Low-temperature extraction technology-based elymus pumila functional component extraction method

Abstract

The invention belongs to the technical field of natural product extraction, discloses an elymus pumila functional component extraction method based on a low-temperature extraction technology, and aims to solve the problems that heat-sensitive functional components are easy to damage and extraction is not thorough in a traditional extraction method. The method comprises the following steps: carrying out ultralow-temperature pretreatment on raw materials; a first deep eutectic solvent (DES-I) with enzymatic compatibility is adopted for synchronous enzymolysis, and high-polarity components are extracted; then through in-situ liquid-liquid microextraction, medium and weak polar components are extracted by using a hydrophobic second deep eutectic solvent (DES-II); performing subcritical water dynamic extraction on the residues to obtain residual functional components; and grading and recycling. According to the method, the whole-process mild, efficient and high-fidelity obtaining of the all-component functional substances of the elymus pubescens can be achieved, the process is simplified, and the method is green and environmentally friendly.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction technology, and specifically relates to a method for extracting functional components of Leymus chinensis based on low-temperature extraction technology. Background Technology

[0002] With the booming development of modern life sciences and the health industry, the discovery, extraction, and application of functional components with specific physiological activities from natural plant resources has become a core driving force for technological innovation in the pharmaceutical, health food, and high-end cosmetic fields. *Elymus breviaristatus* subsp. *breviaristatus* 'Tongde', a distinctive herbaceous plant from a specific region, possesses a diverse array of structurally significant and bioactive secondary metabolites, such as flavonoids, polysaccharides, alkaloids, and phenolic acids, due to its unique growth environment and genetic background, demonstrating enormous application potential. Therefore, how to efficiently and accurately isolate and purify these functional components from the complex *Elymus breviaristatus* plant matrix is ​​directly related to its subsequent industrial development value and remains a focus of ongoing research in the fields of phytochemistry and natural product engineering for this species.

[0003] In existing techniques for extracting functional components from *Elymus truncatula* matrix, researchers have generally recognized that relying solely on solvent extraction is inefficient and results in insufficient dissolution of target components due to the physical barrier formed by the plant cell walls (mainly composed of cellulose, hemicellulose, and pectin) hindering solvent penetration. Therefore, a relatively mature technical paradigm has gradually emerged for the extraction of this plant: enzymatic pretreatment followed by gradient solvent extraction. Specifically, this paradigm first utilizes complex enzyme preparations such as cellulase and pectinase to specifically degrade the cell wall structure of *Elymus truncatula* under mild biological reaction conditions, thereby breaking down the barrier and fully releasing the functional components encapsulated within the cells, significantly improving the solvent contact efficiency and mass transfer efficiency in subsequent extraction processes. Based on this, the principle of "like dissolves like" is applied, using solvents of different polarities for sequential extraction. For example, highly polar water or ethanol solutions are first used to extract water-soluble components, followed by less polar organic solvents or supercritical fluids (such as supercritical carbon dioxide) to extract lipid-soluble components. This combined strategy of "enzymatic pretreatment + gradient solvent extraction" did, in a specific historical period, improve the total extraction rate of target components in *Elymus truncatula* to a certain extent by combining the high efficiency of biocatalysis with the selectivity of physicochemical separation, representing an important technological advancement in the field to improve the extraction efficiency of this plant.

[0004] However, as research into the activity, structural integrity, and multi-component synergistic effects of the functional components of *Elymus truncatula* has deepened, the technical community has placed unprecedentedly stringent demands on the temperature and fidelity of its extraction process. Against this backdrop, the aforementioned classic technical paradigm of "enzymatic pretreatment + gradient solvent extraction" for this plant, with its inherent discontinuous stages and incompatibility with energy input, has gradually become a core technical bottleneck restricting the final quality of its extract. The reason for this lies in a profound inherent contradiction in the process flow: its front end employs highly mild, low-energy-consumption bio-enzymatic hydrolysis technology, aiming to maximize the protection of the natural conformation and bioactivity of the target molecules in *Elymus truncatula*; however, the subsequent traditional gradient solvent extraction often inevitably introduces harsh physicochemical conditions. For example, if organic solvent extraction is used, the subsequent solvent removal process usually requires heated vacuum distillation, which can easily cause irreversible decomposition or polymerization of heat-sensitive functional components in *Elymus truncatula* (such as certain flavonoid glycosides and phenolic esters with specific structures), leading to a loss of activity. For example, while supercritical carbon dioxide extraction avoids the high-temperature problem, the process requires extremely high pressure (typically tens of megapascals). This high-pressure environment may physically damage certain pressure-sensitive macromolecules or compounds with fine spatial conformations in *Elymus truncatula*. This technological break from "mild biological reaction" to "violent physical separation" severely weakens or even negates the "high-fidelity release" advantage of the enzymatic hydrolysis step for this plant in subsequent separation and purification stages. Consequently, the final *Elymus truncatula* extract may contain functional components whose types, contents, and activity states differ significantly from their original state within the living plant. This undoubtedly represents a huge waste of this rare plant resource and limits its development for high-end applications.

[0005] Therefore, overcoming the technical incompatibility between the enzymatic pretreatment and subsequent extraction and separation steps of *Elymus truncatula* in existing technologies, and constructing a continuous extraction system that can maintain low temperature and low energy consumption throughout the entire process, and achieving efficient, selective, and high-fidelity sequential separation of different polar functional components in its enzymatic hydrolysis products through the ingenious design and combination of extraction media, thereby maximizing the preservation of the natural activity and synergistic effect of functional components in *Elymus truncatula*, has become a key challenge and an urgent technical problem for those skilled in the art in their research on this species. Summary of the Invention

[0006] The technical problem this invention aims to solve is to overcome the inherent technical incompatibility of the existing "enzymatic pretreatment + gradient solvent extraction" paradigm in the extraction of *Elymus truncatula*, namely, the process breakpoint between the gentle enzymatic hydrolysis step and the subsequent drastic physicochemical separation step. This breakpoint leads to the structural damage and activity loss of heat-sensitive and pressure-sensitive functional components (such as specific flavonoid glycosides and phenolic esters) in the *Elymus truncatula* plant matrix. Existing processes, in pursuing high extraction rates, sacrifice the natural authenticity of the extract, failing to achieve a complete, efficient, and gentle overall acquisition of the endogenous active substances profile of *Elymus truncatula*.

[0007] To achieve the aforementioned objectives, this invention provides a method for extracting functional components from *Elymus truncatula* based on low-temperature extraction technology. The core technical concept of this method lies in constructing a low-temperature, continuous, and internally synergistic extraction system tailored to the cellular structure characteristics and physicochemical properties of the functional components of *Elymus truncatula*. By designing and applying a functionalized eutectic solvent system with enzymatic reaction compatibility and polarity controllability, multiple processes, including enzymatic cell wall disruption, gradient dissolution of the target product, and stepwise separation of components with different polarities, are integrated into a mild and continuous physicochemical environment. This fundamentally eliminates the destructive operating conditions introduced by the incompatibility of stages in traditional processes, achieving efficient retention and extraction of the functional components of *Elymus truncatula*.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A method for extracting functional components from *Elymus truncatum* based on low-temperature extraction technology includes the following steps: S1, Raw material ultra-low temperature pretreatment step: The raw material of *Elymus truncatula* is freeze-dried, then cryogenically pulverized and sieved to obtain ultrafine plant powder; S2, First stage simultaneous enzymatic hydrolysis-extraction step: Taking advantage of the fact that the cell walls of *Elymus truncatula* are rich in cellulose, pectin and other components, choline chloride, which acts as a hydrogen bond acceptor, and betaine, which acts as a hydrogen bond donor, are prepared into a first eutectic solvent. The ultrafine plant powder is mixed with the first eutectic solvent to form a first suspension system. A compound enzyme preparation composed of cellulase, pectinase and β-glucosidase is added for simultaneous enzymatic hydrolysis and extraction to obtain the first extract L1 and solid residue R1. S3, Second stage in-situ liquid-liquid microextraction step: The solid residue R1 is remixed with the fresh first eutectic solvent to form a second suspension system. L-menthol, as a phase change trigger, is added to it to react in situ with the residual choline chloride in the solid residue R1 to form a hydrophobic second eutectic solvent phase composed of L-menthol and choline chloride that is immiscible with the hydrophilic system. This allows for the targeted extraction of moderately polar and weakly polar functional components from *Elymus truncatula* into this hydrophobic phase, resulting in a second extract L2 and a secondary solid residue R2. S4, Third stage subcritical water dynamic extraction step: The secondary solid residue R2 is loaded into the high-pressure extraction vessel. After rinsing to remove residual solvent components, preheated deionized water as the extractant is introduced under pressure, and a dynamic heating extraction program is executed to linearly raise the temperature inside the vessel from 110°C to 160°C. The effluent is collected to obtain the third extract L3. S5, graded product recovery step: the first extract L1 is separated using macroporous adsorption resin; the second extract L2 is separated by adding antisolvent and precipitation; the third extract L3 is concentrated under reduced pressure and freeze-dried to recover functional components of different polarities from *Elymus truncatula*.

[0009] Furthermore, the cryogenic pretreatment step of the raw materials in S1 specifically includes: (1) The raw material of *Elymus truncatula* is freeze-dried for 24 to 48 hours at a temperature of -50°C to -45°C and a vacuum of less than 10 Pa to obtain a dried material with a moisture content of less than 2% w / w, so as to avoid the heat-sensitive components in *Elymus truncatula* from being deactivated due to high-temperature drying. (2) The dried material is placed in a liquid nitrogen environment for cryogenic pulverization and then sieved through a sieve with a mesh size of 200 to 400 to obtain the ultrafine plant powder. This particle size can increase the contact area between the *Elymus truncatula* and the subsequent solvent, thereby improving the extraction efficiency.

[0010] Furthermore, the first stage of the simultaneous enzymatic hydrolysis-extraction step in S2 specifically includes: (1) The first eutectic solvent is prepared by heating and stirring choline chloride, which acts as a hydrogen bond acceptor, and betaine, which acts as a hydrogen bond donor, at a molar ratio of 1:1.5 to 1:2.5, with the addition of 15% to 25% w / w of deionized water, at 60°C to 70°C until a homogeneous and transparent liquid is formed. This solvent system has good solubility for the highly polar components of Leymus chinensis. (2) The ultrafine plant powder is mixed with the first eutectic solvent at a solid-liquid ratio of 1:20 to 1:30 (g / mL) to form the first suspension system. This ratio is optimized to suit the characteristics of *Elymus truncatula*. (3) The compound enzyme preparation added to the first suspension system is composed of cellulase, pectinase and β-glucosidase in an enzyme activity unit U ratio of 5:3:2. The total amount of the compound enzyme preparation added is 1.5% w / w of the dry weight of the plant powder. This amount of addition can achieve the best enzymatic hydrolysis effect for the cell wall composition of Leymus chinensis. (4) The isothermal oscillation reaction is carried out at a temperature of 35°C to 45°C and a rotation speed of 150 to 250 rpm for 12 to 24 hours. These reaction conditions are beneficial to protecting the active ingredients in *Elymus truncatula*. (5) After the reaction is completed, the first extract L1 and the solid residue R1 are obtained by centrifugation.

[0011] Furthermore, in the compound enzyme preparation, the cellulase activity is not less than 10,000 U / g, the pectinase activity is not less than 30,000 U / g, and the β-glucosidase activity is not less than 600 U / g. This enzyme activity configuration can efficiently decompose the cell wall components of Leymus chinensis.

[0012] Furthermore, the second stage in-situ liquid-liquid microextraction step in S3 specifically includes: (1) The solid residue R1 is remixed with the fresh first eutectic solvent at a ratio of 1:10 (g / mL) to form the second suspension system, which is suitable for the extraction of the remaining components in the residue of *Elymus truncatula*. (2) Add the L-menthol to the second suspension system as a phase change trigger, wherein the molar ratio of the L-menthol to the choline chloride contained in the solid residue R1 is 2:1 to 3:1; The amount of choline chloride in the solid residue R1 can be determined by the following method: To ensure the accuracy of the results, all wet solid residue R1 should be thoroughly mixed in a sealed container for at least 5 minutes by mechanical scraping and stirring until its texture and moisture are visually uniform. Then, approximately 5.0 grams of sample should be taken from three different locations (top, middle, and bottom) of the mixed residue, and the average value should be used for calculation after parallel determination. The sample should be placed in a beaker, 100 ml of deionized water added, and the mixture magnetically stirred for 15 minutes before centrifugation. This washing operation should be repeated three times. The residue after the third wash should be washed a fourth time. The fourth wash solution should be tested with silver nitrate solution. Only after confirming the absence of chloride ions can it be considered that choline chloride has been completely eluted. All supernatants should be combined and the volume adjusted to 500 ml. The concentration of chloride ions C_Cl in the solution after volume adjustment was determined by silver nitrate titration or ion chromatography, thereby calculating the molar amount of choline chloride n_sample in the sample, and converting it to the total amount in all solid residues R1 according to the formula n_total = (n_sample / m_sample) × M_total (where M_total is the total wet weight of R1); (3) The reaction is carried out at a temperature of 40°C to 50°C and a speed of 300 to 400 rpm for 2 to 4 hours under constant temperature stirring, so that the L-menthol and the choline chloride form in situ a hydrophobic second eutectic solvent phase (DES-II) rich in menthol that is immiscible with the aqueous betaine phase. This process constitutes in-situ liquid-liquid microextraction, in which the hydrophobic second eutectic solvent phase selectively extracts the moderately and weakly polar functional components of Leymus chinensis, such as flavonoids and phenolic acids, remaining in the solid residue R1. Those skilled in the art should understand that the occurrence of this phase separation is crucial to the total water content in the system. To ensure reliable and clear phase separation, the total water content of the second suspension system should be controlled within the range of 15% to 30% w / w. This water content can be achieved by adjusting the amount of water added to the fresh first eutectic solvent or by adding a small amount of deionized water. At this water content, the addition of L-menthol will effectively compete for binding with choline chloride due to its hydrophobicity, thereby driving the formed DES-II system to 'salt out' from the highly hydrophilic betaine-water system, forming a stable liquid-liquid two-phase system. To ensure the repeatability of this process, those skilled in the art can control and judge it in the following way: after adding L-menthol, continuously stir and monitor the turbidity change of the system using an online turbidimeter. When the turbidity reaches its peak and then begins to decrease and tends to stabilize, it indicates that the phase separation process has been basically completed. This invention has been experimentally verified that when the total water content of the system is controlled at 20% to 25% w / w, the phase separation interface is the clearest and the volume of the separated hydrophobic phase is the largest. Under these conditions, after standing for 30 minutes, a clearly stratified two-phase liquid can be obtained by centrifugation. (4) After the reaction is completed, the second extract L2 and the secondary solid residue R2 are obtained by centrifugation.

[0013] Furthermore, the third stage subcritical water dynamic extraction step in S4 specifically includes: (1) After filling the secondary solid residue R2, the material in the reactor is first rinsed at room temperature and pressure with a betaine aqueous solution of concentration of 0.1 mol / L at a flow rate of 5-15 mL / min until no chloride ions are detected in the effluent. The purpose of using betaine aqueous solution instead of pure water is to utilize its similar osmotic pressure environment with the residual first eutectic solvent system, which can gently replace the residual solvent, while avoiding secondary problems that may be caused by the damage to the cell structure of Leymus chinensis due to drastic changes in osmotic pressure. (2) The high-pressure extraction vessel is sealed and pressurized to 5 to 10 MPa, and preheated deionized water is introduced as a subcritical water extractant. (3) The dynamic heating extraction program is set as follows: the temperature inside the vessel is linearly increased from 110℃ to 160℃ at a heating rate of 2℃ / min, while the flow rate of deionized water as the extractant is maintained at 5 to 15 mL / min; during this dynamic heating process, the polarity of subcritical water changes continuously, thereby sequentially extracting residual functional components with a wider polarity range and tighter binding in the *Elymus truncatula* matrix, such as alkaloids and some deeply embedded phenolic compounds; (4) Collect the effluent from the entire process to obtain the third extract L3.

[0014] Furthermore, the fractionation product recovery step in S5 specifically includes: (1) Treatment of the first extract L1: passing it through a pretreated solution with an average pore size of 8 to 12 nm and a specific surface area of ​​500 to 600 m². 2A styrene-divinylbenzene macroporous adsorption resin column of / g was loaded with a flow rate of 2 column volumes / hour. After loading, the sample was eluted sequentially with 5 column volumes of deionized water and 8 column volumes of 70% ethanol solution. The water elution fraction and the ethanol elution fraction were collected separately to separate and purify the highly polar components in *Elymus truncatula*. (2) Treatment of the second extract L2: At 4°C, add 5 to 10 times the volume of pre-cooled deionized water as an antisolvent, stir vigorously for 30 minutes, and let stand for 2 hours to extract the medium and weak polar functional components of the lysimachia christinae. Then, obtain the powder product by filtration, washing and freeze drying. (3) Treatment of the third extract L3: Under nitrogen protection, most of the water is removed by vacuum distillation, and then freeze-drying is performed to obtain a powder product in order to retain the activity of the components in *Elymus truncatula* that are susceptible to high temperature.

[0015] Furthermore, before loading the macroporous adsorption resin that treats the first extract L1, it is sequentially activated and pretreated with an equal volume of hydrochloric acid solution (4% w / v), sodium hydroxide solution (4% w / v), and ethanol (95% ethanol), and finally rinsed with deionized water until neutral. This rigorous pretreatment process aims to completely remove impurities from the resin pores and achieve optimal adsorption activity and selectivity to better separate the highly polar components of Leymus chinensis.

[0016] Furthermore, in the recovery treatment of the third extract L3, the vacuum degree of the vacuum distillation is controlled at 0.08 to 0.09 MPa, and the water bath temperature does not exceed 50°C, so as to gently remove water and prevent any possible structural changes to the heat-sensitive alkaloids in *Elymus truncatula* during the concentration process.

[0017] The beneficial effects of this invention are: (1) This invention addresses the structural characteristics of *Elymus truncatula* by constructing a functionalized, enzyme-compatible eutectic solvent system (DES-I), achieving for the first time the simultaneous enzymatic hydrolysis of the cell wall and the extraction of highly polar functional components from *Elymus truncatula*. This eutectic solvent serves not only as an extractant but also as a reaction medium, providing a stable working environment for the enzyme. This allows for the completion of operations that traditionally require two or more steps in a single, mild, low-temperature step, significantly simplifying the extraction process for *Elymus truncatula* and reducing energy consumption.

[0018] (2) This invention innovatively proposes and implements an in-situ liquid-liquid microextraction technique for *Elymus truncatula*. By precisely adding a phase change trigger (L-menthol) to the system after the first-stage reaction, a hydrophobic eutectic solvent phase (DES-II) that is immiscible with the original aqueous system is generated in situ, achieving gradient extraction of moderately polar and weakly polar functional components in *Elymus truncatula*. This method completely avoids the use of volatile organic solvents, fundamentally solving the problems of solvent residue, environmental pollution, and thermal damage during subsequent desolvation, and achieving green and efficient separation of functional components from *Elymus truncatula*.

[0019] (3) This invention organically integrates the first two steps of low-temperature extraction with the subsequent dynamic subcritical water extraction, forming an extraction system specifically suitable for *Elymus truncatula*. Through effective cleaning of the residue and precise temperature control, subcritical water extraction becomes a final, in-depth method for extracting the strongly bound components in *Elymus truncatula*. The entire methodology covers a wide range of components in *Elymus truncatula*, from highly polar to weakly polar, forming a complete and seamless gradient extraction spectrum, thereby enabling the maximum and high-fidelity extraction of all functional substances in *Elymus truncatula*.

[0020] (4) The entire process of this invention is carried out under low temperature or mild conditions (the maximum temperature of subcritical water extraction is also strictly controlled), and does not involve severe high pressure (such as supercritical extraction) or highly toxic reagents. Compared with traditional processes, this invention increases the retention rate of the characteristic heat-sensitive component 'flavonoid glycoside A' from 35.8% to over 92.5%, and its antioxidant activity IC 50 The concentration decreased from 28.3 μg / mL to nearly 12.5 μg / mL, which is close to the purity of the product, irrefutably demonstrating the decisive advantage of the mild processing throughout the entire process of this invention. This ensures that all the functional components of *Elymus truncatula* obtained, especially its unique, structurally unstable, and easily degradable flavonoid glycosides, phenolic esters, and specific alkaloids, can maintain their natural molecular conformation and biological activity. The resulting extract spectrum is closer to its original state in *Elymus truncatula* in vivo, greatly improving the quality and application value of the *Elymus truncatula* extract.

[0021] In summary, this invention provides a systematic, mild, and highly integrated extraction method specifically for *Elymus truncatula*. It creatively utilizes the unique physicochemical properties of functionalized eutectic solvents, resolving the fundamental contradictions inherent in existing techniques for extracting *Elymus truncatula*. This provides a novel technical solution with significant industrial application prospects for obtaining the full spectrum of functional components from *Elymus truncatula* with high fidelity and efficiency. Detailed Implementation

[0022] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0023] Example 1 (1) Raw material pretreatment: 1000 grams of fresh whole *Elymus truncatum* (a type of grass) harvested from a meadow at an altitude of 3500 meters in Tongde, Qinghai Province, were quickly placed in an FD-50 vacuum freeze dryer. The temperature was set to -50℃ and the vacuum degree to 5 Pa, and the drying was carried out continuously for 36 hours. After removal, the dried material was weighed, yielding 245.2 grams (moisture content approximately 1.8%).

[0024] All the dried material was transferred to a 5L planetary ball mill with a liquid nitrogen jacket, and the material was submerged in liquid nitrogen and ground at -196°C for 90 minutes. The pulverized material was then sieved through a 300-mesh standard vibrating screen, and the undersize material was collected to obtain 241.5 grams of ultrafine plant powder.

[0025] (2) First stage simultaneous enzymatic hydrolysis-extraction: Accurately weigh 139.5 g (1 mol) of choline chloride and 234.3 g (2 mol) of betaine, add 94.8 g (20% of the total mass) of deionized water, and magnetically stir in a 65°C water bath until a homogeneous and transparent first eutectic solvent (DES-I) is formed.

[0026] The 241.5 g of ultrafine plant powder obtained in step (1) was added to the above DES-I with a volume of 6037.5 mL (solid-liquid ratio 1:25 g / mL) to form the first suspension system.

[0027] A compound enzyme preparation was added to the system, which included cellulase (enzyme activity 12000 U / g), pectinase (enzyme activity 35000 U / g) and β-glucosidase (enzyme activity 650 U / g), with the three enzymes compounded in an enzyme activity ratio of 5:3:2. The total amount added was 1.5% of the dry weight of the plant powder, i.e., 3.62 grams.

[0028] The system was placed in a constant temperature shaker and reacted at 40°C and 200 rpm for 18 hours.

[0029] After the reaction was completed, the mixture was centrifuged at 4°C and 8000g for 30 minutes, and the supernatant was collected to obtain the first extract L1, with a volume of approximately 5950 mL. The precipitate was the solid residue R1, with a wet weight of approximately 450 g.

[0030] (3) Second stage in-situ liquid-liquid microextraction: The solid residue R1 was remixed with 2415 mL of freshly prepared DES-I to form a second suspension system. The amount of choline chloride adsorbed and carried in R1 was determined to be approximately 0.2 mol. Therefore, 78.1 g (0.5 mol) of L-menthol was added to the system, resulting in a molar ratio of L-menthol to choline chloride adsorbed and carried in R1 of 2.5:1.

[0031] The system was stirred at 45°C and 350 rpm for 3 hours.

[0032] After the reaction was complete, the system was milky. Centrifuged at 4°C and 8000g for 30 minutes, the upper layer was an oily second extract L2, with a volume of approximately 150 mL. The bottom layer was a secondary solid residue R2, with a wet weight of approximately 380 g.

[0033] (4) Third-stage subcritical water dynamic extraction: The secondary solid residue R2 was loaded into a 2L high-pressure extraction vessel.

[0034] Wash with 0.1 mol / L betaine aqueous solution at a flow rate of 10 mL / min at room temperature until no AgCl precipitate can be detected in the effluent with 0.1 mol / L silver nitrate solution.

[0035] Seal the extraction vessel and use a high-pressure pump to raise the system pressure to 8 MPa. Start the extraction program: introduce deionized water at a flow rate of 10 mL / min, and simultaneously linearly raise the vessel temperature from 110°C to 160°C at a rate of 2°C / min. Collect the eluent throughout the process, collecting approximately 3000 mL, which is the third extract, L3.

[0036] (5) Recovery of graded products: For L1: The average pore size after activation treatment is 10 nm and the specific surface area is 550 m². 2 The sample was loaded onto a styrene-divinylbenzene macroporous adsorption resin column (50 mm × 500 mm, column volume approximately 1 L) at a flow rate of 2 L / h.

[0037] First, the product was eluted with 5L of deionized water, the washed fraction was collected, and then freeze-dried to obtain product P1 (mainly polysaccharides and amino acids), weighing 28.5g.

[0038] The product was then eluted with 8L of 70% ethanol. The ethanol eluent was collected, concentrated by vacuum evaporation, and then freeze-dried to obtain product P2 (mainly polar phenolic glycosides), weighing 4.2g.

[0039] For L2: At 4°C, add 1200 mL (8 times the volume) of deionized water at 4°C to 150 mL of L2, stir vigorously at 4°C for 30 minutes, and then let stand for 2 hours. Filter, wash three times with ice water, and freeze-dry to obtain product P3 (mainly flavonoids and phenolic acids), weighing 11.8 g.

[0040] For L3: Under nitrogen protection, the product was concentrated to about 300 mL by rotary evaporation in a water bath at 50 °C and a vacuum of 0.085 MPa. After freeze-drying, product P4 (mainly alkaloids and strongly bound phenols) was obtained, weighing 2.1 g.

[0041] Example 2 (1) Raw material pretreatment: Same as in Example 1, take 1000g of fresh Tongde short awned wheatgrass, freeze dry (-48℃, 8Pa, 30h) and cryogenically pulverize (300 mesh) to obtain 242.1g of ultrafine plant powder.

[0042] (2) First stage simultaneous enzymatic hydrolysis-extraction: Prepare DES-I (molar ratio 1:1.5) of choline chloride (1 mol) and betaine (1.5 mol), add deionized water (22% of total mass), and stir at 60°C until transparent.

[0043] Mix the powder with DES-I at a solid-liquid ratio of 1:20 (g / mL), add the compound enzyme preparation (same as in Example 1, total addition amount 1.5%), and react at 35℃ and 180 rpm for 12 hours.

[0044] Centrifugation yielded the first extract L1 (5890 mL) and solid residue R1 (wet weight 445 g).

[0045] (3) Second stage in-situ liquid-liquid microextraction: R1 was mixed with fresh DES-I at a ratio of 1:10, and the choline chloride content in R1 was measured to be 0.18 mol. L-menthol (0.36 mol, molar ratio 2:1) was added, and the mixture was stirred at 40°C and 320 rpm for 2.5 hours. Centrifugation yielded a second extract L2 (145 mL) and a secondary residue R2 (wet weight 375 g).

[0046] (4) Third-stage subcritical water dynamic extraction: After R2 is loaded into the reactor, it is rinsed with 0.1 mol / L betaine aqueous solution until no chloride ions are present. The pressure is increased to 6 MPa, and deionized water is introduced at a flow rate of 5 mL / min. The temperature is increased from 110℃ to 160℃ at a rate of 2℃ / min. The effluent L3 (approximately 2900 mL) is collected.

[0047] (5) Recovery of graded products: Same as in Example 1, products P1 (27.8g), P2 (4.0g), P3 (11.2g), and P4 (2.0g) were obtained.

[0048] Example 3 (1) Raw material pretreatment: Same as in Example 1, 240.8 g of ultrafine plant powder was obtained.

[0049] (2) First stage simultaneous enzymatic hydrolysis-extraction: Prepare DES-I (molar ratio 1:2.5) of choline chloride (1 mol) and betaine (2.5 mol), add deionized water (25% of total mass), and stir at 70°C until transparent. Mix at a solid-liquid ratio of 1:30 (g / mL), add the compound enzyme preparation, and react at 45°C and 220 rpm for 24 hours with shaking.

[0050] Centrifugation yielded L1 (6120 mL) and R1 (wet weight 455 g).

[0051] (3) Second stage in-situ liquid-liquid microextraction: R1 was mixed with fresh DES-I, and 0.22 mol of choline chloride was measured. L-menthol (0.66 mol, molar ratio 3:1) was added, and the mixture was stirred at 50°C and 380 rpm for 4 hours.

[0052] Centrifugation yielded L2 (155 mL) and R2 (wet weight 385 g).

[0053] (4) Third-stage subcritical water dynamic extraction: After rinsing, pressurize to 10 MPa, pass water at a flow rate of 15 mL / min, increase the temperature at 2 °C / min, and collect L3 (approximately 3100 mL).

[0054] (5) Recovery of graded products: Same as in Example 1, P1 (29.1g), P2 (4.3g), P3 (12.1g), and P4 (2.2g) were obtained.

[0055] Comparative Example 1 This comparative example uses the traditional method of "enzymatic pretreatment + gradient solvent extraction".

[0056] (1) Raw material pretreatment: Same as in Example 1, take 1000g of fresh Tongde short awned wheatgrass, freeze dry and cryogenically pulverize to obtain 241.5g of 300-mesh ultrafine plant powder.

[0057] (2) Enzymatic pretreatment: 241.5 g of plant powder was suspended in 4830 mL of citrate-sodium citrate buffer (0.1 M, pH 4.8) (solid-liquid ratio 1:20 g / mL). The same type and amount of compound enzyme preparation as in Example 1 (total 3.62 g) was added. The reaction was carried out at 50 °C and 200 rpm for 12 hours with shaking (this is the optimal condition for conventional enzymatic hydrolysis).

[0058] (3) Solvent extraction: After enzymatic hydrolysis, the mixture was filtered to obtain a moist solid residue. The residue was transferred to a 5L round-bottom flask and 3000 mL of 80% (v / v) ethanol aqueous solution was added. The mixture was refluxed at 80°C for 2 hours each time. The three ethanol extracts were combined.

[0059] (4) Product recovery: The filtrate obtained from enzymatic hydrolysis in step (2) is mixed with the combined ethanol extract in step (3). The mixture is rotary evaporated at 60°C water bath and 0.07MPa vacuum until no ethanol odor is detected, and a concentrated solution is obtained. The concentrated solution is freeze-dried to obtain the final total extract powder, weighing 39.6 grams.

[0060] Comparative Example 2 This comparative example uses supercritical CO2 extraction.

[0061] (1) Raw material pretreatment: Same as in Example 1, to obtain 241.5g of ultrafine powder.

[0062] (2) Supercritical extraction: The powder was loaded into a 5L extraction vessel and extracted for 3 hours at 35 MPa and 45°C using CO2 as the solvent. The entrainer was 95% ethanol (10% of the amount used). The separation pressure was 8 MPa and the temperature was 35°C.

[0063] (3) Product recovery: The extract was collected, the ethanol was removed by vacuum evaporation and then freeze-dried to obtain 28.3g of total extract.

[0064] Comparative Example 3 This comparative example uses a simple water-alcohol precipitation method.

[0065] (1) Raw material pretreatment: Same as in Example 1, 241.5g of powder was obtained.

[0066] (2) Water extraction: Add 20 times the amount of deionized water to the powder and reflux at 80°C for 3 times (2 hours each time), and combine the filtrates.

[0067] (3) Alcohol precipitation: Add ethanol to the filtrate to a concentration of 70%, let stand at 4°C for 12 hours, filter and dry to obtain 31.5g of extract.

[0068] Comparative Example 4 This comparative example uses the method of "room temperature enzymatic hydrolysis + high temperature solvent extraction".

[0069] (1) Raw material pretreatment: Same as in Example 1, take 1000g of fresh Tongde short awned wheatgrass, freeze dry (-50℃, 5Pa, 36h) and cryogenically pulverize (300 mesh) to obtain 241.5g of ultrafine plant powder.

[0070] (2) First-stage simultaneous enzymatic hydrolysis-extraction (using the same DES-I and enzymatic hydrolysis conditions as in this invention): Accurately weigh 139.5 g (1 mol) of choline chloride and 234.3 g (2 mol) of betaine, add 94.8 g of deionized water (20% of total mass), and stir at 65°C to form DES-I.

[0071] Mix the powder with DES-I at a solid-liquid ratio of 1:25 (g / mL), add the compound enzyme preparation (same as in Example 1, total addition amount 1.5%), and shake at 40℃ and 200 rpm for 18 hours.

[0072] Centrifugation yielded the first extract L1 (5940 mL) and solid residue R1 (wet weight 448 g).

[0073] (3) Second stage high-temperature organic solvent extraction (disrupting the "low temperature" condition): The solid residue R1 was mixed with 3000 mL of 80% ethanol aqueous solution and transferred to a 5 L round-bottom flask. The mixture was then refluxed at 70 °C for 2 hours (as an alternative to the low-temperature in-situ liquid-liquid microextraction of this invention).

[0074] After cooling and filtration, a second extract L2 (approximately 2800 mL) was obtained. The residue was washed to obtain a secondary solid residue R2 (wet weight 370 g).

[0075] (4) Third stage: high-temperature water extraction (continuing high-temperature conditions): Mix the secondary solid residue R2 with 20 times the amount of deionized water, reflux at 80°C for 2 hours, and filter to obtain the third extract L3 (approximately 3200 mL).

[0076] (5) Recovery of graded products: Combine L1, L2, and L3, remove ethanol and some water by vacuum distillation at 60℃, and freeze-dry to obtain 42.3 g of total extract.

[0077] Effect verification The components and activities of the products obtained in the examples (P1, P2, P3, P4) and the total extract obtained in the comparative examples were analyzed and evaluated. The results are summarized in Table 1.

[0078] The total flavonoid content was determined using rutin as a standard by the NaNO2-Al(NO3)3-NaOH colorimetric method; the total phenolic acid content was determined using gallic acid as a standard by the Folin-Ciocalteu method.

[0079] The total polysaccharide content was determined using glucose as a standard by the phenol-sulfuric acid method.

[0080] The retention rate of flavonoid A (a characteristic heat-intolerant flavonoid glycoside isolated from Leymus chinensis) was determined by HPLC as the ratio of its content in the final product to its content in the methanol cold extract of the untreated liquid nitrogen-ground powder. IC 50 The free radical scavenging rate was determined by the DPPH free radical scavenging method, which involves reacting extract solutions of varying concentrations with DPPH working solution, measuring the absorbance at 517 nm using an enzyme-linked immunosorbent assay (ELISA) reader, calculating the free radical scavenging rate, and fitting a dose-response curve to obtain the extract concentration corresponding to a scavenging rate of 50%. The activity retention rate of characteristic flavonoid glycoside A is measured by its IC50 value in the final product. 50 The IC50 value of the pure characteristic flavonoid glycoside A 50 The ratio of values ​​is calculated, that is, (pure IC) 50 / Sample IC 50 ) × 100%.

[0081] Table 1 Evaluation indicators Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Total polysaccharide yield 11.80 11.52 12.13 8.52 3.25 9.81 9.20 Total flavonoid yield 4.88 4.63 5.06 2.95 3.18 2.23 3.15 Total phenolic acid yield 2.40 2.36 2.59 1.66 1.83 1.46 1.76 Characteristic flavonoid glycoside A 92.5 91.2 93.1 35.8 78.5 42.3 58.3 Characteristic flavonoid glycoside A 12.5 13.1 12.1 28.3 18.6 32.5 22.4 Characteristic flavonoid glycoside A 93.2 91.5 94.8 41.7 65.3 36.2 53.9 Volatile organic solvents (VOCs) 3.32 3.30 3.41 12.43 8.69 15.3 10.5 DES recovery rate (%) > 96 > 96 > 96 / / / / Note: The DPPH free radical scavenging IC50 of the pure characteristic flavonoid glycoside A. 50 It was 11.8 μg / mL.

[0082] As shown in the table, compared with the traditional "enzymatic pretreatment + gradient solvent extraction" process used in Comparative Example 1, the supercritical CO2 extraction method of Comparative Example 2, the simple water extraction-alcohol precipitation method of Comparative Example 3, and the "room temperature enzymatic hydrolysis + high temperature extraction" process of Comparative Example 4, the technical solutions of the present invention represented by Examples 1-3 show significant advantages in all key indicators of extracting *Elymus truncatula*. Specifically, in terms of the yield of functional components, the yield of total polysaccharides in *Elymus truncatula* can reach 11.80%-12.13%, which is 38%-42% higher than the traditional process (8.52%), and far exceeds supercritical extraction (3.25%), simple water extraction (9.81%), and Comparative Example 4. High-temperature extraction yielded 9.20%; the total flavonoid yield was 4.63%-5.06%, which is 56%-71% higher than the traditional process (2.95%), and superior to supercritical extraction (3.18%), water extraction alone (2.23%) and Comparative Example 4 (3.15%); the total phenolic acid yield reached 2.36%-2.59%, which is 42%-56% higher than the traditional process (1.66%), and also higher than supercritical extraction (1.83%), water extraction alone (1.46%) and Comparative Example 4 (1.76%), which fully demonstrates that the method of the present invention has higher extraction efficiency for various functional components in *Elymus truncatula*.

[0083] Crucially, the method of this invention achieves a retention rate of 91.2%-93.1% for the heat-labile characteristic flavonoid A in *Elymus truncatula*, and its DPPH scavenging IC50 value is also high. 50 The activity concentration (12.1-13.1 μg / mL) is close to that of the pure product (11.8 μg / mL), with an activity retention rate of 91.5%-94.8%; while the traditional high-temperature reflux extraction method leads to severe degradation (retention rate 35.8%), IC50... 50 The concentration increased to 28.3 μg / mL, with activity only 41.7% of the pure product. Supercritical fluid extraction, due to the high-pressure environment, also reduced the retention rate to 78.5%. IC50 50 The concentration was increased to 18.6 μg / mL, with the activity only 65.3% of the pure product. High-temperature treatment with simple water extraction further reduced the retention rate to only 42.3%. IC50 50 The concentration was increased to 32.5 μg / mL, with the activity only 36.2% of the pure product. Although Comparative Example 4 initially used the same low-temperature enzymatic hydrolysis as this invention, subsequent high-temperature extraction led to partial degradation of flavonoid glycoside A (retention rate 58.3%), and its IC50 concentration was significantly lower. 50 The concentration (22.4 μg / mL) was significantly higher than in the example, and the activity retention rate decreased to 53.9%. This irrefutably confirms the decisive advantage of the mild processing throughout the entire process of the present invention in maintaining the natural structure of the active ingredients in *Elymus truncatula*.

[0084] Furthermore, this invention completely avoids the use of volatile organic solvents in the core steps of extracting *Elymus truncatula*, using only recyclable ethanol in the post-treatment recovery stage. The total amount of ethanol (3.30-3.41 L / kg dry powder) is far lower than that of traditional processes (12.43 L / kg), supercritical extraction (8.69 L / kg), simple water extraction (15.3 L / kg), and Comparative Example 4 (10.5 L / kg). Moreover, the DES recovery rate is higher than 96%, fully demonstrating its green, efficient, and environmentally friendly characteristics in the extraction of *Elymus truncatula*.

Claims

1. A method for extracting functional components from *Elymus truncatum* based on low-temperature extraction technology, characterized in that, Includes the following steps: S1, Raw material ultra-low temperature pretreatment step: The raw material of *Elymus truncatula* is freeze-dried, then cryogenically pulverized and sieved to obtain ultrafine plant powder; S2, First stage simultaneous enzymatic hydrolysis-extraction step: Taking advantage of the fact that the cell walls of *Elymus truncatula* are rich in cellulose, pectin and other components, choline chloride, which acts as a hydrogen bond acceptor, and betaine, which acts as a hydrogen bond donor, are prepared into a first eutectic solvent. The ultrafine plant powder is mixed with the first eutectic solvent to form a first suspension system. A compound enzyme preparation composed of cellulase, pectinase and β-glucosidase is added for simultaneous enzymatic hydrolysis and extraction to obtain the first extract L1 and solid residue R1. S3, Second stage in-situ liquid-liquid microextraction step: The solid residue R1 is remixed with the fresh first eutectic solvent to form a second suspension system. L-menthol, as a phase change trigger, is added to it to react in situ with the residual choline chloride in the solid residue R1 to form a hydrophobic second eutectic solvent phase composed of L-menthol and choline chloride that is immiscible with the hydrophilic system. This allows for the targeted extraction of moderately polar and weakly polar functional components from *Elymus truncatula* into this hydrophobic phase, resulting in a second extract L2 and a secondary solid residue R2. S4, Third stage subcritical water dynamic extraction step: The secondary solid residue R2 is loaded into the high-pressure extraction vessel. After rinsing to remove residual solvent components, preheated deionized water as the extractant is introduced under pressure, and a dynamic heating extraction program is executed to linearly raise the temperature inside the vessel from 110°C to 160°C. The effluent is collected to obtain the third extract L3. S5, Step S5 for graded product recovery: The first extract L1 is separated using macroporous adsorption resin; the second extract L2 is separated by adding an antisolvent; the third extract L3 is concentrated under reduced pressure and freeze-dried to recover functional components of different polarities from *Elymus truncatula*.

2. The method for extracting functional components of *Elymus truncatula* based on low-temperature extraction technology according to claim 1, characterized in that, The cryogenic pretreatment step for raw materials in S1 specifically includes: (1) The raw material of *Elymus truncatula* is freeze-dried for 24 to 48 hours at a temperature of -50°C to -45°C and a vacuum of less than 10 Pa to obtain a dried material with a moisture content of less than 2% w / w. (2) The dried material is placed in a liquid nitrogen environment for cryogenic pulverization and then sieved through a sieve with a pore size of 200 to 400 mesh to obtain the ultrafine plant powder.

3. The method for extracting functional components of *Elymus truncatula* based on low-temperature extraction technology according to claim 1, characterized in that, The first stage of the simultaneous enzymatic hydrolysis-extraction step in S2 specifically includes: (1) The first eutectic solvent is prepared by heating and stirring choline chloride, which acts as a hydrogen bond acceptor, and betaine, which acts as a hydrogen bond donor, at a molar ratio of 1:1.5 to 1:2.5, with the addition of 15% to 25% w / w of deionized water, at 60°C to 70°C until a homogeneous transparent liquid is formed. (2) The ultrafine plant powder is mixed with the first eutectic solvent at a solid-liquid ratio of 1:20 to 1:30 to form the first suspension system; (3) The compound enzyme preparation added to the first suspension system is composed of cellulase, pectinase and β-glucosidase in an enzyme activity unit U ratio of 5:3:2, and the total amount of the compound enzyme preparation added is 1.5% w / w of the dry weight of the plant powder. (4) The isothermal oscillation reaction is carried out at a temperature of 35°C to 45°C and a rotation speed of 150 to 250 rpm for 12 to 24 hours; (5) After the reaction is completed, the first extract L1 and the solid residue R1 are obtained by centrifugation.

4. The method for extracting functional components of *Elymus truncatula* based on low-temperature extraction technology according to claim 2, characterized in that, In S2: In the compound enzyme preparation, the cellulase activity is not less than 10,000 U / g, the pectinase activity is not less than 30,000 U / g, and the β-glucosidase activity is not less than 600 U / g.

5. The method for extracting functional components of *Elymus truncatula* based on low-temperature extraction technology according to claim 1, characterized in that, The second stage in-situ liquid-liquid microextraction step in S3 specifically includes: (1) The solid residue R1 is remixed with the fresh first eutectic solvent at a ratio of 1:10 to form the second suspension system; (2) Add the L-menthol to the second suspension system as a phase change trigger, wherein the molar ratio of the L-menthol to the choline chloride contained in the solid residue R1 is 2:1 to 3:1; (3) The reaction is carried out at a temperature of 40°C to 50°C and a speed of 300 to 400 rpm for 2 to 4 hours with constant temperature stirring, so that the L-menthol and the choline chloride form the hydrophobic second eutectic solvent phase in situ; (4) After the reaction is completed, the second extract L2 and the secondary solid residue R2 are obtained by centrifugation.

6. The method for extracting functional components of *Elymus truncatula* based on low-temperature extraction technology according to claim 1, characterized in that, The third stage of subcritical water dynamic extraction in S4 specifically includes: (1) After filling the secondary solid residue R2, first use a betaine aqueous solution with a concentration of 0.1 mol / L to rinse the material in the reactor at room temperature and pressure at a flow rate of 5-15 mL / min until no chloride ions are detected in the outflow. (2) The high-pressure extraction vessel is sealed and pressurized to 5 to 10 MPa, and preheated deionized water is introduced as a subcritical water extractant; (3) The dynamic heating extraction program is set as follows: the temperature inside the vessel is linearly increased from 110℃ to 160℃ at a heating rate of 2℃ / min, while the flow rate of deionized water as the extractant is maintained at 5 to 15 mL / min. (4) Collect the effluent from the entire process to obtain the third extract L3.

7. The method for extracting functional components of *Elymus truncatula* based on low-temperature extraction technology according to claim 1, characterized in that, The specific steps for recovering graded products in S5 include: (1) Treatment of the first extract L1: passing it through a pretreated solution with an average pore size of 8 to 12 nm and a specific surface area of ​​500 to 600 m². 2 A styrene-divinylbenzene macroporous adsorption resin column of / g was loaded with a flow rate of 2 column volumes / hour. After loading, the column was eluted sequentially with 5 column volumes of deionized water and 8 column volumes of 70% ethanol solution. The water elution fraction and the ethanol elution fraction were collected separately. (2) Treatment of the second extract L2: At 4°C, add 5 to 10 times the volume of pre-cooled deionized water as an antisolvent, stir vigorously for 30 minutes, and let stand for 2 hours to allow the medium polarity and weak polarity functional components to be extracted. Then, obtain the powder product by filtration, washing and freeze drying. (3) Treatment of the third extract L3: Under nitrogen protection, most of the water is removed by vacuum distillation, and then freeze-drying is performed to obtain the powder product.

8. The method for extracting functional components of *Elymus truncatula* based on low-temperature extraction technology according to claim 7, characterized in that, In S5: Before loading the macroporous adsorption resin that treats the first extract L1, it is activated and pretreated sequentially with an equal volume of hydrochloric acid solution with a concentration of 4% w / v, a sodium hydroxide solution with a concentration of 4% w / v, and ethanol with a volume fraction of 95%, and finally rinsed with deionized water until neutral. In the recovery treatment of the third extract L3, the vacuum degree of the vacuum distillation is controlled at 0.08 to 0.09 MPa, and the water bath temperature does not exceed 50°C.