Extraction process of effective components of traditional Chinese medicine

Abstract

The application relates to the technical field of traditional Chinese medicine preparation, and particularly discloses an extraction process of effective components of traditional Chinese medicine, which comprises the following steps: after traditional Chinese medicinal materials are pretreated by impurity reduction, the pretreated traditional Chinese medicinal materials are soaked in a pretreatment solution containing betaine and activated by composite enzymes, then subjected to stress induction, solid-state fermentation and standing conversion in sequence, so that endogenous organic acids, amino acids and the like form natural deep eutectic solvents in situ with the embedded betaine; subsequently, the traditional Chinese medicinal materials are soaked in a composite extraction solvent, subjected to pulsed negative pressure extraction, ultrasonic treatment and microwave treatment, and then subjected to microfiltration, ultrafiltration, macroporous adsorption resin gradient elution, reduced-pressure concentration and spray drying, so that the effective component extract of traditional Chinese medicine is obtained. Through the synergistic process of the pretreatment of stress induction-solid-state fermentation-in-situ deep eutectic solvent conversion and the multi-field combined extraction, efficient synchronous extraction of multi-polar components is realized, the activity of heat-sensitive components is effectively protected, and the co-extraction of impurities is significantly reduced; the effective component extract of traditional Chinese medicine obtained has high purity and good stability.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine preparation technology, specifically to an extraction process for effective components of traditional Chinese medicine. Background Technology

[0002] The extraction of active ingredients from traditional Chinese medicine (TCM) is a crucial step in the development of TCM preparations, natural drugs, health foods, and functional products. TCM herbs typically contain a variety of chemical components, including flavonoids, phenolic acids, saponins, alkaloids, quinones, volatile oils, and polysaccharides. These components vary considerably in polarity, solubility, stability, and tissue distribution. Therefore, improving the extraction efficiency of target components, reducing impurity content, and ensuring the feasibility of industrial production while maintaining the stability of active ingredients has always been a key focus in this field.

[0003] In existing technologies, water extraction, alcohol extraction, reflux extraction, as well as ultrasound-assisted extraction and microwave-assisted extraction are commonly used to process Chinese medicinal materials. Although these methods are widely used in production, they still have certain shortcomings. First, the adaptability of a single extraction solvent to components of different polarities is limited, making it difficult to simultaneously and efficiently extract multiple types of active ingredients, which can easily lead to insufficient extraction of some target components. Second, traditional extraction processes usually increase the extraction rate by raising the temperature, extending the extraction time, or increasing the amount of solvent. However, some active ingredients in Chinese medicinal materials are prone to oxidation, decomposition, or structural transformation under high temperature or long-term extraction conditions, thus affecting the extraction effect and product stability.

[0004] Furthermore, the cell walls and intercellular layers of traditional Chinese medicine (TCM) herbs hinder the release of active ingredients, resulting in low mass transfer efficiency in conventional extraction methods, leading to long extraction cycles and high energy consumption. Simultaneously, impurities such as tannins, gums, pectin, proteins, and polysaccharides in the herbs are easily carried out during extraction, increasing the viscosity of the extract, making filtration difficult, increasing the burden on subsequent purification, and increasing the risk of loss of active ingredients. Based on the above, this invention provides an extraction process for active ingredients from TCM herbs. Summary of the Invention

[0005] To address the shortcomings of existing technologies, such as insufficient extraction of multipolar active ingredients, easy degradation of heat-sensitive components, severe co-extraction of impurities, and low extraction efficiency, this invention provides an extraction process for active ingredients of traditional Chinese medicine.

[0006] In a first aspect, the present invention provides an extraction process for effective components of traditional Chinese medicine, employing the following technical solution: An extraction process for effective components of traditional Chinese medicine includes the following steps: S1. After washing, drying, and crushing the Chinese medicinal materials, add them to the impurity reduction pretreatment solution, stir, filter, rinse, and drain to obtain the impurity-reduced medicinal materials. S2. After soaking the impurity-reduced medicinal materials in the tissue activation solution, drain them to obtain pre-soaked medicinal materials. Add compound enzymes for activation treatment to obtain enzymatically hydrolyzed medicinal materials. S3. The enzymatically hydrolyzed medicinal materials are subjected to stress induction, solid-state fermentation of inoculated bacterial suspension, and static transformation in sequence to obtain transformed medicinal materials; S4. After soaking the transformed medicinal materials in the compound extraction solvent, the soaking system is obtained. The soaking system is then subjected to pulse negative pressure extraction, ultrasonic treatment and microwave treatment in sequence, and centrifuged to obtain the Chinese herbal extract. S5. The extract of traditional Chinese medicine is sequentially subjected to microfiltration and ultrafiltration and then loaded onto a macroporous adsorption resin column for dynamic adsorption. After gradient elution, the eluent is concentrated under reduced pressure and spray-dried to obtain the extract of effective components of traditional Chinese medicine.

[0007] Preferably, the medicinal material used in step S1 is Astragalus membranaceus.

[0008] Preferably, the impurity reduction pretreatment solution in step S1 is composed of citric acid, sodium chloride, disodium ethylenediaminetetraacetate, and deionized water in a mass ratio of 0.5-1:1-2:0.3-0.5:100.

[0009] Preferably, in step S2, the tissue activation solution is composed of betaine, propylene glycol, and deionized water in a mass ratio of 5-15:0.5-2:100; the complex enzyme is composed of cellulase with an enzyme activity ≥20000U / g and pectinase with an enzyme activity ≥15000U / g in a mass ratio of 1:2-3.

[0010] Preferably, the amount of compound enzyme added in step S2 is 1-3 wt% of the pre-soaked medicinal materials.

[0011] Preferably, the activation parameters in step S2 include: adjusting the activation pH to 5.0-6.0, the activation temperature to 30-40℃, and the activation time to 2-4h.

[0012] Preferably, the parameters for stress induction in step S3 include: temperature 28-35℃, relative humidity 85-95%, oxygen volume concentration 30-40%, and stress induction time 8-12h, during which the animal is turned over once every 1-2h; the parameters for static conversion include: temperature 40-60℃, relative humidity 60-85%, and static time 6-8h.

[0013] Preferably, in step S3, the bacterial suspension is composed of Aspergillus oryzae and Lactobacillus plantarum at an effective viable count ratio of 1-3:1, with a total viable count of 10. 7 -10 9 CFU / mL.

[0014] Preferably, the parameters for solid-state fermentation in step S3 include: temperature 30-37℃, relative humidity 70-85%, and solid-state fermentation time 24-48h.

[0015] Preferably, the preparation method of the composite extraction solvent in step S4 is as follows: A pharmaceutical eutectic solvent was mixed with an ethanol solution to obtain a mixture, which was then mixed with 1-butyl-3-methylimidazolium glycinate. The mixture was then ultrasonically dispersed and allowed to stand for aging to obtain a composite extraction solvent.

[0016] Preferably, the specific preparation method of the composite extraction solvent in step S4 is as follows: A pharmaceutical eutectic solvent and a 50-70% ethanol solution are mixed at a volume ratio of 2-4:3-5 to obtain a mixture. The mixture is then mixed with 1-butyl-3-methylimidazolium glycinate at a mass ratio of 100:1-2. The mixture is ultrasonically dispersed at 20-50 kHz and 200-500 W for 10-30 min, and then allowed to stand and mature at 25-45℃ for 8-12 h to obtain a composite extraction solvent.

[0017] Preferably, the pharmaceutical eutectic solvent is composed of hydrogen bond acceptors, hydrogen bond donors, and deionized water in a mass ratio of 1:2-4:2-3; the hydrogen bond acceptors are selected from one or more of choline chloride and betaine; and the hydrogen bond donors are selected from one or more of glycerol, lactic acid, and propylene glycol.

[0018] Preferably, the hydrogen bond acceptor is betaine; and the hydrogen bond donor is lactic acid.

[0019] Preferably, the parameters for pulse negative pressure extraction in step S4 include: negative pressure -0.02MPa to -0.09MPa, negative pressure holding time 5-10min, normal pressure recovery holding time 5-15min, and number of cycles 2-6; the parameters for ultrasonic treatment include: ultrasonic frequency 20-60kHz, ultrasonic power 200-400W, and ultrasonic time 10-30min; the parameters for microwave treatment include: microwave power 300-500W, microwave time 100-150s, and microwave temperature 40-50℃.

[0020] Preferably, the specific operation steps of step S5 are as follows: The herbal extract was first filtered through a microfiltration membrane with a pore size of 0.5-1.0 μm, and then ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 10-20 kDa. The filtrate was collected. The filtrate was loaded into an AB-8 macroporous adsorption resin column and dynamically adsorbed at a flow rate of 3-5 BV / h until saturation. Then, it was eluted sequentially with an ethanol aqueous solution with a volume fraction of 20-40%, 50-70%, and 80-95%, and the eluent was collected in fractions. The eluents of the target component fraction were combined to obtain the enriched fraction. The enriched fraction was concentrated under reduced pressure at 40-50℃ and -0.04 MPa to -0.09 MPa to a relative density of 1.05-1.30 to obtain the concentrate. The concentrate was spray-dried at an inlet air temperature of 120-190℃ and an outlet air temperature of 60-95℃ for 1-2 hours to obtain the extract of the effective components of the herbal medicine.

[0021] Secondly, the present invention provides an extract of effective components of traditional Chinese medicine prepared using the above-mentioned extraction process.

[0022] Thirdly, the present invention provides an application of the above-mentioned extracts of effective components of traditional Chinese medicine in the preparation of traditional Chinese medicine preparations, pharmaceutical compositions, health foods or functional products.

[0023] In summary, the present invention has the following beneficial effects: 1. Achieved synergistic and efficient extraction of multipolar active ingredients. This invention constructs a pretreatment chain of "stress induction-solid-state fermentation-in-situ eutectic solvent conversion": First, in S2, exogenous betaine is pre-embedded into the medicinal material by wetting with a pretreatment solution containing betaine; then, endogenous organic acids and amino acids are released by enzymatic decomposition; next, stress induction stimulates the stress-induced secondary metabolic response of the isolated tissue; solid-state fermentation utilizes microbial metabolism to further produce organic acids and enzyme conversion products; finally, in the static conversion stage of S3, the endogenous organic acids and amino acids accumulated in the previous steps, together with the exogenous betaine introduced in S2, form a natural eutectic solvent in situ within the medicinal material, creating a micro-regional multipolar dissolution environment. Combined with a subsequent composite extraction solvent consisting of a pharmaceutical eutectic solvent, ethanol, and 1-butyl-3-methylimidazolium glycinate, and a pulsed negative pressure-ultrasound-microwave combined extraction technology, simultaneous and efficient dissolution of components from strong polarity to weak polarity is achieved, significantly improving the total extraction rate of effective components in the extract.

[0024] 2. Effectively protects the activity of heat-sensitive components. This invention employs a low- or medium-temperature process throughout. The enzymatic activation temperature is 30-40℃, the stress induction temperature is 28-35℃, the solid-state fermentation temperature is 30-37℃, the static conversion temperature is 40-60℃, the microwave temperature during extraction is controlled at 40-50℃, and the concentration temperature is controlled at 40-50℃. Compared to traditional high-temperature decoction or reflux extraction, the temperature window of this invention is strictly controlled below 50℃, minimizing the degradation or volatilization loss of heat-sensitive components such as flavonoids, phenolic acids, and volatile oils. The resulting extract more closely resembles the original active ingredient profile of the medicinal material, with more complete preservation of its biological activity.

[0025] 3. Significantly reduced the co-extraction of impurities and improved the purity of the target components. This invention employs a multi-stage impurity removal barrier: the pretreatment solution in S1 removes inorganic salts, heavy metals, and some proteinaceous interfering substances at the source by using citric acid to complex metal ions, sodium chloride to adjust osmotic pressure, and EDTA-2Na to chelate polyvalent cations; S5 uses microfiltration membranes to remove suspended particles and colloids, ultrafiltration membranes to retain large molecular proteins and polysaccharides, and macroporous adsorption resin columns to achieve fine separation of the target component from impurities with different polarities through gradient elution. This combination of staged impurity removal and segmented purification significantly improves the purity of the effective components in the final extract compared to conventional processes, reducing the purification burden in subsequent formulation stages.

[0026] 4. Improved extraction efficiency and shortened the process cycle. The pulsed negative pressure extraction of this invention uses alternating negative and normal pressure to cause repeated expansion and contraction of gas in the pores of medicinal materials, forming a "pumping effect" that accelerates the penetration of solvent into the medicinal materials and the diffusion of active ingredients outward. The cavitation effect of ultrasonic treatment further disrupts the residual structure of cell walls. Microwave treatment utilizes dielectric heating to achieve rapid heating from the inside out. The combination of these three methods forms a highly efficient mass transfer chain of "penetration-cell wall disruption-accelerated dissolution". At the same time, the pretreatment of stress induction-solid-state fermentation-static conversion at the front end has created a micro-regional dissolution environment of natural eutectic solvent in the medicinal materials, further reducing the mass transfer resistance of subsequent extraction and shortening the overall process cycle.

[0027] 5. The obtained extract is safe and reliable, and has a wide range of applications. This invention uses food-grade citric acid and EDTA-2Na in the impurity reduction pretreatment, pharmaceutical-grade betaine and propylene glycol in the tissue activation solution, and a pharmaceutical-grade eutectic solvent with choline chloride or betaine as hydrogen bond acceptors and glycerol or lactic acid as hydrogen bond donors in the composite extraction solvent. No highly toxic organic solvents such as benzene, chloroform, or methanol are introduced throughout the process, and the reduced pressure concentration in S5 effectively removes residual ethanol. The resulting extract has low heavy metal content and controllable organic solvent residues, making it suitable for various applications such as traditional Chinese medicine preparations, pharmaceutical compositions, health foods, and functional products, and has promising prospects for industrial application. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0029] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0030] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0031] Citric acid (food grade) was purchased from Hefei Shengrun Biological Products Co., Ltd., CAS No.: 5949-29-1; Cellulase was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number: S10041; Pectinase was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number: S10007; Aspergillus oryzae ( Aspergillus oryzae Purchased from Wuhan Gray Algae Biotechnology Co., Ltd., product number: HZB307838; Lactobacillus plantarum ( Lactobacillus plantarum Purchased from China Center for Type Culture Collection, accession number: CCTCC M 206032; 1-Butyl-3-methylimidazolium glycinate was purchased from Xi'an Qiyue Biotechnology Co., Ltd., product number: Q-0180434.

[0032] Examples 1-3 provide an extraction process for the effective components of traditional Chinese medicine.

[0033] Example 1 An extraction process for effective components of traditional Chinese medicine includes the following steps: S1. Wash Astragalus membranaceus three times with deionized water, dry at 40℃ until the moisture content is 5%, and pulverize to 40 mesh to obtain pretreated medicinal material; add the pretreated medicinal material to the impurity reduction pretreatment solution (composed of citric acid, sodium chloride, disodium EDTA and deionized water in a mass ratio of 0.5:1:0.3:100) at a mass ratio of 1:6, stir at 25℃ and 100 rpm for 30 min, filter, rinse with deionized water until the conductivity of the washing solution is 40 μS / cm, and drain for 10 min to obtain impurity-reduced medicinal material; S2. The impurity-reduced medicinal materials were soaked in a tissue activation solution (composed of betaine, propylene glycol, and deionized water in a mass ratio of 5:0.5:100) for 1 hour at a mass ratio of 1:6, and then drained for 10 minutes to obtain pre-soaked medicinal materials. A compound enzyme (composed of cellulase with an enzyme activity of 50,000 U / g and pectinase with an enzyme activity of 50,000 U / g in a mass ratio of 1:2) was added to the pre-soaked medicinal materials at a mass ratio of 1 wt%. The activation pH was adjusted to 5.0 with 0.2 mol / L citric acid solution, and the materials were activated at 30°C for 2 hours to obtain enzymatically hydrolyzed medicinal materials. S3. The enzymatically hydrolyzed medicinal materials were subjected to stress induction at 28℃, 85% relative humidity, and 30% oxygen concentration for 8 hours, with the materials turned over every hour during this period. After the stress induction was completed, the materials were inoculated with a bacterial suspension (composed of Aspergillus oryzae and Lactobacillus plantarum in a 1:1 ratio of viable viable bacteria, with a total viable bacteria count of 10) at 5 wt% of the enzymatically hydrolyzed medicinal materials. 7 The material was fermented in solid state at 30℃ and 70% relative humidity for 24 hours (CFU / mL); after solid-state fermentation, it was allowed to stand for 6 hours at 40℃ and 60% relative humidity to obtain the transformed medicinal material. S4. Add the transformed medicinal materials to the compound extraction solvent at a mass ratio of 1:10 and soak for 30 min to obtain the soaking system. Extract the soaking system under negative pressure of -0.02MPa pulsed negative pressure, where the negative pressure is maintained for 5 min, and the normal pressure is restored and maintained for 5 min, and the cycle is repeated twice. Then, it is ultrasonically treated at 20kHz and 200W for 10 min, followed by microwave treatment at 300W and 40℃ for 100 s. Finally, it is centrifuged at 6000rpm for 5 min to obtain the Chinese herbal extract. S5. The herbal extract is first filtered through a microfiltration membrane with a pore size of 0.5 μm, and then ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa. The filtrate is collected. The filtrate is loaded into an AB-8 macroporous adsorption resin column and dynamically adsorbed at a flow rate of 3 BV / h until saturation. Then, it is eluted sequentially with 20%, 50%, and 80% (v / v) ethanol aqueous solution. The eluents are collected in fractions, and the eluents containing the target components are combined to obtain the enriched fraction. The enriched fraction is concentrated at 40℃ and -0.04 MPa under reduced pressure to a relative density of 1.05 to obtain the concentrate. The concentrate is spray-dried at an inlet air temperature of 120℃ and an outlet air temperature of 60℃ for 1 h to obtain the extract of the effective components of the herbal medicine.

[0034] The specific preparation method of the composite extraction solvent in step S4 is as follows: A pharmaceutical eutectic solvent (composed of betaine, lactic acid, and deionized water in a mass ratio of 1:2:2) was mixed with a 50% ethanol solution in a volume ratio of 2:3 to obtain a mixture. The mixture was then mixed with 1-butyl-3-methylimidazolium glycinate in a mass ratio of 100:1 and ultrasonically dispersed at 20 kHz and 200 W for 10 min. The mixture was then allowed to stand at 25 °C for 8 h to mature, resulting in a composite extraction solvent.

[0035] Example 2 An extraction process for effective components of traditional Chinese medicine includes the following steps: S1. Wash Astragalus membranaceus three times with deionized water, dry at 45℃ until the moisture content is 10%, and pulverize to 60 mesh to obtain pretreated medicinal material; add the pretreated medicinal material to the impurity reduction pretreatment solution (composed of citric acid, sodium chloride, disodium EDTA and deionized water in a mass ratio of 0.75:1.5:0.4:100) at a mass ratio of 1:8, stir at 30℃ and 200 rpm for 40 min, filter, rinse with deionized water until the conductivity of the washing solution is 40 μS / cm, and drain for 20 min to obtain impurity-reduced medicinal material; S2. The impurity-reduced medicinal materials were soaked in a tissue activation solution (composed of betaine, propylene glycol, and deionized water in a mass ratio of 10:1:100) for 2 hours at a mass ratio of 1:7, and then drained for 20 minutes to obtain pre-soaked medicinal materials. 2 wt% of a compound enzyme (composed of cellulase with an enzyme activity of 50000 U / g and pectinase with an enzyme activity of 50000 U / g in a mass ratio of 1:2.5) was added to the pre-soaked medicinal materials. The activation pH was adjusted to 5.5 with 0.2 mol / L citric acid solution, and the materials were activated at 35℃ for 3 hours to obtain enzymatically hydrolyzed medicinal materials. S3. The enzymatically hydrolyzed medicinal materials were subjected to stress induction at 30℃, 90% relative humidity, and 35% oxygen concentration for 10 hours, with the materials turned over every 1.5 hours during this period. After the stress induction was completed, the materials were inoculated with a bacterial suspension (composed of Aspergillus oryzae and Lactobacillus plantarum in a viable viable count ratio of 2:1, with a total viable count of 10 wt%). 8 The material was fermented in solid state at 35℃ and 80% relative humidity for 36 hours (CFU / mL); after solid-state fermentation, it was allowed to stand for 7 hours at 50℃ and 70% relative humidity to obtain the transformed medicinal material. S4. Add the transformed medicinal materials to the compound extraction solvent at a mass ratio of 1:15 and soak for 40 min to obtain the soaking system. Extract the soaking system under negative pressure of -0.05MPa pulsed negative pressure, where the negative pressure is maintained for 8 min, and the normal pressure is restored and maintained for 10 min. Repeat this cycle 4 times. Then, treat with ultrasound at 50kHz and 300W for 20 min, followed by microwave treatment at 400W and 45℃ for 120 s. Finally, centrifuge at 7000rpm for 10 min to obtain the Chinese herbal extract. S5. The herbal extract is first filtered through a microfiltration membrane with a pore size of 0.8 μm, and then ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 15 kDa. The filtrate is collected. The filtrate is loaded into an AB-8 macroporous adsorption resin column and dynamically adsorbed at a flow rate of 4 BV / h until saturation. Then, it is eluted sequentially with 30%, 60%, and 85% (v / v) ethanol aqueous solution. The eluents are collected in fractions, and the eluents containing the target components are combined to obtain the enriched fraction. The enriched fraction is concentrated at 45℃ and -0.05 MPa reduced pressure to a relative density of 1.20 to obtain the concentrate. The concentrate is spray-dried at an inlet air temperature of 150℃ and an outlet air temperature of 70℃ for 1.5 h to obtain the extract of the effective components of the herbal medicine.

[0036] The specific preparation method of the composite extraction solvent in step S4 is as follows: A pharmaceutical eutectic solvent (composed of betaine, lactic acid, and deionized water in a mass ratio of 1:3:2.5) was mixed with a 60% ethanol solution in a volume ratio of 3:4 to obtain a mixture. The mixture was then mixed with 1-butyl-3-methylimidazolium glycinate in a mass ratio of 100:1.5. The mixture was ultrasonically dispersed at 40 kHz and 300 W for 20 min, and then allowed to stand at 30 °C for 10 h to mature, thus obtaining a composite extraction solvent.

[0037] Example 3 An extraction process for effective components of traditional Chinese medicine includes the following steps: S1. Wash Astragalus membranaceus three times with deionized water, dry it at 50℃ until the moisture content is 12%, and pulverize it to 80 mesh to obtain pretreated medicinal material; add the pretreated medicinal material to the impurity reduction pretreatment solution (composed of citric acid, sodium chloride, disodium EDTA and deionized water in a mass ratio of 1:2:0.5:100) at a mass ratio of 1:10, stir at 300 rpm for 50 min at 40℃, filter, rinse with deionized water until the conductivity of the washing solution is 40 μS / cm, and drain for 30 min to obtain impurity-reduced medicinal material; S2. The impurity-reduced medicinal materials were soaked in a tissue activation solution (composed of betaine, propylene glycol, and deionized water in a mass ratio of 15:2:100) for 3 hours at a mass ratio of 1:8, and then drained for 30 minutes to obtain pre-soaked medicinal materials. A compound enzyme (composed of cellulase with an enzyme activity of 50,000 U / g and pectinase with an enzyme activity of 50,000 U / g in a mass ratio of 1:3) was added to the pre-soaked medicinal materials at a mass ratio of 3 wt%. The activation pH was adjusted to 6.0 with 0.2 mol / L citric acid solution, and the materials were activated at 40℃ for 4 hours to obtain enzymatically hydrolyzed medicinal materials. S3. The enzymatically hydrolyzed medicinal materials were subjected to stress induction at 35℃, 95% relative humidity, and 40% oxygen concentration for 12 hours, with the materials turned over every 2 hours during this period. After the stress induction was completed, the materials were inoculated with a bacterial suspension (composed of Aspergillus oryzae and Lactobacillus plantarum in a viable viable count ratio of 3:1, with a total viable count of 10) at 15 wt% of the enzymatically hydrolyzed medicinal materials. 9 The material was fermented in solid state at 37℃ and 85% relative humidity for 48 hours (CFU / mL); after solid-state fermentation, it was allowed to stand for 8 hours at 60℃ and 85% relative humidity to obtain the transformed medicinal material. S4. Add the transformed medicinal materials to the compound extraction solvent at a mass ratio of 1:20 and soak for 50 min to obtain the soaking system. Extract the soaking system under negative pressure of -0.09MPa pulsed negative pressure, where the negative pressure is maintained for 10 min, and the normal pressure is restored and maintained for 15 min, and the cycle is repeated 6 times. Then, it is ultrasonically treated at 60kHz and 400W for 30 min, followed by microwave treatment at 500W and 50℃ for 150 s. Finally, it is centrifuged at 8000rpm for 15 min to obtain the Chinese herbal extract. S5. The herbal extract was first filtered through a microfiltration membrane with a pore size of 1.0 μm, and then ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 20 kDa. The filtrate was collected. The filtrate was loaded into an AB-8 macroporous adsorption resin column and dynamically adsorbed at a flow rate of 5 BV / h until saturation. Then, it was eluted sequentially with 40%, 70%, and 95% ethanol aqueous solutions by volume fraction. The eluents were collected in fractions, and the eluents containing the target components were combined to obtain the enriched fraction. The enriched fraction was concentrated under reduced pressure at 50℃ and -0.09 MPa to a relative density of 1.30 to obtain the concentrate. The concentrate was spray-dried at an inlet air temperature of 190℃ and an outlet air temperature of 95℃ for 2 hours to obtain the extract of the effective components of the herbal medicine.

[0038] The specific preparation method of the composite extraction solvent in step S4 is as follows: A pharmaceutical eutectic solvent (composed of betaine, lactic acid, and deionized water in a mass ratio of 1:4:3) was mixed with a 70% ethanol solution in a volume ratio of 4:5 to obtain a mixture. The mixture was then mixed with 1-butyl-3-methylimidazolium glycinate in a mass ratio of 100:2. The mixture was ultrasonically dispersed at 50 kHz and 500 W for 30 min, and then allowed to stand at 45 °C for 12 h to mature, thus obtaining a composite extraction solvent.

[0039] Comparative Example 1 This comparative example provides an extraction process for the effective components of traditional Chinese medicine. The only difference from Example 2 is that the quality of the impurity-reducing medicinal materials is replaced with pre-treated medicinal materials. The other types of raw materials, dosages, and preparation process parameters are completely consistent with those of Example 2.

[0040] Specifically: S1. Wash Astragalus membranaceus three times with deionized water, dry it at 45℃ until the moisture content is 10wt%, and pulverize it to 60 mesh to obtain pretreated medicinal material.

[0041] Comparative Example 2 This comparative example provides an extraction process for the effective components of traditional Chinese medicine. The only difference between this example and Example 2 is that the pretreatment solution is composed of propylene glycol and deionized water; the other raw material types, dosages, and preparation process parameters are completely consistent with those of Example 2.

[0042] Specifically: S2. The impurity-reduced medicinal materials were soaked in tissue activation solution (composed of propylene glycol and deionized water at a mass ratio of 11:100) for 2 hours at a mass ratio of 1:7, and then drained for 20 minutes to obtain pre-soaked medicinal materials. 2 wt% of compound enzyme (composed of cellulase with an enzyme activity of 50000 U / g and pectinase with an enzyme activity of 50000 U / g at a mass ratio of 1:2.5) was added to the pre-soaked medicinal materials. The pH was adjusted to 5.5 with 0.2 mol / L citric acid solution, and the materials were activated at 35℃ for 3 hours to obtain enzymatically hydrolyzed medicinal materials.

[0043] Comparative Example 3 This comparative example provides an extraction process for the effective components of traditional Chinese medicine. The only difference from Example 2 is that the stress-induced treatment is omitted in step S3; the other raw material types, dosages, and preparation process parameters are completely consistent with those of Example 2.

[0044] Specifically: S3. Inoculate with a bacterial suspension comprising 10 wt% of the enzymatically hydrolyzed medicinal material (composed of Aspergillus oryzae and Lactobacillus plantarum in a viable viable count ratio of 2:1, with a total viable count of 10). 8 The material was fermented in solid state at 35℃ and 80% relative humidity for 36 hours (CFU / mL). After solid-state fermentation, it was allowed to stand for 7 hours at 50℃ and 70% relative humidity to obtain the transformed medicinal material.

[0045] Comparative Example 4 This comparative example provides an extraction process for the effective components of traditional Chinese medicine. The only difference between this example and Example 2 is that solid-state fermentation is omitted in step S3; the other raw material types, dosages, and preparation process parameters are completely consistent with those of Example 2.

[0046] Specifically: S3. The enzymatically hydrolyzed medicinal material was subjected to stress induction at 30℃, 90% relative humidity, and 35% oxygen volume concentration for 10 hours, and was turned over every 1.5 hours during the period. After the stress induction was completed, it was placed at 50℃ and 70% relative humidity for 7 hours to obtain the transformed medicinal material.

[0047] Comparative Example 5 This comparative example provides an extraction process for the effective components of traditional Chinese medicine. The only difference from Example 2 is that the static conversion treatment is omitted in step S3; the other raw material types, dosages, and preparation process parameters are completely consistent with Example 2.

[0048] Specifically: S3. The enzymatically hydrolyzed medicinal materials were subjected to stress induction at 30℃, 90% relative humidity, and 35% oxygen concentration for 10 hours, with the materials turned over every 1.5 hours during this period. After the stress induction was completed, the materials were inoculated with a bacterial suspension (composed of Aspergillus oryzae and Lactobacillus plantarum in a viable viable count ratio of 2:1, with a total viable count of 10 wt%). 8 The fermented medicinal material was obtained by solid-state fermentation at 35℃ and 80% relative humidity for 36 hours (CFU / mL).

[0049] Comparative Example 6 This comparative example provides an extraction process for the effective components of traditional Chinese medicine. The only difference between this example and Example 2 is that the compound extraction solvent in step S4 does not contain a pharmaceutical eutectic solvent; the other raw material types, amounts, and preparation process parameters are completely consistent with Example 2.

[0050] Specifically: The specific preparation method of the composite extraction solvent in step S4 is as follows: A 60% (v / v) ethanol solution was mixed with 1-butyl-3-methylimidazolium glycinate at a mass ratio of 100:1.5, ultrasonically dispersed at 40 kHz and 300 W for 20 min, and then aged at 30 °C for 10 h to obtain a composite extraction solvent.

[0051] Comparative Example 7 This comparative example provides an extraction process for the effective components of traditional Chinese medicine. The only difference from Example 2 is that in step S4, the combined extraction method of pulse negative pressure extraction, ultrasonic treatment and microwave treatment is not used, but conventional stirring and soaking is used; the other raw material types, dosages and preparation process parameters are completely consistent with Example 2.

[0052] Specifically: S4. Add the transformed medicinal materials to the compound extraction solvent at a mass ratio of 1:15 and soak for 40 minutes to obtain the soaking system. Extract the soaking system at 45℃, normal pressure, and 200 rpm for 60 minutes by stirring. Finally, centrifuge at 7000 rpm for 10 minutes to obtain the Chinese herbal extract.

[0053] Performance testing To verify the promoting effect of the extraction process of effective components of traditional Chinese medicine according to the present invention on the extract of effective components of Astragalus membranaceus, the extracts of effective components of Astragalus membranaceus prepared in Examples 1-3 and Comparative Examples 1-7 were used as test objects for performance evaluation. The yield of astragaloside A, the content of non-target impurities, and stability were selected as evaluation indicators. Astragaloside A is a representative saponin component of Astragalus membranaceus, and its yield reflects the extraction effect of the target component; the content of non-target impurities reflects the impurity reduction ability of the process; and stability reflects the retention ability of the target component in the extract. All samples were prepared using the same batch of Astragalus membranaceus raw materials, and each group was tested in triplicate. The results are expressed as mean ± standard deviation.

[0054] 1. Detection Method (1) Detection of astragaloside A yield The high-performance liquid chromatography-evaporative light scattering detection method (HPLC-ELSD) was used, and the specific steps are as follows: Preparation of reference solutions: Accurately weigh 10 mg of astragaloside A reference standard (purity ≥98%), place it in a 50 mL volumetric flask, dissolve it in methanol and dilute to the mark, shake well to obtain a reference stock solution with a concentration of 0.2 mg / mL; accurately measure 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mL of the reference stock solution, place them in 10 mL volumetric flasks respectively, dilute to the mark with methanol, shake well to obtain a series of reference solutions of different concentrations.

[0055] Preparation of test solution: Accurately weigh 0.1g of the effective component extract of Astragalus membranaceus in each group, place it in a 50mL volumetric flask, add 40mL of methanol, extract by sonication for 30min (power 300W, frequency 50kHz), cool to room temperature, add methanol to make up to the mark, shake well, let stand for 30min, take the supernatant, filter through a 0.22μm filter membrane to obtain the test solution.

[0056] Determination: An evaporative light scattering detector was used. The chromatographic column was a C18 column (250 mm × 4.6 mm, 5 μm); the mobile phase was acetonitrile-water (acetonitrile to water volume ratio of 32:68); the flow rate was 1.0 mL / min; the column temperature was 30 ℃; the injection volume was 10 μL; the drift tube temperature was 105 ℃; and the carrier gas flow rate was 2.8 L / min. A series of reference solutions and test solutions were accurately pipetted into the high-performance liquid chromatograph, the peak areas were recorded, a standard curve was plotted, and the content of astragaloside A in the test sample was calculated, and then the yield of astragaloside A was calculated.

[0057] Calculation formula: Astragaloside A yield (%) = (mass of astragaloside A in extract / mass of Astragalus raw material) × 100%.

[0058] (2) Detection of non-target impurities Common impurities (soluble inorganic salts and proteins) found in Astragalus membranaceus extract were selected for detection. The specific detection methods are as follows: Soluble inorganic salt content: The conductivity method was used. 0.5g of the effective component extract of Astragalus membranaceus in each group was accurately weighed and dissolved in 50mL of deionized water. The solution was sonicated for 30min. The conductivity of the solution was measured using a conductivity meter. The soluble inorganic salt content (calculated as sodium chloride) was calculated in combination with the standard curve.

[0059] Protein content: The Coomassie brilliant blue method was used. 0.1g of the effective component extract of Astragalus membranaceus in each group was accurately weighed and dissolved in 10mL of deionized water. The extract was ultrasonically extracted for 20min. 1mL of the supernatant was taken, 5mL of Coomassie brilliant blue reagent was added, and the mixture was shaken well. The absorbance was measured at 595nm wavelength, and the protein content was calculated using the standard curve.

[0060] Calculation formula: Non-target impurity content (mg / g) = soluble inorganic salt content + protein content.

[0061] (3) Stability testing The extracts of effective components of Astragalus membranaceus in each group were sealed and stored at 40℃ for 30 days. The content of astragaloside A was measured before storage and after 30 days of storage, and the content change rate was calculated to evaluate the stability of the extracts.

[0062] Calculation formula: Content change rate (%) = (Astragaloside A content after storage - Astragaloside A content before storage) / Astragaloside A content before storage × 100%.

[0063] 2. Test Results The specific test results of Examples 1-3 and Comparative Examples 1-7 are shown in Table 1.

[0064] Table 1 Performance test results of Examples 1-3 and Comparative Examples 1-7 3. Results Analysis As shown in Table 1, the Astragalus extracts obtained in Examples 1-3 all exhibited good overall performance. This indicates that the extraction process of the present invention is effective in improving the extraction efficiency of astragaloside A, controlling the carry-over of non-target impurities, and maintaining the stability of the extract. Among them, Example 2 showed the highest yield of astragaloside A, the lowest content of non-target impurities, and the smallest absolute value of the change rate of astragaloside A content after 30 days of storage.

[0065] Compared with Example 2, Comparative Example 1, after omitting the impurity reduction pretreatment, showed a decrease in the yield of astragaloside A, a significant increase in the content of non-target impurities, and a change rate of -5.87% in the content of astragaloside A after storage at 40°C for 30 days. This indicates that the impurity reduction pretreatment can effectively reduce the carry-out of inorganic salts and protein impurities, and is beneficial to the subsequent extraction and retention of target components.

[0066] Compared with Example 2, Comparative Example 2, after using a pretreatment solution without betaine, showed a decrease in the yield of astragaloside A, an increase in the content of non-target impurities, and a change rate of -4.19% in the content of astragaloside A after storage at 40°C for 30 days. This indicates that betaine pre-soaking is beneficial to improving the extraction efficiency of astragaloside A and helps to improve the stability of the extract.

[0067] Compared with Example 2, Comparative Example 3, after omitting the stress induction treatment, showed a decrease in the yield of astragaloside A, an increase in the content of non-target impurities, and a significant deterioration in stability, indicating that the stress induction step helps to release and retain the target components in Astragalus membranaceus.

[0068] Compared with Example 2, Comparative Example 4, which omitted solid-state fermentation treatment, showed a decrease in the yield of astragaloside A and an increase in the content of non-target impurities. After storage at 40°C for 30 days, the change rate of astragaloside A content was -3.87%, indicating that the solid-state fermentation step can promote the disruption of the Astragalus membranaceus tissue structure and the transformation of related components, thereby improving the extraction effect of the target components.

[0069] Compared with Example 2, Comparative Example 5, after omitting the static conversion treatment, showed a decrease in the yield of astragaloside A, an increase in the content of non-target impurities, and lower stability than Example 2. This indicates that the static conversion step has a positive effect on improving the dissolution of the target component and maintaining the stability of the extract.

[0070] Compared with Example 2, in Comparative Example 6, the yield of astragaloside A decreased, the content of non-target impurities increased, and the stability further decreased after no pharmaceutical eutectic solvent was added to the composite extraction solvent. This indicates that pharmaceutical eutectic solvent is beneficial to improving the extraction efficiency of astragaloside A and has a promoting effect on the stable maintenance of the target component.

[0071] Compared with Example 2, Comparative Example 7, after replacing pulse negative pressure extraction, ultrasonic treatment and microwave treatment with conventional stirring extraction, the yield of astragaloside A decreased and the content of non-target impurities increased. Moreover, the change rate of astragaloside A content after storage at 40℃ for 30 days was -6.21%, which was the worst among all comparative examples. This indicates that the combined enhanced extraction method can significantly improve mass transfer efficiency and the release effect of target components.

[0072] In summary, the extraction process of Astragalus membranaceus in this invention, through the synergistic effect of steps such as impurity reduction pretreatment, betaine pre-soaking, stress induction, solid-state fermentation, static conversion, compound extraction solvent, and combined enhanced extraction, can effectively improve the yield of astragaloside A, reduce the content of non-target impurities, and enhance the stability of the extract, thus exhibiting good process effects.

[0073] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A process for extracting effective components from traditional Chinese medicine, characterized in that, Includes the following steps: S1. After washing, drying, and crushing the Chinese medicinal materials, add them to the impurity reduction pretreatment solution, stir, filter, rinse, and drain to obtain the impurity-reduced medicinal materials. S2. After soaking the impurity-reduced medicinal materials in the tissue activation solution, drain them to obtain pre-soaked medicinal materials. Add compound enzymes for activation treatment to obtain enzymatically hydrolyzed medicinal materials. S3. The enzymatically hydrolyzed medicinal materials are subjected to stress induction, solid-state fermentation of inoculated bacterial suspension, and static transformation in sequence to obtain transformed medicinal materials; S4. After soaking the transformed medicinal materials in the compound extraction solvent, the soaking system is obtained. The soaking system is then subjected to pulse negative pressure extraction, ultrasonic treatment and microwave treatment in sequence, and centrifuged to obtain the Chinese herbal extract. S5. The extract of traditional Chinese medicine is sequentially subjected to microfiltration and ultrafiltration and then loaded onto a macroporous adsorption resin column for dynamic adsorption. After gradient elution, the eluent is concentrated under reduced pressure and spray-dried to obtain the extract of effective components of traditional Chinese medicine. The medicinal material used in step S1 is Astragalus membranaceus; The impurity reduction pretreatment solution in step S1 is composed of citric acid, sodium chloride, disodium ethylenediaminetetraacetate, and deionized water in a mass ratio of 0.5-1:1-2:0.3-0.5:

100. The tissue activation solution in step S2 is composed of betaine, propylene glycol and deionized water in a mass ratio of 5-15:0.5-2:

100. The preparation method of the composite extraction solvent in step S4 is as follows: The pharmaceutical eutectic solvent was mixed with an ethanol solution to obtain a mixture. The mixture was then mixed with 1-butyl-3-methylimidazolium glycinate, ultrasonically dispersed, and allowed to stand for aging to obtain a composite extraction solvent.

2. The extraction process of effective components of traditional Chinese medicine according to claim 1, characterized in that, In step S2, the complex enzyme is composed of cellulase with an enzyme activity ≥20000 U / g and pectinase with an enzyme activity ≥15000 U / g in a mass ratio of 1:2-3.

3. The extraction process of effective components of traditional Chinese medicine according to claim 1, characterized in that, The parameters for stress induction in step S3 include: temperature 28-35℃, relative humidity 85-95%, oxygen volume concentration 30-40%, and stress induction time 8-12h, during which the animal is turned over once every 1-2h; the parameters for static conversion include: temperature 40-60℃, relative humidity 60-85%, and static time 6-8h.

4. The extraction process of effective components of traditional Chinese medicine according to claim 1, characterized in that, In step S3, the bacterial suspension is composed of Aspergillus oryzae and Lactobacillus plantarum at an effective viable count ratio of 1-3:1, with a total viable count of 10. 7 -10 9 CFU / mL.

5. The extraction process of effective components of traditional Chinese medicine according to claim 1, characterized in that, The pharmaceutical eutectic solvent is composed of hydrogen bond acceptors, hydrogen bond donors, and deionized water in a mass ratio of 1:2-4:2-3; the hydrogen bond acceptors are selected from one or more of choline chloride and betaine; the hydrogen bond donors are selected from one or more of glycerol, lactic acid, and propylene glycol.

6. The extraction process of effective components of traditional Chinese medicine according to claim 1, characterized in that, The parameters for pulse negative pressure extraction in step S4 include: negative pressure -0.02MPa to -0.09MPa, negative pressure holding time 5-10min, normal pressure recovery holding time 5-15min, and number of cycles 2-6; the parameters for ultrasonic treatment include: ultrasonic frequency 20-60kHz, ultrasonic power 200-400W, and ultrasonic time 10-30min; the parameters for microwave treatment include: microwave power 300-500W, microwave time 100-150s, and microwave temperature 40-50℃.

7. An extract of effective components of traditional Chinese medicine prepared by the extraction process of effective components of traditional Chinese medicine as described in any one of claims 1-6.

8. The use of the extract of effective components of traditional Chinese medicine as described in claim 7 in the preparation of traditional Chinese medicine preparations, pharmaceutical compositions, health foods or functional products.

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