition: dissolving 5g of the pure gastrodin prepared in the step S3 in 70mL of water to obtain a water phase; dissolving 3g of benzomarie and 2.5g of lecithin in 50mL of ethanol, and stirring and mixing for 10min to prepare an alcohol phase; dropping the alcohol phase into the water phase with a syringe, carrying out hydration reaction for 30min, and filtering with a 0.22 μm microporous filter membrane to obtain the gastrodin composition.
Comparative example 8
In comparison with example 3, the difference is that step S3 is not performed.
The method comprises the following steps: s1, enzymolysis of gastrodia elata: cleaning 10g of gastrodia elata, crushing, adding the crushed gastrodia elata into 85mL of water, adding 1.5g of complex enzyme, and carrying out enzymolysis for 3 hours at 45 ℃ to obtain an enzymolysis complex; the compound enzyme is cellulase and pectase, and the mass ratio is 11:8.
S2, ultrahigh pressure extraction: extracting the enzymolysis complex in the step S1 at an ultrahigh pressure of 110MPa for 20min, filtering, and concentrating the filtrate to obtain an extracting solution with a relative density of 1.07.
S3, fine purification of a molecularly imprinted polymer: purifying the extract obtained in the step S2 by using the molecularly imprinted polymer microsphere prepared in the preparation example 6 to obtain a pure gastrodin product; filling a column by adopting a wet method, filling molecularly imprinted polymer microspheres into a chromatographic column, wherein the column volume is 150mL, adding a gastrodin initial pure product into the column, eluting with an ethanol-acetic acid mixed solution (95:5, v/v) to obtain a mobile phase, wherein the flow rate is 0.3mL/min, and the column temperature is 25 ℃.
S4, preparing a gastrodin composition: dissolving 5g of the pure gastrodin prepared in the step S3 in 70mL of water to obtain a water phase; dissolving 3g of benzomarie and 2.5g of lecithin in 50mL of ethanol, and stirring and mixing for 10min to prepare an alcohol phase; dropping the alcohol phase into the water phase with a syringe, carrying out hydration reaction for 30min, and filtering with a 0.22 μm microporous filter membrane to obtain the gastrodin composition.
Comparative example 9
In comparison with example 3, the difference is that step S4 is not performed.
The method comprises the following steps: s1, enzymolysis of gastrodia elata: cleaning 10g of gastrodia elata, crushing, adding the crushed gastrodia elata into 85mL of water, adding 1.5g of complex enzyme, and carrying out enzymolysis for 3 hours at 45 ℃ to obtain an enzymolysis complex; the compound enzyme is cellulase and pectase, and the mass ratio is 11:8.
S2, ultrahigh pressure extraction: extracting the enzymolysis complex in the step S1 at an ultrahigh pressure of 110MPa for 20min, filtering, and concentrating the filtrate to obtain an extracting solution with a relative density of 1.07.
S3, primary purification: the extract obtained in the step S2 is primarily purified by a chromatographic column, wherein the filler of the chromatographic column is the ferulic acid/abietic acid crosslinked silica gel prepared in the preparation example 3, and a gastrodin primary pure product is obtained; filling a column by a wet method, filling ferulic acid/abietic acid crosslinked silica gel into a stainless steel chromatographic empty column (250 mm multiplied by 5 mm), filling the column for 20min under the pressure of 45MPa, filling the column for 15min under the pressure of 22MPa, unloading the chromatographic column after the column pressure is balanced, filling the column head and a sieve plate, adding the extracting solution into the column, and using acetonitrile-0.1 wt% phosphoric acid (2:98, v/v) as a mobile phase, wherein the flow rate is 0.5mL/min, and the column temperature is 25 ℃.
S4, preparing a gastrodin composition: dissolving 5g of the gastrodin initial pure product prepared in the step S3 in 70mL of water to obtain a water phase; dissolving 3g of benzomarie and 2.5g of lecithin in 50mL of ethanol, and stirring and mixing for 10min to prepare an alcohol phase; dropping the alcohol phase into the water phase with a syringe, carrying out hydration reaction for 30min, and filtering with a 0.22 μm microporous filter membrane to obtain the gastrodin composition.
Comparative example 10
In comparison with example 3, there was no addition of benzomaric acid in step S5.
The method comprises the following steps: s5, preparing gastrodin ethosomes: dissolving 8g of the pure gastrodin prepared in the step S4 in 70mL of water to obtain a water phase; 2.5g of lecithin was dissolved in 50mL of ethanol, and stirred and mixed for 10min to prepare an alcohol phase; dropwise adding the alcohol phase into the water phase by using a syringe, carrying out hydration reaction for 30min, and filtering by using a microporous filter membrane with the diameter of 0.22 mu m to obtain the gastrodin ethosome.
Comparative example 11
Compared with example 3, the difference is that no pure gastrodin is added in step S5.
The method comprises the following steps: s5, preparation of a medicament: dissolving 8g of benzomarie and 2.5g of lecithin in 50mL of ethanol, and stirring and mixing for 10min to prepare an alcohol phase; adding the alcohol phase into water by syringe, hydrating for 30min, and filtering with 0.22 μm microporous membrane to obtain the final product.
Comparative example 12
In comparison with example 3, the difference is that step S5 is not performed.
The method comprises the following steps: s1, enzymolysis of gastrodia elata: cleaning 10g of gastrodia elata, crushing, adding the crushed gastrodia elata into 85mL of water, adding 1.5g of complex enzyme, and carrying out enzymolysis for 3 hours at 45 ℃ to obtain an enzymolysis complex; the compound enzyme is cellulase and pectase, and the mass ratio is 11:8.
S2, ultrahigh pressure extraction: extracting the enzymolysis complex in the step S1 at an ultrahigh pressure of 110MPa for 20min, filtering, and concentrating the filtrate to obtain an extracting solution with a relative density of 1.07.
S3, primary purification: the extract obtained in the step S2 is primarily purified by a chromatographic column, wherein the filler of the chromatographic column is the ferulic acid/abietic acid crosslinked silica gel prepared in the preparation example 3, and a gastrodin primary pure product is obtained; filling a column by a wet method, filling ferulic acid/abietic acid crosslinked silica gel into a stainless steel chromatographic empty column (250 mm multiplied by 5 mm), filling the column for 20min under the pressure of 45MPa, filling the column for 15min under the pressure of 22MPa, unloading the chromatographic column after the column pressure is balanced, filling the column head and a sieve plate, adding the extracting solution into the column, and using acetonitrile-0.1 wt% phosphoric acid (2:98, v/v) as a mobile phase, wherein the flow rate is 0.5mL/min, and the column temperature is 25 ℃.
S4, fine purification of a molecularly imprinted polymer: purifying the crude gastrodin product prepared in the step S3 by using the molecularly imprinted polymer microspheres prepared in the preparation example 6 to obtain the pure gastrodin product; filling a column by adopting a wet method, filling molecularly imprinted polymer microspheres into a chromatographic column, wherein the column volume is 150mL, adding a gastrodin initial pure product into the column, eluting with an ethanol-acetic acid mixed solution (95:5, v/v) to obtain a mobile phase, wherein the flow rate is 0.3mL/min, and the column temperature is 25 ℃.
Test example 1
The crude or pure gastrodin products prepared in examples 1 to 3 and comparative examples 1 to 9 were evaluated, and the extraction yield and purity of gastrodin in each method were evaluated, and the results are shown in Table 1.
TABLE 1
| Group of | Extraction yield of gastrodin (%) | Purity of gastrodin (%) |
| Example 1 | 1.42 | 99.3 |
| Example 2 | 1.44 | 99.1 |
| Example 3 | 1.45 | 99.5 |
| Comparative example 1 | 1.34 | 94.7 |
| Comparative example 2 | 1.32 | 94.1 |
| Comparative example 3 | 1.25 | 91.2 |
| Comparative example 4 | 1.10 | 98.9 |
| Comparative example 5 | 1.09 | 98.2 |
| Comparative example 6 | 0.97 | 98.0 |
| Comparative example 7 | 1.12 | 98.4 |
| Comparative example 8 | 1.39 | 93.2 |
| Comparative example 9 | 1.35 | 89.3 |
As shown in the table above, the pure gastrodin products prepared in examples 1-3 of the invention have high extraction rate and high purity.
Test example 2
SPF-grade KM mice (male and female halves) were randomly divided into a blank group, a model group and examples 1-3, and comparative examples 1-12, 10 mice per group, and the blank group, the model group and examples 1-3, and the comparative examples 1-12 mice were intraperitoneally injected with a 2wt% D-galactose solution for 35 days. On day 11, 50 mg/kg.d of the drug was administered to the patients in the groups of examples 1 to 3 and comparative examples 1 to 12, and the patients were continuously perfused with the same amount of physiological saline as the patients in the blank group and the model group.
1. Bench jump experiment
Bench jump experiments were performed on model mice 1h after day 35 dosing to determine the memory retention capacity of the mice.
Putting a mouse into a passive avoidance condition reflection box, electrifying the bottom of the reflection box, jumping the mouse onto a cork placed at the upper left corner, recording the number of times that the mouse jumps down the cork to be an error reaction after 5min, and recording the time of jumping down the cork for the first time as a latency exceeding 5min as 60min. The mice are trained 24 hours before the experiment starts, the mice are put into the reflecting box for 1min and then are electrified during the training, the time that the mice jump down the platform for the first time during the training is the reaction time, the situation during the training is recorded as the learning score of the mice, and the formal experiment situation is recorded as the memorizing score of the mice. The results are shown in Table 2.
TABLE 2
Annotation: * P <0.05 compared to the blank; # is P <0.05 compared to model group.
As shown in the table above, the gastrodin compositions prepared in examples 1-3 of the present invention can significantly prolong the reaction time and latency time and reduce the number of errors.
2. Morris water maze experiment
① Positioning navigation experiment: the study and memory ability of the mice was determined by a pilot voyage experiment performed 2h after the 35 th day of administration. The water maze is divided into 4 quadrants, the safety platform is arranged in one quadrant, water is injected into the water maze to be 1cm higher than the platform, and the whole camera connected with the display system is adjusted to enable the camera to clearly shoot the navigation track of the mouse. Training each group of mice before the formal experiment starts, recording the escape latency and the navigation track of the mice after the mice are launched from the launching to the boarding of the safety platform and lasting for 2s, if the mice are not launched on the safety platform for more than 60s, guiding the mice to the safety platform for 5s by an experimenter so as to enable the mice to memorize the position of the platform, and recording the escape latency of the mice as 60s; the results are shown in Table 3.
TABLE 3 Table 3
| Group of | Mouse escape latency(s) |
| Blank group | 13.2±2.4 |
| Model group | 47.5±4.9* |
| Piracetam group | 14.9±1.9# |
| Example 1 | 15.7±2.2# |
| Example 2 | 15.9±2.4# |
| Example 3 | 15.1±1.7# |
| Comparative example 1 | 16.2±3.2 |
| Comparative example 2 | 16.4±3.9 |
| Comparative example 3 | 16.9±3.5 |
| Comparative example 4 | 16.1±3.6 |
| Comparative example 5 | 16.3±4.1 |
| Comparative example 6 | 17.0±4.5 |
| Comparative example 7 | 16.9±4.3 |
| Comparative example 8 | 17.2±4.8 |
| Comparative example 9 | 17.9±5.1 |
| Comparative example 10 | 18.8±6.4 |
| Comparative example 11 | 17.1±5.2 |
| Comparative example 12 | 20.5±6.8 |
Annotation: * P <0.05 compared to the blank; # is P <0.05 compared to model group.
As shown in the table above, the gastrodin compositions prepared in examples 1-3 of the present invention can significantly shorten the escape latency of mice.
② Space search experiment: and removing the safety platform on the basis of the positioning navigation experiment, and keeping other steps consistent with the positioning navigation experiment. The results are shown in Table 4.
TABLE 4 Table 4
| Group of | Latency(s) | Stay original platform quadrant duration(s) | Number of passes through the original platform (times) |
| Blank group | 14.8±3.1 | 18.9±2.4 | 4.21±0.79 |
| Model group | 42.1±12.4* | 10.3±1.9* | 2.13±0.32* |
| Piracetam group | 20.5±5.2# | 15.2±2.1# | 3.45±0.29# |
| Example 1 | 17.4±2.9# | 14.9±2.3# | 3.12±0.24# |
| Example 2 | 18.1±2.2# | 15.1±2.0# | 3.27±0.31# |
| Example 3 | 17.0±1.8# | 15.6±1.7# | 3.34±0.19# |
| Comparative example 1 | 22.1±3.2 | 13.9±2.7 | 3.02±0.35 |
| Comparative example 2 | 22.4±3.5 | 13.1±2.9 | 2.97±0.34 |
| Comparative example 3 | 22.9±3.1 | 12.4±3.1 | 2.82±0.42 |
| Comparative example 4 | 21.4±4.2 | 12.9±3.5 | 2.90±0.45 |
| Comparative example 5 | 21.0±3.9 | 13.3±3.0 | 2.88±0.41 |
| Comparative example 6 | 22.7±5.2 | 13.7±2.9 | 2.82±0.38 |
| Comparative example 7 | 22.9±4.9 | 13.0±3.4 | 2.75±0.42 |
| Comparative example 8 | 23.4±5.3 | 13.2±3.2 | 2.79±0.44 |
| Comparative example 9 | 24.5±5.1 | 12.8±3.6 | 2.71±0.56 |
| Comparative example 10 | 29.7±7.2 | 12.0±3.1 | 2.62±0.43 |
| Comparative example 11 | 20.4±5.3 | 14.2±3.9 | 3.02±0.47 |
| Comparative example 12 | 32.2±6.8 | 11.5±2.9 | 2.53±0.52 |
Annotation: * P <0.05 compared to the blank; # is P <0.05 compared to model group.
As shown in the table above, the gastrodin compositions prepared in examples 1-3 of the present invention can significantly prolong the latency period, increase the time for staying in the quadrant of the original platform, and increase the times for crossing the original platform.
3. Preparation of mouse serum and hippocampal tissue
The eyeball blood of each group of mice is taken to be about 0.5mL after the administration for 1h on the 35 th day, and the mice are centrifuged, and the upper serum is taken for freezing storage at the temperature of minus 20 ℃. Immediately after blood collection, the brain is collected, washed, homogenized, and the supernatant is collected, and the TNF-alpha, T-SOD in the serum of the mice and the T-SOD in the tissues of the hippocampus are detected by ELISA. The results are shown in Table 5.
TABLE 5
| Group of | TNF-alpha content (ng/L) | Serum T-SOD content (U/mL) | Hippocampus T-SOD content (U/mL) |
| Blank group | 489.52±52.21 | 98.28±12.24 | 142.53±22.19 |
| Model group | 824.91±74.99* | 90.44±14.41* | 118.42±19.52* |
| Piracetam group | 798.35±63.84# | 103.25±10.48# | 136.19±20.41# |
| Example 1 | 524.48±60.81# | 97.11±14.23# | 137.04±24.21# |
| Example 2 | 529.81±58.41# | 96.92±13.29# | 136.82±21.48# |
| Example 3 | 520.48±62.24# | 99.05±12.48# | 139.14±22.84# |
| Comparative example 1 | 552.15±69.84 | 94.56±15.23 | 131.24±26.82 |
| Comparative example 2 | 555.34±71.12 | 95.06±15.11 | 133.39±27.11 |
| Comparative example 3 | 561.21±70.56 | 95.72±15.62 | 135.09±26.71 |
| Comparative example 4 | 567.82±65.21 | 92.25±17.82 | 130.12±25.78 |
| Comparative example 5 | 569.71±62.25 | 92.57±16.69 | 128.94±26.77 |
| Comparative example 6 | 576.25±59.75 | 92.06±17.51 | 126.25±25.22 |
| Comparative example 7 | 571.42±65.51 | 93.42±18.32 | 124.21±27.31 |
| Comparative example 8 | 568.92±62.24 | 93.10±16.86 | 125.04±28.15 |
| Comparative example 9 | 562.04±60.26 | 92.86±17.67 | 120.45±26.82 |
| Comparative example 10 | 579.62±62.55 | 91.67±16.88 | 120.62±28.35 |
| Comparative example 11 | 533.25±65.52 | 95.02±18.94 | 134.41±24.11 |
| Comparative example 12 | 588.65±70.31 | 91.06±15.72 | 119.24±24.48 |
Annotation: * P <0.05 compared to the blank; # is P <0.05 compared to model group.
As shown in the table above, the gastrodin compositions prepared in examples 1-3 of the present invention can significantly reduce TNF-alpha and increase the T-SOD content in serum and hippocampal tissues.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (9)
1. The preparation method of the gastrodin composition is characterized by comprising the following steps:
S1, enzymolysis of gastrodia elata: cleaning rhizoma Gastrodiae, pulverizing, adding into water, and adding complex enzyme for enzymolysis to obtain enzymolysis complex;
S2, ultrahigh pressure extraction: extracting the enzymolysis complex in the step S1 under ultrahigh pressure, filtering, and concentrating the filtrate to obtain an extracting solution;
S3, primary purification: the extract obtained in the step S2 is subjected to primary purification through a chromatographic column to obtain a gastrodin primary pure product;
the filler added into the chromatographic column is ferulic acid/abietic acid crosslinked silica gel, and the preparation method comprises the following steps:
t1. Modified silica gel: adding silica gel powder into ethanol, adding a silane coupling agent KH570, heating and stirring for reaction, filtering, washing and drying to obtain modified silica gel;
T2. Preparation of ferulic acid/abietic acid crosslinked silica gel: adding ferulic acid, abietic acid and the modified silica gel prepared in the step T1 into water, uniformly dispersing by ultrasonic, adding an initiator under the protection of inert gas, heating, stirring, reacting, filtering, washing and drying to obtain ferulic acid/abietic acid crosslinked silica gel;
s4, fine purification of a molecularly imprinted polymer: purifying the crude gastrodin product prepared in the step S3 by using molecular engram polymer microspheres to obtain the pure gastrodin product;
S5, preparing a gastrodin composition: dissolving the pure gastrodin product obtained in the step S4 in water to obtain a water phase; dissolving the benzomarie and lecithin in ethanol, and uniformly mixing to obtain an alcohol phase; dropping the alcohol phase into the water phase with a syringe, carrying out hydration reaction, and filtering with a microporous filter membrane to obtain the gastrodin composition.
2. The preparation method of claim 1, wherein in the step S1, the compound enzyme is cellulase and pectase, the mass ratio is 10-12:7-10, the enzymolysis temperature is 40-50 ℃, the time is 2-4h, and the solid-to-liquid ratio of the gastrodia elata to the water is 1:7-10g/mL.
3. The method according to claim 1, wherein the pressure of the ultra-high pressure extraction in step S2 is 100-120MPa, the extraction time is 15-30min, and the relative density of the extract is 1.05-1.1.
4. The preparation method according to claim 1, wherein the temperature of the heating and stirring reaction in the step T1 is 40-50 ℃ for 1-2 hours, and the mass ratio of the silica gel powder to the silane coupling agent KH570 is 10:1-2; in the step T2, the mass ratio of the ferulic acid to the abietic acid to the modified silica gel to the initiator is 4-6:3-5:15-20:0.1-0.2, the initiator is at least one of potassium persulfate, sodium persulfate and ammonium persulfate, the power of ultrasonic is 800-1000W, the temperature of the heating and stirring reaction is 65-75 ℃, and the time is 3-5h.
5. The method according to claim 1, wherein the method for preparing the molecularly imprinted polymer microsphere in step S4 comprises the following steps: mixing gastrodin, monomer, cross-linking agent, emulsifier and pore-forming agent, adding into acetonitrile, mixing uniformly, dripping into water, emulsifying, adding initiator under the protection of inert gas, heating, stirring, reacting, filtering the product, adding into Soxhlet extractor, extracting with mixed solvent until gastrodin is not detected, washing, and drying to obtain the molecularly imprinted polymer microsphere.
6. The preparation method according to claim 5, wherein the gastrodin, the monomer, the cross-linking agent, the emulsifying agent, the pore-forming agent and the initiator are in a mass ratio of 1-2:15-18:0.5-1:1-2:0.3-0.6:0.1-0.2, the initiator is a mixture of azobisisobutyronitrile and ammonium persulfate, the cross-linking agent is ethylene glycol dimethacrylate, the emulsifying agent is at least one selected from span-20, span-40, span-60, span-80 and span-85, the pore-forming agent is cetyl trimethyl ammonium chloride, the monomer is a mixture of methyl methacrylate, 4-vinyl pyridine, acrylamide and styrene, the temperature of the heating and stirring reaction is 60-70 ℃ for 3-5h, and the mixed solvent is a mixed solution of ethanol and acetic acid according to a volume ratio of 8-10:1.
7. The preparation method according to claim 1, wherein the mass ratio of the gastrodin pure product, the benzomarirope and the lecithin in the step S5 is 4-6:2-4:2-3, and the hydration reaction time is 20-40min.
8. A gastrodin composition prepared by the method of any one of claims 1-7.
9. Use of a gastrodin composition according to claim 8 for the preparation of a medicament for the prevention and treatment of senile dementia and parkinson's disease.
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