Preparation method for synthesizing gastrodin and gastrodigenin through double-enzyme catalysis and application of gastrodin and gastrodigenin

Abstract

The invention discloses a preparation method for synthesizing gastrodin and gastrodin through double-enzyme catalysis and application of the gastrodin and the gastrodin, and belongs to the technical field of compound extraction. The technical problems to be solved are that the chemical synthesis process of gastrodin and gastrodigenin is relatively high in safety risk, more in toxic and harmful reagent solvent residue, easy to generate by-products, limited in yield and low in purity, time-consuming and labor-consuming in extraction and separation from traditional Chinese medicinal materials, long in extraction time, low in extraction yield and purity, and low in effective content in medicines. According to the technical scheme, the method is characterized in that immobilized cellulase and lipase are adopted as biocatalysts to catalyze parishin compounds in the gastrodia elata extract to be converted into gastrodin and gastrodin, and the content of gastrodin and gastrodin in the gastrodia elata extract is increased, so that the drug effect is improved. The preparation process is green and environment-friendly, high selectivity is achieved, large-scale production can be achieved, the extraction efficiency and purity are improved, and the cost is reduced.

Description

Technical Field

[0001] This invention belongs to the field of compound extraction technology, specifically, it relates to a method for preparing gastrodin and gastrodin aglycone through dual-enzyme catalysis and their applications. Background Technology

[0002] Gastrodia elata contains polyphenolic compounds, polysaccharides, proteins, amino acids, and trace elements. Its chemical components include gastrodin, gastrodin aglycone (p-hydroxybenzyl alcohol), p-hydroxybenzaldehyde, barisonoside A, barisonoside B, barisonoside C, barisonoside D, and barisonoside E. Among these, gastrodin and gastrodin aglycone are chemical components that must be tested according to the Chinese Pharmacopoeia, with a combined content ≥0.25%. Gastrodin possesses various pharmacological activities, showing significant potential in neuroprotection, anti-epileptic activity, anti-inflammation, antidepressant activity, anti-tumor activity, and blood sugar regulation. Gastrodin aglycone exhibits anti-epileptic, neuroprotective, sedative, anti-inflammatory, antioxidant, free radical scavenging, neuroprotective, and tumor cell growth-inhibiting effects. After being absorbed into the bloodstream, gastrodin is broken down into gastrodin aglycone and glucose. Gastrodin and gastrodin aglycone have similar pharmacological effects and share the same main material basis for their pharmacological actions. They are widely used clinically in the treatment of diseases of the nervous, cardiovascular, and immune systems, with no significant toxic side effects.

[0003] Gastrodin has the chemical structure of p-hydroxymethylphenyl β-D-glucopyranoside, consisting of one molecule of β-D-glucopyranoside and one molecule of gastrodinogene linked by a glycosidic bond. Gastrodinogene has the chemical structure of p-hydroxybenzyl alcohol, containing a benzene ring (C6H5) with two hydroxyl (-OH) groups. In addition to the two chemical components specified in the Chinese Pharmacopoeia, Gastrodia elata also contains several other active components, including barisonoside A, barisonoside B, barisonoside C, barisonoside D, and barisonoside E, which also have high research value. Barisonoside A belongs to the glycoside class of compounds, consisting of one molecule of citric acid replacing three molecules of gastrodin via ester bonds at positions 1, 2, and 3. Barisonoside B consists of one molecule of citric acid replacing two molecules of gastrodin via ester bonds at positions 1 and 2. Barisonoside C has the same molecular formula as Barisonoside B, but differs in that Barisonoside B contains two glucose molecules, while Barisonoside C contains both glucose and rhamnose, and the glycosidic bonds are not entirely identical. Barisonoside D is obtained by esterification of one molecule of citric acid with two molecules of gastrodin. Barisonoside E consists of one molecule of citric acid replacing one molecule of gastrodin via an ester bond at position 1. These Barisonoside compounds are typically formed by the condensation of one molecule of citric acid with multiple gastrodin groups, belonging to the polyphenol or polyphenolic glycoside class of compounds. Barisonoside compounds and gastrodin can interconvert. Based on the fact that the chemical structure of Barison-like substances contains glycosidic bonds and / or ester bonds, these substances are chemically unstable and are easily destroyed and deactivated during processing.

[0004] Currently, gastrodin and gastrodin aglycone are mainly prepared through extraction and separation from traditional Chinese medicinal materials and chemical synthesis. However, both extraction and separation, and chemical synthesis have drawbacks. The extraction and separation of gastrodin and gastrodin from Gastrodia elata often involves decoction or soaking, which frequently uses organic solvents. This process is not only time-consuming and labor-intensive, but also results in long extraction times, low yields, and a large amount of waste liquid with high waste liquid treatment costs. Furthermore, the extracted substances contain not only the effective components but also other ineffective components. As the effective active ingredients, gastrodin and gastrodin aglycone cannot be fully extracted; in some cases, prolonged decoction or soaking can even cause the medicinal effects to dissipate, significantly reducing their efficacy in treating hypertension and protecting blood vessels. This inevitably leads to higher dosages, causing inconvenience for patients and limiting their market application. The chemical synthesis of gastrodin and gastrodin aglycone involves multiple reaction steps, uses toxic and harmful reagents, leaves significant solvent residues, easily generates byproducts, has limited yield, affects the purity of the final product, and is extremely costly.

[0005] Relevant patent documents retrieved:

[0006] This document, published in China (CN102206682A) on October 5, 2011, discloses a method for preparing gastrodin from Gastrodia elata. The method involves first impregnating Gastrodia elata powder with dilute hydrochloric acid, then performing enzymatic hydrolysis using a combination of β-glucosidase, pectinase, and cellulase as a complex enzyme. After drying, the mixture is subjected to supercritical CO2 extraction, followed by dissolution and crystallization using methanol upon heating. This invention has the advantages of simple operation and low energy consumption. However, this technique only yields gastrodin, while the pharmacologically active components in Gastrodia elata include gastrodin as well as gastrodinin.

[0007] This document, published in China (CN101531689B) on April 27, 2011, discloses a method for bio-enzymatic extraction of gastrodin. The method includes the following steps: pulverizing dried gastrodia elata, sieving it, adjusting the pH of the system, mixing it with a complex enzyme, adding alcohol, filtering, concentrating, and drying to obtain the final product. The complex enzyme comprises plant cell wall-breaking enzyme, amylase, pectinase, and protease. This extraction method is simple, lowers the extraction system temperature, reduces heating energy consumption and environmental pollution, and preserves some heat-volatile small-molecule pharmacologically active components, resulting in a more complete efficacy of gastrodin. However, this technique uses four enzymes, which obviously increases the extraction cost. Furthermore, the pharmacologically active components in gastrodia elata include gastrodin aglycone in addition to gastrodin.

[0008] Currently, there is a lack of a technical method that can simultaneously increase the content of gastrodin and gastrodin.

[0009] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: 1. Existing bio-enzymatic extraction methods for gastrodin use, which employ complex enzymes (plant cell wall-breaking enzymes, amylase, pectinase, and protease) to specifically degrade impurities such as starch, pectin, and protein in gastrodia, have different effective components and yields depending on the extraction method, resulting in insufficient versatility.

[0010] 2. Direct extraction with alcohol after enzymatic hydrolysis did not take into account the impact of residual enzyme proteins, polysaccharides and other impurities in the hydrolysate on the dissolution efficiency of gastrodin. This may result in some gastrodin encapsulated in the residue not being fully released, and there is still room for improvement in the extraction rate.

[0011] 3. Multiple heating and crystallization processes with methanol are required to improve product purity, but this also leads to a loss in product yield.

[0012] Other content that is useful for understanding, searching, and examining this invention: This technology overcomes the shortcomings of chemical synthesis, which involves separate preparation and organic solvent extraction. It directly decomposes barisoniside compounds from Gastrodia elata into gastrodin and gastrodin, thereby increasing the content of active pharmaceutical ingredients. Summary of the Invention

[0013] The purpose of this invention is to provide: A method for preparing gastrodin and gastrodin aglycone by dual-enzyme catalysis, their application, and related technologies are disclosed to address technical problems such as high safety risks, large amounts of toxic and harmful reagent and solvent residues, easy generation of by-products, limited yield and low purity in the chemical synthesis process of gastrodin and gastrodin aglycone, and time-consuming, labor-intensive, long extraction time, low extraction yield and low purity in traditional Chinese medicine, as well as low effective content in the drugs, or a combination thereof.

[0014] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0015] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0016] Unless otherwise stated, conventional methods within the scope of the art, such as transmission electron microscopy, X-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy, shall be used.

[0017] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0018] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0019] The term "Gastrodia elata" as used in this article refers to: Gastrodia elata, a plant belonging to the Orchidaceae family (Gastrodia elata). Gastrodia elata Bl. The dried tubers of Gastrodia elata. Gastrodia elata has the effects of calming the liver and relieving wind, and stopping spasms. It is used to treat headaches, dizziness, numbness of limbs, infantile convulsions, epileptic seizures, tetanus, and other symptoms.

[0020] The term "gastrodin" as used in this article refers to: p-hydroxymethylphenyl β-D-glucopyranoside, which is composed of one molecule of β-D-glucopyranoside and one molecule of gastrodin linked by a glycosidic bond. It is one of the effective chemical components in Gastrodia elata that must be tested according to the Chinese Pharmacopoeia. It has a variety of pharmacological activities, including neuroprotection, anti-epileptic, anti-inflammatory, antidepressant, anti-tumor, and blood sugar regulation.

[0021] The term "gastrodin" used in this article refers to p-hydroxybenzyl alcohol, which has a chemical structure containing one benzene ring (C6H5) with two hydroxyl (-OH) groups on the benzene ring. It is also known as p-hydroxybenzyl alcohol and is one of the effective chemical components in Gastrodia elata that must be tested according to the Chinese Pharmacopoeia. It has similar pharmacological effects to gastrodin and has anti-epileptic, neuroprotective, sedative, anti-inflammatory, antioxidant, free radical scavenging, and tumor cell growth inhibitory effects.

[0022] The term "barisonoside compounds" as used in this article refers to a class of active ingredients found in Gastrodia elata, including barisonoside A, barisonoside B, barisonoside C, barisonoside D, and barisonoside E. They are usually formed by the condensation of one molecule of citric acid with multiple molecules of gastrodin groups. They belong to the class of polyphenols or polyphenol glycosides, and their chemical structure contains glycosidic bonds and / or ester bonds. They are chemically unstable and can interconvert with gastrodin.

[0023] As used herein, the term "lipase" refers to a highly selective and environmentally friendly biocatalyst capable of hydrolyzing ester bonds in lipid molecules, converting lipid molecules into free fatty acids and glycerol (or other alcohols). In this invention, it can break the ester bonds of barisonin to obtain one molecule of citric acid and multiple gastrodins.

[0024] The term "cellulase" as used in this article refers to an enzyme that converts cellulose (a polysaccharide composed of glucose units linked by β-1,4-glycosidic bonds) by hydrolyzing glycosidic bonds. This disrupts the integrity and permeability of the cell wall, making it easier for encapsulated phenolic components (such as gastrodin) to dissolve.

[0025] The term "enzyme immobilization technology" used in this article refers to a technology that uses physical or chemical methods to position free enzymes in a defined spatial region, maintain their activity, and allow them to be reused repeatedly. It has advantages such as increasing enzyme density per unit volume, enhancing enzyme tolerance to the environment (such as pH, temperature, time, etc.), easy enzyme recovery, and reusability.

[0026] In a first aspect, the present invention provides: a method for preparing gastrodin and gastrodin by dual-enzyme catalysis, comprising: immobilizing free cellulase and lipase to obtain immobilized cellulase and immobilized lipase; obtaining freeze-dried powder of gastrodin extract using gastrodin as raw material; and then, using the freeze-dried powder of gastrodin extract as raw material, synthesizing gastrodin and gastrodin by catalyzing the immobilized cellulase and immobilized lipase into barisonin substances in gastrodin extract.

[0027] As a preferred embodiment, the method for preparing the immobilized lipase includes the following steps: (1.1) Dissolve Fe3O4 nanoparticles in water, sonicate, and then add dopamine hydrochloride to obtain solution 1; (1.2) Adjust the pH of solution 1 prepared in step (1.1), stir, and obtain precipitate 1; (1.3) Wash the precipitate 1 obtained in step (1.2) several times until the supernatant is clear to obtain Fe3O4@dopamine hydrochloride, freeze dry and set aside; (1.4) The Fe3O4@dopamine hydrochloride and lipase prepared in step (1.3) are added to phosphate buffer and fixed for a period of time to obtain precipitate 2; (1.5) Wash and freeze-dry the precipitate 2 obtained in step (1.4) for later use.

[0028] Preferably, the molar ratio of Fe3O4 nanoparticles and dopamine hydrochloride in step (1.1) is selected from any value or range between 1-5:1-5, specifically from: 1:1, 1:2, 1:3, 1:4, 1:5, 3:4, 4:2, 2:3, 5:4, 5:5, or any two of them.

[0029] More preferably, the molar ratio of Fe3O4 nanoparticles and dopamine hydrochloride in step (1.1) is selected from any value or range between 1-3:1-3.

[0030] More preferably, the mass ratio of the Fe3O4 nanoparticles in step (1.1) to the lipase in step (1.4) is 1:1.

[0031] Preferably, the water in step (1.1) is deionized water.

[0032] Preferably, the mass of water added in step (1.1) is 100 times that of the Fe3O4 nanoparticle solution.

[0033] Preferably, the power of the ultrasonic treatment in step (1.1) is 200W.

[0034] Preferably, the time for the ultrasonic treatment in step (1.1) is selected from any value or range between 8 and 12 minutes.

[0035] More preferably, the ultrasound time in step (1.1) is selected from any value or range between 8 and 12 minutes, specifically from: 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes or any two of them.

[0036] More preferably, the ultrasound time in step (1.1) is selected from any value or range between 8 and 12 minutes, specifically from 8 minutes, 10 minutes, 12 minutes or any two of them.

[0037] More preferably, the ultrasound time in step (1.1) is 10 minutes.

[0038] Preferably, the specific operation of adjusting pH in step (1.2) is as follows: use 0.1 mol / L NaOH solution to adjust the pH of solution 1 prepared in step (1.1) to 8.5.

[0039] Preferably, the stirring speed in step (1.2) is selected from any value or range between 150-300 r / min.

[0040] More preferably, the stirring speed in step (1.2) is selected from any value or range between 150-300 r / min, specifically from: 150 r / min, 160 r / min, 170 r / min, 180 r / min, 190 r / min, 200 r / min, 220 r / min, 230 r / min, 240 r / min, 260 r / min, 280 r / min, 300 r / min or any two of them.

[0041] More preferably, the stirring speed in step (1.2) is selected from any value or range between 150-300 r / min, specifically from: 150 r / min, 200 r / min, 300 r / min or any two of them.

[0042] More preferably, the stirring speed in step (1.2) is 200 r / min.

[0043] Preferably, the stirring time in step (1.2) is selected from any value or range between 22 and 24 hours.

[0044] More preferably, the stirring time in step (1.2) is selected from any value or range between 22-24h, specifically from 22h, 23h, 24h or any two of them.

[0045] More preferably, the stirring time in step (1.2) is 23 hours.

[0046] Preferably, the precipitate 1 in step (1.2) is obtained by magnetic adsorption.

[0047] Preferably, the washing operation in step (1.3) is as follows: the precipitate 1 obtained in step (1.2) is washed multiple times with deionized water until the supernatant is clear.

[0048] Preferably, the number of times the washing is performed in step (1.3) is 1-3 times.

[0049] More preferably, the washing process in step (1.3) is performed three times.

[0050] Preferably, the freeze-drying process described in step (1.3) can be stored in a sealed container at 4°C.

[0051] Preferably, the mass-volume ratio of Fe3O4@dopamine hydrochloride, lipase and phosphate buffer in step (1.4) is selected from any value or range between 1-5:1-5:1-5, specifically from: 1:1:1, 3:1:1, 3:4:3, 4:1:1, 4:3:2, 4:3:5, 4:5:5, 5:1:1, 5:2:1, 5:2:5, 5:4:5 or any two of them.

[0052] More preferably, the mass-volume ratio of Fe3O4@dopamine hydrochloride, lipase and phosphate buffer in step (1.4) is selected from any value or range between 1-3:1-3:1-3.

[0053] More preferably, the mass-to-volume ratio of Fe3O4@dopamine hydrochloride, lipase and phosphate buffer in step (1.4) is 1:1:1.

[0054] Preferably, the pH of the phosphate buffer solution described in step (1.4) is 6.0.

[0055] Preferably, the specific operation of the fixation in step (1.4) is as follows: during the fixation period, the stirring is continuously carried out at a speed of 250 r / min.

[0056] Preferably, the fixed time mentioned in step (1.4) is selected from any value or range between 5 and 10 hours, specifically from: 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours or any two of them.

[0057] More preferably, the fixed time mentioned in step (1.4) is selected from any value or range between 5 and 7 hours.

[0058] Preferably, the fixed time in step (1.4) is 6 hours.

[0059] Preferably, the precipitate 2 in step (1.4) is obtained by magnetic adsorption.

[0060] Preferably, the specific operation described in step (1.5) is as follows: wash the precipitate 2 repeatedly with deionized water until the supernatant no longer contains protein, freeze-dry it, and then seal and store it at 4°C for later use.

[0061] As the most preferred embodiment, the method for preparing the immobilized lipase includes the following steps: (1.1) Dissolve Fe3O4 nanoparticles in dialysis equilibrium in 100 times the amount of deionized water, sonicate for 10 min, and then add dopamine hydrochloride. The molar ratio of Fe3O4 nanoparticles to dopamine hydrochloride is 1:1 to obtain solution 1. (1.2) Adjust the pH of solution 1 prepared in step (1.1) to 8.5 with 0.1 mol / L NaOH solution, stir at room temperature for 23 h at a speed of 200 r / min, and then obtain precipitate 1 by magnetic means. (1.3) Wash the precipitate 1 obtained in step (1.2) three times until the supernatant is clear to obtain Fe3O4@dopamine hydrochloride. After freeze-drying, store it in a sealed container at 4°C for later use. (1.4) Add the Fe3O4@dopamine hydrochloride and lipase prepared in step (1.3) to the reaction flask and then add phosphate buffer with pH=6.0. The mass-volume ratio of Fe3O4@dopamine hydrochloride, lipase and phosphate buffer is 1:1:1. Fix for 6h. Stir continuously during the immobilization period at a speed of 250r / min. After the immobilization is completed, use magnetism to obtain precipitate 2. (1.5) Wash the precipitate 2 obtained in step (1.4) with deionized water multiple times until the supernatant no longer contains protein. After freeze-drying, seal and store at 4°C for later use.

[0062] As a preferred embodiment, the method for preparing the immobilized cellulase includes the following steps: Fe2O3 nanoparticles were added to a cellulase solution containing polyvinyl alcohol, stirred, rapidly frozen, thawed, and repeatedly frozen and thawed. NaCl was then added for precipitation, followed by adsorption separation, washing, precipitation, and freeze-drying for later use.

[0063] Preferably, the stirring temperature is selected from any value or range between 1 and 10°C, specifically from: 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C or any two of them.

[0064] More preferably, the stirring temperature is selected from any value or range between 1 and 5°C.

[0065] More preferably, the stirring temperature is 4°C.

[0066] Preferably, the stirring time is selected from any value or range between 5 and 15 hours, specifically from: 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours or any two of these ranges.

[0067] More preferably, the stirring time is selected from any value or range between 7 and 12 hours.

[0068] Preferably, the preparation method of the immobilized cellulase is as follows: Fe2O3 nanoparticles of equal mass to the cellulase and in dialysis equilibrium are added to a cellulase solution containing 0.5% polyvinyl alcohol at a mass fraction of 30 mg / mL. After stirring at 4°C for 10 h, the solution is rapidly frozen at -80°C, then thawed on ice. This freeze-thaw cycle is repeated 3 times. 0.5 mol / L NaCl is added for precipitation, and the solution is separated by magnetic adsorption. The solution is washed 3 times with ultrapure water, the precipitate is collected, freeze-dried, and stored at 4°C for later use.

[0069] As a preferred embodiment, the method for preparing the freeze-dried powder of Gastrodia elata extract includes the following steps: (3.1) Select fresh Gastrodia elata, wash, peel, slice, steam, dry, and grind to obtain Gastrodia elata powder for later use; (3.2) Add water to the gastrodia powder obtained in step (3.1), reflux to extract, let stand, centrifuge to obtain supernatant, extract the remaining residue again, combine the filtrates to obtain solution A; (3.3) Add activated carbon to solution A from step (3.2), stir, let stand, filter and decolorize to obtain filtrate B; (3.4) After freezing and solidifying the filtrate B, freeze-dry it to obtain freeze-dried powder of Gastrodia elata extract.

[0070] Preferably, the specific operation of step (3.1) is as follows: select fresh, disease-free Gastrodia elata tubers, wash them, peel them, slice them into 3-5 mm pieces, steam them for 15-30 minutes, dry them at low temperature for 24-48 hours, pulverize them through a 100-mesh sieve to obtain Gastrodia elata powder, and store it at 4℃ for later use.

[0071] Preferably, the ratio of gastrodia elata powder to water in step (3.2) is selected from any value or range between 1g:30-44mL.

[0072] More preferably, the ratio of gastrodia elata powder to water in step (3.2) is selected from any value or range between 1g:30-44mL, specifically from: 1g:30mL, 1g:31mL, 1g:32mL, 1g:33mL, 1g:34mL, 1g:35mL, 1g:36mL, 1g:37mL, 1g:38mL, 1g:39mL, 1g:40mL, 1g:41mL, 1g:42mL, 1g:43mL, 1g:44mL or any two of them.

[0073] More preferably, the ratio of gastrodia elata powder to water in step (3.2) is selected from any value or range between 1g:30-44mL, specifically from: 1g:30mL, 1g:37mL, 1g:44mL or any two of them.

[0074] More preferably, the ratio of gastrodia elata powder to water in step (3.2) is 1g:37mL.

[0075] Preferably, the reflux extraction temperature in step (3.2) is selected from any value or range between 90-110°C.

[0076] More preferably, the reflux extraction temperature in step (3.2) is selected from any value or range between 90-110℃, specifically from: 90℃, 91℃, 92℃, 93℃, 94℃, 95℃, 96℃, 97℃, 98℃, 99℃, 100℃, 101℃, 102℃, 103℃, 104℃, 105℃, 106℃, 107℃, 108℃, 109℃, 110℃ or any two of them.

[0077] More preferably, the reflux extraction temperature in step (3.2) is selected from any value or range between 90-110℃, specifically from: 90℃, 91℃, 94℃, 95℃, 96℃, 97℃, 100℃, 101℃, 102℃, 103℃, 108℃, 109℃, 110℃ or any two of them.

[0078] More preferably, the reflux extraction temperature in step (3.2) is selected from any value or range between 90-110℃, specifically from: 90℃, 100℃, 110℃ or any two of them.

[0079] More preferably, the reflux extraction temperature in step (3.2) is 100°C.

[0080] Preferably, in step (3.2), the reflux extraction time for each reflux extraction is selected from any value or range between 1 and 3 hours.

[0081] More preferably, in step (3.2), the reflux extraction time for each reflux extraction is selected from any value or range between 1 and 3 hours, specifically from: 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours or any two of them.

[0082] More preferably, in step (3.2), the reflux extraction time for each reflux extraction is selected from any value or range between 1 and 3 hours, specifically from: 1 hour, 1.5 hours, 3 hours or any two of them.

[0083] More preferably, the reflux extraction in step (3.2) takes 1.5 hours each time.

[0084] Preferably, the centrifugation temperature in step (3.2) is selected from any value or range between 2-8°C.

[0085] More preferably, the centrifugation temperature in step (3.2) is selected from any value or range between 2 and 8°C, specifically from: 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C or any two of them.

[0086] More preferably, the centrifugation temperature in step (3.2) is selected from any value or range between 2-8℃, specifically from: 2℃, 4℃, 8℃ or any two of them.

[0087] More preferably, the centrifugation temperature in step (3.2) is 4°C.

[0088] Preferably, the centrifugation speed in step (3.2) is selected from any value or range between 3000-5000 r / min.

[0089] More preferably, the centrifugation speed in step (3.2) is selected from any value or range between 3000-5000 r / min, specifically from: 3000 r / min, 3500 r / min, 4000 r / min, 4500 r / min, 5000 r / min or any two of them.

[0090] More preferably, the centrifugation speed in step (3.2) is selected from any value or range between 3000-5000 r / min, specifically from: 3000 r / min, 4000 r / min, 5000 r / min or any two of them.

[0091] More preferably, the centrifugation speed in step (3.2) is 4000 r / min.

[0092] Preferably, the centrifugation time in step (3.2) is selected from any value or range between 5 and 15 min.

[0093] More preferably, the centrifugation time in step (3.2) is selected from any value or range between 5 and 15 min, specifically from: 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min or any two of them.

[0094] More preferably, the centrifugation time in step (3.2) is selected from any value or range between 5 and 15 min, specifically from 5 min, 10 min, 15 min or any two of them.

[0095] More preferably, the centrifugation time in step (3.2) is 10 minutes.

[0096] Preferably, the number of times the extraction is repeated in step (3.2) is 3.

[0097] Preferably, the mass of activated carbon added in step (3.3) is selected from 1-3% of the mass of solution A.

[0098] More preferably, the mass of activated carbon added in step (3.3) is selected from 1-3% of the mass of solution A, specifically from 1%, 2%, 3% or any two of them.

[0099] More preferably, the mass of activated carbon added in step (3.3) is selected from 1% of the mass of solution A.

[0100] Preferably, the stirring temperature in step (3.3) is selected from any value or range between 10-30°C.

[0101] More preferably, the stirring temperature in step (3.3) is selected from any value or range between 10-30℃, specifically from: 10℃, 21℃, 30℃ or any two of them.

[0102] More preferably, the stirring temperature in step (3.3) is 21°C.

[0103] Preferably, the stirring time in step (3.3) is selected from any value or range between 30 and 40 minutes.

[0104] More preferably, the stirring time in step (3.3) is selected from any value or range between 30 and 40 min, specifically from 30 min, 35 min, 40 min or any two of them.

[0105] More preferably, the stirring time in step (3.3) is 35 minutes.

[0106] Preferably, the freezing temperature in step (3.4) is selected from any value or range between -80°C and -60°C.

[0107] More preferably, the freezing temperature in step (3.4) is selected from any value or range between -80℃ and -60℃, specifically from: -80℃, -75℃, -70℃, -65℃, -60℃ or any two of them.

[0108] More preferably, the freezing temperature in step (3.4) is selected from any value or range between -80℃ and -60℃, specifically from -80℃, -70℃, -60℃ or any two of them.

[0109] More preferably, the freezing temperature in step (3.4) is -80°C.

[0110] Preferably, the freezing time in step (3.4) is selected from any value or range between 10 and 14 hours.

[0111] More preferably, the freezing time in step (3.4) is selected from any value or range between 10 and 14 hours, specifically from: 10 hours, 10.5 hours, 11 hours, 11.5 hours, 12 hours, 12.5 hours, 13 hours, 13.5 hours, 14 hours or any two of them.

[0112] More preferably, the freezing time in step (3.4) is selected from any value or range between 10-14h, specifically from: 10h, 11h, 12h, 13h, 14h or any two of them.

[0113] Preferably, the freezing time in step (3.4) is 12 hours.

[0114] Preferably, the drying time in step (3.4) is selected from any value or range between 45 and 50 hours.

[0115] More preferably, the drying time in step (3.4) is selected from any value or range between 45 and 50 hours, specifically from 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours or any two of them.

[0116] More preferably, the drying time in step (3.4) is selected from any value or range between 45 and 50 hours, specifically from 45 hours, 48 ​​hours, 50 hours or any two of them.

[0117] More preferably, the drying time in step (3.4) is 48 hours.

[0118] As the preferred embodiment, the method for preparing the freeze-dried powder of Gastrodia elata extract includes the following steps: (3.1) Select fresh, disease-free Gastrodia elata tubers, wash, peel, slice into 3-5 mm pieces, steam for 15-30 min, dry at low temperature for 24-48 h, and pulverize through a 100-mesh sieve to obtain Gastrodia elata powder, which is stored at 4℃ for later use. (3.2) Weigh 200g of dried Gastrodia elata powder and place it in a round-bottom flask. Add water at a ratio of 1g:37mL (w / v) and reflux at 100℃ for 1.5h. After the sample returns to room temperature, centrifuge at 4000r / min for 10min at 4℃ and collect the supernatant. Extract the remaining residue again using the above method. Repeat this process 3 times and combine the filtrates from the above 3 times to obtain solution A. (3.3) Add 1% activated carbon to solution A in step (3.2), stir at 21°C, let stand for 35 min, filter and decolorize to obtain filtrate B; (3.4) After freezing and solidifying the filtrate B at -80℃ for 12 h, freeze-dry it for 48 h to obtain the freeze-dried powder of Gastrodia elata extract; As a preferred embodiment, the synthesis of gastrodin and gastrodinogen from barisonin in Gastrodia elata extract specifically includes the following steps: (4.1) The immobilized lipase, immobilized cellulase and freeze-dried powder of Gastrodia elata extract were mixed with water, the pH was adjusted, and then heated and shaken for a period of time before being reacted at a constant temperature to obtain material C; (4.2) Centrifuge material C and collect the supernatant to recover the product.

[0119] Preferably, the molar ratio of the freeze-dried Gastrodia elata extract powder, the immobilized cellulase and the immobilized lipase in step (4.1) is selected from any value or range between 100:2.0-5.0:1.0-2.0.

[0120] More preferably, the molar ratio of the freeze-dried Gastrodia elata extract powder, the immobilized cellulase, and the immobilized lipase in step (4.1) is selected from any value or range between 100:2.0-5.0:1.0-2.0, specifically from: 100:2.0:1.0, 100:3.0:1.0, 100:4.0:1.0, 100:5.0:1.0, 100:2.0:2.0, 100:3.0:2.0, 100:4.0:2.0, 100:5.0:2.0, or any two of them.

[0121] More preferably, the molar ratio of the freeze-dried Gastrodia elata extract powder, the immobilized cellulase, and the immobilized lipase in step (4.1) is selected from any value or range between 100:2.0-5.0:1.0-2.0, specifically from: 100:2.0:1.0, 100:2.0:2.0, 100:3.0:1.0, 100:5.0:1.0, 100:5.0:2.0, or any two of them.

[0122] More preferably, the molar ratio of the freeze-dried Gastrodia elata extract powder, the immobilized cellulase and the immobilized lipase in step (4.1) is 100:5.0:1.0.

[0123] Secondly, the present invention provides: a gastrodin and gastrodinogen prepared by the above-described preparation method.

[0124] Thirdly, the present invention provides the use of gastrodin and gastrogenin in the preparation of medicaments for treating diseases of the nervous system, cardiovascular system or immune system.

[0125] Preferably, the nervous system is Alzheimer's disease, Parkinson's disease, or epilepsy.

[0126] Preferably, the cardiovascular system is ischemic brain injury, vascular dementia, ischemic myocardial injury, hypertension, or arteriosclerosis.

[0127] Preferably, the immune system disease is rheumatoid arthritis.

[0128] The beneficial effects of this invention are as follows: The present invention has at least the following beneficial effects: 1. This invention promotes the conversion of barisonin in Gastrodia elata extract into gastrodin and gastrodin by using lipase and cellulase in combination. This is beneficial for improving efficiency, reducing costs, reducing pollution, and increasing the content of gastrodin and gastrodin in large-scale production.

[0129] 2. This invention improves enzyme density per unit volume by positioning lipase and cellulase in a defined spatial region to maintain their activity. It also enhances the enzyme's tolerance to environmental factors (such as pH, temperature, and time), facilitates enzyme recovery, and allows for repeated use.

[0130] 3. The preparation method of the present invention has a high yield, is easy to synthesize, and is a green and simple process with significant practical application effects.

[0131] 4. The immobilized lipase prepared by this invention has a more compact particle structure without changing the particle shape; the prepared immobilized cellulase is a solidified complex similar to a gel cluster, which is more flexible. Attached Figure Description

[0132] Figure 1 Diagram of lipase and cellulase cleavage sites of barisonoside bonds.

[0133] Figure 2 Transmission electron microscopy images of immobilized enzymes, where A is Fe3O4; B is Fe3O4@dopamine hydrochloride; C is Fe3O4@dopamine hydrochloride@lipase; D is Fe2O3; and E is Fe2O3@polyvinyl alcohol@cellulase.

[0134] Figure 3 Particle size analysis diagram of immobilized enzymes, where A is Fe3O4; B is Fe3O4@dopamine hydrochloride; C is Fe3O4@dopamine hydrochloride@lipase; D is Fe2O3; E is Fe2O3@polyvinyl alcohol@cellulase.

[0135] Figure 4 XPS analysis of Fe3O4.

[0136] Figure 5 XPS analysis chromatogram of Fe3O4@dopamine hydrochloride.

[0137] Figure 6 XPS analysis of Fe3O4@dopamine hydrochloride@lipase.

[0138] Figure 7 X-ray photoelectron C-spectrum of Fe3O4@dopamine hydrochloride@lipase.

[0139] Figure 8 The N-spectrum of X-ray photoelectrons of Fe3O4@dopamine hydrochloride@lipase.

[0140] Figure 9 X-ray photoelectron spectroscopy of Fe3O4@dopamine hydrochloride@lipase.

[0141] Figure 10 Fourier transform infrared spectroscopy analysis of immobilized enzymes, where (a) is Fe3O4; (b) is Fe3O4@dopamine hydrochloride; (c) is Fe3O4@dopamine hydrochloride@lipase; (d) is Fe2O3; and (e) is Fe2O3@polyvinyl alcohol@cellulase.

[0142] Figure 11 X-ray diffraction analysis of immobilized enzymes, where A is Fe3O4; B is Fe3O4@dopamine hydrochloride; and C is Fe3O4@dopamine hydrochloride@lipase.

[0143] Figure 12 Thermogravimetric analysis of immobilized enzymes, where A: Fe3O4; B: Fe3O4@dopamine hydrochloride; C: Fe3O4@dopamine hydrochloride@lipase; D: dopamine hydrochloride; E: Fe2O3; F: Fe2O3@polyvinyl alcohol@cellulase; G: polyvinyl alcohol.

[0144] Figure 13 Chromatograms of gastrodin and gastrodinogenin prepared in Example 1. Detailed Implementation

[0145] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0146] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0147] Table 1

[0148] Table 2 Main Experimental Instruments

[0149] Example 1: Preparation of gastrodin and gastrodin aglycone (1) A method for preparing immobilized lipase, comprising the following steps: (1.1) An equal volume of Fe3O4 nanoparticle solution in dialysis equilibrium with lipase was placed in 100 times the amount of deionized water, sonicated for 10 min, and then dopamine hydrochloride was added. The molar ratio of Fe3O4 nanoparticles to dopamine hydrochloride was 1:1 to obtain solution 1. (1.2) Adjust the pH of solution 1 prepared in step (1.1) to 8.5 with 0.1 mol / L NaOH solution, stir at room temperature for 23 h at a speed of 200 r / min, and then obtain precipitate 1 by magnetic means. (1.3) Wash the precipitate 1 obtained in step (1.2) several times until the supernatant is clear to obtain Fe3O4@dopamine hydrochloride. After freeze-drying, store it in a sealed container at 4°C for later use. (1.4) Add the Fe3O4@dopamine hydrochloride and lipase prepared in step (1.3) to the reaction flask and then add phosphate buffer with pH=6.0. The mass-volume ratio of Fe3O4@dopamine hydrochloride, lipase and phosphate buffer is 1:1:1. Fix for 6h. Stir continuously during the immobilization period at a speed of 250r / min. After the immobilization is completed, use magnetism to obtain precipitate 2. (1.5) Wash the precipitate 2 obtained in step (1.4) with deionized water multiple times until the supernatant no longer contains protein. After freeze-drying, seal and store at 4°C for later use.

[0150] The method for preparing immobilized cellulase includes the following steps: An equal amount of dialysis-equilibrated Fe2O3 nanoparticles, along with cellulase, were added to a cellulase solution containing 0.5% polyvinyl alcohol (30 mg / mL). The mixture was stirred at 4°C for 10 h, then rapidly frozen at -80°C, thawed on ice, and subjected to three freeze-thaw cycles. A 0.5 mol / L NaCl solution was added to precipitate the precipitate, followed by adsorption separation using a magnet. The precipitate was washed three times with ultrapure water, freeze-dried, and stored at 4°C for later use.

[0151] (3) The preparation method of freeze-dried powder of Gastrodia elata extract includes the following steps: (3.1) Select fresh, disease-free Gastrodia elata tubers, wash, peel, slice into 3-5 mm pieces, steam for 15-30 min, dry at low temperature for 24-48 h, and pulverize through a 100-mesh sieve to obtain Gastrodia elata powder, which is stored at 4℃ for later use. (3.2) Weigh the Gastrodia elata powder obtained in step (3.1) and add purified water, wherein the mass-volume ratio of Gastrodia elata powder to purified water is 1g:37mL. Reflux extract at 100℃ for 1.5h. After extraction, let stand until the sample returns to room temperature. Then centrifuge at 4000r / min for 10min at 4℃ to obtain supernatant. Repeat the reflux extraction on the remaining filter residue 3 times. Combine the filtrates to obtain solution A. (3.3) Add 1% activated carbon to solution A in step (3.2), control the temperature at 21℃, stir with a magnetic stirrer for 35 min, let stand after stirring, filter and decolorize to obtain filtrate B; (3.4) Filtrate B was frozen at -80℃ for 12 hours, and then dried for 48 hours to obtain freeze-dried powder of Gastrodia elata extract. (4) The preparation method of gastrodin and gastrodin aglycone includes the following steps: (4.1) Add water to the immobilized lipase, immobilized cellulase and freeze-dried powder of Gastrodia elata extract and stir. The molar ratio of freeze-dried powder of Gastrodia elata extract, cellulase and lipase is 100:5:1. Adjust the pH to 8.0, heat to 40°C, place in a fingertip turbine shaker and shake, then place in a constant temperature incubator for 7 hours to obtain material C. (4.2) After the reaction is complete, place the material C in a high-speed centrifuge at a temperature of 4℃ and a speed of 8000r / min for 10min. Take the supernatant and recover the solvent to obtain the product.

[0152] Example 2 Preparation of gastrodin and gastrodin aglycone (1) A method for preparing immobilized lipase, comprising the following steps: (1.1) An equal volume of Fe3O4 nanoparticle solution in dialysis equilibrium with lipase was placed in 100 times the amount of deionized water, sonicated for 8 min, and then dopamine hydrochloride was added. The molar ratio of Fe3O4 nanoparticles to dopamine hydrochloride was 1:1 to obtain solution 1. (1.2) Adjust the pH of solution 1 prepared in step (1.1) to 8.5 with 0.1 mol / L NaOH solution, stir at room temperature for 24 h at a speed of 150 r / min, and then obtain precipitate 1 by magnetic means. (1.3) Wash the precipitate 1 obtained in step (1.2) three times until the supernatant is clear to obtain Fe3O4@dopamine hydrochloride. After freeze-drying, store it in a sealed container at 4°C for later use. (1.4) Add the Fe3O4@dopamine hydrochloride and lipase prepared in step (1.3) to the reaction flask and then add phosphate buffer with pH=6.0. The mass-volume ratio of Fe3O4@dopamine hydrochloride, lipase and phosphate buffer is 1:1:1. Fix for 6h. Stir continuously during the immobilization period at a speed of 250r / min. After the immobilization is completed, use magnetism to obtain precipitate 2. (1.5) Wash the precipitate 2 obtained in step (1.4) with deionized water multiple times until the supernatant no longer contains protein. After freeze-drying, seal and store at 4°C for later use.

[0153] (2) The method for preparing immobilized cellulase includes the following steps: An equal amount of dialysis-equilibrated Fe2O3 nanoparticles, along with cellulase, were added to a cellulase solution containing 0.5% polyvinyl alcohol (30 mg / mL). The mixture was stirred at 4°C for 10 h, then rapidly frozen at -80°C, thawed on ice, and subjected to three freeze-thaw cycles. A 0.5 mol / L NaCl solution was added to precipitate the precipitate, followed by adsorption separation using a magnet. The precipitate was washed three times with ultrapure water, freeze-dried, and stored at 4°C for later use.

[0154] (3) The preparation method of freeze-dried powder of Gastrodia elata extract includes the following steps: (3.1) Select fresh, disease-free Gastrodia elata tubers, wash, peel, slice into 3-5 mm pieces, steam for 15-30 min, dry at low temperature for 24-48 h, and pulverize through a 100-mesh sieve to obtain Gastrodia elata powder, which is stored at 4℃ for later use. (3.2) Weigh the Gastrodia elata powder obtained in step (3.1) and add purified water, wherein the mass-volume ratio of Gastrodia elata powder to purified water is 1g:30mL. Reflux extraction is performed at 90℃ for 3h. After the extraction, let it stand until the sample returns to room temperature. Then, centrifuge at 5000r / min for 5min at 2℃ to obtain the supernatant. Repeat the reflux extraction on the remaining filter residue 3 times. Combine the filtrates to obtain solution A. (3.3) Add 2% activated carbon to solution A in step (3.2), control the temperature at 10℃, stir with a magnetic stirrer for 40 min, let stand after stirring, filter and decolorize to obtain filtrate B; (3.4) Filtrate B was frozen at -70℃ for 10 hours, and then dried for 45 hours to obtain freeze-dried powder of Gastrodia elata extract. (4) The preparation method of gastrodin and gastrodin aglycone includes the following steps: (4.1) Add water to the immobilized lipase, immobilized cellulase and freeze-dried powder of Gastrodia elata extract and stir. The molar ratio of freeze-dried powder of Gastrodia elata extract, cellulase and lipase is 100:2:2. Adjust the pH to 8.0, heat to 40°C, place in a fingertip turbine shaker and shake, then place in a constant temperature incubator for 7 hours to obtain material C. (4.2) After the reaction is complete, place the material C in a high-speed centrifuge at a temperature of 4℃ and a speed of 8000r / min for 10min. Take the supernatant and recover the solvent to obtain the product.

[0155] Example 3 Preparation of gastrodin and gastrodin aglycone (1) A method for preparing immobilized lipase, comprising the following steps: (1.1) An equal volume of Fe3O4 nanoparticle solution in dialysis equilibrium with lipase was placed in 100 times the amount of deionized water, sonicated for 12 min, and then dopamine hydrochloride was added. The molar ratio of Fe3O4 nanoparticles to dopamine hydrochloride was 1:1 to obtain solution 1. (1.2) Adjust the pH of solution 1 prepared in step (1.1) to 8.5 with 0.1 mol / L NaOH solution, stir at room temperature for 22 h at a speed of 300 r / min, and then obtain precipitate 1 by magnetic means. (1.3) Wash the precipitate 1 obtained in step (1.2) three times until the supernatant is clear to obtain Fe3O4@dopamine hydrochloride. After freeze-drying, store it in a sealed container at 4°C for later use. (1.4) Add the Fe3O4@dopamine hydrochloride and lipase prepared in step (1.3) to the reaction flask and then add phosphate buffer with pH=6.0. The mass-volume ratio of Fe3O4@dopamine hydrochloride, lipase and phosphate buffer is 1:1:1. Fix for 6h. Stir continuously during the immobilization period at a speed of 250r / min. After the immobilization is completed, use magnetism to obtain precipitate 2. (1.5) Wash the precipitate 2 obtained in step (1.4) with deionized water multiple times until the supernatant no longer contains protein. After freeze-drying, seal and store at 4°C for later use.

[0156] (2) The method for preparing immobilized cellulase includes the following steps: An equal amount of dialysis-equilibrated Fe2O3 nanoparticles, along with cellulase, were added to a cellulase solution containing 0.5% polyvinyl alcohol (30 mg / mL). The mixture was stirred at 4°C for 10 h, then rapidly frozen at -80°C, thawed on ice, and subjected to three freeze-thaw cycles. A 0.5 mol / L NaCl solution was added to precipitate the precipitate, followed by adsorption separation using a magnet. The precipitate was washed three times with ultrapure water, freeze-dried, and stored at 4°C for later use.

[0157] (3) The preparation method of freeze-dried powder of Gastrodia elata extract includes the following steps: (3.1) Select fresh, disease-free Gastrodia elata tubers, wash, peel, slice into 3-5 mm pieces, steam for 15-30 min, dry at low temperature for 24-48 h, and pulverize through a 100-mesh sieve to obtain Gastrodia elata powder, which is stored at 4℃ for later use. (3.2) Weigh the Gastrodia elata powder obtained in step (3.1) and add purified water, wherein the mass-volume ratio of Gastrodia elata powder to purified water is 1g:44mL. Reflux extraction is performed at 110℃ for 1h. After the extraction is completed, let it stand until the sample returns to room temperature. Then, centrifuge at 3000r / min for 10min at 4℃ to obtain the supernatant. Repeat the reflux extraction on the remaining filter residue 3 times. Combine the filtrates to obtain solution A. (3.3) Add 3% activated carbon to solution A in step (3.2), control the temperature at 30℃, stir with a magnetic stirrer for 30 min, let stand after stirring, filter and decolorize to obtain filtrate B; (3.4) Filtrate B was frozen at -60℃ for 14 hours, and then dried for 50 hours to obtain freeze-dried powder of Gastrodia elata extract. (4) The preparation method of gastrodin and gastrodin aglycone includes the following steps: (4.1) Add water to the immobilized lipase, immobilized cellulase and freeze-dried powder of Gastrodia elata extract and stir. The molar ratio of freeze-dried powder of Gastrodia elata extract, cellulase and lipase is 100:3:1. Adjust the pH to 8.0, heat to 40°C, place in a fingertip turbine shaker and shake, then place in a constant temperature incubator for 7 hours to obtain material C. (4.2) After the reaction is complete, place the material C in a high-speed centrifuge at a temperature of 4℃ and a speed of 8000r / min for 10min. Take the supernatant and recover the solvent to obtain the product.

[0158] Comparative Example 1 The difference from Example 1 is that the lipase in step (1) is replaced with acidic protease, while the other dosages and steps are the same as in Example 1.

[0159] Comparative Example 2 The difference from Example 1 is that the cellulase in step (2) is replaced with a complex enzyme of pectinase and β-glucosidase in a mass ratio of 1:3, while the remaining amounts and steps are the same as in Example 1.

[0160] Comparative Example 3 The difference from Example 1 is that the molar ratio of the freeze-dried powder of Gastrodia elata extract, cellulase and lipase in step (4.1) is changed to 100:1.5:2.5, while the other dosages and steps are the same as in Example 1.

[0161] Comparative Example 4 The difference from Example 1 is that the molar ratio of the freeze-dried powder of Gastrodia elata extract, cellulase and lipase in step (4.1) is changed to 100:5.5:0.8, while the other dosages and steps are the same as in Example 1.

[0162] Comparative Example 5 The difference from Example 1 is that step (2) of cellulase fixation is omitted, while the remaining dosages and steps are the same as in Example 1.

[0163] Comparative Example 6 The difference from Example 1 is that free cellulase and lipase were used directly without immobilization treatment, specifically: (1) The preparation method of freeze-dried powder of Gastrodia elata extract includes the following steps: (1.1) Select fresh, disease-free Gastrodia elata tubers, wash, peel, slice into 3-5 mm pieces, steam for 15-30 min, dry at low temperature for 24-48 h, and pulverize through a 100-mesh sieve to obtain Gastrodia elata powder, which is stored at 4℃ for later use. (1.2) Weigh the Gastrodia elata powder obtained in step (1.1) and add purified water, wherein the mass-volume ratio of Gastrodia elata powder to purified water is 1g:37mL. Reflux extraction is performed at 100℃ for 1.5h. After extraction, let stand until the sample returns to room temperature. Then, centrifuge at 4000r / min for 10min at 4℃ to obtain the supernatant. Repeat the extraction three times with the remaining filter residue. Combine the filtrates to obtain solution A. (1.3) Add 1% activated carbon to solution A in step (1.2), control the temperature at 21℃, stir with a magnetic stirrer for 35 min, let stand after stirring, filter and decolorize to obtain filtrate B; (1.4) Filtrate B was frozen at -80℃ for 12 hours, and then dried for 48 hours to obtain freeze-dried powder of Gastrodia elata extract. (2) The preparation method of gastrodin and gastrodin aglycone includes the following steps: (2.1) Add water to free lipase, cellulase and freeze-dried powder of Gastrodia elata extract and stir. The molar ratio of freeze-dried powder of Gastrodia elata extract, cellulase and lipase is 100:5:1. Adjust the pH to 8.0, heat to 40°C, place in a fingertip turbine shaker and shake, then place in a constant temperature incubator for 7 hours to obtain material C. (2.2) After the reaction is complete, place the material C in a high-speed centrifuge at a temperature of 4℃ and a speed of 8000 r / min for 10 min. Take the supernatant and recover the solvent to obtain the product. The remaining dosages and steps are the same as in Example 1.

[0164] Example 1: Characterization of Immobilized Enzyme Performance 1. Experimental Methods: Take Fe3O4@dopamine hydrochloride, Fe3O4@dopamine hydrochloride@lipase and Fe3O4@dopamine hydrochloride@lipase prepared in Example 1. 4、 Fe2O3, Fe2O3@polyvinyl alcohol@cellulase were characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy.

[0165] 2. Experimental Results: from Figure 2 As can be seen, both Fe3O4 and Fe3O4@dopamine hydrochloride appear as irregular spheres under transmission electron microscopy, with each nanoparticle measuring approximately 20-30 nm in size. In the electron micrograph of Fe3O4@dopamine hydrochloride, a light gray "shell" is clearly visible encasing the darker particles; the black particles are Fe3O4, and this "shell" is a polydopamine layer with adhesive properties formed by dopamine on the surface of Fe3O4 under weak alkaline and air conditions. Under the influence of this polydopamine layer, the Fe3O4 particles become more compact. Figure 2 As can be seen from C, after immobilizing the lipase, the particles became more compact, without changing the shape of the particles.

[0166] Similar to Fe3O4@dopamine hydrochloride@lipase, after adsorption and repeated freeze-thaw cycles, Fe2O3 (such as...) Figure 2 The D) particles form a solidified complex of about 1 μm, similar to gel clusters, with polyvinyl alcohol and cellulase (e.g. Figure 2 (E), this microgel cluster structure makes the immobilized cellulose more flexible.

[0167] Particle size analysis of the immobilized enzymes revealed that the average particle size of Fe3O4@dopamine hydrochloride and Fe3O4@dopamine hydrochloride@lipase increased significantly after dopamine was loaded onto the Fe3O4 particles, reaching 24.73±3.57 nm, which is significantly larger than the 18.65±1.41 nm of Fe3O4. This indicates the formation of a polydopamine layer with a thickness of approximately 3.04 nm on the Fe3O4 surface. Furthermore, when enzyme was further loaded onto the Fe3O4@dopamine hydrochloride particles, the average particle size of Fe3O4@dopamine hydrochloride@lipase reached 32.95±4.41 nm, indicating successful immobilization of the lipase on the nanocarrier with a thickness of approximately 4.11 nm.

[0168] A comparison of the particle sizes of Fe2O3 and Fe2O3@polyvinyl alcohol@cellulase revealed that when polyvinyl alcohol and cellulase were loaded onto Fe2O3 particles, the average particle size of Fe2O3@polyvinyl alcohol@cellulase increased significantly to about 1 μm, which is significantly larger than the 14.32±1.54 nm of Fe2O3. This indicates that a very large gel layer was formed on the surface of Fe2O3.

[0169] To further determine whether the enzyme successfully attached to the carrier, XPS was used to analyze the surface elements of Fe3O4, Fe3O4@dopamine hydrochloride, and Fe3O4@dopamine hydrochloride@lipase. Figures 4-6 The XPS analysis chromatograms show Fe3O4, Fe3O4@dopamine hydrochloride, and Fe3O4@dopamine hydrochloride@lipase, respectively. Figure 4 As can be seen, the Fe3O4 surface contains three main characteristic peaks: C1s at 282.00 eV, O1s at 527.00 eV, and Fe2p at 708.00 eV. The peak area of ​​C1s is 1033.36, that of O1s is 2583.83, and that of Fe2p is 4199.83. The presence of the characteristic C peaks on the Fe3O4 surface may be due to exposure to air during preparation or storage. Carbon in the air (such as CO2, hydrocarbon molecules, etc.) can be adsorbed onto the sample surface, resulting in the presence of carbon signals.

[0170] from Figure 5As can be seen, the characteristic peaks of Fe3O4@dopamine hydrochloride include the addition of nitrogen (N) and the N1s peak with a bond energy of 397.00 eV, which is due to the introduction of the amino group (-NH2) in dopamine hydrochloride. Furthermore, the peak area of ​​C1s increases significantly to 2351.30, because the dopamine hydrochloride molecule contains eight carbon atoms forming a benzene ring structure and an alcohol group. The peak area of ​​O1s does not increase due to the presence of the hydroxyl group (-OH) in dopamine hydrochloride, and the peak area of ​​Fe2p also decreases significantly. This is because the "shell" structure formed on the Fe3O4 surface reduces the signals of O and Fe to some extent. This indicates that dopamine successfully attaches to the Fe3O4 surface. Compared to Fe3O4@dopamine hydrochloride, the XPS diagram of Fe3O4@dopamine hydrochloride@lipase shows no increase in the number of peaks, but the peak area of ​​the N1s peak increases, precisely because lipase is the sole source of the increased N peak signal. The changes in element type and content are direct evidence of successful modification and immobilization, indicating that the lipase was successfully immobilized on the surface of Fe3O4@dopamine hydrochloride.

[0171] To further clarify the changes in chemical bonds and functional groups of the carrier and enzyme before and after immobilization, Fourier transform infrared spectroscopy was used to characterize Fe3O4, Fe3O4@dopamine hydrochloride, and Fe3O4@dopamine hydrochloride@lipase. Observations revealed that all three curves were at 573 cm⁻¹. -1 A strong absorption peak appeared at the wavenumber, mainly due to the Fe-O functional groups. Fe3O4 showed an absorption peak at 1640 cm⁻¹. -1 The peak at the wavenumber is mainly due to the vibration of hydroxyl groups adsorbed in water on the Fe3O4 surface. The peak at 1640 cm⁻¹ for Fe3O4@dopamine hydrochloride is also significant. -1 The peak at the wavenumber is significantly larger than that of Fe3O4, which is due to the stretching vibration peak of the -COOH group in dopamine. The hydroxyl groups adsorbed on the Fe-O surface react with the hydroxyl groups of catechol in dopamine hydrochloride to form an ester. Compared to Fe3O4, Fe3O4@dopamine hydrochloride shows a higher peak at 795 cm⁻¹. -1 The peak at the wavenumber corresponds to the out-of-plane deformation vibration peak of the CH group of the benzene ring, at 1040 cm⁻¹. -1 The peak at wavenumber is caused by the CN functional group in lipase, at 1270 cm⁻¹. -1 The peak at wavenumber is caused by the stretching vibration of the phenolic hydroxyl group in the lipase, at 1510 cm⁻¹. -1 The peaks at the wavenumber are caused by the C=C in the benzene ring of dopamine hydrochloride. The appearance of these three peaks proves that dopamine has successfully attached to the surface of Fe3O4. Compared to Fe3O4@dopamine hydrochloride@lipase, no new peaks were generated in Fe3O4@dopamine hydrochloride@lipase, but the peak at 1510 cm⁻¹... -1 C=C peak at wavenumber, 1270 cm⁻¹ -1The hydroxyl peak at wavenumber and at 1040 cm⁻¹ -1 The peak area of ​​CN peaks at different wavenumbers is significantly increased, especially at 1040 cm⁻¹. -1 The increase in peak area of ​​CN peak at wavenumber is particularly significant, and lipase is the only source of this peak during the immobilization process, indicating that lipase has been successfully immobilized on Fe3O4@dopamine hydrochloride.

[0172] Fourier transform infrared spectroscopy characterization of Fe2O3@polyvinyl alcohol and Fe2O3@polyvinyl alcohol@cellulase revealed that, due to the presence of citric acid, at 1673 cm⁻¹... -1 A C=O stretching vibration peak appears at 3350 cm⁻¹. After the addition of cellulase, the C=O stretching vibration peak becomes sharper due to the influence of the amide group. This is also due to the influence of the NH bond in the cellulase. -1 The absorption peak of the -OH stretching vibration at the site is broader and its intensity is weaker. This indicates that the colloid formed by Fe2O3 and polyvinyl alcohol after repeated freeze-thaw cycles successfully immobilized the cellulase.

[0173] To clarify whether the immobilization of dopamine onto Fe3O4 magnetic nanoparticles affected the crystal structure of Fe3O4, XRD analysis was performed on Fe3O4, Fe3O4@dopamine hydrochloride, and Fe3O4@dopamine hydrochloride@lipase. The XRD pattern of Fe3O4 showed a series of characteristic diffraction peaks consistent with its crystal structure. Six diffraction peaks appeared at 2θ=30.2°(220), 2θ=35.4°(311), 2θ=43.3°(400), 2θ=53.5°(422), 2θ=57.5°(511), and 2θ=62.9°(440). Dopamine hydrochloride is an organic compound with a relatively large molecular weight; its X-ray diffraction peaks were not obvious because organic compounds typically exhibit weak signals in X-ray diffraction patterns. Dopamine hydrochloride can have a certain impact on the crystal structure of Fe3O4 particles, causing slight changes in the position or intensity of some peaks. Careful observation is needed to determine the effect. Figure 11 In step B, a weak diffraction peak was observed at 2θ = 21.1°, while the other peaks remained unchanged, indicating that dopamine hydrochloride successfully adhered to Fe3O4 without affecting its structure. Further introduction of lipase revealed no significant difference between the XRD patterns of Fe3O4@dopamine hydrochloride@lipase and Fe3O4@dopamine hydrochloride, suggesting that enzyme immobilization did not significantly affect the Fe3O4 structure.

[0174] Thermogravimetric analysis (TGA) of immobilized enzymes showed that Fe3O4, a magnetic iron oxide nanoparticle, typically exhibits high thermal stability. The TGA curves of Fe3O4 remained stable across low, medium, and high temperature ranges with almost no mass loss, demonstrating extremely high stability and maintaining 98.1% of its original mass. Fe3O4@dopamine hydrochloride underwent slow pyrolysis and degradation in the medium temperature range (100-350℃), potentially disrupting its benzene ring structure and resulting in a slight mass loss, maintaining 93.5% of its original mass. In the medium temperature range (100-350℃), the presence of lipase caused partial protein pyrolysis and denaturation, releasing water and light organic matter. Similarly, dopamine hydrochloride also underwent transformation, ultimately maintaining 87.4% of its original mass. Ordinary dopamine retained only 5.4% of its original mass. The dehydration of dopamine mainly occurs in three stages: the first stage at low temperatures (<100℃), where dopamine hydrochloride loses its own free water, resulting in a mass loss of 11.1%; the second stage occurs in the intermediate temperature range (150℃-350℃), where the benzene ring and other organic structures transform; finally, at temperatures above 350℃, degradation and carbonization form stable residues. Analysis of the mass loss of each sample revealed that the lipase immobilization products exhibited good thermal stability. Fe3O4@dopamine hydrochloride lost 4.6% more mass than Fe3O4, and Fe3O4@dopamine hydrochloride@lipase lost 6.1% more mass than Fe3O4@dopamine hydrochloride. Since no other substances were introduced during the preparation of these two nanoparticles, this indicates that dopamine and lipase were successfully immobilized on the Fe3O4 and Fe3O4@dopamine hydrochloride nanoparticles, respectively.

[0175] Similarly, Fe2O3, as a stable metal oxide, showed almost no significant mass loss in the TGA curve. In the Fe2O3@polyvinyl alcohol@cellulase mixture, the addition of Fe2O3 improved the overall thermal stability of the polymer, shifting the pyrolysis temperature to higher temperatures. Fe2O3 also inhibited polymer pyrolysis, and the mass loss in the TGA curve was concentrated in water evaporation (below approximately 100℃). However, thermogravimetric analysis of polyvinyl alcohol showed water evaporation (<100℃); PVA decomposed (200-400℃), producing small molecule compounds (ethanol, vinyl alcohol, etc.), but the process was unstable, accompanied by chain breakage and the release of volatile small molecules; complete pyrolysis (>400℃) yielded inorganic residues (such as carbon black). Analysis of the mass loss of each sample revealed that the immobilized cellulase product exhibited good thermal stability, and the cellulase was successfully immobilized in the polyvinyl colloid and Fe2O3 complex.

[0176] Example 2: Yields of gastrodin and gastrodin aglycone 1. Experimental method: The solids prepared in Examples 1-4 and Comparative Examples 1-6 were dissolved in 1.5 mL of deionized water, and the solution was drawn up with a 1 mL syringe, filtered through a 0.22 μm microporous membrane into a liquid chromatography sample vial, and then detected by HPLC.

[0177] The specific HPLC method is as follows: Column: Thermo ODS HYPERSIL C 18 The column (250 mm × 4.6 mm, 5 μm), mobile phase: acetonitrile (A) - 0.1% phosphoric acid - aqueous solution (B), gradient elution (0 min: 97% B; 21 min: 95% B; 42 min: 86% B; 56 min: 86% B; 60 min: 80% B; 65 min: 97% B; 70 min: 97% B), column temperature: 30 °C, detection wavelength: 220 nm, flow rate: 1 mL / min, injection volume: 10 μL.

[0178] 2. Experimental Results The standard curves for the six Gastrodia elata standards are shown in Table 3 below: Table 3 Standard curves for Gastrodia elata standard samples

[0179] 2. Experimental Results According to Table 4 below, the yields of baicalin and gastrodin are significantly higher than those of Comparative Examples 1 and 6 (1.3%), indicating that the synergistic effect of specific immobilized cellulase and lipase is key to the efficient conversion of baicalin to gastrodin and gastrodin. Example 1 achieved a yield of 1.5%, representing the optimal yield group. Its core advantage lies in the specific ratio of freeze-dried Gastrodia elata extract, immobilized cellulase, and immobilized lipase. The dual enzymes synergistically and precisely break the glycosidic and ester bonds of baicalin, achieving efficient conversion. Furthermore, the high stability of the immobilized enzyme further ensures catalytic efficiency, ultimately achieving the highest yield.

[0180] Table 4. Yields of gastrodin and gastrodin aglycone

[0181] Example 3: Determination of immobilized enzyme activity 1. Experimental Methods: Take 3 mL centrifuge tubes and add 100 mg of Gastrodia elata extract prepared in Example 1, 3 mg of immobilized cellulase, 1 mg of immobilized lipase, and 1.5 mL of ultrapure water. Shake with a fingertip turbine for 2 min to ensure complete substrate dispersion. Incubate at 40°C for 6 h. After the reaction, centrifuge at 8000 rpm for 10 min at 4°C. Use a 1 mL syringe to aspirate the supernatant, filter through a 0.22 μm microporous membrane into a HPLC sample vial, and perform HPLC analysis. Repeat the test 10 and 20 times.

[0182] 2. Experimental Results After 10 batches of immobilized enzyme reuse, the relative enzyme activity remained above 80%, demonstrating good operational stability and reusability. After 20 batches of immobilized enzyme reuse, the relative enzyme activity remained above 68%, demonstrating good operational stability and reusability.

[0183] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing gastrodin and gastrodin by dual-enzyme catalysis, characterized in that, Free cellulase and lipase were immobilized to obtain immobilized cellulase and immobilized lipase; Gastrodia elata extract freeze-dried powder was obtained using Gastrodia elata as raw material; then, using the freeze-dried powder of Gastrodia elata extract as raw material, under the catalysis of immobilized cellulase and immobilized lipase, the barisonin substances in Gastrodia elata extract were synthesized into gastrodin and gastrodin aglycone.

2. The preparation method according to claim 1, characterized in that, The method for preparing the immobilized lipase includes the following steps: (1.1) Dissolve Fe3O4 nanoparticles in water, sonicate, and then add dopamine hydrochloride to obtain solution 1; (1.2) Adjust the pH of solution 1 prepared in step (1.1), stir, and obtain precipitate 1; (1.3) Wash the precipitate 1 obtained in step (1.2) several times until the supernatant is clear to obtain Fe3O4@dopamine hydrochloride, freeze dry and set aside; (1.4) The Fe3O4@dopamine hydrochloride and lipase prepared in step (1.3) are added to phosphate buffer and fixed for a period of time to obtain precipitate 2; (1.5) Wash and freeze-dry the precipitate 2 obtained in step (1.4) for later use.

3. The preparation method according to claim 1, characterized in that, The method for preparing the immobilized cellulase includes the following steps: Fe2O3 nanoparticles were added to a cellulase solution containing polyvinyl alcohol, stirred, rapidly frozen, thawed, and repeatedly frozen and thawed. NaCl was then added for precipitation, followed by adsorption separation, washing, precipitation, and freeze-drying for later use.

4. The preparation method according to claim 1, characterized in that, The preparation method of the freeze-dried powder of Gastrodia elata extract includes the following steps: (3.1) Select fresh Gastrodia elata, wash, peel, slice, steam, dry, and grind to obtain Gastrodia elata powder for later use; (3.2) Add water to the gastrodia powder obtained in step (3.1), reflux to extract, let stand, centrifuge to obtain supernatant, extract the remaining residue again, combine the filtrates to obtain solution A; (3.3) Add activated carbon to solution A from step (3.2), stir, let stand, filter and decolorize to obtain filtrate B; (3.4) After freezing and solidifying the filtrate B, freeze-dry it to obtain freeze-dried powder of Gastrodia elata extract.

5. The preparation method according to claim 4, characterized in that, The mass-to-volume ratio of Gastrodia elata powder to water in step (3.2) is 1g:30-44mL; the reflux extraction temperature is 90-110℃.

6. The preparation method according to claim 4, characterized in that, The reflux extraction in step (3.2) takes 1-3 hours each time; the centrifugation temperature is 2-8℃.

7. The preparation method according to claim 1, characterized in that, The synthesis of gastrodin and gastrodinogenin from barine glycosides in Gastrodia elata extract specifically includes the following steps: (4.1) The immobilized lipase, immobilized cellulase and freeze-dried powder of Gastrodia elata extract were mixed with water, the pH was adjusted, and then heated and shaken for a period of time before being reacted at a constant temperature to obtain material C; (4.2) Centrifuge material C and collect the supernatant to recover the product.

8. The preparation method according to claim 7, characterized in that, The molar ratio of the freeze-dried Gastrodia elata extract powder, the immobilized cellulase, and the immobilized lipase in step (4.1) is 100:2.0-5.0:1.0-2.

0.

9. Gastrodin and gastrodinogenin prepared by the preparation method according to any one of claims 1-8.

10. The use of gastrodin and gastrodin as described in claim 9 in the preparation of medicaments for treating diseases of the nervous system, cardiovascular system or immune system.

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