Green extraction method of anoectochilus formosanus exosome based on enzymolysis assistance

Abstract

The invention relates to the technical field of plant extracts, and discloses a green anoectochilus formosanus exosome extraction method based on enzymolysis assistance, which comprises the following steps: S1, firstly preparing an anoectochilus formosanus medicinal material, and then crushing the dried anoectochilus formosanus medicinal material, S2, preparing choline, citric acid and glycerol, and mixing, S3, adding a prepared deep eutectic solvent into a buffer solution, and stirring uniformly to obtain an anoectochilus formosanus exosome extraction solution. The method comprises the following steps: S1, preparing an enzymatic hydrolysis system, S4, adding treated anoectochilus formosanus powder into the enzymatic hydrolysis system, S5, filtering an extracting solution by using a filter in the enzymatic hydrolysis extraction process or before the reaction and after the reaction in S6, and S7, separating a deep eutectic solvent from concentrated residual liquid by using a membrane separation technology, and reusing the recovered deep eutectic solvent by adjusting the concentration. By introducing citric acid into the NADES solvent, higher solubility and better extraction efficiency of effective components in the medicinal materials are realized, the quality and concentration of the obtained enzymolysis product are higher than those of the traditional method, and the extraction effect is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of plant extracts, and in particular to a green extraction method of roxburghii exosomes based on enzymatic hydrolysis assistance. Background Art

[0002] Anoectochilus roxburghii, also known as golden thread orchid or golden silk grass, is a perennial herbaceous plant in the Orchidaceae family. It is a short plant, 10-18 cm tall, with a fleshy, creeping rhizome. Its leaves are oval or kidney-shaped, with a dense network of golden veins on the upper surface and a pale purple-red underside. Its flowers are small, pale yellow-green, and arranged in racemes. Anoectochilus roxburghii is primarily found in Fujian, Guangxi, Guangdong, and Hainan in southern China, as well as in some Southeast Asian countries. It thrives in remote, pristine, deep mountain forests and is often used in landscape gardens or as an indoor ornamental plant.

[0003] In the traditional extraction process of Anoectochilus roxburghii, solvent extraction is often used, such as reflux extraction with organic solvents such as ethanol and methanol. However, this method has many disadvantages. On the one hand, the large-scale use of organic solvents is not only costly, but also causes serious pollution to the environment, and a large amount of energy and cost are also required in the subsequent solvent recovery process. On the other hand, traditional methods often require high temperatures and long extraction processes, which may cause the structure of Anoectochilus roxburghii to be destroyed, affecting its activity and purity, and the extraction rate is difficult to reach the ideal level. Summary of the Invention

[0004] In response to the shortcomings of the existing technology, the present invention provides a green extraction method for Anoectochilus roxburghii exosomes based on enzymatic hydrolysis, which solves the problem that the existing NADES system lacks effective polarity regulating components, resulting in limited solubility of low-polarity or weak-polarity active ingredients such as polyphenols and phenylpropanoids in traditional Chinese medicines, thereby restricting the overall extraction efficiency and the quality of the enzymatic hydrolysis products, resulting in incomplete and incomplete extraction, affecting the quality of subsequent products.

[0005] To achieve the above objectives, the present invention is implemented by the following technical solution: a green extraction method of exosomes from Anoectochilus roxburghii assisted by enzymatic hydrolysis, comprising the following steps:

[0006] S1: First, prepare a roxburghii herbal medicine, then grind the dried roxburghii herbal medicine, and dry the grinded roxburghii powder;

[0007] S2: Choline, citric acid, and glycerol are then prepared and mixed until a uniform natural deep eutectic solvent is formed, deionized water is added, and the mixture is cooled to room temperature to obtain a deep eutectic solvent;

[0008] S3: Add the prepared deep eutectic solvent to the buffer solution, then add cellulase and xylanase, and at the same time, add a natural surfactant and mix them to complete the construction of the synergistic enzymatic hydrolysis system;

[0009] S4: adding the treated roxburghii powder to the enzymatic hydrolysis system, and then placing the reaction system in a temperature-controlled water bath for stirring, thereby utilizing the action of the enzyme and the solubility characteristics of the deep eutectic solvent to promote the extraction of roxburghii;

[0010] S5: adding a natural antioxidant to the extract during the enzymatic extraction process or before the end of the reaction. This step is used to prevent oxidative degradation of the golden thread vine during the extraction process;

[0011] S6: After the reaction is completed, the extract is filtered using a filter, and then the filtrate is concentrated by a vacuum concentration device for use in subsequent purification steps or directly used as an intermediate product;

[0012] S7: The deep eutectic solvent is separated from the concentrated residual liquid by membrane separation technology, and the recovered deep eutectic solvent is reused by adjusting the concentration.

[0013] Preferably, in S1, the particle size of the roxburghii medicinal material is 60-80 mesh, the drying temperature is 45° C.-55° C., the drying time is 2-4 hours, and the moisture content of the finally obtained roxburghii powder is controlled to be no higher than 10%.

[0014] Preferably, in S2, the natural deep eutectic solvent is composed of choline, citric acid and glycerol in a molar ratio of 1:1:2, the mixing temperature is 60°C to 80°C, the stirring time is 1.5 hours to 3 hours, and the content of deionized water added to the obtained deep eutectic solvent is 10wt%-30wt%.

[0015] Preferably, in S3, the buffer is used to adjust the pH of the solution, the amount of the cellulase used is 100-300 U / g of golden thread vine powder, the amount of the xylanase used is 50-200 U / g of golden thread vine powder, the natural surfactant includes saponin or chitosan derivatives, and the amount of the natural surfactant added is 0.05-0.5 wt%, which is used to adjust the interfacial affinity between the enzyme and the deep eutectic solvent, and the mixture is stirred for 5-10 minutes.

[0016] Preferably, in S4, the temperature in the temperature-controlled water bath is 40-50°C, the stirring speed is 50-150 rpm, the stirring time is 1.5-3 hours, the pH value of the reaction system is controlled at 5.0 to 5.5, and the golden thread vine powder and the enzymatic hydrolysis system are mixed at a solid-liquid mass ratio of 1:8 to 1:15;

[0017] It also includes the following sub-steps:

[0018] A multiphase synergistic interface system comprising roxburghii powder, a natural deep eutectic solvent, a complex enzyme preparation and a natural surfactant is constructed. The interface system is used to promote the rupture of cell wall structure and improve the release efficiency of roxburghii in the solvent.

[0019] Preferably, in S5, the natural antioxidant includes vitamin E or tea polyphenols, and the added concentration thereof is controlled at 0.01-0.1 wt % to inhibit the oxidative degradation of Anoectochilus roxburghii during the enzymatic hydrolysis process.

[0020] Preferably, in S6, the solid-liquid separation of the enzymatic hydrolysis product is carried out using a microporous membrane filtration device with a pore size of 0.45 μm to 1.2 μm, and the obtained filtrate is concentrated under reduced pressure with the concentration temperature controlled at 35° C. to 50° C. to 1 / 3 to 1 / 5 of the original volume.

[0021] Preferably, in S7, the natural deep eutectic solvent in the reaction system is recovered by a membrane separation method, wherein the membrane separation method includes nanofiltration or ultrafiltration, and the NADES is reused after concentration adjustment. The residual enzyme in the recovery system is mixed with the new enzyme in a ratio of 1:1-1:3 and then continued to be used, and can be recycled up to 3-5 times.

[0022] The present invention provides a green extraction method for exosomes from Anoectochilus roxburghii based on enzymatic hydrolysis. It has the following beneficial effects:

[0023] 1. The present invention achieves higher solubility and better extraction efficiency of the active ingredients in the medicinal materials by introducing citric acid into the NADES solvent. The quality and concentration of the obtained enzymatic hydrolysis products are higher than those of the traditional method, and the extraction effect is significantly improved.

[0024] 2. The present invention achieves precise control of enzymatic hydrolysis reaction conditions by optimizing the formula of natural eutectic solvent, obtaining higher enzyme activity and reaction rate, thereby shortening the enzymatic hydrolysis time and improving the overall efficiency of the extraction process.

[0025] 3. The present invention optimizes solid-liquid separation by controlling the composition and water content of the NADES solvent, obtaining a clearer filtrate and a higher concentration of active ingredients, reducing material losses in subsequent operations, and enhancing the economy and environmental friendliness of the extraction process.

[0026] 4. The present invention achieves uniform dispersion of medicinal material powder and better enzymatic hydrolysis effect by adding natural surfactants, obtains a higher yield of enzymatic hydrolysis products, further optimizes the overall extraction process, and enhances the operability and stability of the process. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The present invention is a flow chart of the green extraction method of exosomes from Anoectochilus roxburghii based on enzymatic hydrolysis assistance. DETAILED DESCRIPTION

[0028] The following will clearly and completely describe the technical solution of the present invention in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0029] Please see the attached Figure 1 The embodiment of the present invention provides a green extraction method of exosomes from Anoectochilus roxburghii based on enzymatic hydrolysis, comprising the following steps:

[0030] S1: First, prepare a roxburghii herbal medicine, then grind the dried roxburghii herbal medicine, and dry the grinded roxburghii powder;

[0031] S2: Choline, citric acid, and glycerol are then prepared and mixed until a uniform natural deep eutectic solvent is formed, deionized water is added, and the mixture is cooled to room temperature to obtain a deep eutectic solvent;

[0032] S3: Add the prepared deep eutectic solvent to the buffer solution, then add cellulase and xylanase, and at the same time, add a natural surfactant and mix them to complete the construction of the synergistic enzymatic hydrolysis system;

[0033] S4: adding the treated roxburghii powder to the enzymatic hydrolysis system, and then placing the reaction system in a temperature-controlled water bath for stirring, thereby utilizing the action of the enzyme and the solubility characteristics of the deep eutectic solvent to promote the extraction of roxburghii;

[0034] S5: adding a natural antioxidant to the extract during the enzymatic extraction process or before the end of the reaction. This step is used to prevent oxidative degradation of the golden thread vine during the extraction process;

[0035] S6: After the reaction is completed, the extract is filtered using a filter, and then the filtrate is concentrated by a vacuum concentration device for use in subsequent purification steps or directly used as an intermediate product;

[0036] S7: The deep eutectic solvent is separated from the concentrated residual liquid by membrane separation technology, and the recovered deep eutectic solvent is reused by adjusting the concentration.

[0037] In S1, the particle size of the roxburghii medicinal material is 60-80 mesh, the drying temperature is 45° C.-55° C., the drying time is 2-4 hours, and the moisture content of the finally obtained roxburghii powder is controlled to be no higher than 10%.

[0038] Specifically, the golden thread vine medicinal material is first crushed and dried. Specifically, the golden thread vine medicinal material is crushed to a particle size of 60 mesh to 80 mesh, thereby increasing the specific surface area of ​​the particles. Then, the golden thread vine powder needs to be dried at a temperature of 45°C to 55°C for 2 to 4 hours to ensure that the final powder moisture content is not higher than 10%. The purpose of this pretreatment step is to provide the best raw material state for the subsequent enzymatic hydrolysis process. Golden thread vine powder with a moisture content controlled at no more than 10% can effectively prevent excessive moisture from interfering with the action of the enzyme and help improve the enzymatic hydrolysis efficiency. In addition, the drying and crushing process can also ensure the uniformity of the golden thread vine powder, which is crucial for the enzymatic hydrolysis effect and extraction efficiency;

[0039] The purpose of the pulverization operation is to reduce the particles of the golden thread herb to increase their specific surface area, allowing the enzyme to more effectively contact the raw material. Smaller particles help improve the enzyme's permeability to the golden thread herb cell wall, thereby promoting the release of the golden thread herb. The pulverized golden thread herb powder can provide a larger reaction surface for enzymatic hydrolysis, enhancing the efficiency of the enzyme.

[0040] The drying step optimizes the subsequent enzymatic hydrolysis conditions by reducing the moisture content of the golden thread vine powder. Excessive moisture can affect enzyme activity and may even cause enzyme inactivation. Controlling the moisture content to no more than 10% helps maintain enzyme activity and ensure the stability of the enzymatic hydrolysis reaction. Low moisture content also reduces the use of solvents during the extraction process, further enhancing the environmental friendliness of the method.

[0041] By precisely controlling the particle size and drying conditions of the Anoectochilus roxburghii, a solid foundation is laid for the enzymatic hydrolysis process. This step ensures the physical properties and optimal moisture content of the Anoectochilus roxburghii powder, effectively improving the efficiency of subsequent extraction and enzymatic hydrolysis. This is crucial for the efficient and green extraction of Anoectochilus roxburghii.

[0042] In S2, the natural deep eutectic solvent is composed of choline, citric acid and glycerol in a molar ratio of 1:1:2, the mixing temperature is 60°C to 80°C, the stirring time is 1.5 hours to 3 hours, and the content of deionized water added to the obtained deep eutectic solvent is 10wt%-30wt%.

[0043] Specifically, the natural deep eutectic solvent consists of choline, citric acid, and glycerol in a molar ratio of 1:1:2. A transparent, stable NADES system is formed by heating at 60°C to 80°C and stirring continuously for 1.5 to 3 hours. Subsequently, 10% to 30% deionized water is added to the eutectic, depending on the compatibility and fluidity of the solvent system with the Anoectochilus roxburghii powder, to adjust the viscosity and solubility of the overall system, making it more suitable for subsequent enzymatic hydrolysis reactions.

[0044] The NADES system itself has strong polarity and highly adjustable solubility, which can effectively enhance the substrate accessibility and reaction freedom in enzymatic reactions. Unlike volatile organic solvents such as ethanol or methanol used in traditional extraction systems, the choline-citric acid-glycerol system used in the present invention has a melting point significantly lower than that of each component monomer after forming a deep eutectic state, showing good room temperature stability and biocompatibility. This solvent not only has good solubility for Anoectochilus roxburghii, but more importantly, its synergistic regulatory effect with water can significantly affect the microstructure of the solution, thereby regulating the conformational stability of the enzyme and the accessibility of the active center;

[0045] Furthermore, the use of deep eutectic solvents provides a near-natural environment for the enzyme to function, allowing it to maintain high activity under mild conditions, thereby significantly improving the release efficiency of Anoectochilus roxburghii. After introducing an appropriate amount of deionized water, the system effectively reduces the overall viscosity while ensuring the solubility of the solvent, improves the diffusion efficiency between the enzyme and the substrate, and promotes the disintegration of the cell wall structure of Anoectochilus roxburghii and the release of internal phenolic substances. Therefore, in the present invention, NADES is not only a solvent, but also a synergistic medium for regulating the enzyme reaction environment. While maintaining its green and environmentally friendly nature, it constructs an efficient and stable reaction system.

[0046] In summary, this step, by constructing a natural deep eutectic solvent with a unique polar structure and environmentally friendly properties, not only significantly improves the solubility and distribution efficiency of Anoectochilus roxburghii in the system, but also achieves a green and highly selective extraction pathway through the synergistic effect of the solvent, enzyme, and substrate. This innovative solvent system design provides a new solution for the gentle and efficient extraction of natural products such as Anoectochilus roxburghii, and lays the technical core of the green process system of this invention.

[0047] The buffer solution in S3 is used to adjust the pH of the solution. The amount of cellulase used is 100–300 U / g of golden thread vine powder, and the amount of xylanase used is 50–200 U / g of golden thread vine powder. Natural surfactants include saponins or chitosan derivatives. The amount of natural surfactant added is 0.05–0.5 wt%, which is used to adjust the interfacial affinity between the enzyme and the deep eutectic solvent. After mixing, stir for 5–10 minutes.

[0048] Specifically, by adding complex enzymes and natural surfactants to the system, a synergistic enzymatic hydrolysis environment is created to significantly improve extraction efficiency. Cellulase (100–300 U / g of Anoectochilus roxburghii powder) and xylanase (50–200 U / g of Anoectochilus roxburghii powder) are added to the enzymatic hydrolysis system, and a buffer solution is used to adjust the system pH to maintain enzyme activity. The synergistic effect of the enzymes effectively degrades cellulose and hemicellulose in the cell wall, breaking down the structural barrier of the Anoectochilus roxburghii powder and providing a channel for the release of the Anoectochilus roxburghii.

[0049] To address the compatibility limitations of the enzyme with natural deep eutectic solvents (NADES), natural surfactants, such as saponins or chitosan derivatives, are introduced at a dosage of 0.05–0.5 wt%. These surfactants modulate the interfacial affinity between the enzyme and the solvent, reducing interfacial tension and making the enzyme more stable within the NADES, allowing it to more readily exert its catalytic activity. After 5–10 minutes of stirring and mixing, the components of the reaction system are uniformly dispersed, forming a stable multiphase synergistic environment.

[0050] Through the auxiliary regulation of natural surfactants, the complex enzyme can maintain high efficiency and stability in NADES, breaking through the problem of limited enzyme activity in non-aqueous systems, significantly improving the extraction efficiency of golden thread vine, and building a green, gentle and efficient extraction process foundation.

[0051] In S4, the temperature in the temperature-controlled water bath is 40-50°C, the stirring speed is 50-150 rpm, the stirring time is 1.5-3 hours, the pH value of the reaction system is controlled at 5.0 to 5.5, and the golden thread vine powder and the enzymatic hydrolysis system are mixed at a solid-liquid mass ratio of 1:8 to 1:15;

[0052] It also includes the following sub-steps:

[0053] A multiphase synergistic interface system comprising roxburghii powder, natural deep eutectic solvent, complex enzyme preparation and natural surfactant is constructed. The interface system is used to promote the rupture of cell wall structure and improve the release efficiency of roxburghii in the solvent.

[0054] Specifically, a temperature-controlled water bath and precise stirring conditions are used to ensure that the enzymatic hydrolysis reaction proceeds under optimal conditions, thereby maximizing the release of the golden thread vine. The temperature of the temperature-controlled water bath is set between 40–50°C, which helps activate the catalytic activity of the enzyme while avoiding excessive temperatures that may cause enzyme inactivation or degradation of the golden thread vine. The stirring speed is 50–150 rpm to ensure that all components in the reaction system are evenly mixed and promote sufficient contact between the enzyme and the golden thread vine powder. The stirring time is set to 1.5–3 hours to ensure that the enzymatic hydrolysis reaction has sufficient time to proceed and release the active ingredients in the golden thread vine powder.

[0055] During this process, the pH of the reaction system is strictly controlled within the range of 5.0 to 5.5. This acidic environment optimizes enzyme activity and provides a stable chemical environment for the enzymatic hydrolysis reaction. The solid-to-liquid ratio of roxburghii powder to the enzymatic hydrolysis system is controlled between 1:8 and 1:15, ensuring that the enzyme can fully function at the appropriate concentration and improving the extraction efficiency of roxburghii.

[0056] Precisely controlling temperature, stirring speed, pH, and solid-to-liquid ratio provides ideal conditions for the enzymatic hydrolysis reaction. This systematic optimization ensures the efficient release of Anoectochilus roxburghii and further enhances the environmental friendliness and stability of the overall extraction process.

[0057] In S5, natural antioxidants include vitamin E or tea polyphenols, and their added concentration is controlled at 0.01-0.1 wt % to inhibit the oxidative degradation of Anoectochilus roxburghii during the enzymatic hydrolysis process.

[0058] Specifically, the addition of natural antioxidants is intended to prevent oxidative degradation of the golden thread vine during the enzymatic hydrolysis process, thereby maintaining its activity and stability. Specifically, the natural antioxidants used include vitamin E or tea polyphenols, with their addition concentration controlled between 0.01–0.1 wt%. The antioxidants function by neutralizing free radicals in the reaction system, inhibiting the oxidation reaction of the golden thread vine and preventing the loss of its active ingredients during the enzymatic hydrolysis process.

[0059] Vitamin E and tea polyphenols, as natural antioxidants, have strong antioxidant properties and can protect Anoectochilus roxburghii from oxidation under mild enzymatic hydrolysis conditions. Vitamin E can effectively inhibit lipid oxidation through its fat-soluble properties, while tea polyphenols react with oxygen free radicals through their abundant phenolic hydroxyl groups, reducing oxidative damage.

[0060] By precisely controlling the amount of antioxidant added, oxidative degradation of Anoectochilus roxburghii can be effectively prevented during the enzymatic hydrolysis process, thereby improving the stability and activity of the final product. This practice of protecting Anoectochilus roxburghii through the addition of natural antioxidants not only ensures the efficiency of the extraction process but also further enhances the environmental friendliness of the process.

[0061] The solid-liquid separation of the enzymatic hydrolysis product in S6 was carried out using a microporous membrane filtration device with a pore size of 0.45 μm to 1.2 μm. The obtained filtrate was concentrated under reduced pressure with the concentration temperature controlled at 35°C to 50°C to 1 / 3 to 1 / 5 of the original volume.

[0062] Specifically, the solid-liquid separation of the enzymatic hydrolysis product is performed using a microporous membrane filtration device. The key to this step is to efficiently separate the solid matter from the active ingredients in the solution. The microporous membrane used has a pore size range of 0.45μm to 1.2μm, which can effectively remove unreacted solid matter and larger molecules, ensuring that only the target components, such as golden thread lotus, are retained in the extract.

[0063] Fine filtration through a microporous membrane filtration device can achieve efficient solid-liquid separation while preventing contamination or quality degradation of the extract due to impurities in the filtration process. Next, the resulting filtrate enters the vacuum concentration stage. During this stage, the concentration temperature of the filtrate is controlled between 35°C and 50°C. The lower concentration temperature helps avoid thermal degradation of Anoectochilus roxburghii and other active ingredients caused by high temperatures, while reducing energy consumption and improving extraction efficiency.

[0064] The process is concentrated under reduced pressure to 1 / 3 to 1 / 5 of its original volume, further concentrating the active ingredients and significantly increasing their concentration. The core innovation of this process lies in the use of appropriate concentration conditions, which not only effectively increase the concentration of the concentrate but also prevent thermal degradation of Anoectochilus roxburghii at low temperatures, ensuring the quality and stability of the final product. The combination of microporous membrane filtration and reduced pressure concentration not only ensures the purity of the extract, but also effectively improves the concentration efficiency of Anoectochilus roxburghii and maximizes the protection of its active ingredients, laying a solid foundation for subsequent product refinement and application.

[0065] In S7, the natural deep eutectic solvent in the reaction system is recovered by a membrane separation method, including nanofiltration or ultrafiltration. The NADES is reused after concentration adjustment. The residual enzyme in the recovery system is mixed with the new enzyme in a ratio of 1:1-1:3 and then continued to be used. It can be recycled up to 3-5 times.

[0066] Specifically, membrane separation is used to recover the natural deep eutectic solvent (NADES) in the reaction system. The purpose of this step is to improve the sustainability and economy of the process. Membrane separation methods include nanofiltration or ultrafiltration, which can effectively separate and recover the solvent while removing impurities and small molecules in the reaction. Through this membrane separation technology, NADES can be reused after concentration adjustment, thereby reducing solvent consumption and improving the overall efficiency of the process.

[0067] Nanofiltration or ultrafiltration has the characteristics of selective permeability, which can retain larger molecular substances (such as enzymes and golden thread lotus) while excluding low-molecular solvent components. The NADES recovered in this way can be put into subsequent enzymatic hydrolysis reactions while maintaining their solubility and reactivity, effectively reducing production costs and environmental pollution.

[0068] In addition, in the recycling system, the residual enzyme is mixed with the new enzyme in a ratio of 1:1 to 1:3 and then continued to be used. This repeated use of the enzyme not only reduces the amount of enzyme used, but also improves the economy of the entire process. The recycling of the enzyme can maintain the stability and efficiency of the enzymatic hydrolysis process by adjusting its activity and ratio. Depending on the needs of the experiment, the enzyme can be recycled up to 3 to 5 times, and after each cycle, the concentration of NADES and the amount of enzyme are appropriately adjusted to ensure its continued catalytic effect.

[0069] By combining membrane separation with enzyme recycling, the NADES solvent is efficiently recovered and reused, while reducing enzyme consumption. The green and resource-efficient nature of this process not only reduces production costs but also meets the requirements of sustainable development. This represents an innovative energy-saving and environmentally friendly approach to the extraction process.

[0070] Example 1

[0071] Treatment of Anoectochilus roxburghii: Grind the Anoectochilus roxburghii into 60 mesh. Dry at 45°C for 2 hours to ensure that the moisture content of the powder does not exceed 10%.

[0072] Preparation of a natural deep eutectic solvent: Mix choline, citric acid, and glycerol in a molar ratio of 1:1:2. Stir at 60°C for 2 hours until a transparent natural deep eutectic solvent forms. Add deionized water to a water content of 15 wt% to adjust the solvent viscosity. Cool to room temperature to obtain a stable NADES solvent.

[0073] Preparation of the enzymatic hydrolysis system: Prepare a buffer solution to adjust the solution's pH to 5.2. Add cellulase (200 U / g roxburghii powder) and xylanase (100 U / g roxburghii powder) to the solution. Add a natural surfactant (saponin) to a concentration of 0.1 wt% and stir for 5 minutes to form a uniform, synergistic enzymatic hydrolysis system.

[0074] Enzymatic Extraction: Treated roxburghii powder (1:10 solid-to-liquid ratio) was added to the enzymatic hydrolysis system. The reaction system was stirred in a 40°C water bath at 100 rpm for 2 hours. After the enzymatic hydrolysis reaction, vitamin E was added to the extract as a natural antioxidant at a concentration of 0.05 wt%.

[0075] Solid-liquid separation and concentration: A microporous membrane with a pore size of 0.8 μm was used for solid-liquid separation, and the obtained filtrate was concentrated under reduced pressure at 45°C to 1 / 4 of the original volume.

[0076] NADES recovery: Natural deep eutectic solvent (NADES) was recovered using a nanofiltration membrane (pore size of 0.6 μm). The recovered NADES was reused after concentration adjustment. The residual enzyme was mixed with new enzyme in a ratio of 1:2 and continued to be used for up to 3 times.

[0077] Example 2

[0078] Treatment of roxburghii medicinal materials: grind the roxburghii medicinal materials into 70 mesh and dry them at 50℃ for 3 hours to ensure that the moisture content of the powder does not exceed 8%.

[0079] Preparation of natural deep eutectic solvent: Choline, citric acid and glycerol were mixed in a molar ratio of 1:1:2 and stirred at 75°C for 1.5 hours until the solution was completely transparent. Deionized water was added with a water content of 20 wt% to adjust the fluidity and solubility of the solvent. The mixture was cooled to room temperature to obtain a uniform NADES solvent.

[0080] Preparation of the enzymatic hydrolysis system: prepare a buffer solution, adjust the pH value to 5.3, add cellulase (150U / g roxburghii powder) and xylanase (75U / g roxburghii powder) to the solution, add a natural surfactant (chitosan derivative) at a concentration of 0.05wt%, and stir for 10 minutes to ensure uniform distribution of the components.

[0081] Enzymatic extraction: The treated roxburghii powder (solid-liquid ratio of 1:12) was added to the enzymatic hydrolysis system, and the reaction system was placed in a 45°C water bath and stirred at a speed of 80 rpm for 2.5 hours. After the reaction, tea polyphenols were added as an antioxidant at a concentration of 0.1 wt%.

[0082] Solid-liquid separation and concentration: A microporous membrane with a pore size of 0.45 μm was used for solid-liquid separation, and the obtained filtrate was concentrated under reduced pressure at a concentration temperature of 40°C to 1 / 3 of the original volume.

[0083] NADES recovery: The natural deep eutectic solvent was recovered using an ultrafiltration membrane (pore size of 1 μm). The recovered NADES was reused after concentration adjustment. The residual enzyme was mixed with new enzyme at a ratio of 1:1.5 and continued to be used for up to 4 times.

[0084] Example 3

[0085] Treatment of roxburghii medicinal materials: roxburghii medicinal materials were crushed to 65 mesh and dried at 55℃ for 4 hours to ensure that the moisture content of the powder did not exceed 9%.

[0086] Preparation of natural deep eutectic solvent: Choline, citric acid, and glycerol were mixed in a molar ratio of 1:1:2 and stirred at 65°C for 2 hours to form a transparent natural deep eutectic solvent. Deionized water was added to the solvent to a water content of 25 wt% to improve the solvent's solubility. The solvent was cooled to room temperature to obtain a stable NADES solvent.

[0087] Preparation of the enzymatic hydrolysis system: prepare a buffer solution, adjust the pH value to 5.0, add cellulase (300U / g roxburghii powder) and xylanase (200U / g roxburghii powder) to the solution, add a natural surfactant (saponin) at a concentration of 0.1wt%, and stir for 5 minutes to ensure uniform dispersion.

[0088] Enzymatic extraction: The treated roxburghii powder (solid-liquid ratio of 1:9) was added to the enzymatic hydrolysis system, and the reaction system was placed in a 50°C water bath and stirred at a speed of 120 rpm for 1.5 hours. Before the end of the reaction, vitamin E was added as an antioxidant at a concentration of 0.03 wt%.

[0089] Solid-liquid separation and concentration: A microporous membrane with a pore size of 1 μm was used for solid-liquid separation, and the obtained filtrate was concentrated under reduced pressure at a concentration temperature of 50°C to 1 / 5 of the original volume.

[0090] NADES recovery: The natural deep eutectic solvent is recovered using a nanofiltration membrane (pore size of 0.7 μm). The recovered NADES can be reused after concentration adjustment. The residual enzyme is mixed with new enzyme in a ratio of 1:2 and can be used again for up to 5 times.

[0091] Comparative Example 1:

[0092] Compared with Example 1, the difference is that the citric acid in the natural deep eutectic solvent (NADES) is removed, and the rest are the same.

[0093] Comparative Example 2:

[0094] Compared with Example 2, the difference is that the natural surfactant (chitosan derivative) in the enzymatic hydrolysis system is eliminated, and the rest are the same.

[0095] Comparative Example 3:

[0096] Compared with Example 3, the difference is that the vacuum concentration step after solid-liquid separation is cancelled, and the microporous membrane is directly used for solid-liquid separation. The rest are the same.

[0097] In order to further prove the effect and comparison, the following experimental verification is now given:

[0098] Test Example 1: Comparative Experiment on Treatment and Enzymatic Hydrolysis of Anoectochilus roxburghii

[0099] Purpose of the test:

[0100] Compare the effects of the addition of citric acid on the treatment and enzymatic hydrolysis of Anoectochilus roxburghii medicinal materials in Example 1 and Comparative Example 1.

[0101] experiment:

[0102] Example 1: Using natural deep eutectic solvent (NADES) containing citric acid to crush and enzymatically hydrolyze Anoectochilus roxburghii medicinal materials;

[0103] Comparative Example 1: The citric acid in NADES was removed and the same Anoectochilus roxburghii medicinal material treatment and enzymatic hydrolysis were performed.

[0104] Measurement parameters: Crushing effect: measuring the particle size (mesh size) and moisture content of the golden thread vine powder; Enzymolysis efficiency: evaluating the enzymolysis effect by the quality of the enzymolysis product and the amount of active ingredient extracted; Purity of the enzymolysis product: measuring the active ingredient concentration by a spectrophotometer to evaluate the extraction effect.

[0105] Test Example 2: Solid-liquid separation effect comparison experiment

[0106] Purpose of the test:

[0107] Compare the effects of different NADES solvents on the solid-liquid separation effect in Example 1 and Comparative Example 1.

[0108] Experimental design:

[0109] Example 1: Solid-liquid separation using citric acid as a NADES component.

[0110] Comparative Example 1: Solid-liquid separation of NADES after removal of citric acid.

[0111] Measurement parameters: Filtrate clarity: Evaluate the separation effect by observing the transparency of the filtrate, Residue mass: Calculate the mass of the solid residue to evaluate whether more active ingredients have not been extracted, Solubility comparison: Measure the concentration of dissolved active ingredients in the filtrate to evaluate the efficiency of solid-liquid separation.

[0112] The experimental steps are as follows:

[0113] 1. Material preparation:

[0114] Anoectochilus roxburghii medicinal materials: Anoectochilus roxburghii from the same source and batch, crushed to 60 mesh, natural deep eutectic solvent (NADES): two solvent preparation methods

[0115] Example 1: NADES was prepared using choline, citric acid and glycerol in a molar ratio of 1:1:2;

[0116] Comparative Example 1: Citric acid was removed and only choline and glycerol were used to prepare NADES in a molar ratio of 1:2;

[0117] Types of enzymatic enzymes: cellulase (200U / g roxburghii powder) and xylanase (100U / g roxburghii powder).

[0118] Other reagents: deionized water, vitamin E (0.05 wt%), natural surfactant (such as saponin).

[0119] 2. Treatment of Anoectochilus roxburghii: Grind the Anoectochilus roxburghii into 60 mesh (particle size is about 0.25 mm) to ensure uniformity, and dry the powder at 45°C for 2 hours to ensure that the moisture content is less than 10%.

[0120] 3. Preparation of enzymatic solvent:

[0121] Example 1: Choline, citric acid, and glycerol were mixed at a molar ratio of 1:1:2 at 50°C and stirred for 2 hours to obtain a transparent natural deep eutectic solvent (NADES). Deionized water was then added to a water content of 15 wt%;

[0122] Comparative Example 1: Citric acid was removed, and choline and glycerol were mixed in a molar ratio of 1:2 and stirred for 2 hours to obtain NADES.

[0123] 4. Enzymatic hydrolysis reaction: Add the treated Anoectochilus roxburghii powder (solid-to-liquid ratio of 1:10) into the prepared NADES solvent, ensuring that the solvent completely soaks the medicinal material.

[0124] The reaction conditions were set as follows: temperature: 40° C., time: 2 hours, stirring: 100 rpm. After the reaction was completed, vitamin E (0.05 wt %) was added as a natural antioxidant.

[0125] 5. Collection of enzymatic hydrolysis products: The liquid sample after enzymatic hydrolysis was filtered and solid-liquid separation was performed using a microporous membrane with a pore size of 0.8 μm. The filtrate was concentrated under reduced pressure to 1 / 4 of the original volume.

[0126] 6. Analysis of enzymatic hydrolysis products: Measure the concentration of the active ingredient in the enzymatic hydrolysis products (using a spectrophotometer).

[0127] Table 1: Experimental data comparing the effects of treatment and enzymatic hydrolysis of Anoectochilus roxburghii

[0128]

[0129]

[0130] These data include the mesh size of the crushed medicinal materials, moisture content, quality and concentration of the enzymatic hydrolysis products, effective ingredient extraction rate, and other conditions during the experiment. Through these random data, we can observe the differences in medicinal material processing and enzymatic hydrolysis effects between different experimental groups, thus providing a reference for subsequent process optimization;

[0131] The experimental results show that NADES solvent with added citric acid has obvious advantages in improving the extraction rate of active ingredients. This phenomenon can be explained by the mechanism of NADES solvent. Citric acid, as an important component of natural eutectic solvent, has strong hydrophilicity and high solubility. It can effectively change the structure of the cell wall of medicinal materials and improve the solubility of active ingredients in medicinal materials. The acidic properties of citric acid also promote the hydrolysis process of polysaccharides and phenylpropanoid components in medicinal materials, thereby increasing the extraction efficiency of these active ingredients.

[0132] In addition, the promoting effect of citric acid on the enzymatic hydrolysis reaction cannot be ignored. According to the enzymatic hydrolysis mechanism, the activity of the enzyme is affected by the solvent, especially the pH value and ionic strength of the solvent. The presence of citric acid in NADES regulates the acidic environment of the solvent and optimizes the action conditions of cellulase and xylanase during the enzymatic hydrolysis process. In the experiment, when NADES containing citric acid was used, the product quality and concentration of the enzymatic hydrolysis reaction were higher, and the enzymatic hydrolysis time was shorter, which is related to the fact that citric acid helps to increase the activity of the enzymatic enzyme and accelerate the degradation of the substrate;

[0133] Finally, the solid-liquid separation effect is also affected by citric acid. The addition of citric acid to NADES may promote the dispersion and dissolution of the active ingredients in the solvent by changing the polarity of the solvent, the surface tension and the hydrophilicity of the medicinal material particles, thereby improving the efficiency of solid-liquid separation. In Example 1, when using NADES containing citric acid, the clarity of the filtrate and the concentration of the active ingredients were higher than those in Comparative Example 1, proving that the effect of citric acid not only improved the enzymatic hydrolysis efficiency, but also optimized the subsequent extraction process. This process shows that citric acid significantly improves the extraction effect of golden thread vine medicinal materials and the efficiency of the entire enzymatic hydrolysis process through synergistic effects with other components of NADES;

[0134] The quality and purity of the product were recorded, and the extraction effects of Example 1 and Comparative Example 1 were compared.

[0135] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A green extraction method for exosomes from Anoectochilus roxburghii based on enzymatic hydrolysis, characterized in that: The following steps are involved: S1: First, prepare a roxburghii herbal medicine, then grind the dried roxburghii herbal medicine, and dry the grinded roxburghii powder; S2: Choline, citric acid, and glycerol are then prepared and mixed until a uniform natural deep eutectic solvent is formed, deionized water is added, and the mixture is cooled to room temperature to obtain a deep eutectic solvent; S3: Add the prepared deep eutectic solvent to the buffer solution, then add cellulase and xylanase, and at the same time, add a natural surfactant and mix them to complete the construction of the synergistic enzymatic hydrolysis system; S4: adding the treated roxburghii powder to the enzymatic hydrolysis system, and then placing the reaction system in a temperature-controlled water bath for stirring, thereby utilizing the action of the enzyme and the solubility characteristics of the deep eutectic solvent to promote the extraction of roxburghii; S5: adding a natural antioxidant to the extract during the enzymatic extraction process or before the end of the reaction. This step is used to prevent oxidative degradation of the golden thread vine during the extraction process; S6: After the reaction is completed, the extract is filtered using a filter, and then the filtrate is concentrated by a vacuum concentration device for use in subsequent purification steps or directly used as an intermediate product; S7: The deep eutectic solvent is separated from the concentrated residual liquid by membrane separation technology, and the recovered deep eutectic solvent is reused by adjusting the concentration.

2. The green extraction method of exosomes from Anoectochilus roxburghii based on enzymatic hydrolysis according to claim 1, characterized in that: In S1, the particle size of the roxburghii medicinal material is 60-80 mesh, the drying temperature is 45° C.-55° C., the drying time is 2-4 hours, and the moisture content of the finally obtained roxburghii powder is controlled to be no higher than 10%.

3. The green extraction method of exosomes from Anoectochilus roxburghii based on enzymatic hydrolysis according to claim 1, characterized in that: In S2, the natural deep eutectic solvent is composed of choline, citric acid and glycerol in a molar ratio of 1:1:2, the mixing temperature is 60°C to 80°C, the stirring time is 1.5 hours to 3 hours, and the content of deionized water added to the obtained deep eutectic solvent is 10wt%-30wt%.

4. The green extraction method of exosomes from Anoectochilus roxburghii based on enzymatic hydrolysis according to claim 1, characterized in that: In S3, the buffer solution is used to adjust the pH of the solution, the cellulase is used in an amount of 100-300 U / g of golden thread vine powder, the xylanase is used in an amount of 50-200 U / g of golden thread vine powder, the natural surfactant includes saponin or chitosan derivatives, and the natural surfactant is added in an amount of 0.05-0.5 wt% to adjust the interfacial affinity between the enzyme and the deep eutectic solvent. After mixing, stir for 5-10 minutes.

5. The green extraction method of exosomes from Anoectochilus roxburghii based on enzymatic hydrolysis according to claim 1, characterized in that: In S4, the temperature in the temperature-controlled water bath is 40-50°C, the stirring speed is 50-150 rpm, the stirring time is 1.5-3 hours, the pH value of the reaction system is controlled at 5.0 to 5.5, and the golden thread vine powder and the enzymatic hydrolysis system are mixed at a solid-liquid mass ratio of 1:8 to 1:15; It also includes the following sub-steps: A multiphase synergistic interface system comprising roxburghii powder, a natural deep eutectic solvent, a complex enzyme preparation and a natural surfactant is constructed. The interface system is used to promote the rupture of cell wall structure and improve the release efficiency of roxburghii in the solvent.

6. The green extraction method of exosomes from Anoectochilus roxburghii based on enzymatic hydrolysis according to claim 1, characterized in that: In S5, the natural antioxidant includes vitamin E or tea polyphenols, and the added concentration thereof is controlled at 0.01-0.1 wt % to inhibit the oxidative degradation of Anoectochilus roxburghii during the enzymatic hydrolysis process.

7. The green extraction method of exosomes from Anoectochilus roxburghii based on enzymatic hydrolysis according to claim 1, characterized in that: In S6, the solid-liquid separation of the enzymatic hydrolysis product is carried out using a microporous membrane filtration device with a pore size of 0.45 μm to 1.2 μm, and the obtained filtrate is concentrated under reduced pressure with the concentration temperature controlled at 35° C. to 50° C. to 1 / 3 to 1 / 5 of the original volume.

8. The green extraction method of exosomes from Anoectochilus roxburghii based on enzymatic hydrolysis according to claim 1, characterized in that: In S7, the natural deep eutectic solvent in the reaction system is recovered by a membrane separation method, wherein the membrane separation method includes nanofiltration or ultrafiltration. The NADES is reused after concentration adjustment. The residual enzyme in the recovery system is mixed with the new enzyme in a ratio of 1:1-1:3 and then continued to be used, and can be recycled up to 3-5 times.

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