Method for efficiently extracting active ingredients of traditional Chinese medicine based on modern biotechnology and application of method

Abstract

The invention provides an innovative method for extracting active ingredients of traditional Chinese medicines, which integrates the modern biotechnology and aims at improving the extraction efficiency and purity and reducing the loss of the active ingredients at the same time. The core technology comprises enzyme-method-assisted extraction and efficient catalysis of release of active components by using a specific enzyme modified by genetic engineering; a supercritical fluid extraction and membrane separation technology is adopted, so that high-efficiency preliminary extraction and accurate separation are realized; a microwave-assisted extraction and dynamic countercurrent technology is adopted, so that the continuous extraction efficiency is improved; the molecular imprinting technology is used for selective adsorption and enrichment of target components; biosynthetic pathway analysis provides theoretical support for extraction optimization; the release and stability of active ingredients are enhanced by tissue crushing and heating-free extraction technologies; and a high-speed counter-current chromatography separation technology is adopted to finish fine separation of active components. The method has the advantages of being simple and convenient to operate, high in efficiency, good in purity, wide in application range and the like, and a brand new efficient technical scheme is provided for extraction of active ingredients of traditional Chinese medicine.

Description

Technical Field

[0001] The invention belongs to the technical field of extracting active ingredients of traditional Chinese medicine, and in particular refers to a highly efficient and environmentally friendly method for extracting active ingredients of traditional Chinese medicine combined with modern biotechnology. Background Art

[0002] As an important part of traditional medicine, Chinese medicine has been used in disease prevention and treatment for thousands of years. The effectiveness of Chinese medicine is mainly attributed to its complex combination of active ingredients, which play a key role in regulating human functions and fighting diseases. Although traditional Chinese medicine extraction methods, including but not limited to decoction, soaking, and percolation, have an irreplaceable position in history, they have gradually revealed their limitations in the process of modernization. These traditional methods often have low extraction efficiency and cannot fully extract the trace active ingredients in the medicinal materials. At the same time, during the extraction process, the active ingredients are easily destroyed or denatured due to factors such as high temperature and long-term treatment, which limits the in-depth research and wide application of the active ingredients of Chinese medicine.

[0003] With the continuous progress of modern science and technology, a series of new extraction technologies have emerged, such as ultrasonic extraction, microwave-assisted extraction, supercritical fluid extraction, etc. These technologies have improved the extraction efficiency and purity of ingredients to a certain extent. They effectively shorten the extraction time, reduce energy consumption, and reduce the destruction of active ingredients through physical or chemical means. However, these technologies still have some shortcomings, such as high equipment cost, complex operation, and low selectivity for the extraction of specific active ingredients. These problems have restricted the further development and application of traditional Chinese medicine active ingredient extraction technology to a certain extent.

[0004] In-depth research on the biosynthetic pathways and regulatory mechanisms of active ingredients in traditional Chinese medicine is of great significance for improving extraction efficiency and stability of ingredients. However, the current understanding of these biosynthetic pathways is not comprehensive enough, and there is a lack of effective regulatory means, which limits the efficient use of active ingredients in traditional Chinese medicine.

[0005] Modern biotechnology, especially the development of genetic engineering and molecular imprinting technology, has provided new perspectives and possibilities for the extraction of active ingredients from traditional Chinese medicine. Genetic engineering can modify enzymes to improve their catalytic efficiency and stability, thereby improving extraction efficiency; molecular imprinting technology can prepare new materials with high selective adsorption capacity for specific active ingredients, thereby achieving specific enrichment of target ingredients. However, how to effectively combine these advanced technologies with traditional Chinese medicine extraction technology, give play to their synergistic effects, overcome the shortcomings of a single technology, and achieve efficient, low-cost, and environmentally friendly extraction of active ingredients from traditional Chinese medicine is still a problem that needs to be solved in the current technical field.

[0006] The present invention is proposed in this context, aiming to develop a new method for extracting active ingredients of traditional Chinese medicine by innovatively integrating modern biotechnology and modern extraction technology. This method can not only make full use of the advantages of modern biotechnology to improve extraction efficiency and purity of ingredients, but also reduce extraction costs, reduce energy consumption, protect the environment, provide technical support for the modernization and standardization of traditional Chinese medicine production, and lay the foundation for in-depth research on active ingredients of traditional Chinese medicine and development of new drugs.

[0007] Technical Solution

[0008] The technical solution of the present invention is an innovative method for extracting active ingredients from traditional Chinese medicines. The specific process is as follows:

[0009] Preferably, the pretreatment stage is a prerequisite for ensuring efficient extraction, and the specific steps are as follows: the collected Chinese medicine raw materials are first thoroughly washed with deionized water at 4°C for 30 minutes to remove soil, dust and other impurities on the surface of the medicinal materials and reduce potential contamination in the subsequent extraction process. The washed raw materials are crushed under low temperature conditions and crushed to 200 mesh. This step uses gentle mechanical force, such as cutting, grinding or low-temperature airflow crushing technology, to properly destroy the cell structure and increase the surface area of ​​the medicinal materials, thereby creating conditions for the release of active ingredients. During the crushing process, the strength and time are strictly controlled to avoid excessive crushing and reduce the loss of active ingredients. The crushed Chinese medicine material is dried in a vacuum drying oven at 40°C to a moisture content of less than 10%. This step uses low-temperature drying technology, such as vacuum drying, to prevent hydrolysis and oxidation of active ingredients, while improving the efficiency and quality of subsequent extraction steps. After each pretreatment step, a quality check is performed. This includes a thorough inspection of the washed raw materials, a uniformity inspection of the crushed raw materials, and a fitness and active ingredient integrity inspection of the dried raw materials. The quality changes of medicinal materials during the pretreatment process are monitored through regular sampling and chemical analysis, such as high performance liquid chromatography (HPLC), to ensure that the pretreatment effect meets the requirements of the subsequent extraction process.

[0010] The enzyme-assisted extraction is a delicate biochemical process: a specific commercial enzyme preparation suitable for the target active ingredient is selected, which is genetically engineered to have high affinity and catalytic efficiency for specific chemical bonds of the active ingredients of traditional Chinese medicine. Under laboratory conditions, the optimal activity conditions of the enzyme are determined, such as stirring for 2 hours at pH 7.0 and 50°C to optimize the catalytic efficiency of the enzyme. Under sterile conditions, the optimized enzyme preparation is mixed with the pretreated traditional Chinese medicine material to ensure full contact and promote cell wall decomposition and active ingredient release. The reaction temperature is controlled to be constant at 50°C in a constant temperature water bath, and the pH value is maintained at 7.0 using a pH meter and a buffer solution to provide a stable optimal reaction environment for the enzyme. According to the preliminary experimental results, the reaction time monitoring point is set, such as sampling every 30 minutes, and the reaction progress is evaluated by rapid active ingredient detection. After achieving the predetermined extraction effect, the enzyme is inactivated by heating to 80°C or using a specific chemical agent, and then the enzyme and the extract are separated by filtration or centrifugation. The liquid portion obtained by enzyme-assisted extraction was collected and evaluated using methods such as UV-visible spectroscopy and HPLC, and the active ingredient release efficiency was increased by 30%.

[0011] Supercritical fluid extraction uses supercritical fluid extraction technology to make full use of the solubility characteristics of supercritical carbon dioxide to efficiently extract the active ingredients of traditional Chinese medicine: carbon dioxide is selected as a supercritical fluid because it is non-toxic, harmless, chemically inert, and easy to separate from the extraction system. By adjusting the pressure to above 73.8 bar and the temperature to above 31.1 degrees Celsius, carbon dioxide reaches a supercritical state, at which time carbon dioxide exhibits excellent solubility and fluidity. The carbon dioxide gas is pressurized and heated by a compressor and a heater to reach a supercritical state in preparation for extraction. The traditional Chinese medicine materials that have been extracted with enzyme assistance are evenly loaded into the extraction container to optimize the extraction efficiency. The prepared supercritical carbon dioxide fluid is circulated in the extraction container to extract the active ingredients in the traditional Chinese medicine materials using its high solubility. During the extraction process, the pressure is precisely controlled to 35MPa, the temperature is 40℃, and the fluid flow rate to ensure the stability of the extraction conditions. The active ingredients dissolved from the traditional Chinese medicine materials are collected by a separator. During the decompression process, the supercritical fluid quickly changes to a gaseous state, depositing the active ingredients in the collector. The collected extract containing active ingredients is purified to remove impurities and residual solvents to ensure the purity of the extract. High performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS) and other analytical techniques are used to conduct qualitative and quantitative analysis of the extracted active ingredients to evaluate the extraction efficiency and ingredient purity. For example, after 2 hours of continuous extraction, the extraction rate reaches 75% and the purity is increased to 90%.

[0012] Membrane separation technology finely separates the mixture obtained by supercritical fluid extraction to enrich the target active ingredients: a membrane material suitable for the target active ingredients is selected, such as polyvinylidene fluoride (PVDF), which has good chemical stability and mechanical strength, as well as high selective permeability. The molecular weight cutoff is set to 10kDa, specifically for separating the target active ingredients.

[0013] Before membrane separation, the mixture obtained by supercritical fluid extraction was pretreated by 0.5 micron filtration or centrifugation to remove larger solid particles, and then the pretreated liquid was loaded into the membrane separation system. A membrane separation system was configured, and a flat membrane module was selected. The operating parameters included a flow rate of 0.5 m / s, a pressure of 0.5 MPa, and a temperature of 25 degrees Celsius. The membrane separation system was started, and a pressure pump was used to circulate the mixture through the membrane module for component separation. During the membrane separation process, the permeate and the retentate were collected. The permeate contained smaller molecular active ingredients, while the retentate was rich in larger molecular active ingredients or other high molecular substances. In order to improve the purity of the target active ingredient, a multi-stage membrane separation was implemented, in which the permeate was further separated by passing through a membrane with a smaller molecular weight cutoff, such as a 5kDa membrane. After each use, the membrane was cleaned and regenerated, and an alkaline detergent and an acidic detergent were used to alternately clean the membrane to remove impurities and restore membrane performance. The permeate and retentate obtained by separation were analyzed by high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) to evaluate the membrane separation effect and the enrichment degree of active ingredients. Through these analyses, the active ingredients with target molecular weight were successfully enriched.

[0014] Microwave-assisted extraction The active ingredients in the concentrate after membrane separation were further extracted by microwave radiation: a microwave-assisted extraction device capable of precisely controlling the frequency, power and radiation time of 2.45 GHz was selected to adapt to the concentrates with different characteristics. The pH value of the concentrate obtained by membrane separation was adjusted to 7.0, and the concentration was adjusted as needed to optimize the conditions for microwave-assisted extraction. The microwave power was set to 600 W and the frequency was fixed at 2.45 GHz to achieve optimal penetration and heating efficiency. The adjusted concentrate was placed in the microwave-assisted extraction device and microwave radiation treatment was started for 3 minutes. The high energy of microwave radiation penetrated the concentrate, destroyed the cell wall and other cell structures, and promoted the release of active ingredients. During the microwave-assisted extraction process, the temperature was monitored in real time to remain below 50 °C and the pressure was controlled within 0.1 MPa to ensure the stability of the extraction conditions. The optimal time for microwave radiation was determined to be 3 minutes to avoid the decomposition of active ingredients due to long-term treatment. After the microwave treatment was completed, the extract was quickly collected and cooled to room temperature to prevent thermal degradation. The extract was analyzed using high performance liquid chromatography (HPLC) and mass spectrometry (MS) techniques to evaluate the release of active ingredients and the extraction efficiency, and the results showed that the extraction efficiency of active ingredients increased by 15%. After the extraction was completed, the microwave-assisted extraction equipment was thoroughly cleaned, including disinfection and decontamination with a 70% ethanol solution, and then dried to ensure the cleanliness of the equipment and the efficiency of the next extraction.

[0015] Dynamic countercurrent extraction technology improves extraction efficiency and active ingredient concentration by continuously renewing the extraction solvent: A dynamic countercurrent extraction system is designed. The system controls the solvent pump to continuously replace the extraction solvent with fresh solvent after contacting the Chinese medicine material, forming an effective countercurrent cycle. According to the solubility of the target active ingredient, ethanol was selected as the extraction solvent. It is a common polar organic solvent suitable for extracting a variety of active ingredients in Chinese medicine. The Chinese medicine material was loaded into the extraction column, the solvent pump was started, and the flow rate was set to 1 liter / minute, so that the extraction solvent entered from one end of the extraction column and the used solvent was discharged from the other end to form a countercurrent. During the extraction process, the continuous renewal of the extraction solvent was ensured by controlling the flow rate of the solvent to 1.5 liters / minute and the renewal frequency to once per minute. The solvent forms a dynamic countercurrent circulation in the system, so that every part of the Chinese medicine material can be evenly exposed to the fresh extraction solvent, improving the dissolution and transfer efficiency of the active ingredient. According to the extraction effect, the extraction temperature was adjusted to 40°C and the flow rate to 1.5 liters / minute in real time to optimize the extraction efficiency and the extraction rate of the active ingredient. Through online monitoring technology, such as UV-visible spectroscopy analysis, samples are taken regularly every 30 minutes to monitor the concentration changes of active ingredients in the extract. The extract after countercurrent extraction is collected and concentrated using a rotary evaporator as needed to improve the concentration and purity of the active ingredients. After each extraction, the system is thoroughly cleaned with 70% ethanol solution and properly maintained to remove residues to ensure the cleanliness of the system and the efficiency of the next extraction.

[0016] Molecular imprinting technology carefully selects the target active ingredient as a template, matches it with suitable functional monomers and cross-linkers, and synthesizes specific molecular imprinting polymers under controlled polymerization reaction conditions (such as pH 7.0). Using optimized elution conditions (such as 60% ethanol solution), the template molecules are removed to form highly selective adsorption sites. These polymers are applied to the extract to achieve efficient selective adsorption and enrichment of the target components, and then the enrichment effect is evaluated by HPLC and LC-MS techniques. Finally, the polymer is regenerated through methods such as solvent treatment to restore its adsorption capacity and achieve sustainable use, thereby significantly improving the extraction purity and efficiency of the active ingredients of traditional Chinese medicine.

[0017] Biosynthetic pathway analysis: In-depth understanding and optimization of the biosynthetic mechanism of active ingredients of traditional Chinese medicines through genomics and metabolomics methods: high-throughput sequencing technology, such as the Illumina platform, is used to sequence the genome of organisms derived from traditional Chinese medicines with a coverage of 50X to identify genes and gene clusters related to the biosynthesis of active ingredients. Liquid chromatography-mass spectrometry (LC-MS) technology is used to analyze metabolites in traditional Chinese medicines to identify active ingredients, their precursors and metabolic pathways, with a detection limit of nanomolar level. Combining genomics and metabolomics data, bioinformatics software such as KEGG and MetaCyc are used to infer the biosynthetic pathway of active ingredients. Using CRISPR-Cas9 technology, specific gRNAs are designed to target key enzyme genes, and knockout or knock-in mutations are performed to regulate the biosynthesis of active ingredients. The designed gene editing elements are introduced into organisms or their cells derived from traditional Chinese medicines through Agrobacterium-mediated transformation to construct recombinant organisms. Gene editing events are verified by PCR and Sanger sequencing, and successfully edited recombinant organisms are screened. Biochemical experiments using HPLC and LC-MS are used to verify their ability to biosynthesize active ingredients. Adjust the culture conditions, such as pH to 6.5, temperature to 28°C, and light intensity to 3000 lux, to optimize the biosynthetic efficiency of the recombinant organism. The optimized recombinant organism is cultured on a large scale in a bioreactor, and the active ingredients are collected using supercritical fluid extraction technology. The extracted active ingredients are further purified using HPLC and preparative chromatography techniques to evaluate their potential for application in drug development and treatment. Based on the production data and feedback of the recombinant organism, the culture and extraction conditions are continuously optimized to achieve increased active ingredient yields and sustainability of the production process.

[0018] Tissue crushing extraction technology significantly improves the extraction efficiency of active ingredients of traditional Chinese medicine through ultrasonic and mechanical shearing methods. Using an ultrasonic crusher, set the power to 200W and the frequency to 20kHz, the pretreated traditional Chinese medicine materials were treated for 5 minutes, and the cavitation effect was used to destroy the cell wall and release the active ingredients. Subsequently, mechanical shearing technology was used, and the blade rotated at a speed of 3000 rpm to further refine the cell structure and promote the release of ingredients. Throughout the process, conditions are strictly controlled to maximize the extraction efficiency and protect the active ingredients to avoid thermal damage or chemical degradation. After the treatment is completed, the extract rich in the target active ingredients is separated by filtration or centrifugation separation technology, ensuring the high quality and biological activity of the extract, and providing a solid foundation for subsequent purification and application.

[0019] The heat-free extraction technology is suitable for the extraction of heat-sensitive active ingredients to avoid component loss caused by heat treatment: acetonitrile is selected as the cold solvent because it has good solubility and does not cause denaturation of the target active ingredients. A low-temperature circulating water bath is used to control the temperature of the extraction environment at 4°C to ensure that the active ingredients remain stable during the extraction process. The Chinese medicine raw materials are fully mixed with the cold acetonitrile solvent at a solid-liquid ratio of 1:10 to ensure uniform extraction. Static extraction is performed for 12 hours at 4°C to allow the solvent to fully penetrate the raw material tissue. A dynamic extraction method is used to continuously flow the cold solvent through the raw material bed at a flow rate of 1 liter / minute through a circulating pump. According to the characteristics of the active ingredient, 12 hours is determined to be the optimal static extraction time to dissolve the active ingredient to the greatest extent. After the extraction process, the extract is collected and the undissolved solid residue is removed by 0.2 micron filtration or centrifugation (10,000 rpm). The extract is concentrated to 20% of the original volume using a rotary evaporator at 40°C to increase the concentration of the active ingredient. High performance liquid chromatography (HPLC) and mass spectrometry (MS) techniques are used to qualitatively and quantitatively analyze the active ingredients in the extract to evaluate the extraction effect and the recovery rate of the active ingredients. The acetonitrile solvent is recovered by distillation or solvent recovery system, and the recovery rate after purification reaches 95%, achieving resource conservation and environmental protection.

[0020] High-speed countercurrent chromatography separation technology is used to quickly separate and purify the target active ingredient from a complex extraction mixture: a high-speed countercurrent chromatography system with a high flow rate of at least 5 liters / minute, a resolution of 15,000 theoretical plate height / m, and a high sample load of at least 100 mg was selected. Diatomaceous earth was used as the filling material to fill a chromatographic column with a diameter of 26 mm to ensure uniform and tight filling, and the column bed height reached 25 cm. According to the chemical properties of the target active ingredient, a solvent system consisting of acetonitrile and water was screened, with porous silica gel as the stationary phase and acetonitrile / water = 60:40 (v / v) as the mobile phase. The flow rate was adjusted to 3 ml / min, the pressure to 2 MPa, and the column temperature to room temperature (25°C) to optimize the chromatographic separation process. The extraction mixture was injected into the chromatographic system by static injection, with a sample load of 50 mg. A UV detector (monitoring wavelength 254 nm) and an evaporative light scattering detector were used to monitor the changes in the chromatographic peaks in real time. According to the chromatographic spectrum, an automatic fraction collector was used to accurately collect the chromatographic peaks of the target active ingredient, and the collection time window was determined according to the width of the chromatographic peak. The collected components were analyzed for purity using HPLC and LC-MS / MS techniques, and the purity reached more than 95%. After separation, the chromatographic system was cleaned for 30 minutes using a mixed solvent of methanol and water (50:50, v / v), followed by regeneration with 100% water to remove residual sample and solvent.

[0021] The above technical solution mainly brings the following three specific technical effects:

[0022] 1. High-efficiency extraction: Through supercritical fluid extraction technology, at a pressure of 35MPa and a temperature of 40°C, using carbon dioxide as a solvent, the extraction rate of active ingredients is increased to 75%, which is significantly higher than traditional extraction methods.

[0023] 2. High purity: By using high-speed countercurrent chromatography separation technology and optimizing the flow rate to 3 ml / min and the chromatographic column filling materials, the target active ingredient with a purity of up to 98% was successfully separated, ensuring the high quality and high activity of the extract.

[0024] 3. Environmental friendliness and cost reduction: The heating-free extraction technology uses environmentally friendly solvents such as acetonitrile and performs static extraction at room temperature for 12 hours, avoiding the loss of active ingredients that may be caused by heat treatment. At the same time, through solvent recovery technology, the solvent recovery rate is achieved to exceed 95%, effectively reducing production costs and reducing the impact on the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The extraction and preparation steps of the present invention are shown in FIG. DETAILED DESCRIPTION

[0026] The following will be combined with the attached embodiment of the present invention Figure 1 , the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] The present invention realizes efficient and accurate extraction of active ingredients of traditional Chinese medicine through a series of carefully designed steps. The specific implementation methods and technical effects thereof are as follows:

[0028] S1: Raw material selection and pretreatment, select mature Chinese medicinal plants, and confirm their medicinal value after identification. Wash the raw materials with deionized water at 4°C for 30 minutes. Crush to 200 mesh at low temperature. Dry in a vacuum drying oven at 40°C until the moisture content is less than 10%.

[0029] S2: Enzyme-assisted extraction, select a specific commercial enzyme preparation suitable for the target active ingredient. Add the enzyme preparation and keep stirring for 2 hours at pH 7.0 and 50℃. The active ingredient release efficiency is increased by 30%.

[0030] S3: Supercritical fluid extraction, using supercritical carbon dioxide, extraction at a pressure of 35MPa and a temperature of 40°C. The extraction was continued for 2 hours and the extract was collected. The extraction rate reached 75% and the purity was increased to 90%.

[0031] S4: Membrane separation technology, using an ultrafiltration membrane with a molecular weight cutoff of 10kDa. Membrane separation was performed at a pressure of 0.5MPa. The target molecular weight active ingredient was successfully enriched.

[0032] S5: Microwave assisted extraction, set the microwave frequency to 2.45 GHz and the power to 600 W. The concentrate was subjected to microwave irradiation for 3 minutes. The extraction efficiency of the active ingredient was increased by 15%.

[0033] S6: Dynamic countercurrent extraction, set the solvent renewal frequency to once per minute. Continuous countercurrent extraction for 60 minutes. Active ingredient concentration increased by 20%.

[0034] S7: Molecular imprinting technology, forming a molecular imprinting polymer containing the target active ingredient. The polymer was contacted with the extract for 2 hours at pH 7.0. A 60% ethanol solution was used for elution. The enrichment of the target active ingredient reached 95%.

[0035] S8: Biosynthetic pathway analysis and gene editing, using CRISPR-Cas9 technology to edit the key enzyme genes for the biosynthesis of active ingredients. The expression of key enzymes is increased, and the biosynthesis efficiency is increased by 50%.

[0036] S9: Tissue crushing and extraction, using an ultrasonic crusher, power 200W, processing time 5 minutes. The cell structure is effectively destroyed and the release rate of active ingredients is increased by 10%.

[0037] S10: No heating required, acetonitrile was selected as the cold solvent. Static extraction was performed at room temperature for 12 hours. The loss of heat-sensitive components was avoided.

[0038] S11: High-speed countercurrent chromatography separation, using a chromatographic filler with a diameter of 2.6 μm and a flow rate of 3 ml / min. The target active ingredient with high purity (98%) was successfully separated.

[0039] S12: Post-processing, rotary evaporator at 40℃ to concentrate the extract to 20% of the original volume. Freeze dryer to dry the concentrate into powder. Sieve through 100 mesh screen. HPLC analysis to ensure that the active ingredient content meets the standard. Package in moisture-proof and light-proof container, indicating product information and storage conditions.

[0040] S13: Stability study: conduct accelerated stability test and long-term stability test on the packaged extracts. Determine the shelf life and storage conditions of the product to ensure long-term stability.

[0041] Through the above embodiments, the present invention provides an efficient, mild and environmentally friendly method for extracting active ingredients from traditional Chinese medicine, which achieves optimization of the entire process from raw material selection to final product and ensures the high purity and biological activity of the extract.

[0042] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for extracting active ingredients from traditional Chinese medicine, characterized in that: The method comprises the following steps: Select one or more specific enzymes that have been genetically engineered and have high catalytic ability for chemical bonds of specific active ingredients in traditional Chinese medicine materials, mix the enzymes with the traditional Chinese medicine materials, and carry out catalytic reactions under suitable temperature and pH conditions to improve the release efficiency and extraction rate of the active ingredients; Using supercritical carbon dioxide as a solvent, by adjusting the pressure and temperature to the optimal extraction conditions of the Chinese herbal medicine materials, efficient extraction of active ingredients is achieved, wherein the pressure is controlled at 100-400 bar and the temperature is controlled at 35-60 degrees Celsius; The mixture obtained by supercritical fluid extraction is separated by a membrane with a specific molecular cut-off capacity to enrich the target active ingredient. The molecular weight cut-off of the membrane is selected according to the size of the active ingredient; The separated extract is treated with microwave radiation, and the remaining cell structure is destroyed by adjusting the frequency and power of the microwave to further release the active ingredients; The dynamic countercurrent method is used for continuous extraction, and the extraction efficiency and the concentration of active ingredients are improved by constantly updating the extraction solvent; Molecularly imprinted polymers are used as adsorbents. The polymers have the ability to specifically recognize the target active ingredients, and the target ingredients are enriched and purified through adsorption and elution processes. Through genomic and metabolomics analysis, the biosynthetic pathways of active ingredients in traditional Chinese medicines are analyzed, and gene editing technology is used to modify key enzymes or regulatory elements to enhance the biosynthesis of active ingredients; Use mechanical shearing or physical impact to break the cell structure of traditional Chinese medicine materials to make the active ingredients easier to release; Adopt non-heat treatment solvent extraction method to avoid the destruction of heat-sensitive active ingredients and maintain their biological activity; A high-speed countercurrent chromatography system is used to perform fine separation of active ingredients, and by optimizing chromatographic conditions, rapid separation of high-purity active ingredients is achieved.

2. The extraction method according to claim 1, characterized in that The enzyme-assisted extraction step uses specific enzymes that have been genetically engineered to exhibit significantly improved catalytic efficiency and stability. Specifically, these modified enzymes can maintain high catalytic activity in the pH range of 4 to 10, and have good thermal stability in the temperature range of 0 to 70 degrees Celsius, thereby ensuring that the active ingredients can be efficiently released under a variety of extraction conditions. The modified enzymes have a longer service life, reduce the frequency of replacement during the extraction process, and reduce production costs.

3. The extraction method according to claim 1, characterized in that In the supercritical fluid extraction step, supercritical carbon dioxide is used as an extractant, and by finely adjusting the pressure and temperature parameters, efficient extraction of active ingredients of different polarities is achieved. In the specific operation, the fluid pressure of carbon dioxide is controlled between 100 and 400 bars, and the temperature is maintained at 35 to 60 degrees Celsius. Such conditions can ensure that the supercritical fluid has excellent solubility, thereby selectively extracting the active ingredients in traditional Chinese medicine.

4. The extraction method according to claim 1, characterized in that The microwave assisted extraction step is adjusted by carefully designed microwave parameters to meet the specific extraction requirements of different active ingredients. In this step, the microwave frequency is set to 2.45 GHz, which is a commonly used microwave frequency and can ensure effective transfer and penetration of energy.

5. The extraction method according to claim 1, characterized in that The molecular imprinting material is reusable and has high selectivity and high adsorption capacity, which enables it to specifically adsorb the target active ingredients and effectively reduce the interference of impurities. The design of this material allows it to be used repeatedly in the extraction process, increasing its economy and sustainability.

6. The extraction method according to claim 1, characterized in that In the analysis and application of the biosynthetic pathway, advanced gene editing technologies, such as the CRISPR-Cas9 system, are used to precisely modify the biosynthetic pathway of active ingredients in traditional Chinese medicine. Through this technology, researchers can specifically enhance or inhibit specific metabolic pathways related to the biosynthesis of target active ingredients, thereby effectively increasing the yield of these active ingredients.

7. The extraction method according to claim 1, characterized in that The tissue crushing and extraction technology uses an ultrasonic crusher as a key device to effectively destroy the cell structure of traditional Chinese medicine materials and promote the release of active ingredients. The device can accurately control the force and time of crushing to ensure the optimization of crushing conditions.

8. The extraction method according to claim 1, characterized in that The heat-free extraction technology uses environmentally friendly solvents, such as acetonitrile, which not only has good biodegradability but also has excellent solubility for active ingredients. By performing static extraction at room temperature for 12 hours, the technology avoids the loss of active ingredients that may be caused by traditional heat treatment, ensuring the integrity and biological activity of the extract.

9. A device for implementing the extraction method according to any one of claims 1 to 8, characterized in that: The device integrates key equipment such as enzyme reactor, supercritical fluid extractor, membrane separation unit, etc., and realizes precise operation through sensors and automatic control systems, which simplifies the extraction process and improves efficiency and purity.

10. An application of active ingredients of traditional Chinese medicine extracted by the extraction method or device according to any one of claims 1 to 9, characterized in that: The active ingredient is suitable for the development of medicines, health products and cosmetics, has broad market application potential, and can be used for various effects such as anti-inflammatory, antioxidant, anti-tumor, antiviral and immunomodulatory.

Images