Extraction method for classifying and screening active components

Abstract

The invention relates to the technical field of active ingredient extraction, and particularly provides an extraction method for classifying and screening active ingredients, raw materials are subjected to low-temperature freeze-drying pretreatment by adopting supercritical CO2, and treatment is performed for 4-6 hours under the conditions of minus 40-minus 20 DEG C and 8-12 MPa, so that the active ingredients are reserved to the greatest extent; through gradient ultrasonic-assisted extraction of ultrapure water, a 50% ethanol solution and a 95% ethanol solution, step-by-step separation of water-soluble, alcohol-soluble and fat-soluble components is realized; carrying out multi-dimensional purification by using a composite chromatographic column containing a 0.2 mu m ceramic membrane layer, a macroporous adsorption resin layer and a sephadex layer; a two-channel biosensor array is adopted to synchronously detect a cell response signal and a molecule binding signal, and components with the activity intensity reaching 80% or above are screened; and finally, carrying out vacuum rotary evaporation concentration and spray drying to obtain a finished product, and completing system purity verification through a high performance liquid chromatography-mass spectrometry technology to form a complete quality control system.

Description

Technical Field

[0001] This invention provides an extraction method, and particularly relates to an extraction method for classifying and screening active ingredients. Background Technology

[0002] Active ingredient extraction and screening devices are equipment systems used to separate, purify, and screen specific bioactive components from natural raw materials (such as Chinese medicinal materials, plant tissues, and microbial fermentation products). Their core function is to achieve efficient separation and identification of target active ingredients through pretreatment, extraction, purification, and detection. They are widely used in the fields of medicine, food, and cosmetics, providing key technical support for new drug development and functional ingredient development.

[0003] The basic structure of existing active ingredient extraction and screening devices typically includes: traditional hot air drying equipment, which dehydrates raw materials using high-temperature hot air (60-100℃), easily leading to degradation or inactivation of heat-sensitive active ingredients due to high temperatures; single solvent extraction systems, which often use water or a single concentration of ethanol as the extraction solvent, combined with conventional stirring or static extraction methods, making it difficult to effectively separate different types of components such as water-soluble, alcohol-soluble, and lipid-soluble components, resulting in low extraction efficiency; purification stages often use single chromatography columns (such as simple macroporous adsorption resin columns or dextran gel columns), lacking multi-stage filtration and adsorption synergy, resulting in low purity of purified components and a large amount of impurities remaining; activity screening often relies on a single detection instrument (such as using only a fluorescence spectrophotometer or a single type of sensor), which can only obtain a single-dimensional detection signal, making it difficult to comprehensively reflect the biological activity of the components, thus limiting the accuracy of screening; and most devices are not equipped with a systematic purity verification module, making it impossible to accurately detect the purity of the final product, affecting the reliability of product quality. Summary of the Invention

[0004] To address the aforementioned problems, this application provides an extraction method for classifying and screening active ingredients, thereby overcoming the technical bottlenecks of active ingredient inactivation, incomplete separation, low purity, inaccurate screening, and lack of systematic verification.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: an extraction method for classifying and screening active ingredients, comprising the following steps:

[0006] S1. Raw material pretreatment: The raw material to be extracted is subjected to supercritical CO2 low-temperature freeze drying treatment, with the temperature controlled at -40℃ to -20℃ and the pressure at 8 to 12 MPa, to obtain freeze-dried powder; in the supercritical CO2 low-temperature freeze drying treatment, the CO2 flow rate is 20 to 30 L / h and the treatment time is 4 to 6 h;

[0007] S2. Gradient solvent extraction: The lyophilized powder obtained in step S1 was sequentially extracted with ultrapure water, 50% ethanol solution, and 95% ethanol solution using ultrasonic-assisted extraction. The ultrasonic power was 300-500W, the extraction temperature was 30-50℃, and the extraction time for each solvent was 30-60min, resulting in water-soluble extract, alcohol-soluble extract, and lipid-soluble extract, respectively.

[0008] S3. Multidimensional classification and purification: The three extracts obtained in step S2 are purified by passing them through a composite chromatography column. The composite chromatography column consists of a 0.2 μm ceramic membrane layer, a macroporous adsorption resin layer, and a dextran gel layer from top to bottom. The flow rate is controlled at 1 to 2 mL / min, and the eluent is collected.

[0009] S4. Activity screening: The activity of the purified effluent obtained in step S3 is detected by a dual-channel biosensor array, which includes a cell impedance sensor and a fluorescence quantitative sensor. The cell response signal and molecular binding signal of the active ingredient are acquired simultaneously, and the components with an activity intensity ≥80% are screened out.

[0010] S5. Concentration and Drying: The active components obtained in step S4 are concentrated by vacuum rotary evaporation, with the vacuum degree controlled at 0.08-0.1 MPa and the temperature at 40-50°C. After concentration to a solid content of 30%-40%, the active components are spray-dried to obtain the finished product.

[0011] Preferably, in step S1, the raw material to be extracted is a traditional Chinese medicine, plant tissue or microbial fermentation product, and before supercritical CO2 low-temperature freeze drying, it is first pulverized to a particle size of 100-200 mesh.

[0012] Preferably, in step S2, the ratio of the freeze-dried powder to each extraction solvent is 1:10 to 1:20 (g / mL), and pulsed ultrasound is used in the ultrasonic-assisted extraction process with a pulse frequency of 2 to 5 Hz.

[0013] Preferably, in step S3, the macroporous adsorption resin layer is AB-8 type macroporous adsorption resin, the dextran gel layer is Sephadex G-100 dextran gel, and the volume ratio of the 0.2μm ceramic membrane layer, the macroporous adsorption resin layer, and the dextran gel layer is 1:3 to 5:2 to 3.

[0014] Preferably, in step S4, the cell impedance sensor uses a gold-plated electrode array with a detection frequency of 10–100 kHz, and the fluorescence quantitative sensor uses a fluorescent probe with an excitation wavelength of 488 nm and an emission wavelength of 520 nm.

[0015] Preferably, in step S5, the inlet air temperature of the spray dryer is 160-180°C, the outlet air temperature is 80-90°C, and the atomization pressure is 0.2-0.3 MPa.

[0016] Preferably, the method further includes step S6: using high performance liquid chromatography-mass spectrometry (HPLC-MS) to detect the purity of the active ingredient product obtained in step S5, wherein the mobile phase of the HPLC is a methanol-water mixture, wherein the methanol volume fraction is 30% to 70%, and the flow rate is 0.8 to 1.2 mL / min.

[0017] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages compared with the prior art:

[0018] This invention addresses several problems in existing active ingredient extraction technologies, including the risk of active ingredient inactivation during raw material pretreatment, the inability of single-solvent extraction to effectively separate multiple types of components, cumbersome purification steps resulting in insufficient purity, low accuracy due to reliance on single detection methods for activity screening, and the lack of a systematic purity verification process. It solves these problems one by one through innovative methods: supercritical CO2 low-temperature freeze-drying replaces traditional high-temperature drying, maximizing the preservation of active ingredients in the raw materials under low-temperature conditions of -40℃ to -20℃ and specific pressure and flow rates, while avoiding the damage of heat-sensitive components to high temperatures; and gradient ultrasonic extraction using ultrapure water, 50% ethanol solution, and 95% ethanol solution, combined with pulsed ultrasound and an optimized material-to-liquid ratio, achieves water... The stepwise separation of soluble, alcohol-soluble, and lipid-soluble components overcomes the limitations of single-solvent extraction. A composite chromatography column composed of a 0.2μm ceramic membrane, an AB-8 macroporous adsorption resin layer, and a Sephadex G-100 dextran gel layer is designed to simplify the operation and improve component purity through a multi-stage purification structure. A dual-channel biosensor array consisting of a cell impedance sensor with a gold-plated electrode array and a fluorescence quantitative sensor with a specific fluorescent probe is used to simultaneously acquire cellular response and molecular binding signals, improving the accuracy of activity screening. Finally, high-performance liquid chromatography-mass spectrometry is used to detect the purity of the finished product, forming a complete quality control system and solving the problem of lack of systematic validation in existing technologies.

[0019] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0020] Figure 1 This is a timeline diagram of the overall process flow of an extraction method for classifying and screening active ingredients according to the present invention.

[0021] Figure 2This is a diagram showing the equipment connection topology of an extraction method for classifying and screening active ingredients according to the present invention.

[0022] Figure 3 This is a gradient extraction time sequence diagram of an extraction method for classifying and screening active ingredients according to the present invention.

[0023] Figure 4 This is a flowchart of a biosensor detection process for an extraction method for classifying and screening active ingredients according to the present invention.

[0024] Figure 5 This is a timeline diagram for purity verification of an extraction method for classifying and screening active ingredients according to the present invention. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] It should be noted that the terms "vertical," "horizontal," "up," "down," "left," "right," and similar expressions used in this article are for illustrative purposes only and do not represent the only possible implementation.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to limit the invention; the term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0028] like Figure 1 , 2As shown in Figure 3, an extraction method for classifying and screening active ingredients is characterized by achieving efficient extraction through innovative pretreatment and multidimensional separation technology. The method involves first subjecting the raw material to be extracted to supercritical CO2 low-temperature freeze-drying, controlling the temperature at -40℃ to -20℃, the pressure at 8 to 12 MPa, the CO2 flow rate at 20 to 30 L / h, and the treatment time at 4 to 6 hours to obtain freeze-dried powder. This pretreatment method maximizes the retention of active ingredients. Subsequently, ultrasonic-assisted extraction is performed sequentially using ultrapure water, 50% ethanol solution, and 95% ethanol solution, with treatment at 300 to 500 W power and 30 to 50℃ for 30 to 6 hours. Water-soluble, alcohol-soluble, and lipid-soluble extracts were obtained at 0 min, and the components were initially classified using gradient solvents. The three extracts were then purified by passing them through a composite chromatography column consisting of a 0.2 μm ceramic membrane layer, a macroporous adsorption resin layer, and a dextran gel layer at a flow rate of 1–2 mL / min. The multi-level purification structure was used to improve the purity of the components. Finally, a dual-channel biosensor array, including a cell impedance sensor and a fluorescence quantitative sensor, was used for simultaneous detection to screen for components with an activity intensity ≥80%. The final product was obtained by vacuum rotary evaporation concentration and spray drying. The simultaneous detection technology improved the accuracy of the screening.

[0029] In this implementation scheme, the connections and positions of the components are as follows: the outlet of the supercritical CO2 low-temperature freeze-drying equipment is connected to the inlet of the ultrasonic extraction equipment through a sealed pipeline; the three solvent storage tanks of the ultrasonic extraction equipment are connected to the extraction chamber in sequence through valves; the outlet of the extraction chamber is connected to the top inlet of the composite chromatography column through a pipeline; the bottom outlet of the composite chromatography column is connected to the sample cell of the dual-channel biosensor array through a delivery pump; the outlet of the screened components of the biosensor array is connected to the inlet of the vacuum rotary evaporation concentration equipment; the outlet of the concentration equipment is connected to the inlet of the spray drying equipment; a 0.2μm ceramic membrane layer is located at the top of the composite chromatography column, and a macroporous adsorption resin layer is tightly connected below it; below the macroporous adsorption resin layer is a dextran gel layer, and the three layers are arranged sequentially along the axial direction of the chromatography column. From the perspectives of implementation key points and innovation, the combination of these components allows for several key benefits. Supercritical CO2 low-temperature freeze-drying avoids the inactivation of active ingredients caused by high temperatures, preserving more effective substances for subsequent extraction. Gradient solvent ultrasonic extraction can specifically separate components of different polarities, improving extraction specificity. The three-layer structure of the composite chromatography column sequentially performs filtration, adsorption, and fractionation, synergistically enhancing purification efficiency and achieving higher purity than a single chromatography column. A dual-channel biosensor array simultaneously detects cell responses and molecular binding signals, improving screening accuracy. Vacuum rotary evaporation concentration combined with spray drying efficiently removes solvents at low temperatures, reducing the loss of active ingredients. In addressing existing problems, supercritical CO2 low-temperature freeze-drying solves the problem of active ingredient inactivation caused by traditional drying; gradient extraction solves the problem of incomplete separation by a single solvent; the composite chromatography column solves the problem of insufficient purification purity; the dual-channel sensor solves the problem of inaccurate screening; and the concentration-drying combination solves the problem of low retention rate of active ingredients in traditional thermal concentration. The synergistic effect of these structures comprehensively improves the efficiency and quality of active ingredient extraction and screening.

[0030] like Figure 4 and Figure 5 As shown, an extraction method for classifying and screening active ingredients focuses on optimizing parameters at each stage and expanding detection methods. Its features include: the raw material to be extracted is traditional Chinese medicine, plant tissue, or microbial fermentation product, which is pulverized to 100-200 mesh and then freeze-dried at low temperature using supercritical CO2; during gradient extraction, the ratio of freeze-dried powder to solvent is 1:10-1:20, and 2-5Hz pulsed ultrasound is used; in the composite chromatography column, AB-8 type macroporous adsorption resin and Sephadex G-100 dextran gel are set at a volume ratio of 3-5:2-3, and the ratio of the resin to the ceramic membrane is 1:3-5:2-3; the cell impedance sensor uses a gold-plated electrode array, and the detection frequency is...

[0031] The fluorescence quantitative sensor uses a 488nm excitation / 520nm emission fluorescent probe, operating at 10–100 kHz. The spray drying process controls the inlet air temperature to 160–180℃, the outlet air temperature to 80–90℃, and the nebulization pressure to 0.2–0.3 MPa. Finally, purity is determined by high-performance liquid chromatography-mass spectrometry (HPLC-MS) with a mobile phase of 30%–70% methanol-water and a flow rate of 0.8–1.2 mL / min.

[0032] In this implementation scheme, the connections and positions of the components are as follows: the outlet of the raw material crushing equipment is connected to the inlet of the supercritical CO2 low-temperature freeze-drying equipment via a conveying device; the pulsed ultrasonic generator of the ultrasonic extraction equipment is installed on the side wall of the extraction chamber; the three solvent storage tanks are respectively connected to the supply pipelines of ultrapure water, 50% ethanol solution, and 95% ethanol solution and are connected to the extraction chamber; the AB-8 type macroporous adsorption resin layer in the composite chromatography column is located directly below the 0.2μm ceramic membrane layer, below which is the Sephadex G-100 dextran gel layer, and the three layers are arranged closely along the column axis in proportion; the gold-plated electrode array in the dual-channel biosensor array is embedded at the bottom of the sample cell, and the fluorescent probe detection module is located at the corresponding position above the sample cell; the inlet of the high-performance liquid chromatography-mass spectrometry equipment is connected to the outlet of the spray drying equipment via a pipeline. From the perspectives of implementation key points and innovation, these optimized parameters and structural combinations allow for increased contact area with the solvent when raw materials are pulverized to 100-200 mesh. Combined with a 1:10-1:20 material-to-liquid ratio and pulsed ultrasound, extraction efficiency is improved while avoiding localized overheating. The proportional settings of the composite chromatography column ensure functional compatibility of each layer, enhancing purification specificity. The combination of sensors and probes with specific parameters enhances signal sensitivity and improves detection accuracy. Spray drying parameter control ensures stable retention of active ingredients, while high-performance liquid chromatography-mass spectrometry (HPLC-MS) enables precise quantification of finished product purity. In addressing existing problems, optimized pulverization and extraction parameters solve the problem of low extraction efficiency in traditional methods; the column ratio settings address insufficient purification specificity; optimized sensor and probe parameters solve the problem of weak detection signals; and the combination of spray drying and coupled detection solves the problem of inaccurate verification of finished product quality. These components synergistically improve the stability and reliability of the method.

[0033] When using this device, existing raw material cleaning equipment (such as a high-pressure spray cleaner) is required to pre-treat Chinese medicinal materials (such as honeysuckle and astragalus), plant tissues (such as ginkgo leaves and wolfberry pulp), or microbial fermentation products (such as Ganoderma lucidum fermentation mycelium) to remove mud and impurities. Solvent preparation requires the use of a precision pipette and electronic balance to prepare ultrapure water, 50% ethanol solution (anhydrous ethanol and ultrapure water mixed at a 1:1 volume ratio), and 95% ethanol solution (anhydrous ethanol and ultrapure water mixed at a 19:1 volume ratio). Extract delivery requires flow control via a peristaltic pump. The regeneration of the composite chromatography column requires alternating rinsing with 0.1 mol / L hydrochloric acid solution and 0.1 mol / L sodium hydroxide solution; cell culture for the cell impedance sensor requires a CO2 incubator (temperature 37℃, CO2 concentration 5%) and DMEM medium; fluorescent probes (such as FITC-labeled specific antibodies) for the quantitative fluorescence sensor need to be diluted with phosphate buffer; the chromatographic column for high performance liquid chromatography-mass spectrometry can be a C18 reversed-phase column (250 mm × 4.6 mm, 5 μm), and the mass spectrometer detector uses an electrospray ionization source (ESI).

[0034] Specifically, during installation, the raw material crushing equipment is fixed 1.5–2 m in front of the feed inlet of the supercritical CO2 low-temperature freeze-drying equipment. The two are connected by a stainless steel screw conveyor with an inclination angle of 30°–45° to avoid raw material accumulation. The three solvent storage tanks of the ultrasonic extraction equipment are installed side by side 0.8–1 m above the extraction chamber and are connected to the extraction chamber through PVC pipes with solenoid valves. The solenoid valves are electrically connected to the equipment control system to achieve automatic switching. The composite chromatography column is vertically fixed on the support, and its top inlet is connected to the outlet of the ultrasonic extraction equipment through a silicone tube. A flow meter is installed on the silicone tube to monitor the flow rate in real time. The sample cell of the dual-channel biosensor array is connected to the bottom outlet of the composite chromatography column through a quick connector. The sensor signal cable is connected to the data acquisition instrument, which is connected to a computer to achieve real-time data display and analysis. When using existing equipment, after turning on the raw material cleaning equipment, place the raw materials such as Chinese medicinal herbs into the cleaning tank, set the spray pressure to 0.2-0.3 MPa and the cleaning time to 3-5 minutes. After cleaning, the raw materials are conveyed to a hot air drying oven (temperature 50-60℃, dried to a moisture content ≤10%). For solvent preparation, accurately weigh anhydrous ethanol using an electronic balance and pour it into a glass beaker. Use a precision pipette to transfer the corresponding volume of ultrapure water and mix. Stir with a magnetic stirrer (300-500 r / min) for 5-10 minutes until homogeneous. The peristaltic pump for extract delivery needs to be calibrated for flow rate beforehand. The rotation speed was set to 10–20 r / min to match the flow rate requirement of 1–2 mL / min for the composite chromatography column. During regeneration of the composite chromatography column, it was first flushed forward with twice the column volume of 0.1 mol / L hydrochloric acid solution (flow rate 2–3 mL / min), allowed to stand for 30 min, then flushed backward with the same volume of 0.1 mol / L sodium hydroxide solution, and finally rinsed with ultrapure water until neutral. Before use, the gold-plated electrode array of the cell impedance sensor needed to be disinfected by immersing it in 75% ethanol for 30 min, rinsed with ultrapure water, and preheated to 37°C in a CO2 incubator. 1 × 10⁶ cells were then inoculated. 5 Target cell suspension of cells / mL was cultured for 24 hours until cell adhesion was achieved before detection. When diluting the fluorescent probe of the quantitative fluorescence sensor, 1 μL of FITC-labeled antibody was added to 99 μL of phosphate buffer (pH 7.4) using a pipette, and mixed for 1 min using a vortex mixer (2000-3000 r / min). The mixture was then stored at 4℃ in the dark for later use. Before high performance liquid chromatography-mass spectrometry (HPLC-MS), the C18 column was activated by rinsing with a methanol-water (9:1) mixture for 30 min, and the mass spectrometer was preheated for 30 min. The electrospray ionization source was set to positive ion mode, with a capillary voltage of 3.5 kV and an ion source temperature of 110℃.

[0035] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for classifying and screening active ingredients, characterized in that, Includes the following steps: S1. Raw material pretreatment: The raw material to be extracted is subjected to supercritical CO2 low-temperature freeze drying treatment, with the temperature controlled at -40℃ to -20℃ and the pressure at 8 to 12 MPa, to obtain freeze-dried powder; in the supercritical CO2 low-temperature freeze drying treatment, the CO2 flow rate is 20 to 30 L / h and the treatment time is 4 to 6 h; S2. Gradient solvent extraction: The lyophilized powder obtained in step S1 was sequentially extracted with ultrapure water, 50% ethanol solution, and 95% ethanol solution using ultrasonic-assisted extraction. The ultrasonic power was 300-500W, the extraction temperature was 30-50℃, and the extraction time for each solvent was 30-60min, resulting in water-soluble extract, alcohol-soluble extract, and lipid-soluble extract, respectively. S3. Multidimensional classification and purification: The three extracts obtained in step S2 are purified by passing them through a composite chromatography column. The composite chromatography column consists of a 0.2 μm ceramic membrane layer, a macroporous adsorption resin layer, and a dextran gel layer from top to bottom. The flow rate is controlled at 1 to 2 mL / min, and the eluent is collected. S4. Activity screening: The activity of the purified effluent obtained in step S3 is detected by a dual-channel biosensor array, which includes a cell impedance sensor and a fluorescence quantitative sensor. The cell response signal and molecular binding signal of the active ingredient are acquired simultaneously, and the components with an activity intensity ≥80% are screened out. S5. Concentration and Drying: The active components obtained in step S4 are concentrated by vacuum rotary evaporation, with the vacuum degree controlled at 0.08-0.1 MPa and the temperature at 40-50°C. After concentration to a solid content of 30%-40%, the active components are spray-dried to obtain the finished product.

2. The extraction method according to claim 1, characterized in that, In step S1, the raw material to be extracted is Chinese medicinal material, plant tissue or microbial fermentation product, and before supercritical CO2 low-temperature freeze drying, it is first pulverized to a particle size of 100-200 mesh.

3. The extraction method according to claim 1, characterized in that, In step S2, the ratio of the freeze-dried powder to each extraction solvent is 1:10 to 1:20 (g / mL), and pulsed ultrasound is used in the ultrasonic-assisted extraction process with a pulse frequency of 2 to 5 Hz.

4. The extraction method according to claim 1, characterized in that, In step S3, the macroporous adsorption resin layer is AB-8 type macroporous adsorption resin, the dextran gel layer is Sephadex G-100 dextran gel, and the volume ratio of the 0.2μm ceramic membrane layer, the macroporous adsorption resin layer, and the dextran gel layer is 1:3 to 5:2 to 3.

5. The extraction method according to claim 1, characterized in that, In step S4, the cell impedance sensor uses a gold-plated electrode array with a detection frequency of 10–100 kHz, and the fluorescence quantitative sensor uses a fluorescent probe with an excitation wavelength of 488 nm and an emission wavelength of 520 nm.

6. The extraction method according to claim 1, characterized in that, In step S5, the inlet air temperature of the spray dryer is 160-180°C, the outlet air temperature is 80-90°C, and the atomization pressure is 0.2-0.3 MPa.

7. The extraction method according to claim 1, characterized in that, The method also includes step S6: using high performance liquid chromatography-mass spectrometry (HPLC-MS) to detect the purity of the active ingredient product obtained in step S5, wherein the mobile phase of the HPLC is a methanol-water mixture, wherein the volume fraction of methanol is 30% to 70%, and the flow rate is 0.8 to 1.2 mL / min.

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