Liver protection gastrodia elata polyphenol extract as well as preparation method and application thereof

Abstract

The invention discloses a liver protection gastrodia elata polyphenol extract as well as a preparation method and application thereof, and belongs to the technical field of natural product extraction. The method aims at solving the problems that in the prior art, gastrodia elata polyphenol extraction efficiency is low, and active ingredients are prone to being damaged by heat. According to the technical scheme, the method is characterized by comprising the following steps: mixing a gastrodia elata raw material with liquid nitrogen after being subjected to freezing and unfreezing circulating pretreatment, and performing cell wall breaking treatment through a high-voltage pulsed electric field; mixing the wall-broken raw material with a buffer solution, carrying out enzymolysis by virtue of an immobilized enzyme reactor, cooling the system after enzymolysis, and carrying out ultrahigh-pressure extraction; centrifugally filtering the extracting solution, purifying through a macroporous adsorption resin column, and collecting ethanol eluent; and finally, carrying out vacuum concentration and spray drying on the eluent to obtain the gastrodia elata polyphenol extract. According to the method, the polyphenol yield is effectively improved, the activity of the components is protected, and the obtained extract can be used for preparing liver protection medicines.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction technology. More specifically, this invention relates to a liver-protective gastrodia polyphenol extract, its preparation method, and its uses. Background Technology

[0002] The preparation of polyphenol extracts from Gastrodia elata is a research direction in the field of natural product extraction. Existing technical solutions face some inherently related technical difficulties in practice.

[0003] First, the polyphenols in Gastrodia elata are quite sensitive to thermal environments. During extraction, commonly used methods such as hot reflux or prolonged high-temperature extraction can cause structural changes in these components. While some physical cell-wall disruption methods, such as ultrasonic treatment, can avoid overall heating to some extent, the instantaneous high temperatures generated by cavitation effects can still affect heat-sensitive components in specific areas. Especially in later stages of extraction, such as concentration and drying, traditional high-temperature operations struggle to balance efficiency and component preservation. Therefore, establishing an effective low-temperature or mild-temperature environment throughout the entire process, while ensuring extraction efficiency, to prevent polyphenol deactivation due to thermal effects in key stages such as cell-wall disruption, extraction, and drying, is a problem that requires comprehensive consideration.

[0004] Secondly, when enzymatic hydrolysis is applied to plant extraction, it often involves directly adding free enzymes to the reaction system. In this method, the enzyme preparation cannot be recovered, increasing the cost per processing run; simultaneously, enzyme proteins remain in the extract after the reaction, adding an extra burden to subsequent separation and purification. Immobilized enzyme technology offers a solution to the enzyme recovery problem, but the catalytic efficiency of immobilized enzymes prepared by conventional methods tends to decrease during continuous or repeated use. This phenomenon may be caused by enzyme molecules detaching from the carrier, impurities in the reaction system adsorbing and covering the carrier surface, or changes in the enzyme protein's conformation during repeated use. Maintaining the stability of immobilized enzymes during operation to meet the requirements of continuous multi-batch production is a factor affecting the economic viability and practicality of this technology.

[0005] Third, when purifying extracts using macroporous adsorption resins, elution with a single concentration of ethanol solution is a common practice. However, the selectivity of this method is sometimes less than ideal, potentially leading to insufficient separation of the target polyphenol from impurities with similar physicochemical properties, thus limiting the purity of the final product. While adding elution steps or optimizing the elution curve can improve the separation effect to some extent, this may introduce new problems such as increased process complexity, increased solvent consumption, and prolonged operation time. Therefore, achieving efficient separation of polyphenols from coexisting impurities within a reasonable range of process complexity is a key focus in purification process research.

[0006] In summary, existing technologies for preparing Gastrodia elata polyphenols typically face challenges such as systematically controlling the heat effect to protect component activity, achieving stability and reusability of the enzymatic hydrolysis process, and improving the purity of the final product while ensuring process efficiency. These factors collectively constrain the quality and preparation efficiency of Gastrodia elata polyphenol extracts. Summary of the Invention

[0007] One objective of this invention is to provide a liver-protective gastrodia polyphenol extract, its preparation method, and its uses. Ultimately, a gastrodia polyphenol extract with sufficient retention of active ingredients and high purity is obtained, providing a reliable material basis for its subsequent applications.

[0008] To achieve these objectives and other advantages of the present invention, according to one aspect of the present invention, a method for preparing a liver-protective Gastrodia elata polyphenol extract is provided, comprising the following steps: Step 1: Freeze the Gastrodia elata raw material at -25℃ to -30℃ for 2-3 hours, and then thaw it rapidly at 35-40℃. Repeat this freeze-thaw cycle 3 times. Step 2: Mix the processed Gastrodia elata raw material with liquid nitrogen at a mass ratio of 1:2-1:3, and carry out cell wall disruption treatment in a high-voltage pulse electric field device. The electric field strength is 20-30kV / cm, the pulse width is 20-30μs, and the pulse frequency is 100-200Hz. During the treatment, the material temperature is controlled to not exceed 15℃ through a cooling system. Step 3: Mix the cell-wall-broken raw material with a buffer solution preheated to 45-55℃ at a mass-volume ratio of 1:15-1:20 to form a mixed system; allow the mixed system to flow through an immobilized enzyme reactor and perform enzymatic hydrolysis for 90-120 min at a pH of 4.5-5.5 and a temperature of 45-55℃; after enzymatic hydrolysis, uniformly reduce the temperature of the mixed system to 20-25℃ within 5-10 min, and then perform ultra-high pressure extraction at a pressure of 100-150MPa for 15-20 min, while controlling the material temperature to be maintained within the range of 20-25℃ during the pressure holding time; Step 4: Centrifuge and filter the mixture after ultra-high pressure extraction at 20-25℃, and purify the filtrate by passing it through an AB-8 macroporous adsorption resin column. Step 5: Concentrate the ethanol eluent under reduced pressure at a vacuum of -0.08 to -0.1 MPa and a temperature of 40-50℃ until the relative density reaches 1.05-1.15; spray dry the concentrated liquid, controlling the inlet temperature at 120-150℃ and the outlet temperature at 60-80℃ to obtain the Gastrodia elata polyphenol extract.

[0009] Preferably, the mixing process of the treated Gastrodia elata raw material and liquid nitrogen in step two is as follows: the Gastrodia elata raw material after the freeze-thaw cycle is taken out from the thawing environment and transferred to an insulated container that has been pre-cooled to -50°C to -70°C within 1-3 minutes. Liquid nitrogen is introduced into the insulated container in two stages: In the first stage, liquid nitrogen accounting for 40%-50% of the total liquid nitrogen mass is injected by atomization spray to rapidly freeze the surface of the Gastrodia elata raw material to form a protective layer, which lasts for 1-2 minutes; In the second stage, the remaining 50%-60% of liquid nitrogen is added by immersion to fully mix the Gastrodia elata raw material and liquid nitrogen at a total mass ratio of 1:2-1:3; After mixing is completed, the material temperature is maintained below -50°C in the insulated container, and the material is transferred to a high-voltage pulse electric field device for cell wall disruption within 5 minutes after mixing is completed.

[0010] Preferably, the high-voltage pulse electric field processing in step two uses a bipolar exponentially decaying pulse waveform and is performed in two stages according to the following procedure: The first stage of processing conditions are: electric field strength 20-25 kV / cm, pulse width 20-25 μs, pulse frequency 100-150 Hz, and processing time 30-60 s. The second stage processing conditions are: electric field strength 25-30 kV / cm, pulse width 25-30 μs, pulse frequency 150-200 Hz, and processing time 60-90 s; The two-stage processing interval is 10-20 seconds.

[0011] Preferably, the specific process for immobilizing the enzyme reactor packing material in step three is as follows: Step a: Select porous diatomaceous earth with a particle size of 150-300 μm as a carrier, soak it in a hydrochloric acid solution with a concentration of 0.5-1.0 mol / L for 2-3 h, filter it, wash it with deionized water until neutral, and then dry it at 105-110℃ to constant weight; immerse the dried carrier in an ethanol solution of 5%-8% by mass of γ-aminopropyltriethoxysilane, and stir it at a constant temperature of 60-70℃ for 4-6 h. After the reaction is completed, wash it thoroughly with anhydrous ethanol to obtain an aminated and activated carrier. Step b: Mix cellulase, pectinase and papain in an enzyme activity ratio of 1:1.5:0.8, dissolve in phosphate buffer with a pH of 6.0-6.5, and prepare a compound enzyme solution with a total enzyme concentration of 25-40 mg / mL. Step c: Mix the aminated activated carrier obtained in step a with the composite enzyme solution prepared in step b at a mass ratio of 1:8-1:12, and place it in an environment of 4-8℃ with slow shaking for 2-3 h; then add 1%-2% of genipin as a cross-linking agent as the total mass of the composite enzyme solution, and continue the shaking reaction at 25-30℃ for 8-12 h to fix the enzyme on the carrier through covalent bonds. Step d: After the immobilization reaction is completed, the immobilized enzyme packing is filtered and separated. It is then washed with phosphate buffer solution with a pH of 6.0-6.5 until no protein is detected in the wash solution. Subsequently, it is freeze-dried to obtain the dried immobilized enzyme reactor packing and then filled into the reactor.

[0012] Preferably, in step d, after the immobilized enzyme reactor packing material is filled into the reactor, an activation pretreatment process is performed. The specific steps are as follows: Place the filled immobilized enzyme reactor in a constant temperature environment of 45-55℃; use phosphate-citric acid buffer with a pH of 4.5-5.5 as the activation solution and pass it through the reactor at a constant flow rate of 2-3 column volumes / hour; continuously flush for 3-5 column volumes, or until the pH of the effluent stabilizes in the range of 4.5-5.5 and the conductivity change rate does not exceed ±2% for 3 consecutive minutes; after the activation pretreatment is completed, the reactor can be used for the enzymatic hydrolysis process in step three.

[0013] Preferably, the specific process of filtering the extract in step four is as follows: Centrifuge the enzymatically hydrolyzed and ultra-high pressure treated mixture directly for 15-25 min and collect the supernatant. Maintain the pH of the supernatant in the range of 4.5-5.5, then add 0.2%-0.5% of its mass of activated carbon, and stir and adsorb at 15-25℃ for 20-30 min. After adsorption is complete, the system is filtered sequentially through a filter membrane with a pore size of 1.0-1.5 μm and a pretreated diatomaceous earth filter aid layer, with the flow rate controlled at 2-3 mL / min. The permeate is collected, which is the filtrate after filtration.

[0014] Preferably, when the filtrate in step four passes through the AB-8 type macroporous adsorption resin column, it is eluted according to the following gradient: Elute with 3-5 column volumes of deionized water at a flow rate of 2-3 mL / min, and discard this eluent. Elute with 3-5 column volumes of 20%-25% ethanol aqueous solution at a flow rate of 2-3 mL / min, and discard this eluent. Elute with 5-8 column volumes of 50%-60% ethanol aqueous solution at a flow rate of 1-2 mL / min, and collect this portion of the ethanol eluent.

[0015] Preferably, in step five, before vacuum concentration, 0.5%-1.5% of maltodextrin by mass is added to the ethanol eluent, and the mixture is stirred thoroughly until completely dissolved before subsequent vacuum concentration and spray drying are carried out.

[0016] The present invention also provides a liver-protective gastrodia polyphenol extract obtained by the above preparation method.

[0017] This invention also provides the application of a hepatoprotective gastrodia polyphenol extract in the preparation of hepatoprotective drugs.

[0018] This invention provides at least the following beneficial effects: The liver-protective Gastrodia elata polyphenol extract, its preparation method, and its uses, as described in this invention, involve a cyclical process of deep freezing and rapid thawing. This process causes fatigue damage to the plant cell walls due to repeated ice crystal formation and melting, creating favorable conditions for subsequent cell wall disruption. The combination of staged liquid nitrogen treatment and the sequential action of a bipolar high-voltage pulsed electric field achieves gentle and thorough cell structure disintegration, effectively avoiding compositional changes caused by localized overheating, thus laying the foundation for the efficient release of polyphenols.

[0019] A composite immobilized enzyme reactor, prepared using a specific carrier and cross-linking agent, enables multiple cell wall degrading enzymes to function synergistically and stably. This not only significantly improves enzyme utilization efficiency and economy but also ensures high efficiency and batch-to-batch consistency of the enzymatic hydrolysis reaction through subsequent activation pretreatment and in-situ cleaning procedures, thus extending the reactor's lifespan.

[0020] During the extraction stage, the integration of enzymatic hydrolysis and ultra-high pressure extraction was optimized through precise temperature control. Ultra-high pressure conditions, while maintaining a suitable temperature, further promoted the dissolution of cell contents, thereby increasing the polyphenol transfer rate while avoiding the effects of heat.

[0021] During the purification process, low-temperature adsorption and filtration under specific acid-base conditions were maintained to remove impurities while minimizing the loss of the target analyte. Subsequently, elution was performed using a gradient of increasing ethanol concentrations, effectively improving the separation of the target polyphenol from impurities and thus increasing the purity of the final product.

[0022] In the final processing stage, maltodextrin is introduced into the eluent as a protective agent. Combined with reduced concentration and spray drying temperatures, this constitutes a key protective measure for heat-sensitive components. This design significantly reduces the oxidation and wall adhesion of polyphenols during the drying process, ensuring product yield and physical properties.

[0023] In summary, this preparation method, through the aforementioned interconnected process steps, ultimately yields a Gastrodia elata polyphenol extract with sufficient retention of active ingredients and high purity, providing a reliable material basis for its subsequent applications.

[0024] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0025] Figure 1 This is a comparison of the in vitro hepatoprotective and antioxidant activities of gastrodin (GAS) and gastrodin polyphenol extract (GE-UAE).

[0026] Figure 2 A comparative diagram showing the in vitro anti-inflammatory effects of gastrodin (GAS) and gastrodin polyphenol extract (GE-UAE).

[0027] Figure 3 The graph shows the changes in mouse body weight and liver index.

[0028] Figure 4 This is a graph showing the levels of AST, ALT, and AKP in mouse serum. Detailed Implementation

[0029] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can implement it based on the description.

[0030] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0031] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0032] In the description of this invention, the terms "lateral", "longitudinal", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", and "outer" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0033] This invention provides a method for preparing a liver-protective gastrodia polyphenol extract, comprising the following steps: Step 1: Freeze the Gastrodia elata raw material at -25℃ to -30℃ for 2-3 hours, and then thaw it rapidly at 35-40℃. Repeat this freeze-thaw cycle 3 times. Step 2: Mix the processed Gastrodia elata raw material with liquid nitrogen at a mass ratio of 1:2-1:3, and carry out cell wall disruption treatment in a high-voltage pulse electric field device. The electric field strength is 20-30kV / cm, the pulse width is 20-30μs, and the pulse frequency is 100-200Hz. During the treatment, the material temperature is controlled to not exceed 15℃ through a cooling system. Step 3: Mix the cell-wall-broken raw material with a buffer solution preheated to 45-55℃ at a mass-volume ratio of 1:15-1:20 to form a mixed system; allow the mixed system to flow through an immobilized enzyme reactor and perform enzymatic hydrolysis for 90-120 min at a pH of 4.5-5.5 and a temperature of 45-55℃; after enzymatic hydrolysis, uniformly reduce the temperature of the mixed system to 20-25℃ within 5-10 min, and then perform ultra-high pressure extraction at a pressure of 100-150MPa for 15-20 min, while controlling the material temperature to be maintained within the range of 20-25℃ during the pressure holding time; Step 4: Centrifuge and filter the mixture after ultra-high pressure extraction at 20-25℃, and purify the filtrate by passing it through an AB-8 macroporous adsorption resin column. Step 5: Concentrate the ethanol eluent under reduced pressure at a vacuum of -0.08 to -0.1 MPa and a temperature of 40-50℃ until the relative density reaches 1.05-1.15; spray dry the concentrated liquid, controlling the inlet temperature at 120-150℃ and the outlet temperature at 60-80℃ to obtain the Gastrodia elata polyphenol extract.

[0034] In the above technical solution, during the raw material pretreatment stage, an ultra-low temperature freezer can be used for freezing the Gastrodia elata raw material, and a constant temperature water bath can be used for thawing. The raw material can be fresh or dried Gastrodia elata from Yunnan. The ultra-low temperature freezer can be placed in the pretreatment workshop, and the constant temperature water bath can be placed on a workbench near the ultra-low temperature freezer. The raw material is placed in the ultra-low temperature freezer and kept at -25℃ to -30℃ for 2 to 3 hours, then quickly transferred to a constant temperature water bath pre-set to 35℃ to 40℃ until completely thawed. This process is repeated three times. The raw material that has completed the freeze-thaw cycle can be transferred within 1 to 3 minutes to an insulated container pre-cooled to -50℃ to -70℃, which can be placed near the inlet of the high-voltage pulse electric field equipment. Liquid nitrogen was then introduced in two stages. First, 40% to 50% of the total liquid nitrogen mass was injected through an atomizing nozzle for 1 to 2 minutes. Then, the remaining liquid nitrogen was added by immersion, so that the total mass ratio of Gastrodia elata to liquid nitrogen was 1:2 to 1:3. This stage of treatment weakened the cell wall structure of Gastrodia elata due to the repeated formation and melting of ice crystals, creating favorable conditions for the subsequent cell wall breaking step.

[0035] In the cell disruption and enzymatic extraction stages, a high-voltage pulsed electric field treatment device can be selected, with its treatment chamber equipped with a bipolar exponentially decaying pulse waveform generator and a water-cooled jacket. A column-type immobilized enzyme reactor can be used, filled with immobilized enzyme packing material using porous diatomaceous earth as a carrier. An ultra-high pressure homogenizer can be used for ultra-high pressure extraction. The high-voltage pulsed electric field device can be installed downstream of the insulated container's outlet, the immobilized enzyme reactor can be connected in series after the mixing tank, and the ultra-high pressure homogenizer can be installed on the enzymatic reaction outlet pipeline. The pretreated Gastrodia elata raw material was mixed with liquid nitrogen and sent into a high-voltage pulsed electric field treatment chamber. In the first stage, the material was treated with parameters of electric field strength 20 to 25 kV / cm, pulse width 20 to 25 μs, and pulse frequency 100 to 150 Hz for 30 to 60 seconds. After an interval of 10 to 20 seconds, in the second stage, the material was treated with parameters of electric field strength 25 to 30 kV / cm, pulse width 25 to 30 μs, and pulse frequency 150 to 200 Hz for 60 to 90 seconds. During this period, the material temperature was controlled to not exceed 15℃ using a water cooling system. The cell-wall-broken raw material was mixed with phosphate citrate buffer solution preheated to 45℃ to 55℃ at a mass-to-volume ratio of 1:15 to 1:20 and then pumped into an immobilized enzyme reactor. Enzymatic hydrolysis was carried out for 90 to 120 minutes at a pH of 4.5 to 5.5 and a temperature of 45℃ to 55℃. After enzymatic hydrolysis, the material is cooled to 20°C to 25°C within 5 to 10 minutes, and then enters an ultra-high pressure homogenizer for extraction at 100 to 150 MPa for 15 to 20 minutes, while maintaining the material temperature between 20°C and 25°C. This stage, through the synergistic effect of multiple physical fields and biological enzymes, achieves thorough disintegration of cell structures and efficient dissolution of contents.

[0036] During the purification and drying stages, a high-speed tubular centrifuge can be used for solid-liquid separation, an AB-8 type macroporous adsorption resin column can be used for purification, a rotary evaporator can be used for vacuum concentration, and a small spray drying tower can be used for drying. The centrifuge can be installed downstream of the outlet of the ultra-high pressure homogenizer, the resin column can be placed after the centrifuge, and the rotary evaporator and spray drying tower can be arranged sequentially in the purification area. The mixture after ultra-high pressure extraction is centrifuged at 4000 to 5000 rpm for 15 to 25 minutes at 20°C to 25°C, and the supernatant is collected. This supernatant is directly loaded onto an AB-8 type macroporous adsorption resin column, first eluted with 5 to 8 column volumes of deionized water at a flow rate of 2 to 3 mL per minute, then eluted with 4 to 6 column volumes of 30% to 40% ethanol aqueous solution at a flow rate of 1 to 2 mL per minute, and the ethanol eluent is collected. The eluent was concentrated to a relative density of 1.05 to 1.15 under a vacuum of -0.08 to -0.1 MPa and a temperature of 40°C to 50°C. Finally, it was spray-dried, with the inlet temperature controlled at 120°C to 150°C and the outlet temperature at 60°C to 80°C, to obtain the Gastrodia elata polyphenol extract. This stage, with its mild purification and drying conditions, minimizes the loss of heat-sensitive components and helps maintain the product's activity.

[0037] This method effectively protects heat-sensitive components by systematically constructing a low-temperature extraction environment and combining a synergistic cell wall disruption strategy with a stable enzymatic hydrolysis system. Specifically, the combined use of liquid nitrogen pretreatment and a high-voltage pulsed electric field enhances cell wall disruption; the optimized design of the immobilized enzyme reactor ensures the controllability and repeatability of the enzymatic hydrolysis process; and the combination of gradient elution and low-temperature drying helps maintain product activity, thereby obtaining a stable quality Gastrodia elata polyphenol extract.

[0038] In some other technical solutions, the mixing process of the processed Gastrodia elata raw material with liquid nitrogen in step two is as follows: the Gastrodia elata raw material after the freeze-thaw cycle is taken out from the thawing environment and transferred to an insulated container that has been pre-cooled to -50°C to -70°C within 1-3 minutes. Liquid nitrogen is introduced into the insulated container in two stages: In the first stage, liquid nitrogen accounting for 40%-50% of the total liquid nitrogen mass is injected by atomization spray to rapidly freeze the surface of the Gastrodia elata raw material to form a protective layer, which lasts for 1-2 minutes; In the second stage, the remaining 50%-60% of liquid nitrogen is added by immersion to fully mix the Gastrodia elata raw material and liquid nitrogen at a total mass ratio of 1:2-1:3; After mixing is completed, the material temperature is maintained below -50°C in the insulated container, and the material is transferred to a high-voltage pulse electric field device for cell wall disruption within 5 minutes after mixing is completed.

[0039] In the above technical solution, the main equipment for liquid nitrogen pretreatment can be a vacuum-insulated container with a sealed lid, equipped with a liquid nitrogen storage tank and an atomizing spray device. The vacuum-insulated container can be placed next to the feeding station of the high-voltage pulse electric field equipment, and the liquid nitrogen storage tank is connected to the atomizing spray device through an insulated pipe. The gastrodia elata raw material that has completed the freeze-thaw cycle is transferred to the vacuum-insulated container pre-cooled to -50°C to -70°C within 1 to 3 minutes, and the first stage of liquid nitrogen treatment begins immediately.

[0040] In the staged liquid nitrogen processing, the supply method and flow rate of liquid nitrogen can be adjusted through a control system. The atomizing spray device can be installed on top of the insulated container, and the submersible injection pipe can be installed on the side wall of the container. In the first stage, 40% to 50% of the total mass of liquid nitrogen is injected in a fine mist through the top atomizing nozzle for 1 to 2 minutes, forming a uniform cryogenic protective layer on the surface of the raw material. In the second stage, the remaining 50% to 60% of the liquid nitrogen is added directly through the side wall pipe, completely submerging the raw material and achieving a mixing state with a raw material to liquid nitrogen mass ratio of 1:2 to 1:3.

[0041] In the material transfer stage, a dedicated cryogenic conveying device can be configured to connect the insulated container and the high-voltage pulsed electric field equipment. The conveying device can be an insulated screw feeder or a vacuum suction system. After mixing, the material temperature is maintained below -50°C within the insulated container, and the cryogenic material is conveyed into the high-voltage pulsed electric field treatment chamber within 5 minutes via the conveying device. This staged liquid nitrogen treatment method helps form a complete protective layer on the raw material surface and maintains the material's cryogenic brittle state through rapid transfer, providing suitable material properties for subsequent cell wall disruption.

[0042] This liquid nitrogen pretreatment scheme utilizes a synergistic effect of staged temperature control and rapid transfer. The first stage, atomized spraying, rapidly forms a continuous protective ice shell on the raw material surface. This structure effectively blocks direct impact between liquid nitrogen and the material during subsequent immersion treatment, while ensuring uniform low-temperature conduction. The second stage, immersion treatment, achieves deep freezing of the material on top of the protective layer, resulting in uniform micro-ice crystals inside and outside the cells. This staged treatment, combined with rapid transfer completed within five minutes, allows the material to enter the electric field cell-wall breaking stage in its complete low-temperature brittle state, creating ideal material conditions for the subsequent high-voltage pulsed electric field. The entire process, through precise temperature and time control, avoids the material cracking problems that are common in traditional liquid nitrogen treatment, while ensuring that the cell structure maintains its optimal low-temperature brittle state, thus laying a solid foundation for the subsequent cell-wall breaking steps.

[0043] In other technical solutions, the high-voltage pulse electric field processing in step two uses a bipolar exponentially decaying pulse waveform and is performed in two stages according to the following procedure: The first stage of processing conditions are: electric field strength 20-25 kV / cm, pulse width 20-25 μs, pulse frequency 100-150 Hz, and processing time 30-60 s. The second stage processing conditions are: electric field strength 25-30 kV / cm, pulse width 25-30 μs, pulse frequency 150-200 Hz, and processing time 60-90 s; The two-stage processing interval is 10-20 seconds.

[0044] In the above technical solution, a bipolar exponentially decaying pulse generator can be selected as the core equipment for the high-voltage pulsed electric field treatment, and a parallel plate electrode treatment chamber and a circulating water cooling system can be provided. The pulse generator can be installed in the upstream control cabinet of the treatment chamber, the parallel plate electrode can be made of food-grade stainless steel, and the heat exchange unit of the cooling system can be integrated into the jacket of the treatment chamber. The inlet of the treatment chamber is connected to the liquid nitrogen pretreatment section via a conveying pipeline, and the outlet is connected to the mixing tank of the enzymatic hydrolysis section.

[0045] Regarding pulse processing program settings, two-stage parameters can be configured through the pulse generator's control interface. The first stage sets the electric field strength to 20-25 kV / cm, pulse width to 20-25 μs, pulse frequency to 100-150 Hz, and processing time to 30-60 s. The second stage sets the electric field strength to 25-30 kV / cm, pulse width to 25-30 μs, pulse frequency to 150-200 Hz, and processing time to 60-90 s. A 10-20 s interval is set between the two stages, during which pulse output is stopped while the cooling system continues to operate.

[0046] During the process, the material pretreated with liquid nitrogen continuously passes through the parallel plate electrode processing chamber. The first stage uses relatively mild parameters to induce reversible electroporation of the cell membrane, while the second stage enhances the electric field parameters to promote complete cell disintegration. Throughout the treatment, a circulating water cooling system maintains the material temperature below 15°C. This phased, progressive treatment method, combined with continuous cooling, helps achieve uniform and effective cell disruption.

[0047] This staged pulsed electric field treatment scheme optimizes cell disruption through a combination of progressive parameter settings and intermittent cooling. The use of a bipolar exponentially decaying waveform helps reduce electrolytic effects during processing, while the two-stage parameter design allows the cell membrane to undergo a gradual process from initial perforation to complete disintegration. The interval setting allows sufficient time for the cell structure to respond while continuous cooling prevents the accumulation of thermal effects. This treatment method effectively maintains a low-temperature environment in the material system while ensuring efficient cell disruption, providing a suitable material state for subsequent processes.

[0048] In other technical solutions, the specific process for immobilizing the enzyme reactor packing material in step three is as follows: Step a: Select porous diatomaceous earth with a particle size of 150-300 μm as a carrier, soak it in a hydrochloric acid solution with a concentration of 0.5-1.0 mol / L for 2-3 h, filter it, wash it with deionized water until neutral, and then dry it at 105-110℃ to constant weight; immerse the dried carrier in an ethanol solution of 5%-8% by mass of γ-aminopropyltriethoxysilane, and stir it at a constant temperature of 60-70℃ for 4-6 h. After the reaction is completed, wash it thoroughly with anhydrous ethanol to obtain an aminated and activated carrier. Step b: Mix cellulase, pectinase and papain in an enzyme activity ratio of 1:1.5:0.8, dissolve in phosphate buffer with a pH of 6.0-6.5, and prepare a compound enzyme solution with a total enzyme concentration of 25-40 mg / mL. Step c: Mix the aminated activated carrier obtained in step a with the composite enzyme solution prepared in step b at a mass ratio of 1:8-1:12, and place it in an environment of 4-8℃ with slow shaking for 2-3 h; then add 1%-2% of genipin as a cross-linking agent as the total mass of the composite enzyme solution, and continue the shaking reaction at 25-30℃ for 8-12 h to fix the enzyme on the carrier through covalent bonds. Step d: After the immobilization reaction is completed, the immobilized enzyme packing is filtered and separated. It is then washed with phosphate buffer solution with a pH of 6.0-6.5 until no protein is detected in the wash solution. Subsequently, it is freeze-dried to obtain the dried immobilized enzyme reactor packing and then filled into the reactor.

[0049] In the above technical solution, during the pretreatment stage of the immobilized enzyme carrier, an acid-resistant reactor can be used for the activation treatment of the diatomaceous earth carrier, and a vacuum filtration device and an electrically heated drying oven can be equipped. The porous diatomaceous earth can be a food-grade product with a particle size of 150 to 300 micrometers, and the γ-aminopropyltriethoxysilane can be a chemically pure reagent. The acid-treated reactor can be placed in a fume hood, and the drying oven can be installed in a fixed position in the pretreatment area. The diatomaceous earth carrier is placed in the reactor, and a hydrochloric acid solution of 0.5 to 1.0 mol / L is added for 2 to 3 hours. Solid-liquid separation is then performed using a vacuum filtration device, and the carrier is washed with deionized water until the filtrate is neutral. The washed carrier is transferred to a drying oven and dried at 105 to 110°C to constant weight. The dried support was immersed in a 5% to 8% (w / w) γ-aminopropyltriethoxysilane ethanol solution and stirred at a constant temperature of 60 to 70°C for 4 to 6 hours. After the reaction was completed, the support was thoroughly washed with anhydrous ethanol to obtain an amino-activated support.

[0050] In the immobilization stage of the complex enzyme, a constant-temperature shaking incubator can be used for the enzyme immobilization reaction, equipped with a pH meter and a UV spectrophotometer. Food-grade enzyme preparations can be used for cellulase, pectinase, and papain, while genipin can be a biochemical reagent. The constant-temperature shaking incubator can be placed in a clean working area, and the pH meter can be used to monitor the buffer preparation process. The three enzymes are compounded at an enzyme activity ratio of 1:1.5:0.8 and dissolved in phosphate buffer with a pH of 6.0 to 6.5 to prepare a complex enzyme solution with a total enzyme concentration of 25 to 40 mg / mL. The aminated activation carrier is mixed with the complex enzyme solution at a mass ratio of 1:8 to 1:12 and placed in an environment at 4 to 8°C with slow shaking for 2 to 3 hours for adsorption. Then, 1% to 2% of the total mass of the complex enzyme solution is added as a cross-linking agent, and the reaction is continued at 25 to 30°C with shaking for 8 to 12 hours, allowing the enzyme to be covalently immobilized on the carrier.

[0051] In the post-treatment stage of the immobilized enzyme packing material, a Buchner funnel can be used for solid-liquid separation, and a freeze dryer can be used for drying. Analytical grade reagents can be used to prepare the phosphate buffer. The Buchner funnel can be mounted on a vacuum filtration flask, and the freeze dryer can be placed in the drying area. After the immobilization reaction is complete, the immobilized enzyme packing material is separated by filtration through a Buchner funnel and washed with phosphate buffer at pH 6.0 to 6.5 until no protein absorption peak is detected in the eluent at 280 nm. The washed immobilized enzyme packing material is then freeze-dried to obtain dried immobilized enzyme reactor packing material, which is then packed into a column reactor for later use.

[0052] This immobilized enzyme preparation method achieves stable immobilization of enzyme proteins through a combination of carrier surface modification and cross-linking agent selection. The pore structure and surface amination treatment of the diatomaceous earth carrier provide suitable immobilization sites for enzyme molecules, while the use of genipin cross-linking agent facilitates the formation of stable covalent links. The synergistic immobilization of the complex enzyme system allows multiple cell wall degrading enzymes to function together, while strict rinsing and drying conditions ensure the cleanliness and stability of the immobilized enzyme packing material. This preparation method contributes to obtaining immobilized enzyme reactor packing materials with good enzyme activity retention and a long service life.

[0053] In other technical solutions, step d involves loading the immobilized enzyme reactor packing material into the reactor followed by an activation pretreatment process. The specific steps are as follows: Place the filled immobilized enzyme reactor in a constant temperature environment of 45-55℃; use phosphate-citric acid buffer with a pH of 4.5-5.5 as the activation solution and pass it through the reactor at a constant flow rate of 2-3 column volumes / hour; continuously flush for 3-5 column volumes, or until the pH of the effluent stabilizes in the range of 4.5-5.5 and the conductivity change rate does not exceed ±2% for 3 consecutive minutes; after the activation pretreatment is completed, the reactor can be used for the enzymatic hydrolysis process in step three.

[0054] In the above technical solution, during the activation pretreatment stage of the immobilized enzyme reactor, a constant-temperature circulating water bath can be used to maintain the reactor temperature, and a constant-flow pump can be equipped to control the flow rate of the activation solution. The phosphate-citric acid buffer solution can be prepared using analytical grade reagents, and a pH meter and conductivity meter can be used for monitoring the effluent. The constant-temperature circulating water bath can be enclosed outside the reactor, and the constant-flow pump can be installed between the activation solution storage tank and the reactor inlet. The filled immobilized enzyme reactor is placed in a constant-temperature environment of 45 to 55°C, using a phosphate-citric acid buffer solution with a pH of 4.5 to 5.5 as the activation solution, and the solution is continuously pumped through the reactor at a constant flow rate of 2 to 3 column volumes per hour.

[0055] During the activation process monitoring, effluent parameters can be monitored using online detection devices or timed sampling. pH meter and conductivity meter sensors can be installed in the reactor outlet pipeline, and the data acquisition system can continuously record parameter changes. Continuously flush with 3 to 5 column volumes of activation solution, or monitor in real time until the effluent pH stabilizes within the range of 4.5 to 5.5, while the conductivity change rate does not exceed ±2% for 3 consecutive minutes. The activation pretreatment can be terminated once either condition is met, at which point the reactor's internal environment has reached a state compatible with the subsequent enzymatic hydrolysis process.

[0056] After the activation process is complete, the constant flow pump can be stopped and the activation solution delivery line disconnected. The reactor is maintained within a certain temperature range and can be directly used for subsequent enzymatic hydrolysis processes. This activation pretreatment brings the temperature, pH, and ionic environment inside the reactor to equilibrium, providing a suitable environment for the immobilized enzyme to function.

[0057] This activation pretreatment scheme establishes a reaction environment consistent with enzymatic hydrolysis conditions, enabling the immobilized enzyme reactor to reach its optimal operating state. Maintaining a constant temperature environment ensures conformational stability of the enzyme molecules, while continuous rinsing with a specific pH buffer not only balances the internal environment of the reactor but also helps remove any impurities that may remain from the immobilization process. Real-time monitoring of the pH and conductivity parameters of the effluent allows for accurate determination of whether the reactor's internal state has reached equilibrium. This objective parameter-based method helps ensure consistency in pretreatment effects across different batches, providing a reliable guarantee for the stable execution of subsequent enzymatic hydrolysis reactions.

[0058] In other technical solutions, the specific process of filtering the extract in step four is as follows: Centrifuge the enzymatically hydrolyzed and ultra-high pressure treated mixture directly for 15-25 min and collect the supernatant. Maintain the pH of the supernatant in the range of 4.5-5.5, then add 0.2%-0.5% of its mass of activated carbon, and stir and adsorb at 15-25℃ for 20-30 min. After adsorption is complete, the system is filtered sequentially through a filter membrane with a pore size of 1.0-1.5 μm and a pretreated diatomaceous earth filter aid layer, with the flow rate controlled at 2-3 mL / min. The permeate is collected, which is the filtrate after filtration.

[0059] In the above technical solution, a high-speed refrigerated centrifuge can be used for solid-liquid separation in the primary treatment stage of the extract, and a pH meter can be equipped for acid-base monitoring. Food-grade powdered activated carbon can be used, and the centrifuge tubes can be made of polypropylene. The high-speed refrigerated centrifuge can be installed in the downstream working area of ​​the ultra-high pressure treatment equipment, and the constant temperature stirring device can be placed next to the centrifuge. The mixture after enzymatic hydrolysis and ultra-high pressure treatment is directly transferred into the centrifuge tubes and centrifuged at 4000 to 5000 rpm for 15 to 25 minutes at 20 to 25°C. The supernatant is carefully collected, and its pH value is maintained within the range of 4.5 to 5.5.

[0060] In the adsorption and impurity removal stage, a temperature-controlled mechanical stirrer can be used for activated carbon adsorption, and a precision electronic balance can be used to weigh the activated carbon. The mechanical stirrer can be placed near the centrifuge bench, and a glass beaker can be used as the stirring container. Accurately weigh 0.2% to 0.5% of the activated carbon by mass of the supernatant, and stir and adsorb at 200 to 300 rpm for 20 to 30 minutes at 15 to 25°C. After stirring, let it stand for 5 minutes to allow the activated carbon to settle naturally.

[0061] In the fine filtration stage, a plate and frame filter can be used in combination with filter media of different specifications, and a peristaltic pump can be used to control the filtration flow rate. The microporous membrane can be a mixed cellulose ester material with a pore size of 1.0 to 1.5 micrometers, and the diatomaceous earth filter aid can be a food-grade product. The plate and frame filter can be connected in series after the adsorption process, and the peristaltic pump can be installed in the inlet pipe of the filter. The system treated with activated carbon is first pre-filtered through a microporous membrane, and then finely filtered through a 2 to 3 mm thick layer of diatomaceous earth filter aid. The filtration flow rate is controlled within the range of 2 to 3 ml per minute, and the collected permeate is the final filtrate.

[0062] This filtration and purification scheme achieves gradual clarification of the extract through a staged process. Centrifugation effectively removes most solid particles, activated carbon selectively adsorbs pigments and small molecule impurities under specific pH and temperature conditions, and the combined filtration method ensures the final clarity of the filtrate. This stepwise purification method protects the target components from damage and maintains the stability of the system through mild operating conditions, providing a suitable material basis for subsequent purification processes.

[0063] In other technical solutions, when the filtrate in step four passes through an AB-8 type macroporous adsorption resin column, it is eluted according to the following gradient: Elute with 3-5 column volumes of deionized water at a flow rate of 2-3 mL / min, and discard this eluent. Elute with 3-5 column volumes of 20%-25% ethanol aqueous solution at a flow rate of 2-3 mL / min, and discard this eluent. Elute with 5-8 column volumes of 50%-60% ethanol aqueous solution at a flow rate of 1-2 mL / min, and collect this portion of the ethanol eluent.

[0064] In the above technical solution, a glass chromatography column can be used as the separation device in the resin column elution stage, and a constant flow pump can be equipped to control the elution flow rate. The AB-8 type macroporous adsorption resin can be of 20 to 60 mesh size, and the ethanol can be a food-grade product. The glass chromatography column can be vertically fixed on a support, and the constant flow pump can be connected between the eluent storage tank and the column. After loading the pretreated filtrate onto the resin column, elution is first performed using 3 to 5 column volumes of deionized water at a flow rate of 2 to 3 ml per minute. The eluting liquid in this stage mainly consists of highly polar impurities and should be discarded.

[0065] In the elution stage for moderately polar impurities, a 20% to 25% (v / v) aqueous ethanol solution can be prepared as the eluent. This concentration of ethanol solution can be prepared by accurately measuring anhydrous ethanol and deionized water using a graduated cylinder. Elution is continued using 3 to 5 column volumes of this concentration of ethanol solution at a flow rate of 2 to 3 ml per minute. The effluent from this stage contains moderately polar impurities and should be collected and discarded.

[0066] During the elution stage of the target component, a 50% to 60% (v / v) aqueous ethanol solution needs to be used. The appropriate concentration of ethanol solution can be prepared fresh, and a new collection container should be used. Elution is performed at a slow flow rate of 1 to 2 mL per minute, passing 5 to 8 column volumes of this concentration of ethanol solution. The effluent from this stage contains the target polyphenol component and should be collected for later use.

[0067] This gradient elution scheme achieves effective separation of materials through an elution strategy of increasing polarity. The deionized water elution stage removes highly polar impurities, a medium-concentration ethanol solution further elutes some medium-polarity impurities, while a higher-concentration ethanol solution effectively desorbs the target polyphenol component. This stepwise elution method helps obtain the target product with high purity, while flow rate adjustment ensures the elution process is thorough. The parameter settings for the entire elution process consider a balance between separation efficiency and solvent usage.

[0068] In other technical solutions, in step five, before vacuum concentration, 0.5%-1.5% of maltodextrin by mass is added to the ethanol eluent, and the mixture is stirred thoroughly until completely dissolved before subsequent vacuum concentration and spray drying are carried out.

[0069] In the above technical solution, during the additive pretreatment stage, an electronic balance can be used for accurate weighing of maltodextrin, and a mechanical stirrer can be used to achieve uniform mixing of the solution. The maltodextrin can be a food-grade product with a DE value of 15 to 20, and the stirring container can be a glass beaker or a stainless steel container. The electronic balance can be placed in a dry and clean weighing area, and the mechanical stirrer can be installed in the pretreatment area before the concentration process. Accurately weigh 0.5% to 1.5% of the maltodextrin equivalent to the mass of the ethanol eluent, slowly add the weighed maltodextrin to the eluent, and stir at 200 to 300 rpm for 20 to 30 minutes at room temperature until the maltodextrin is completely dissolved and the solution is homogeneous.

[0070] In the subsequent concentration and drying process, a rotary evaporator can be used for vacuum concentration, and a spray dryer can be used for drying and granulation. The rotary evaporator can be placed in a fume hood, and the spray dryer can be installed downstream of the concentration equipment. The mixed solution with added maltodextrin is transferred to the rotary evaporator and concentrated to a relative density of 1.05 to 1.15 under a vacuum of -0.08 to -0.1 MPa and a temperature of 40 to 50°C. The concentrate is then fed into the spray dryer, with the inlet temperature controlled at 120 to 150°C and the outlet temperature at 60 to 80°C. After the drying process is completed, the final extract powder is collected.

[0071] This additive application method improves the physical properties of the material in subsequent processing by adding maltodextrin. The molecular network structure formed by maltodextrin in solution helps protect heat-sensitive components, and the carrier matrix formed during drying reduces material adhesion to the walls. This treatment method provides more suitable material conditions for subsequent concentration and drying processes, helping to maintain the physical stability and chemical integrity of the product.

[0072] The present invention also provides a liver-protective gastrodia polyphenol extract obtained by the above preparation method.

[0073] This invention also provides the application of a hepatoprotective gastrodia polyphenol extract in the preparation of hepatoprotective drugs.

[0074] Example 1 A method for preparing a liver-protective gastrodia polyphenol extract includes the following steps: Step 1: Raw material pretreatment Take 1 kg of dried Gastrodia elata from Yunnan Province and place it in an ultra-low temperature freezer. Freeze it at -28°C for 2.5 hours, then transfer it to a constant temperature water bath at 38°C for rapid thawing. Repeat this freeze-thaw cycle three times. Transfer the treated raw material to a vacuum-insulated container pre-cooled to -60°C within 2 minutes. Inject liquid nitrogen (45% of the total liquid nitrogen mass) through an atomizing nozzle for 1.5 minutes to form a protective surface layer. Then, add the remaining liquid nitrogen by immersion to achieve a raw material to liquid nitrogen mass ratio of 1:2.5.

[0075] Step 2: Cell disruption treatment The low-temperature material was transferred to a high-voltage pulsed electric field device within 5 minutes and processed using a bipolar exponentially decaying pulse waveform. The first stage was set with an electric field strength of 23 kV / cm, a pulse width of 22 μs, a pulse frequency of 120 Hz, and a processing time of 45 s. After a 15-s interval, the second stage began, with an electric field strength of 28 kV / cm, a pulse width of 28 μs, a pulse frequency of 180 Hz, and a processing time of 75 s. During the process, a circulating water cooling system was used to control the material temperature to not exceed 15℃.

[0076] Step 3: Enzymatic hydrolysis and extraction The raw material after cell wall disruption was mixed with phosphate-citric acid buffer preheated to 50°C at a mass-to-volume ratio of 1:18 and pumped into the immobilized enzyme reactor. The reactor packing material was prepared as follows: porous diatomaceous earth with a particle size of 200 micrometers was soaked in 0.8 mol / L hydrochloric acid solution for 2.5 hours, washed until neutral, and dried at 108°C; it was then treated with 6% γ-aminopropyltriethoxysilane ethanol solution at 65°C for 5 hours to obtain an aminated carrier. Cellulase, pectinase, and papain were compounded at an enzyme activity ratio of 1:1.5:0.8 to prepare a composite enzyme solution with a total concentration of 32 mg / mL, which was mixed with the carrier at a mass ratio of 1:10. The mixture was shaken and adsorbed at 6°C for 2.5 hours, 1.5% genipin crosslinking agent was added, and the reaction was continued at 28°C for 10 hours. After washing and lyophilization, the mixture was packed into the reactor. Enzymatic hydrolysis was carried out at pH 5.0 and 50°C for 105 minutes. Then, the material was cooled to 22°C within 8 minutes and transferred to an ultra-high pressure device for extraction at 130MPa for 18 minutes, with the temperature controlled at 22°C.

[0077] Step 4: Purification The extraction system was centrifuged at 4500 rpm for 20 minutes at 22°C, and the supernatant was collected while maintaining the pH at 5.0. 0.35% (by weight of the supernatant) of food-grade activated carbon was added, and the mixture was stirred and adsorbed at 20°C for 25 minutes. The solution was then passed sequentially through a 1.2-micron microporous membrane and a 2-millimeter thick diatomaceous earth filter aid layer at a flow rate of 2.5 mL / min, and the filtrate was collected. The filtrate was loaded onto an AB-8 macroporous resin column, eluted first with 4 column volumes of deionized water at a flow rate of 2.5 mL / min, and the eluent was discarded; then eluted with 4 column volumes of 23% ethanol solution at the same flow rate, and the eluent was discarded; finally, eluted with 6 column volumes of 55% ethanol solution at a flow rate of 1.5 mL / min, and this eluent was collected.

[0078] Step 5: Concentration and Drying Add 1.0% (by weight) of maltodextrin to the ethanol eluent, stir until completely dissolved, and then concentrate under reduced pressure at -0.09 MPa and 45°C to a relative density of 1.10. Spray dry the concentrate at an inlet temperature of 135°C and an outlet temperature of 70°C to obtain the Gastrodia elata polyphenol extract.

[0079] Comparative Example 1: Traditional Mechanical Crushing and Thermal Reflux Extraction Method One kg of dried Gastrodia elata from the same source was mechanically crushed to 60 mesh powder using a high-speed pulverizer. The powder was mixed with 70% ethanol solution at a mass-to-volume ratio of 1:15 and placed in a reflux extraction apparatus. The mixture was heated and refluxed at 80°C for 2 hours. The extract was collected by filtration, and the residue was extracted once more. The two extracts were combined. The extract was concentrated under reduced pressure at 65°C until no alcohol odor remained. After redissolving in water, it was passed through an AB-8 macroporous resin column and purified under the same conditions as in Example 1. Finally, the extract was concentrated under reduced pressure at 65°C and spray-dried at an inlet temperature of 180°C and an outlet temperature of 95°C to obtain Comparative Sample 1.

[0080] Comparative Example 2: Free Enzymatic Hydrolysis and Room Temperature Extraction Method One kg of dried Gastrodia elata from the same source was taken, pretreated by freezing and thawing under the same conditions, and then mixed with phosphate-citrate buffer (pH 5.0) at a mass-to-volume ratio of 1:18. A free complex enzyme with the same activity as in Example 1 (the same ratio of cellulase, pectinase, and papain) was directly added, and enzymatic hydrolysis was performed at 50°C for 105 minutes. After enzymatic hydrolysis, instead of high-pressure extraction, slow stirring and soaking were performed at 25°C for 12 hours. Subsequent purification and drying conditions were the same as in Example 1, yielding control sample 2.

[0081] Comparative Example 3: Single Concentration Elution vs. Carrier-Free Drying Method The resin purification step was performed according to the method in Example 1. The filtrate was loaded onto an AB-8 macroporous resin column, and eluted directly with 8 column volumes of 40% ethanol solution at a flow rate of 2 mL / min without gradient elution. All eluent was collected. Maltodextrin was not added to the eluent, and it was concentrated under reduced pressure at 45°C to a relative density of 1.10. The eluent was then spray-dried at an inlet temperature of 180°C and an outlet temperature of 95°C to obtain control sample 3.

[0082] Comparative Example 4: Traditional Full-Process Technology One kg of dried Gastrodia elata from the same source was crushed to 60 mesh powder using a high-speed pulverizer. The powder was mixed with 70% ethanol solution at a mass-to-volume ratio of 1:15, and extracted by reflux at 80℃ for 2 hours. The extraction was repeated once. The extracts were combined, concentrated under reduced pressure at 65℃, and then passed through an AB-8 macroporous resin column, eluted with 40% ethanol solution. The eluent was concentrated directly without any carrier and spray-dried at an inlet temperature of 180℃ and an outlet temperature of 95℃ to obtain control sample 4.

[0083] Identification of phenolic compounds The Gastrodia elata polyphenol extract prepared in Example 1 was used to identify the main phenolic compounds in the extract by UPLC-QTOF-MS. A total of 26 compounds were preliminarily identified from the extract, and the results are shown in Table 1.

[0084] Table 1 NO. RT(min) Formula Mass CAS Name 1 0.789 <![CDATA[C6H 14 N2O2]]> 146.1054 56-87-1 L-Lysine 2 0.88 <![CDATA[C4H7NO4]]> 133.0374 56-84-8 L-Aspartic acid 3 0.971 <![CDATA[C4H6O5]]> 134.0212 6915-15-7 malic acid 4 1.32 <![CDATA[C4H4O4]]> 116.0106 110-17-8 fumaric acid 5 1.457 <![CDATA[C9H 11 NO3]]> 181.0736 60-18-4 L-tyrosine 6 1.516 <![CDATA[C6H8O7]]> 192.0268 77-92-9 Citric acid 7 1.539 <![CDATA[C5H5N5]]> 135.0547 73-24-5 adenine 8 1.584 <![CDATA[C6H 13 NO2]]> 131.0945 61-90-5 L-Leucine 9 2.189 <![CDATA[C 13 H 18 O7]]> 286.1 62499-27-8 Gastrodin 10 2.661 <![CDATA[C7H6O3]]> 138.12 139-85-5 3,4-Dihydroxybenzaldehyde 11 3.161 <![CDATA[C 15 H 22 O 10 ]]> 362.1212 2415-24-9 Zichun 12 4.093 <![CDATA[C7H6O2]]> 120.0437 123-08-0 p-Hydroxybenzaldehyde 13 4.57 <![CDATA[C 10 H 10 O4]]> 194.0579 131-11-3 Dimethyl phthalate 14 4.629 <![CDATA[C6H6O6]]> 174.0163 4023-65-8 trans-aconitine 15 5.897 <![CDATA[C 19 H 24 O 13 ]]> 460.39 952068-57-4 Bartholinoside E 16 9.22 <![CDATA[C 12 H 16 N2O]]> 204.1262 486-86-2 alkaloids 17 10.274 <![CDATA[C9H 10 O5]]> 198.0526 831-61-8 Ethyl gallate 18 11.266 <![CDATA[C7H6O2]]> 122.0369 65-85-0 benzoic acid 19 11.707 <![CDATA[C 10 H 13 N5O4]]> 267.0968 58-61-7 adenosine 20 12.948 <![CDATA[C 32 H 40 O 19 ]]> 728.2156 174972-80-6 Barison Glycoside C 21 14.395 <![CDATA[C 32 H 40 O 19 ]]> 727.2166 174972-79-3 Bartholinoside B 22 15.358 <![CDATA[C7H8O2]]> 124.0524 623-05-2 p-Hydroxybenzyl alcohol 23 16.767 <![CDATA[C 11 H 12 N2O2]]> 204.0899 73-22-3 L-Tryptophan 24 18.184 <![CDATA[C 45 H 56 O 25 ]]> 996.3107 62499-28-9 Bartholin A 25 19.185 <![CDATA[C 14 H 14 O3]]> 230.0942 484-14-0 4,4'-Dihydroxydibenzyl ether 26 25.094 <![CDATA[C 12 H 14 O4]]> 222.0893 4046-02-0 Ethyl ferulic acid The results showed that 26 polyphenolic compounds were identified in the sample of Example 1, including gastrodin, barisonin AE and other major active ingredients.

[0085] Hepatoprotective effect test The gastrodia polyphenol extract (GE-UAE) prepared in Example 1 was compared with commercially available pure gastrodin (GAS), and its health efficacy was evaluated through cell experiments. Figure 1As shown, the cytotoxicity of Gastrodia elata polyphenol extract (GE-UAE) and gastrodin in the concentration range of 50-400 μg / mL was detected, and the results showed that neither had significant toxicity. To evaluate the hepatoprotective effect, a 3% ethanol-induced alcoholic liver injury model of HepG2 cells was established. It was found that the levels of AST and ALT in the supernatant of the culture medium in the model group were significantly increased, while the levels of these two enzymes were significantly reduced after treatment with Gastrodia elata polyphenol extract (GE-UAE) and gastrodin, indicating that both had a protective effect against hepatocyte injury, and that gastrodin was more effective than Gastrodia elata polyphenol extract. Regarding antioxidant activity, the levels of SOD and GSH in the model group were lower than those in the control group, while the level of MDA was higher than that in the control group. The positive group and the sample group significantly increased the levels of SOD and GSH and decreased the level of MDA, showing a dose-response effect, indicating that Gastrodia elata polyphenol extract and gastrodin can effectively scavenge free radicals and reduce oxidative stress. Figure 2 As shown, to evaluate anti-inflammatory activity, an LPS-induced Raw 264.7 cell anti-inflammatory model was used. Results showed that the expression levels of TNF-α and IL-6 were increased, while the expression level of IL-10 was decreased in the model group. After intervention with Gastrodia elata polyphenol extract and gastrodin, the expression levels of TNF-α and IL-6 decreased, while the expression level of IL-10 increased, indicating that both can inhibit the inflammatory response by regulating the release of inflammatory factors. In conclusion, Gastrodia elata polyphenol extract and gastrodin exhibit significant hepatoprotective, antioxidant, and anti-inflammatory activities in vitro.

[0086] In vivo activity assessment of Gastrodia elata polyphenol extract (GE-UAE) Thirty-five healthy, physiologically normal 8-week-old male C57BL / 6J mice (20-22g) were selected to establish an acute alcoholic liver injury model. The mice were randomly divided into five groups: control group, model group, high-dose Gastrodia elata polyphenol extract group (TMH, 2 g / kg BW), low-dose Gastrodia elata polyphenol extract group (TML, 1 g / kg BW), and a combination of Gastrodia elata polyphenol extract and selenomethionine (Sep, polyphenol extract 2g / kg + selenomethionine 1mg / kg).

[0087] like Figure 3 As shown, after modeling, the body weight of mice in the other groups decreased significantly compared to the control group, preliminarily proving the success of modeling. The organ indices of the model group mice increased significantly, further indicating the successful establishment of the alcoholic liver injury model; the liver index of the drugs administered showed a certain degree of decrease compared to the model group. Figure 4 As shown, the serum AST, ALT, and AKP levels in the model group mice were significantly higher than those in the blank control group. Conversely, the serum AST, ALT, and AKP levels in the treatment group mice were significantly lower than those in the model group. This indicates that the polyphenolic extracts of Gastrodia elata can alleviate acute alcoholic liver injury.

[0088] The gastrodia polyphenol extract prepared in Example 1 and samples from Comparative Examples 1-4 were subjected to the following tests: Polyphenol yield determination: The Folin-Ciocalteu method was used, with gallic acid as the standard. The polyphenol yield was calculated using the following formula, with the calculation basis being the mass percentage of dry Gastrodia elata raw material, and the result expressed as gallic acid equivalent; Polyphenol yield (%) = (mass of polyphenols in the extract / mass of dry Gastrodia elata raw material) × 100%; Polyphenol purity analysis: The content of major polyphenol components was determined by HPLC. Antioxidant activity assay: DPPH radical scavenging rate: Refer to the method of Brand-Williams et al.; ABTS+ free radical scavenging rate: Refer to the method of Miller et al.; FRAP value: Refer to the method of Benzie et al.; The results are shown in Table 2.

[0089] Table 2 Polyphenol yield (%) Polyphenol purity (%) DPPH scavenging rate IC50 (μg / mL) ABTS clearance rate (%)* <![CDATA[FRAP value (mM FeSO4 / g)]]> Example 1 4.25±0.18 85.3±1.2 12.8±0.9 93.5±1.6 2.78±0.14 Comparative Example 1 2.13±0.21 65.8±2.1 29.3±1.4 71.2±2.5 1.38±0.17 Comparative Example 2 3.32±0.19 76.4±1.8 18.6±1.2 84.3±2.1 2.08±0.16 Comparative Example 3 3.85±0.17 79.2±1.5 15.4±1.1 88.7±1.9 2.42±0.15 Comparative Example 4 1.82±0.23 58.6±2.4 34.7±1.8 67.5±2.8 1.21±0.19 Note: *The test concentration for ABTS free radical scavenging rate is 100 μg / mL.

[0090] The results show that the integrated preparation method of Gastrodia elata polyphenol extract provided by the present invention exhibits significant advantages in terms of extraction efficiency, product purity, and antioxidant activity.

[0091] Specifically, the polyphenol yield in Example 1 reached 4.25%, significantly higher than that of the comparative examples (1.82%-3.85%), demonstrating that the present invention achieves more complete cell structure disintegration and content release through the synergistic effect of "freezing-thawing, liquid nitrogen embrittlement, and high-voltage pulsed electric field cell disruption". The polyphenol purity of the product was 85.3%, also significantly better than that of the comparative examples (58.6%-79.2%), thanks to the precise enzymatic hydrolysis by immobilized enzymes and the effective removal of impurities by gradient elution purification.

[0092] Regarding functional activity, Example 1 exhibited the strongest in vitro antioxidant capacity, with the best DPPH radical scavenging activity (IC50 of 12.8 μg / mL), ABTS radical scavenging rate (93.5%), and FRAP value (2.78 mM FeSO4 / g). This indicates that the low-temperature or mild temperature control strategy employed in this invention effectively protects the active structure of the polyphenolic compounds.

[0093] In summary, the experimental data fully demonstrate that this invention, through the sequential integration of multiple technologies and the design of full-process activity protection, has achieved significant improvements over traditional processes in multiple dimensions, providing a reliable and efficient technical solution for obtaining high-yield, high-purity, and high-activity Gastrodia elata polyphenol extract.

[0094] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. A method for preparing a liver-protective gastrodia polyphenol extract, characterized in that, Includes the following steps: Step 1: Freeze the Gastrodia elata raw material at -25℃ to -30℃ for 2-3 hours, and then thaw it at 35-40℃. Repeat this freeze-thaw cycle 3 times. Step 2: Mix the processed Gastrodia elata raw material with liquid nitrogen at a mass ratio of 1:2-1:3, and carry out cell wall disruption treatment in a high-voltage pulse electric field device. The electric field strength is 20-30kV / cm, the pulse width is 20-30μs, and the pulse frequency is 100-200Hz. During the treatment, the material temperature is controlled to not exceed 15℃ through a cooling system. Step 3: Mix the cell-wall-broken raw material with a buffer solution preheated to 45-55℃ at a mass-volume ratio of 1:15-1:20 to form a mixed system; allow the mixed system to flow through an immobilized enzyme reactor and perform enzymatic hydrolysis for 90-120 min at a pH of 4.5-5.5 and a temperature of 45-55℃; after enzymatic hydrolysis, uniformly reduce the temperature of the mixed system to 20-25℃ within 5-10 min, and then perform ultra-high pressure extraction at a pressure of 100-150MPa for 15-20 min, while controlling the material temperature to be maintained within the range of 20-25℃ during the pressure holding time; Step 4: Centrifuge and filter the mixture after ultra-high pressure extraction at 20-25℃, and purify the filtrate by passing it through an AB-8 macroporous adsorption resin column. Step 5: Concentrate the ethanol eluent under reduced pressure at a vacuum of -0.08 to -0.1 MPa and a temperature of 40-50℃ until the relative density reaches 1.05-1.15; spray dry the concentrated liquid, controlling the inlet temperature at 120-150℃ and the outlet temperature at 60-80℃ to obtain the Gastrodia elata polyphenol extract.

2. The method for preparing a liver-protective gastrodia polyphenol extract as described in claim 1, characterized in that, The mixing process of the processed Gastrodia elata raw material with liquid nitrogen in step two is as follows: the Gastrodia elata raw material after the freeze-thaw cycle is taken out from the thawing environment and transferred to an insulated container that has been pre-cooled to -50°C to -70°C within 1-3 minutes. Liquid nitrogen is introduced into the insulated container in two stages: In the first stage, liquid nitrogen accounting for 40%-50% of the total liquid nitrogen mass is injected by atomization spray to freeze the surface of the Gastrodia elata raw material to form a protective layer, which lasts for 1-2 minutes; In the second stage, the remaining 50%-60% of liquid nitrogen is added by immersion to fully mix the Gastrodia elata raw material and liquid nitrogen at a total mass ratio of 1:2-1:3; After mixing is completed, the material temperature is maintained below -50°C in the insulated container, and the material is transferred to a high-voltage pulse electric field device for cell wall disruption within 5 minutes after mixing is completed.

3. The method for preparing a liver-protective gastrodia polyphenol extract as described in claim 1, characterized in that, In step two, the high-voltage pulse electric field processing uses a bipolar exponentially decaying pulse waveform and is performed in two stages according to the following procedure: The first stage of processing conditions are: electric field strength 20-25 kV / cm, pulse width 20-25 μs, pulse frequency 100-150Hz, and processing time 30-60 s. The second stage processing conditions are: electric field strength 25-30 kV / cm, pulse width 25-30 μs, pulse frequency 150-200Hz, and processing time 60-90 s. The two-stage processing interval is 10-20 seconds.

4. The method for preparing a liver-protective gastrodia polyphenol extract as described in claim 1, characterized in that, The specific process for immobilizing the enzyme reactor packing material in step three is as follows: Step a: Select porous diatomaceous earth with a particle size of 150-300 μm as a carrier, soak it in a hydrochloric acid solution with a concentration of 0.5-1.0 mol / L for 2-3 h, filter it, wash it with deionized water until neutral, and then dry it at 105-110℃ to constant weight; immerse the dried carrier in an ethanol solution of 5%-8% by mass of γ-aminopropyltriethoxysilane, and stir it at a constant temperature of 60-70℃ for 4-6 h. After the reaction is completed, wash it thoroughly with anhydrous ethanol to obtain an aminated and activated carrier. Step b: Mix cellulase, pectinase and papain in an enzyme activity ratio of 1:1.5:0.8, dissolve in phosphate buffer with a pH of 6.0-6.5, and prepare a compound enzyme solution with a total enzyme concentration of 25-40 mg / mL. Step c: Mix the aminated activated carrier obtained in step a with the composite enzyme solution prepared in step b at a mass ratio of 1:8-1:12, and place it in an environment of 4-8℃ with slow shaking for 2-3 h; then add 1%-2% of genipin as a cross-linking agent as the total mass of the composite enzyme solution, and continue the shaking reaction at 25-30℃ for 8-12 h to fix the enzyme on the carrier through covalent bonds. Step d: After the immobilization reaction is completed, the immobilized enzyme packing is filtered and separated. It is then washed with phosphate buffer solution with a pH of 6.0-6.5 until no protein is detected in the wash solution. Subsequently, it is freeze-dried to obtain the dried immobilized enzyme reactor packing and then filled into the reactor.

5. The method for preparing a liver-protective gastrodia polyphenol extract as described in claim 4, characterized in that, In step d, after the immobilized enzyme reactor packing material is filled into the reactor, an activation pretreatment process is carried out. The specific steps are as follows: Place the filled immobilized enzyme reactor in a constant temperature environment of 45-55℃; use phosphate-citric acid buffer with a pH of 4.5-5.5 as the activation solution and pass it through the reactor at a constant flow rate of 2-3 column volumes / hour; continuously flush for 3-5 column volumes, or until the pH of the effluent stabilizes in the range of 4.5-5.5 and the conductivity change rate does not exceed ±2% for 3 consecutive minutes; after the activation pretreatment is completed, the reactor can be used for the enzymatic hydrolysis process in step three.

6. The method for preparing a liver-protective gastrodia polyphenol extract as described in claim 1, characterized in that, The specific process of filtering the extract in step four is as follows: Centrifuge the enzymatically hydrolyzed and ultra-high pressure treated mixture directly for 15-25 min and collect the supernatant. Maintain the pH of the supernatant in the range of 4.5-5.5, then add 0.2%-0.5% of its mass of activated carbon, and stir and adsorb at 15-25℃ for 20-30 min. After adsorption is complete, the system is filtered sequentially through a filter membrane with a pore size of 1.0-1.5 μm and a pretreated diatomaceous earth filter aid layer, with the flow rate controlled at 2-3 mL / min. The permeate is collected, which is the filtrate after filtration.

7. The method for preparing a liver-protective gastrodia polyphenol extract as described in claim 1, characterized in that, When the filtrate described in step four passes through an AB-8 macroporous adsorption resin column, it is eluted according to the following gradient: Elute with 3-5 column volumes of deionized water at a flow rate of 2-3 mL / min, and discard this eluent. Elute with 3-5 column volumes of 20%-25% ethanol aqueous solution at a flow rate of 2-3 mL / min, and discard this eluent. Elute with 5-8 column volumes of 50%-60% ethanol aqueous solution at a flow rate of 1-2 mL / min, and collect this portion of the ethanol eluent.

8. The method for preparing a liver-protective gastrodia polyphenol extract as described in claim 7, characterized in that, In step five, before vacuum concentration, 0.5%-1.5% of maltodextrin by mass is added to the ethanol eluent, and the mixture is stirred thoroughly until completely dissolved before subsequent vacuum concentration and spray drying.

9. A liver-protective gastrodia polyphenol extract, characterized in that, It is prepared by any one of the methods for preparing a liver-protective gastrodia polyphenol extract according to claims 1-8.

10. The use of the liver-protecting Gastrodia elata polyphenol extract as described in claim 9 in the preparation of liver-protecting drugs.

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