Method for extracting active ingredients from plants by supercooling ultrasonic solvent-free implantation

Abstract

The application discloses a method for extracting plant active components by using ultralow-temperature ultrasonic solvent-free planting extraction, and belongs to the technical field of plant active component extraction. The method comprises the following steps: S1, gradient ultralow-temperature freezing pretreatment; S2, liquid nitrogen assisted physical wall breaking; S3, solvent-free variable-frequency ultrasonic extraction; S4, extract collection; the extract obtained in the step S3 is collected; S5, purification treatment; the extract collected in the step S4 is purified to obtain a plant active extract product. In the application, no water or exogenous solvent is added during extraction, the dependence on organic solvents and even water in the traditional extraction method is completely abandoned, no exogenous chemical substances are introduced in the extraction process, the obtained extract has no solvent residue, and the product is high in purity.

Description

Technical Field

[0001] This invention relates to a method for solvent-free extraction of plant active ingredients using ultra-low temperature ultrasonic extraction, specifically a method for solvent-free extraction of plant active ingredients using ultra-low temperature ultrasonic extraction, belonging to the field of plant active ingredient extraction technology. Background Technology

[0002] Plant active ingredients refer to natural compounds in plants that possess specific biological activities or functions, such as polyphenols, flavonoids, saponins, terpenes, and alkaloids. They have wide applications in food, medicine, cosmetics, and other fields. Currently, the main methods for extracting active ingredients from plant raw materials include solvent extraction, steam distillation, microwave-assisted extraction, and enzyme-assisted extraction.

[0003] For example, CN121846723A discloses a method for producing plant extracts using ultrasound-assisted low-temperature extraction. This method includes the following steps: raw material pretreatment, preparation of a composite solvent system, ultrasound-assisted low-temperature extraction, and low-temperature enrichment and purification. Plant raw materials are pretreated with a composite pre-modifier prepared using an enzyme-mediator-oligosaccharide system. Then, a composite system of hydrophilic eutectic solvent-low-carbon alcohol-natural co-solvent is used combined with a two-stage ultrasound extraction process of wetting and disruption. The plant extract is obtained after natural clarification, low-temperature vacuum concentration, and purification with macroporous resin. This invention protects heat-sensitive components through full-chain low-temperature control, enhances plant extraction efficiency through composite pre-modification and hydrophilic eutectic solvent, and improves product purity through secondary purification. Furthermore, the solvent is environmentally friendly and biodegradable, and the process is easily industrialized. This effectively solves the shortcomings of existing technologies, such as easy degradation of heat-sensitive components and low extraction efficiency, and is suitable for the extraction of active plant components.

[0004] Traditional solvent extraction methods, while simple to operate, consume large amounts of reagents, pollute the environment, and have low recovery rates. The extensive use of organic solvents not only increases the burden of subsequent purification but may also lead to organic solvent residues in the product, affecting its safety and application range. In recent years, although green alternatives such as supercritical CO2 extraction have emerged, supercritical extraction equipment requires significant investment and is not truly solvent-free. Moreover, in existing technologies, extraction efficiency and activity retention are often difficult to balance. While extraction efficiency can be improved through end-to-end low-temperature control and composite solvent systems, these systems rely on a composite system of "hydrophilic deep eutectic solvent - low-carbon alcohol - natural co-solvent," failing to achieve solvent-free extraction. Summary of the Invention

[0005] The purpose of this invention is to provide a method for extracting active plant components using ultra-low temperature ultrasonic solvent-free extraction in order to solve the above-mentioned problems. This method enables solvent-free extraction and produces pure and safe products.

[0006] This invention achieves the above objective through the following technical solution: a method for extracting active plant components using ultra-low temperature ultrasonic solvent-free extraction, comprising the following steps: S1. Gradient ultra-low temperature freezing pretreatment: Plant materials are pre-frozen at -20℃ to -10℃ for 0.5 to 1 hour, and then deep-frozen at -60℃ to -40℃ for 1 to 3 hours, so that ice crystals in plant cells grow slowly and fully pierce the cell wall. S2. Liquid nitrogen-assisted physical cell wall disruption: The plant material after freezing treatment in step S1 is ultra-finely pulverized in a liquid nitrogen atmosphere, with the particle size controlled to be ≤50μm and the cell wall disruption rate to be 95%~99%. S3. Solvent-free variable frequency ultrasonic extraction: The plant raw material after cell wall breaking treatment in step S2 is directly placed in an ultrasonic extraction device without adding organic solvent. Extraction is carried out under low temperature conditions of 10℃~25℃ using dual-frequency alternating variable frequency ultrasonic waves. The ultrasonic frequency alternates between 20~30KHz and 40~50KHz, the ultrasonic power is 150~350W, and the extraction time is 15~35min to obtain the extract. S4. Extract collection: Collect the extract obtained in step S3; S5. Purification: The extract collected in step S4 is purified to obtain the finished plant active extract.

[0007] Preferably, in order to improve the pretreatment effect, the cooling rate of the gradient ultra-low temperature freezing in step S1 is 0.5 to 2℃ / min, and the plant raw material in step S1 also includes a thawing-refreezing cycle between pre-freezing and deep freezing, wherein the thawing temperature is 0℃ to 4℃ and the thawing time is 0.5 to 1h.

[0008] Preferably, in order to efficiently release intracellular active ingredients, the temperature of liquid nitrogen-assisted physical cell disruption in step S2 is -196℃ to -80℃, and the pulverization time is 1 to 5 minutes.

[0009] Preferably, in order to achieve efficient extraction at low temperatures, the alternation period of the dual-frequency alternating ultrasonic waves in step S3 is once every 30 to 120 seconds, the ultrasonic extraction in step S3 is carried out under vacuum conditions with a vacuum degree of 0.5 to 10 kPa, and the ultrasonic extraction in step S3 is carried out in pulse mode with a pulse duty cycle of 30% to 70%.

[0010] Preferably, in order to achieve graded collection of the product, the extract in step S4 is collected by gravity flow combined with low temperature condensation to collect volatile active ingredients, and by centrifugation to collect non-volatile active ingredients.

[0011] Preferably, in order to avoid secondary damage to the active ingredients by high temperature and to ensure the bioactivity of the final product, the purification process in step S5 includes low-temperature membrane separation and freeze drying. The temperature of the low-temperature membrane separation is 5℃~15℃, and the temperature of the freeze drying is -50℃~-30℃.

[0012] The beneficial effects of this invention are: 1. This invention does not add any organic solvents during extraction, completely eliminating the dependence on organic solvents in traditional extraction methods. The extraction process does not introduce any exogenous chemical substances, and the resulting extract has no solvent residue and high product purity.

[0013] This invention utilizes a purely physical combination of gradient freeze-thaw disruption and liquid nitrogen ultrafine pulverization to fully expose intracellular active ingredients, enabling efficient release without the need for any solvent.

[0014] This invention employs dual-frequency alternating variable-frequency ultrasound for extraction. While single-frequency ultrasound cavitation effects are directional and limited by standing waves, dual-frequency alternation generates a synergistic cavitation effect, lowering the cavitation threshold and increasing cavitation yield, thus significantly improving extraction efficiency. Highly efficient extraction can be achieved at low temperatures of 10℃ to 25℃, avoiding the damage to heat-sensitive components caused by high temperatures and solving the problem of uneven cavitation in conventional single-frequency ultrasound. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the overall process of the present invention.

[0016] Figure 2 This is a multi-dimensional comparison diagram of Embodiment 1 of the present invention. Detailed Implementation

[0017] 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.

[0018] Please see Figures 1-2 As shown, the method for extracting active plant components using ultra-low temperature ultrasonic solvent-free extraction includes the following steps: S1. Gradient ultra-low temperature freezing pretreatment: Plant materials are pre-frozen at -20℃ to -10℃ for 0.5 to 1 hour, and then deep-frozen at -60℃ to -40℃ for 1 to 3 hours, so that ice crystals in plant cells grow slowly and fully pierce the cell walls; the cooling rate of gradient ultra-low temperature freezing is 0.5 to 2℃ / min, and the plant materials include a thawing-refreezing cycle between pre-freezing and deep freezing, with a thawing temperature of 0℃ to 4℃ and a thawing time of 0.5 to 1 hour.

[0019] S2, Liquid nitrogen-assisted physical cell wall disruption: The plant material after freezing treatment in step S1 is subjected to ultra-fine pulverization in a liquid nitrogen atmosphere, with the particle size controlled to be ≤50μm and the cell wall breakage rate to be 95%~99%; the temperature of liquid nitrogen-assisted physical cell wall disruption is -196℃~-80℃, and the pulverization time is 1~5min.

[0020] S3. Solvent-free frequency-conversion ultrasonic extraction: The plant material after cell wall disruption in step S2 is directly placed in an ultrasonic extraction device without adding organic solvents. Extraction is carried out at a low temperature of 10℃~25℃ using dual-frequency alternating ultrasonic waves. The ultrasonic frequencies alternate between 20~30KHz and 40~50KHz, the ultrasonic power is 150~350W, and the extraction time is 15~35min to obtain the extract. The alternation cycle of the dual-frequency alternating ultrasonic waves is once every 30~120s. Ultrasonic extraction is carried out under vacuum conditions with a vacuum degree of 0.5~10KPa. Ultrasonic extraction is carried out in pulse mode with a pulse duty cycle of 30%~70%.

[0021] S4. Extract collection: Collect the extract obtained in step S3; the extract is collected by gravity flow combined with low temperature condensation to collect volatile active ingredients, and by centrifugation to collect non-volatile active ingredients.

[0022] S5. Purification: The extract collected in step S4 is purified to obtain the finished plant active extract. The purification process includes low-temperature membrane separation and freeze drying. The temperature of low-temperature membrane separation is 5℃~15℃, and the temperature of freeze drying is -50℃~-30℃.

[0023] Plant raw materials are selected from at least one plant tissue selected from roots, stems, leaves, flowers, fruits, and seeds.

[0024] like Figure 1 As shown, all operation steps of this invention are completed under low temperature conditions, forming a complete low-temperature protection chain: S1 stage: -60℃~-20℃, the active ingredients are in a completely frozen state, the enzymatic oxidation reaction is completely inhibited, and the chemical degradation reaction rate approaches zero.

[0025] S2 stage: -196℃~-80℃ liquid nitrogen atmosphere, the material is in a brittle state, and the inert environment prevents oxidation.

[0026] S3 stage: 10℃~25℃, which is far below the thermal degradation temperature of heat-sensitive active ingredients, avoiding component isomerization, oxidation and polymerization reactions caused by high-temperature extraction.

[0027] S5 stage: membrane separation at 5℃~15℃ and freeze drying at -50℃~-30℃, completely avoiding heat damage during product post-processing.

[0028] Working Principle: In this invention, the plant cell wall is the primary natural barrier preventing the release of intracellular active ingredients. Its structure is mainly composed of cellulose microfibrils, hemicellulose, pectin, and a small amount of structural proteins, giving it high mechanical strength. Step S1 of this invention employs a gradient ultra-low temperature freezing scheme, namely pre-freezing (-20℃ to -10℃, 0.5 to 1 h) - deep freezing (-60℃ to -40℃, 1 to 3 h). Water in plant cells exists in two forms: free water and bound water. In the pre-freezing stage (-20℃ to -10℃), under the slow cooling condition controlled at a rate of 0.5 to 2℃ / min, free water in the intercellular spaces and cells first undergoes heterogeneous nucleation, forming small ice crystals. Due to the slow cooling rate, the ice crystals have sufficient time to preferentially grow under thermodynamic drive, forming larger, regularly shaped ice crystals. As the temperature continues to drop to the deep freezing stage (-60℃ to -40℃), the bound water also gradually undergoes a phase transition, and the ice crystals further grow and expand, with a volume expansion rate of approximately 9%. The volume expansion of ice crystals exerts pressure and dehydration on the cell wall, causing the cellulose microfibrils in the cell wall to loosen and their tensile strength to decrease. Subsequently, during deep freezing at -60℃, the residual bound water undergoes a phase transition, generating expansion stress and inducing microcracks in the cell wall. These microcracks become stress concentration points during subsequent S2 liquid nitrogen ultrafine grinding, causing mechanical impact force to be directionally transmitted along the cracks, thereby achieving efficient cell wall disruption. The cell wall rupture rate of plant cells after gradient freezing is approximately 25-40% higher than that after quick freezing.

[0029] Step S2 involves ultrafine pulverization in a liquid nitrogen atmosphere (-196℃ to -80℃). The plant material, after gradient freezing, already exhibits numerous microcracks and structurally weakened regions at the cell wall level, placing it in a pre-damaged state. The ultra-low temperature environment of liquid nitrogen (-196℃) drastically lowers the overall temperature of the material below its glass transition temperature, causing the plant tissue to enter a fully brittle state. Under this condition, the molecular thermal motion within the material is significantly reduced, and the material's fracture toughness decreases markedly. Under the combined action of the mechanical impact, shearing, and grinding forces of the ultrafine pulverization equipment, the material preferentially fractures along the microcracks in the cell wall, rapidly breaking into ultrafine powder with a particle size ≤50μm, achieving a cell wall breakage rate of 95%~99%. The liquid nitrogen atmosphere serves three functions in this process: first, it provides an ultra-low temperature environment to embrittle the material; second, as an inert protective gas, it reduces the oxygen volume fraction in the pulverizing chamber to below 2%, effectively preventing the active ingredients from undergoing oxidative degradation during pulverization; and third, the rapid vaporization of liquid nitrogen absorbs a large amount of heat, ensuring that the material temperature remains below -50℃ throughout the pulverization process, avoiding the conversion of mechanical energy into thermal energy and causing localized temperature rise. Through the two-stage physical cell wall disruption combination of "gradient freezing pre-damage + liquid nitrogen ultrafine pulverization," the plant cell walls are completely disintegrated, and the intracellular active ingredients are fully exposed in a free state, laying the material foundation for subsequent solvent-free extraction.

[0030] Step S3 is the core extraction step of this invention. No organic solvent is added. Extraction is carried out at a low temperature of 10℃ to 25℃ using dual-frequency alternating variable frequency ultrasound (20-30KHz and 40-50KHz alternating, alternation period of 30-120s, power of 150-350W, time of 15-35min).

[0031] When ultrasound propagates in a liquid medium, the sound pressure changes periodically. In the negative pressure phase of the sound wave, tiny gas nuclei in the liquid rapidly expand to form cavitation bubbles due to the local pressure dropping below the saturated vapor pressure. In the subsequent positive pressure phase, the bubbles are compressed and eventually undergo implosive collapse. At the moment of bubble collapse, local high temperature, high pressure, and intense shock waves are generated within an extremely small spatial and temporal scale, accompanied by strong microjets and shear stress fields. These extreme physical effects strip, disperse, emulsify, and dissolve the active ingredients attached to the surface of the broken cell fragments into the cell's own water and intracellular fluid. The cavitation effect of conventional single-frequency ultrasound exhibits significant spatial inhomogeneity. The distribution of cavitation bubbles in the sound field is affected by the standing wave effect, with weaker cavitation effects at wave nodes, forming cavitation dead zones. This invention employs an alternating low-frequency (20–30 kHz) and high-frequency (40–50 kHz) ultrasonic wave scheme. The synergistic mechanism lies in the following: low-frequency ultrasound has a lower cavitation threshold, readily generating cavitation bubbles. The shock waves generated when these bubbles collapse are intense and have a wide range of effect, primarily performing macroscopic stripping. High-frequency ultrasound has a higher cavitation threshold, but produces a greater number of uniformly distributed cavitation bubbles, generating more microjets upon collapse, primarily performing microscopic dispersion. When the two alternate with a period of 30–120 seconds, low-frequency ultrasound continuously generates new cavitation bubbles and maintains the cavitation field intensity, while high-frequency ultrasound homogenizes and modulates the cavitation field. The cavitation bubbles generated by both form a cascade effect upon collapse, significantly increasing cavitation yield. Experimental studies show that the extraction efficiency of dual-frequency alternating ultrasound is approximately 20–35% higher than that of single-frequency ultrasound.

[0032] This invention achieves completely solvent-free extraction by relying on the orderly connection of two prerequisites: First, through the secondary physical disruption in steps S1-S2, the plant cell walls are broken at a rate ≥98%, and the active ingredients are fully exposed on the surface of the cell fragments, eliminating the need for the rate-limiting step of solvent penetration into the cell walls in traditional extraction. Second, the water, intracellular fluid, and cytoplasm released after the plant cells are broken constitute the liquid continuous medium necessary for ultrasound propagation. The ultrasonic cavitation effect can occur normally in this natural medium and effectively act on the stripping and dispersion of active ingredients. Different plant materials have varying water contents; for materials with low water content, appropriate rehydration treatment can be performed beforehand to provide sufficient ultrasonic propagation medium.

[0033] Example 1 Raw material selection and processing: Select fresh hawthorn fruits, remove the pits, wash, and cut into 1-2cm pieces of pulp. Hawthorn is rich in triterpenoid compounds, mainly including ursolic acid, oleanolic acid, and hawthorn acid, which have various biological activities such as lowering blood lipids, anti-oxidation, and anti-inflammation.

[0034] Extraction process: S1. Gradient ultra-low temperature freezing pretreatment: Place the cut hawthorn pulp in a temperature-controlled freezer and cool it to -18℃ at a cooling rate of 1℃ / min, then keep it at this temperature for 45 minutes; then continue to cool it to -55℃ at a cooling rate of 1℃ / min, and keep it at this temperature for 2 hours.

[0035] S2. Liquid nitrogen-assisted physical cell wall disruption: Frozen hawthorn pieces were transferred to a liquid nitrogen ultrafine pulverizer and ultrafinely pulverized under continuous liquid nitrogen spraying. The temperature of the pulverizing chamber was controlled to be constant between -120℃ and -90℃, and the pulverizing time was 3 minutes, yielding hawthorn ultrafine powder. Laser particle size analyzer analysis showed that particles with a diameter ≤50μm accounted for ≥98% of the powder; microscopic observation showed a cell wall breakage rate of 98.5%.

[0036] S3. Solvent-free frequency conversion ultrasonic extraction: The cell-wall-broken hawthorn ultrafine powder is directly added to the extraction chamber of the ultrasonic extraction equipment without adding organic solvents. The circulating cooling system is turned on to maintain the temperature of the extraction chamber at 20±2℃. The dual-frequency alternating frequency conversion ultrasonic system is started, with the ultrasonic frequency alternating between 25KHz and 45KHz every 60s, the ultrasonic power is 220W, and the total extraction time is 30min. During the extraction process, the water and intracellular fluid released by the hawthorn cells act as the natural medium for ultrasonic propagation, and the triterpene acid components stripped by the cavitation effect are dispersed in the extract.

[0037] S4. Extract Collection: After extraction, open the valve at the bottom of the extraction chamber to allow the extract to flow out naturally under gravity. Simultaneously, start the low-temperature condenser to collect volatile aroma components. The remaining solid residue is separated by low-speed centrifugation (3000 rpm, 10 min), and the extracts are combined.

[0038] S5. Purification: The combined extracts were pumped into a low-temperature membrane separation system. Membrane separation was performed using a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 1000 Da at an operating temperature of 8°C and an operating pressure of 0.2 MPa. The permeate was freeze-dried at -45°C to constant weight to obtain a grayish-white hawthorn triterpene acid extract powder.

[0039] The extraction rate of hawthorn triterpenoids was determined to be 9.26% (dry weight) by high performance liquid chromatography. The content of ursolic acid was 5.82%, oleanolic acid was 2.14%, and hawthorn acid was 1.30%. Compared with the 70℃ hot reflux extraction method, the extraction rate of this invention was increased by 28.6%, and the antioxidant activity was increased by approximately 36.9%.

[0040] Example 2 Raw material selection and processing: Select fresh ginger, wash, peel, and cut into 3mm thick slices. Ginger is rich in gingerol compounds, which are the main source of its pungent flavor and have anti-inflammatory, antioxidant, and anti-tumor biological activities.

[0041] Extraction process: S1. Gradient ultra-low temperature freezing pretreatment: Place ginger slices in a programmable temperature-controlled freezer and cool to -15℃ at a rate of 0.8℃ / min, holding for 1 hour. Then, continue cooling to -50℃ at a rate of 0.8℃ / min and holding for 2 hours. Between pre-freezing and deep freezing, remove the material and thaw it in a 2℃ refrigerator environment for 45 minutes, then freeze it again in a -15℃ environment for 1 hour (thawing-refreezing cycle), and then perform deep freezing treatment.

[0042] S2. Liquid nitrogen-assisted physical cell wall disruption: Frozen ginger slices were transferred to a liquid nitrogen ultrafine pulverizer. Liquid nitrogen was continuously sprayed to maintain the temperature of the pulverizing chamber at -130℃ to -100℃. The pulverizing time was 2 minutes, resulting in ultrafine ginger powder. Testing showed that ≥98% of the powder particles had a diameter ≤50μm, and the cell wall breakage rate was 98.1%.

[0043] S3. Solvent-free frequency conversion ultrasonic extraction: The broken-cell-wall ginger ultrafine powder is directly added to the ultrasonic extraction device without adding any organic solvent. The extraction chamber temperature is controlled at 15±2℃. The dual-frequency alternating frequency conversion ultrasonic system is activated, alternating between 20KHz and 40KHz every 90 seconds, with an ultrasonic power of 280W and a total extraction time of 25 minutes. Simultaneously, the vacuum system is activated to maintain the vacuum degree of the extraction chamber at 3-5KPa.

[0044] S4. Extract Collection: After extraction, the extract is discharged by gravity, and volatile terpenoid aroma components are collected by low-temperature condensation (condensation temperature -15℃). The solid residue is separated by centrifugation (4000 rpm, 8 min), and the extracts are combined.

[0045] S5. Purification: The combined solution was purified by a low-temperature membrane separation system (molecular weight cutoff 500 Da, operating temperature 10℃). The retentate was freeze-dried at -45℃ to obtain a light yellow gingerol extract powder.

[0046] The extraction rate of gingerol was 2.98% (dry weight) as determined by UV-Vis spectrophotometry. High-performance liquid chromatography (HPLC) analysis showed that the content of 6-gingerol was 1.56%, 8-gingerol was 0.42%, and 10-gingerol was 0.28%. Compared with the traditional hot reflux extraction method, the retention rate of 6-gingerol in the extract obtained by the method of this invention was 92.5%, while that of the hot reflux method was only 58.7%, representing a relative increase of 57.6%.

[0047] Example 3 Raw material selection and processing: Select dried black fungus, remove the stems, soak in 25℃ deionized water for 4 hours to rehydrate, drain the surface moisture, and set aside. Black fungus polysaccharides are the main active ingredients of black fungus, possessing pharmacological activities such as immunomodulation, anticoagulation, hypoglycemia, and antitumor effects.

[0048] Extraction process: S1. Gradient ultra-low temperature freezing pretreatment: The rehydrated black fungus is placed in a temperature-controlled freezer and cooled to -20℃ at a cooling rate of 0.6℃ / min, and kept at this temperature for 1 hour for pre-freezing; then the temperature is further reduced to -60℃ and kept at this temperature for 3 hours for deep freezing. A thawing-refreezing cycle is performed between pre-freezing and deep freezing (thawing temperature 0℃, thawing time 1 hour).

[0049] S2. Liquid nitrogen-assisted physical cell wall disruption: Frozen black fungus was ultra-finely pulverized in a liquid nitrogen atmosphere. The temperature of the pulverizing chamber was maintained at -150℃ to -120℃, and the pulverizing time was 5 minutes, resulting in ultra-fine black fungus powder. Testing showed that ≥98% of the powder particles had a diameter ≤50μm, and the cell wall breakage rate was 98.9%.

[0050] S3. Solvent-free frequency conversion ultrasonic extraction: Place the broken-cell-wall black fungus ultrafine powder into an ultrasonic extraction device without adding any organic solvent. The extraction chamber temperature is controlled at 10±2℃. Start the dual-frequency alternating frequency conversion ultrasonic system, alternating between 30KHz and 50KHz every 120s, with an ultrasonic power of 320W and an extraction time of 35min. Turn on the vacuum system to maintain a vacuum level of 1-3KPa, and simultaneously activate the pulse mode with a pulse duty cycle of 50%.

[0051] S4. Extract collection: After extraction, the extract is centrifuged (5000 rpm, 10 min) to remove residue and the supernatant is collected.

[0052] S5. Purification: The supernatant was fractionated and purified using a low-temperature membrane separation system (molecular weight cutoff 10000 Da, operating temperature 5℃). The retentate was freeze-dried at -50℃ to constant weight to obtain a grayish-white black fungus polysaccharide extract.

[0053] The extraction rate of polysaccharides from black fungus reached 7.71% (dry weight), as determined by the phenol-sulfuric acid method. The weight-average molecular weight of the polysaccharides obtained by the traditional hot water extraction method is approximately 1.2 × 10⁻⁶. 5 Da, the polysaccharide obtained by the method of the present invention has a Mw of 2.96 × 10⁻⁶. 5 The molecular weight increased by approximately 146.7%, indicating that the present invention effectively inhibits polysaccharide chain degradation caused by high temperature.

[0054] In summary, the extraction rates of hawthorn triterpenic acid reached 9.26%, gingerol from ginger reached 2.98%, and polysaccharides from black fungus reached 7.71%. Comparative experiments verified that the extraction rates were significantly better than the traditional hot reflux method and slightly better than or equivalent to the conventional ultrasonic extraction method with solvent. The entire process was conducted at low temperatures, resulting in an activity retention rate >95%. For heat-sensitive components, the method of this invention improved the relative retention rate by 57.6% compared to the traditional hot reflux method; for large polysaccharide molecules, the molecular weight retention was improved by 146.7% compared to the traditional hot water method. No water or exogenous solvents were added throughout the process, and the solvent residue in the extract was undetectable. The product purity reached over 96%, meeting the application standards of high-safety fields such as food, pharmaceuticals, and cosmetics. The examples verified the extraction effects of three different types of active ingredients: triterpenoids (hawthorn), gingerols (ginger), and polysaccharides (black fungus), demonstrating that the method of the present invention has good universality and can be further extended to the extraction of various plant active ingredients such as flavonoids, saponins, polyphenols, and volatile oils.

[0055] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0056] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for solvent-free extraction of plant active ingredients using ultra-low temperature ultrasonic extraction, characterized in that: Includes the following steps: S1. Gradient ultra-low temperature freezing pretreatment: Plant materials are pre-frozen at -20℃ to -10℃ for 0.5 to 1 hour, and then deep-frozen at -60℃ to -40℃ for 1 to 3 hours, so that ice crystals in plant cells grow slowly and fully pierce the cell wall. S2. Liquid nitrogen-assisted physical cell wall disruption: The plant material after freezing treatment in step S1 is ultra-finely pulverized in a liquid nitrogen atmosphere, with the particle size controlled to be ≤50μm and the cell wall disruption rate to be 95%~99%. S3. Solvent-free variable frequency ultrasonic extraction: The plant raw material after cell wall breaking treatment in step S2 is directly placed in an ultrasonic extraction device without adding organic solvent. Extraction is carried out under low temperature conditions of 10℃~25℃ using dual-frequency alternating variable frequency ultrasonic waves. The ultrasonic frequency alternates between 20~30KHz and 40~50KHz, the ultrasonic power is 150~350W, and the extraction time is 15~35min to obtain the extract. S4. Extract collection: Collect the extract obtained in step S3; S5. Purification: The extract collected in step S4 is purified to obtain the finished plant active extract.

2. The method for extracting active plant components using ultra-low temperature ultrasonic solvent-free extraction according to claim 1, characterized in that: The cooling rate of the gradient ultra-low temperature freezing in step S1 is 0.5 to 2 °C / min.

3. The method for extracting active plant components using ultra-low temperature ultrasonic solvent-free extraction according to claim 1, characterized in that: The plant material described in step S1 includes a thawing-refreezing cycle between pre-freezing and deep freezing, wherein the thawing temperature is 0℃~4℃ and the thawing time is 0.5~1h.

4. The method for extracting plant active ingredients using ultra-low temperature ultrasonic solvent-free extraction according to claim 1, characterized in that: In step S2, the temperature for liquid nitrogen-assisted physical cell disruption is -196℃ to -80℃, and the pulverization time is 1 to 5 minutes.

5. The method for extracting active plant components using ultra-low temperature ultrasonic solvent-free extraction according to claim 1, characterized in that: The alternation period of the dual-frequency alternating ultrasonic wave in step S3 is a frequency switch every 30 to 120 seconds.

6. The method for extracting plant active ingredients using ultra-low temperature ultrasonic solvent-free extraction according to claim 1, characterized in that: The ultrasonic extraction described in step S3 is carried out under vacuum conditions, with a vacuum degree of 0.5 to 10 kPa.

7. The method for extracting plant active ingredients using ultra-low temperature ultrasonic solvent-free extraction according to claim 1, characterized in that: The ultrasonic extraction described in step S3 is performed in pulse mode with a pulse duty cycle of 30% to 70%.

8. The method for extracting plant active ingredients using ultra-low temperature ultrasonic solvent-free extraction according to claim 1, characterized in that: The extracts described in step S4 are collected by gravity flow combined with low temperature condensation to collect volatile active ingredients, and by centrifugation to collect non-volatile active ingredients.

9. The method for extracting plant active ingredients using ultra-low temperature ultrasonic solvent-free extraction according to claim 1, characterized in that: The purification process in step S5 includes low-temperature membrane separation and freeze drying. The temperature of the low-temperature membrane separation is 5℃ to 15℃, and the temperature of the freeze drying is -50℃ to -30℃.

10. The method for extracting active plant components using ultra-low temperature ultrasonic solvent-free extraction according to any one of claims 1-9, characterized in that: The plant material is selected from at least one plant tissue selected from roots, stems, leaves, flowers, fruits, and seeds.

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