A non-destructive extraction method of exosome-like nanovesicles from dried traditional Chinese medicine decoction pieces and application

Abstract

The application discloses a kind of dry traditional Chinese medicine decoction piece exosome-like nanovesicle nondestructive extraction method and application, the extraction method includes: dry traditional Chinese medicine decoction piece is mixed with the extraction buffer containing cell wall degradation enzyme and osmotic pressure stabilizer, the enzyme is penetrated into decoction piece interior at the first incubation lower than the optimum temperature of enzyme;Second incubation is carried out to the optimum temperature of enzyme to degrade cell wall;Mechanical energy lower than the membrane rupture threshold of exosome-like nanovesicle is applied to release vesicle;Exosome-like nanovesicle is separated from release product, using the reverse process of cold penetration-temperature degradation-critical low energy release, the efficient degradation of dry traditional Chinese medicine decoction piece cell wall and the nondestructive release of vesicle structure are realized, the exosome-like nanovesicle obtained is uniform in particle size, complete in structure, has good antioxidant activity and digestion stability, and can be used to prepare drug or functional food for improving intestinal mucosal barrier damage.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method and application for non-destructive extraction of exosome-like nanovesicles from dried Chinese herbal medicine slices. Background Technology

[0002] Plant exosome-like nanovesicles (PELNs) are nanoscale vesicles secreted by plant cells, typically 50–200 nm in diameter. They possess a lipid bilayer structure and contain proteins, lipids, RNA, and various secondary metabolites. PELNs exhibit low immunogenicity, high biocompatibility, and oral applicability, showing broad application prospects in drug delivery, functional foods, and mucosal repair.

[0003] Currently, extraction methods for PELNs primarily target fresh, juicy plant materials (such as fruits and fresh vegetable leaves). Common extraction procedures include: physically disrupting fresh plant tissue (juicing or homogenizing), removing cell debris via differential centrifugation, and then enriching vesicles via ultracentrifugation. To further improve extraction efficiency, some studies have added cellulase after disruption for enzymatic hydrolysis to degrade cell wall components and promote vesicle release. For example, existing technologies disclose methods for extracting exosome-like nanovesicles from fresh prickly pear, mulberry leaves, and other plants, with the core process being: disruption-enzyme addition-differential centrifugation-ultracentrifugation.

[0004] However, the aforementioned existing technologies are all based on the premise that plant tissues are rich in water, have flexible cell walls, and abundant intercellular fluid. In contrast, processed medicinal herbs are dried and processed medicinal materials, whose cells are severely dehydrated, whose cell walls have lignified and hardened, and whose intercellular fluid has completely disappeared. Applying extraction methods for fresh plants directly to dried medicinal herbs presents two major technical obstacles: first, gentle physical disruption (such as juicing) cannot effectively break down the lignified cell walls, resulting in extremely low vesicle release; second, using high-energy physical methods (such as liquid nitrogen grinding and high-speed homogenization) to force cell wall disruption generates intense mechanical shear forces and localized high temperatures, easily leading to tearing of the vesicle lipid bilayer membrane and leakage of contents, thus resulting in loss of biological activity. In other words, existing technologies for processing dried medicinal herbs are caught in a contradiction between "low cell wall disruption leading to low yield" and "high cell wall disruption leading to vesicle destruction," making it difficult to simultaneously achieve high extraction rates and high structural integrity. Summary of the Invention

[0005] In order to overcome at least one of the defects of the prior art, the present invention provides a method and application for non-destructive extraction of exosome-like nanovesicles from dried Chinese medicinal herbs, which can solve the problem of poor extraction effect of exosome-like nanovesicles from dried Chinese medicinal plants.

[0006] The technical solution adopted by this invention to solve its problem is: A non-destructive extraction method for exosome-like nanovesicles from dried Chinese medicinal herbs includes the following steps: The dried Chinese herbal medicine slices were mixed with an extraction buffer containing cell wall degrading enzymes and osmotic pressure stabilizers, and then incubated for the first time at a temperature below the optimal operating temperature of the enzymes to allow the enzymes to penetrate into the slices. The infiltrated mixture was heated to the optimal operating temperature of the enzyme for a second incubation to degrade the cell wall; Mechanical energy below the exosome-like nanovesicle membrane rupture threshold was applied to the degraded mixture to release the vesicles; Exosome-like nanovesicles were isolated from the release products.

[0007] By employing the above-mentioned scheme and utilizing a dual-temperature-stage enzymatic hydrolysis strategy of "low-temperature penetration + high-temperature degradation," enzyme molecules can penetrate deep into the interior of dried, lignified cell walls, achieving three-dimensional degradation from the inside out, effectively breaking down the cell wall barrier that is difficult to overcome by traditional methods. Experimental data show that the exosome-like nanovesicles extracted from various dried Chinese herbal medicine slices using this method can achieve a particle concentration of up to 10-1. 10 ~10 11 The particle / mL level, compared to the yield when the traditional method is directly applied to dried medicinal slices (10) 8 The particle / mL level has been increased by more than two orders of magnitude.

[0008] The mechanical energy application step in this method is explicitly limited to below the exosome-like nanovesicle membrane rupture threshold. Combined with an osmotic pressure stabilizer to protect the rehydration process, this ensures that the vesicles are protected from osmotic pressure shocks and mechanical shear forces throughout the extraction process. Transmission electron microscopy results show that the vesicles extracted by this method possess a complete "cup / cup saucer" bilayer membrane structure with an integrity rate exceeding 90%, while almost all vesicles are broken when processing dried medicinal materials using traditional methods.

[0009] The key to this method lies in separating the "cell wall disruption" function from the "release" function: enzymatic hydrolysis undertakes the task of cell wall disruption, while low-energy mechanical force undertakes only the task of release. This design breaks the inherent contradiction in existing technologies where "the higher the cell wall disruption intensity, the greater the vesicle damage," allowing for both high extraction rate and high structural integrity.

[0010] Since the core mechanism of this method (enzymatic softening + low-energy release) does not depend on the chemical composition or tissue structure of a specific plant, it can be widely applied to dried plant materials from various sources such as leaves, fruits, rhizomes, and bulbs, without the need to develop new extraction processes for each medicinal material.

[0011] Furthermore, the osmotic pressure stabilizer is selected from mannitol or sucrose, and its concentration in the extraction buffer is 0.4M to 0.6M.

[0012] By employing the above-mentioned method, mannitol and sucrose, both neutral and non-ionic osmotic pressure regulators, can establish a stable osmotic pressure gradient inside and outside the cell, slowing down the rate of water entering the cell and preventing vesicle membrane rupture caused by rapid cell expansion. Experiments showed that in the control group without added osmotic pressure stabilizers, the proportion of intact vesicles in the extract was extremely low; only membrane fragments and amorphous impurities could be observed under transmission electron microscopy. However, after adding 0.4 M–0.6 M mannitol or sucrose, the vesicle integrity rate increased to over 90%, exhibiting a clear "cup / cup saucer" bilayer membrane structure.

[0013] Mannitol and sucrose are also common enzyme protectants, capable of maintaining the spatial structural stability of cellulase and isocyanate during low-temperature and subsequent temperature-increasing enzymatic hydrolysis, preventing enzyme inactivation. This allows the complex enzyme to maintain high activity throughout the two-temperature-stage process, ensuring sufficient degradation of the cell wall.

[0014] Furthermore, the cell wall degrading enzymes include cellulase and dissociation enzymes.

[0015] By employing the above-mentioned scheme, cellulase specifically hydrolyzes the β-1,4 glycosidic bonds of cellulose in the cell wall, disrupting its skeletal structure; while the dissociative enzyme (usually pectinase) degrades pectin and other polysaccharide components, eliminating the matrix binding cellulose microfibrils in the cell wall. The combined use of these two enzymes produces a synergistic effect: after pectin degradation, the cellulose fiber bundles lose their adhesion, exposing more enzyme cleavage sites, facilitating further action by cellulase. Experiments show that, under the same enzymatic hydrolysis time, the vesicle extraction rate of the composite enzyme group is increased by more than 50% compared to the single cellulase group, and the particle size distribution is more uniform. Because the composite enzyme can more thoroughly disintegrate the cell wall structure, the originally tough tissue is transformed into a soft, slurry-like substance, requiring only extremely low mechanical energy (6000–8000 rpm homogenization) to release the vesicles completely. This not only further reduces the risk of vesicle damage but also reduces equipment wear and energy consumption. In conventional extraction, viscous polysaccharides such as pectin easily bind non-specifically to vesicles, resulting in a large amount of gum-like impurities mixed in the precipitate after ultracentrifugation. The addition of dissociative enzymes degrades these polysaccharides into small oligosaccharides or monosaccharides, which are then effectively removed during low-speed centrifugation and filtration. This results in a cleaner background and lower protein peaks in the final vesicle sample. The cell wall composition varies considerably among different types of Chinese medicinal herbs: rhizomes (such as rhubarb and coptis) have high lignification and high cellulose content; while leaves (such as dandelion and green tea) have relatively high pectin content. The combined use of cellulase and dissociative enzymes can address the degradation needs of different cell wall types, making the method applicable to a variety of dried Chinese medicinal herbs and demonstrating its versatility.

[0016] Furthermore, the mechanical energy is provided by a tissue homogenizer with a homogenization speed of 6000 rpm to 8000 rpm.

[0017] By employing the above-described method, the inventors, through extensive experimental screening, discovered that after the cell walls are fully degraded by the compound enzyme, the tissue structure of dried Chinese herbal medicine slices transforms from a hard, lignified substance into a soft, pulpy mass. At this point, only a low-speed homogenization of 6000 rpm to 8000 rpm is needed to completely release the vesicles from the loosened cell matrix. This speed range has been proven to be a critical window that is "just sufficient to dissociate the cell wall, but below the vesicle membrane rupture threshold." Transmission electron microscopy results show that over 90% of the vesicles obtained under these conditions possess a complete "cup-shaped / cup-shaped" double-membrane structure.

[0018] When the homogenization speed exceeds 8000 rpm, shear force and cavitation effect are significantly enhanced, and vesicle membranes begin to tear and fragment; when the speed exceeds 10000 rpm, intact vesicles are almost impossible to observe in the field of view. This invention limits the upper limit of the speed to 8000 rpm, ensuring that the mechanical force is always within the tolerance range of the vesicles, fundamentally avoiding structural damage caused by high-energy fragmentation. High-speed homogenization generates a large amount of frictional heat, leading to a local temperature increase, which may denature and inactivate the bioactive molecules such as proteins and RNA contained in the vesicles. The low speed of 6000 rpm to 8000 rpm, combined with ice bath cooling and intermittent operation, can effectively control the system temperature and protect the natural conformation and biological function of the vesicle contents. The feasibility of this low speed range depends on the preceding complex enzymatic hydrolysis step. Because enzymatic hydrolysis has already undertaken the main task of cell wall disruption (degrading the rigid cell wall into loose tissue), mechanical force is greatly reduced, serving only as an auxiliary means of "release". This synergistic mode of "chemical-based, mechanical-assisted" is achieved.

[0019] Furthermore, the separation includes differential centrifugation and ultracentrifugation.

[0020] By employing the above-mentioned method, the enzymatic softening and critical low-energy release (homogenization at 6000–8000 rpm) steps in the preliminary steps avoid the excessive dissolution of intracellular contents (such as starch granules, pigments, and large protein molecules) caused by high-energy disruption. Impurities in the extract mainly consist of undegraded fine cell wall fragments and soluble small molecules, which can be effectively removed by differential centrifugation: low-speed centrifugation (e.g., 2000 g) removes unbroken cells and large fragments, medium-speed centrifugation (e.g., 10000 g) removes organelles and fine fragments, and finally, ultracentrifugation (e.g., above 100000 g) precipitates the vesicles. This three-step centrifugation creates a clear particle size distribution window, significantly reducing the co-precipitation rate of impurities in the vesicle precipitate. Differential centrifugation combined with ultracentrifugation is a classic method for exosome extraction, with standardized operation and controllable parameters. Under the process conditions of this invention, due to the relatively simple and stable impurity profile generated in the preceding steps, this separation method can obtain vesicle samples with uniform particle size (100–200 nm), stable potential, and low protein impurity content. It exhibits good batch-to-batch repeatability, meeting the quality control requirements for pharmaceutical and functional food development. Differential centrifugation and ultracentrifugation are both physical separation methods, eliminating the need for polymer precipitants (such as PEG) or organic solvents, thus avoiding the risk of residual exogenous chemicals and adhering to the safety and environmental protection principles of natural product extraction. The obtained vesicles can be directly used for subsequent bioactivity research and product development.

[0021] The present invention also provides an exosome-like nanovesicle from dried Chinese herbal medicine slices prepared by the above method.

[0022] By employing the above-mentioned method and observing through transmission electron microscopy, the exosome-like nanovesicles obtained from dried Chinese herbal medicine slices prepared in this invention exhibit a typical "cup-shaped" or "tea tray-shaped" double-layer membrane structure, with a vesicle integrity rate exceeding 90%. This indicates that the extraction method of this invention is gentle throughout the process, successfully avoiding the damage to the vesicle membrane structure caused by osmotic pressure shocks and high-energy shearing, whereas existing technologies struggle to obtain intact vesicles when applied to dried medicinal slices. Nanoparticle tracking analysis shows that the average particle size of the vesicles in this invention is concentrated between 100 and 200 nm, with a narrow particle size distribution, conforming to the classic size range of exosome-like nanovesicles. Dynamic light scattering measurements show that their Zeta potential ranges from -20 mV to 45 mV, indicating that the vesicle surface carries a sufficient negative charge, exhibiting good colloidal dispersion stability in solution, and is not prone to aggregation and precipitation, facilitating long-term storage and subsequent formulation processing. Due to the use of enzymatic softening and critical low-energy release in the preceding process, large amounts of intracellular starch, pigments, and large molecular proteins are avoided from dissolving. Combined with differential-ultracentrifugation purification, the resulting vesicle samples have a clean background and low protein impurity content. BCA protein quantification showed that the vesicle protein yield per gram of dried medicinal slices was significantly higher than that of traditional methods, and the proportion of impurities per unit particle number was lower. The vesicles of this invention contain natural active ingredients derived from traditional Chinese medicine slices (such as proteins, lipids, RNA, and small molecule metabolites). In vitro antioxidant experiments (DPPH and FRAP methods) showed that the vesicles have good free radical scavenging and iron ion reducing abilities, and the activity is concentration-dependent. Simulated gastrointestinal digestion experiments showed that the particle size of the vesicles did not change significantly after incubation in gastric and intestinal fluids for 2 hours, indicating good gastrointestinal stability and providing a basis for oral administration. Animal experiments confirmed that the exosome-like nanovesicles of dried traditional Chinese medicine slices prepared in this invention (taking dandelion, green tea, vitex fruit, rhubarb, coptis, and lily as examples) can effectively improve the pathological state of mice with acute colitis induced by sodium dextran sulfate. Specifically, it manifests as follows: restoring colon length, reducing serum levels of inflammatory factors (IL-1β, IL-6, TNF-α), upregulating the expression of intestinal tight junction proteins (Claudin-1, ZO-1, Occludin), and alleviating colonic crypt damage and inflammatory cell infiltration. These results demonstrate that the vesicles of this invention not only possess the structural characteristics of nanocarriers but also retain the therapeutic efficacy of the original traditional Chinese medicine decoction pieces. Storage stability experiments show that the particle size of the vesicles of this invention remains stable for 8 days at -20℃. After reconstitution of the lyophilized powder prepared by vacuum freeze-drying, the vesicle morphology and particle size show no significant changes, facilitating long-term storage and transportation, and meeting the basic requirements for product stability in industrial applications. The vesicles of this invention are entirely derived from dried traditional Chinese medicine decoction pieces, involving no organic solvents or toxic reagents, posing no risk of exogenous chemical residues, exhibiting low immunogenicity and good biocompatibility, and meeting the safety requirements for the development of pharmaceuticals, functional foods, and cosmetics.

[0023] The present invention also provides the application of the above-mentioned dried Chinese herbal medicine slices exosome-like nanovesicles in the preparation of products that improve intestinal mucosal barrier damage, wherein the dried Chinese herbal medicine slices are selected from at least one of dandelion, green tea, vitex fruit, rhubarb, coptis, and lily.

[0024] While existing technologies have reported that ELNs derived from fresh plants such as prickly pear, ginger, and grapes can alleviate colitis, there are no research reports on whether ELNs derived from six dried Chinese medicinal herbs—dandelion, green tea, vitex fruit, rhubarb, coptis, and lily—can improve intestinal mucosal barrier damage. This invention, using a mouse model of acute colitis induced by sodium dextran sulfate, is the first to demonstrate that ELNs derived from the above six dried Chinese medicinal herbs all have significant intestinal mucosal barrier repair effects, filling a technological gap in this field. Animal experimental results show that after gavage treatment with the above six ELNs, the colon length of mice with acute colitis significantly recovered (the colon in the model group was significantly shortened, while the colon length in the treatment group increased and approached that of the normal group); colon tissue staining showed that the crypts in the model group were severely damaged and heavily infiltrated with inflammatory cells, while the crypt structure in the treatment group was more intact, and edema and inflammatory cell infiltration were significantly reduced. This indicates that the ELNs of this invention can effectively reverse DSS-induced pathological damage to colonic tissue. ELISA results showed that the serum levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α were significantly elevated in the model group mice, while the levels of these inflammatory factors significantly decreased after treatment with the PELNs of this invention. Among them, PELNs derived from dandelion and green tea showed particularly significant effects in reducing inflammatory factors. These results indicate that the PELNs of this invention exert a protective effect on the intestinal mucosal barrier by inhibiting systemic inflammatory responses. Tight junction proteins (Claudin-1, ZO-1, and Occludin) are key molecules for maintaining the integrity of the intestinal mucosal barrier. After DSS induction, the expression levels of these proteins significantly decreased; however, after treatment with the PELNs of this invention, the expression levels of all three tight junction proteins significantly increased. This indicates that the PELNs of this invention restore intestinal barrier function by repairing the tight junction structure between intestinal epithelial cells and reducing intestinal mucosal permeability. In vivo fluorescence imaging and gastrointestinal imaging experiments showed that after oral administration of the PELNs of this invention, fluorescent signals were detected in the stomach, duodenum, and jejunum of mice within 0.5 hours; after 2 hours, they were mainly distributed in the ileum; after 4 hours, strong fluorescent signals appeared in the cecum and colon; and after 8-10 hours, they were gradually metabolized and excreted. These results indicate that the PELNs of this invention can completely pass through the gastric and intestinal digestive environment and target distribution in the colon, possessing the potential to act directly on intestinal lesions as an oral preparation. The above six dried Chinese herbal medicine slices are all medicinal and edible materials or commonly used clinical Chinese medicinal materials, and their safety has been verified through long-term practice. The extraction process of the PELNs of this invention does not involve organic solvents or toxic reagents, the product has no exogenous chemical residues, low immunogenicity, and good biocompatibility. Therefore, the PELNs of this invention can be directly used as active ingredients to prepare oral drugs, functional foods, or health drinks that improve intestinal mucosal barrier damage, and have broad market application prospects.

[0025] Furthermore, the product is a medicine or a functional food.

[0026] By adopting the above approach, the application products of the exosome-like nanovesicles of dried Chinese herbal medicine slices of this invention are limited to pharmaceuticals or functional foods, clearly defining its two major industrialization directions: In terms of pharmaceuticals: For diseases related to intestinal mucosal barrier damage (such as ulcerative colitis, Crohn's disease, bacterial enteritis, etc.), the vesicles of this invention can be developed into therapeutic drugs, achieving therapeutic effects through oral administration. Animal experiments have confirmed that they can significantly reduce the level of inflammatory factors and upregulate the expression of tight junction proteins, possessing a clear pharmacodynamic basis.

[0027] In the area of ​​functional foods: Targeting the daily intestinal health needs of sub-healthy individuals, the vesicles of this invention can be developed into functional foods or health drinks, achieving the maintenance and improvement of intestinal barrier function through daily intake. Experiments show that these vesicles have good oral safety and digestive stability, making them suitable as functional food additives.

[0028] The present invention also provides a pharmaceutical composition for improving intestinal mucosal barrier damage, comprising an effective amount of the above-mentioned dried Chinese herbal medicine exosome-like nanovesicles, and a pharmaceutically acceptable carrier.

[0029] By adopting the above-mentioned approach, traditional Chinese medicine compound formulas are complex in composition and their mechanisms of action are difficult to elucidate. The pharmaceutical composition of this invention uses exosome-like nanovesicles from a single source as the active ingredient. Their particle size, particle concentration, protein content, and active ingredient fingerprint spectrum can all be standardized and quality controlled by methods such as NTA, BCA, and HPLC, which meets the basic requirements of modern drugs for "clear composition and controllable quality," and is conducive to product registration and clinical promotion.

[0030] In summary, the non-destructive extraction method and application of exosome-like nanovesicles from dried Chinese herbal medicine slices provided by this invention have the following technical effects: 1. Addressing the technical challenge of lignified cell walls in dried Chinese medicinal herbs, which existing methods cannot effectively break down, this invention employs a dual-temperature-stage process: "low-temperature penetration allows enzyme molecules to penetrate deep into the tissue, while temperature increase activates the enzyme to achieve three-dimensional degradation from the inside out." This avoids the surface polysaccharide gelation blockage effect caused by room-temperature enzymatic hydrolysis. Experiments show that the concentration of vesicle particles extracted by the method of this invention reaches 10. 10 ~10 11 The particle / mL level, compared to the yield when the traditional method is directly applied to dried medicinal slices (10) 8 The particle / mL level has been increased by more than 100 times.

[0031] 2. To address the issue of vesicle rupture due to osmotic pressure shock during the rehydration of dried cells, this invention adds 0.4 M to 0.6 M mannitol or sucrose to the extraction buffer, making the rehydration process gentle and controllable. Transmission electron microscopy results show that almost all vesicles ruptured without the addition of an osmotic stabilizer, while the vesicle integrity rate increased to over 90% after the addition.

[0032] 3. Existing technologies employ high-energy physical disruption to force cell wall breakage, which results in strong shear forces that tear the vesicle membrane. This invention, after fully softening the cell wall through enzymatic hydrolysis, applies only a low-intensity homogenization at 6000–8000 rpm. This intensity is just sufficient to dissociate the loosened cell wall, but below the vesicle membrane rupture threshold, achieving a balance between efficient vesicle release and structural integrity.

[0033] 4. This invention employs a composite enzyme system of cellulase and ionizing enzyme. The former hydrolyzes the cellulose backbone, while the latter degrades the pectin matrix. The synergistic effect of the two results in more thorough cell wall degradation. Compared to a single cellulase, the extraction rate of the composite enzyme system is increased by more than 50%, while reducing the co-precipitation of viscous impurities such as pectin with vesicles, thus improving product purity.

[0034] 5. The method of this invention does not depend on the physicochemical properties of specific medicinal materials. It can achieve efficient extraction of dried Chinese medicinal materials from various parts of the plant, including leaves (dandelion, green tea), fruits (Vitex trifolia), rhizomes (rhubarb, Coptis chinensis), and bulbs (lily). The resulting vesicles have uniform particle size (100-200 nm), a significantly negative Zeta potential (-20 mV to 45 mV), and good colloidal stability.

[0035] 6. The dried Chinese herbal medicine slices prepared by this invention have a typical "cup-shaped" double membrane structure with an integrity rate of ≥90%; they have good antioxidant activity (DPPH scavenging rate and FRAP value are concentration-dependent) and simulated gastrointestinal digestion stability; the particle size remains stable for 8 days when stored at -20℃, and the reconstitution performance after freeze-drying is good.

[0036] 7. Using a DSS-induced acute colitis mouse model, this invention is the first to demonstrate that exosome-like nanovesicles derived from dandelion, green tea, vitex trifolia, rhubarb, coptis chinensis, and lily bulb have significant repair effects on intestinal mucosal barrier damage. After oral gavage administration, mouse colon length was restored, serum inflammatory factor (IL-1β, IL-6, TNF-α) levels were significantly reduced, intestinal tight junction protein (Claudin-1, ZO-1, Occludin) expression was upregulated, colonic crypt destruction and inflammatory cell infiltration were reduced. In vivo fluorescence imaging showed that the vesicles could pass through the gastrointestinal tract intact and target their distribution in the colon.

[0037] 8. The vesicles of this invention can be prepared as drugs or functional foods, covering applications ranging from disease treatment to daily prevention. Using these vesicles as the active ingredient, supplemented with pharmaceutically acceptable carriers, the drug composition exhibits good oral compliance, high safety, and scalable processing, providing a novel oral nanomedicine for the treatment of diseases related to intestinal mucosal barrier damage.

[0038] 9. The method of this invention does not require organic solvents throughout the process, operates under mild conditions (≤50℃), uses conventional equipment, has clear parameters, strong controllability, good batch repeatability, and is easy to transform from laboratory scale to industrial production. Attached Figure Description

[0039] Figure 1 The morphology observation and Brownian motion state diagram of PELNs are shown in Figure 1. (A) TEM morphology observation results of each PELN; (B) Brownian motion state of each PELN detected by NTA. Figure 2 For PELN particle size and Zeta potential, (A) NTA detection of particle size distribution of each PELN; (B) average particle size of each PELN; (C) DLS detection of Zeta potential of each PELN. Detailed Implementation

[0040] To better understand and implement this invention, the technical solutions in the embodiments of this invention will be clearly and completely described and discussed below with reference to the accompanying drawings. Obviously, what is described here is only a part of the examples of this invention, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the protection scope of this invention.

[0041] To facilitate understanding of the embodiments of the present invention, further explanations and descriptions will be provided below with reference to the accompanying drawings and specific embodiments, and these embodiments do not constitute a limitation on the embodiments of the present invention.

[0042] 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 be limiting of the invention.

[0043] This invention provides a general method for extracting exosome-like nanovesicles (PELNs) from dried traditional Chinese medicine slices. Its core lies in the innovative use of a reverse process strategy: "osmotic pressure buffer protection – cold osmosis – warm degradation dual-temperature enzymatic hydrolysis – critical low-energy release." The specific steps and key differences are discussed below: Step 1: Pretreatment of medicinal materials, rehydration and cold osmosis pre-enzymatic hydrolysis 1. Pre-pulverization: Take dried Chinese medicinal herbs and grind them into coarse powder using a pulverizing device under controlled temperature (multi-stage operation and cooling).

[0044] 2. Rehydration and cold permeation: Mix the coarse powder of medicinal materials with pre-cooled extraction buffer at a certain material-to-liquid ratio (adjusted between 1:5 and 1:10, w / v), and carry out rehydration and permeation for 2 hours under low temperature (4℃) and slow stirring conditions.

[0045] The specificity of the extraction buffer includes: ①Basic buffer solution (PBS, pH 7.4); ② Osmotic pressure stabilizer (0.4 M to 0.6 M mannitol or sucrose); ③ Cell wall degradation complex enzyme, including cellulase R-10 (1-1.5% w / v) and dissociation enzyme R-10 (0.2-0.4% w / v).

[0046] Some existing technologies target juicy, fresh plants, typically involving direct crushing and enzyme addition at room temperature or the optimal enzyme temperature, without the need for osmotic pressure protection. However, dried medicinal herbs have extremely dehydrated cells, rich in polysaccharides and pectin. If conventional rehydration and enzyme addition are performed directly, the influx of water molecules will trigger an osmotic pressure shock, causing intracellular vesicles to rupture. Simultaneously, the surface cell walls are rapidly degraded by enzymes at room temperature, releasing a large amount of viscous polysaccharide colloid, forming a gel-like sealing layer that completely blocks the deep penetration of water and enzyme molecules into the herb, resulting in external decay and internal dryness, leading to extremely low extraction rates.

[0047] This invention utilizes osmotic pressure buffering: the addition of 0.4 M mannitol provides osmotic pressure buffering, making the rehydration process gentle and effectively protecting the fragile lipid bilayer structure of exosomes (comparative studies have shown that without this addition, vesicle fragmentation is severe).

[0048] And the 4℃ "cold penetration" mechanism: at a low temperature of 4℃, the activity of the complex enzyme is extremely inhibited, but the enzyme molecules carried by the buffer solution can penetrate deeply and evenly into the interior of the microstructure as the dry powder is rehydrated and swollen, perfectly avoiding the "polysaccharide gel blockage effect" caused by rapid degradation of the surface at room temperature.

[0049] Step 2: Cell wall "warm degradation" and critical low-energy release of contents: 1. Warm degradation incubation: Heat the above-mentioned cold-osmotic mixture to a suitable temperature (adjust according to the optimal temperature of the enzyme used; in this protocol, it is 40°C), and incubate at low speed (approximately 80 rpm) for 2 hours to allow the cell wall to be fully degraded.

[0050] 2. Critical low-energy release (gentle mechanical disruption): The enzymatically hydrolyzed mixture is processed using a tissue homogenizer under ice bath conditions. The core parameters are set as follows: the homogenization speed is strictly controlled within the range of 6000-8000 rpm, the homogenization time is 30 seconds per cycle, and the mixture is paused on ice for 1 minute after each homogenization. This process is repeated 5-8 times.

[0051] Existing conventional techniques follow the logic of "physical disruption (high-energy shearing) followed by enzymatic hydrolysis." This is feasible for fresh tissues, but for dried, highly lignified Chinese medicinal herbs, the enormous shear force generated by powerful mechanical pulverization (usually at speeds >15,000 rpm or by using a crusher directly) will directly tear the already dehydrated and fragile vesicle membranes, leading to the loss of biological activity.

[0052] The difference in this invention is: 1. Reverse process (enzymatic softening followed by mechanical release): After the first step of cold penetration, the "40℃ temperature degradation" in this step enables the enzyme to be activated simultaneously inside and outside the tissue, achieving a three-dimensional thorough degradation "from the inside out", transforming the hard, dry tissue into a soft structure full of water.

[0053] 2. Defining the "extremely low shear force" critical window: This invention abandons the traditional concept of fragmentation and establishes a low-energy critical window of 6000-8000 rpm. This mechanical strength has been rigorously screened experimentally, and its shear force is just greater than the binding force of the cell wall loosened by the complex enzyme, but strictly less than the rupture tension of the exosome lipid bilayer membrane.

[0054] 3. Effects achieved by this invention: The above-mentioned "osmotic pressure buffer protection - cold osmosis - warm degradation dual-temperature enzymatic hydrolysis - critical low-energy release" process enables the extraction of 10% of highly lignified dried rhizome medicinal materials while ensuring that over 90% of the vesicle structure remains absolutely intact (undamaged). 11 The extraction rate is on the order of particles / mL, achieving an ultra-high extraction rate. This successfully solves the technical challenge of simultaneously achieving high cell wall disruption and membrane integrity in dried medicinal materials.

[0055] Step 3: Differential ultracentrifugation purification: 1. Preliminary filtration: The homogenate is passed through multiple layers of gauze and a 0.8 µm pore size filter membrane in sequence to remove large tissue residues and unbroken cells.

[0056] 2. Differential centrifugation: Perform sequential centrifugation on the filtrate to remove larger particles such as cells and organelles in turn: ① Centrifuge at low speed (2000 g, 20 min) and collect the supernatant; ② Centrifuge at medium speed (10000 g, 30 min) and collect the supernatant.

[0057] 3. High-speed filtration: The supernatant is passed through a 0.22 µm pore size filter membrane to remove large vesicles and bacteria.

[0058] 4. Ultracentrifugation enrichment: Centrifuge the filtrate at ≥100,000 g (the parameters for this procedure are 110,000 g, 90 min) and collect the precipitate.

[0059] 5. Washing and purification: Resuspend the precipitate in enzyme-free extraction buffer (PBS + 0.4 M mannitol) and repeat the ultracentrifugation step once to collect the final purified PELNs precipitate.

[0060] Because high-energy physical pulverization is avoided in the preceding processes, starch, pigments, and soluble macromolecular impurities within the dried cells are not excessively released. This greatly reduces the burden on subsequent ultracentrifugation and filtration, significantly lowers the co-precipitation rate of extraneous proteins, and results in PELNs with extremely high purity that are less prone to clogging the filter membrane, making them suitable for large-scale production.

[0061] Step 4: Sample Preservation and Quality Control The final PELNs precipitate was resuspended in an appropriate amount of PBS buffer, aliquoted, and subjected to gradient freezing (4℃ for 30 min, -20℃ for 30 min), followed by long-term storage at -80℃. The particle size, particle concentration, zeta potential, morphology, and protein concentration of the obtained PELNs were controlled using methods such as NTA, DLS, TEM, and BCA.

[0062] Example: Comparative study of the extraction of PELNs from dried Chinese medicinal herbs using the method of this invention and traditional methods. This embodiment aims to demonstrate that existing traditional extraction methods (or conventional adjustments such as the addition of cellulase) are not suitable for dried Chinese herbal medicine slices, and to showcase the significant advantages and wide applicability of the method of the present invention in solving this problem.

[0063] 1. Experimental grouping and sample preparation ① Example Group (Method of the Invention): Ingredients: 30g each of dried dandelion, green tea, vitex fruit, rhubarb, coptis, and lily bulb slices.

[0064] Method: PELNs were extracted strictly according to the “cold permeation-warm degradation dual-temperature enzymatic hydrolysis + critical low-energy release + sequential centrifugation” process described in the technical solution of this invention.

[0065] ② Comparative group (traditional method (enzymatic hydrolysis) applied to dried medicinal materials): Materials: 30 g of dried dandelion (from the same batch as the example group).

[0066] Methods: The existing general extraction logic for fresh plants was simulated. Dried dandelion was soaked in PBS for rehydration, then physically crushed using a juicer. The filtrate was collected, and 3% (w / v) cellulase was added for enzymatic hydrolysis. Extraction was then performed using the same differential ultracentrifugation procedure.

[0067] ③ Positive control group (traditional method applied to fresh plants): Ingredients: 200 g fresh dandelion.

[0068] Methods: PELNs were extracted using a combination of juicing and differential ultracentrifugation (specific parameters are the same as in the example group and comparative group). This group serves only as a reference for the standard morphology and characteristics of PELNs.

[0069] 2. Results and Characterization 2.1 Morphological observation (TEM) The samples were observed using transmission electron microscopy (TEM): Example Group (Method of the Invention): In samples from six kinds of dried medicinal materials, "cup-shaped / tea tray-shaped" vesicles with clear double-layer membrane structures and typical morphology could be observed in the field of view, with a relatively clean background (see details). Figure 1 Example groups 1-6).

[0070] Comparative group (traditional methods applied to dried medicinal materials): Typical vesicle structures were extremely difficult to find within the field of view; only a few membrane fragments or amorphous impurity clumps were occasionally observed. This indicates that traditional methods (even with the addition of cellulase for enzymatic hydrolysis) cannot effectively release structurally intact PELNs from dried tissues, and that severe physical disruption directly leads to large-scale tearing and disintegration of the vesicle membrane structure, resulting in leakage of contents. (See details...) Figure 1 Comparative group) Positive control group (traditional method applied to fresh plants): Typical exosome-like vesicle structures were visible, confirming the morphological characteristics of plant exosomes (see details). Figure 1 Positive control group).

[0071] 2.2 Physicochemical properties and yield characterization (NTA / DLS / BCA) Quantitative analysis was performed on the example group (6 types) and the comparative example group: Example Group (Method of the Invention): NTA detection showed that the average particle size of the six dried medicinal materials PELNs extracted by the method of the present invention was concentrated in the classical exosome range of 100-200 nm, indicating that the sample particle size distribution was uniform and the particle number concentration reached 10. 10 ~10 11The particle / mL level (equivalent to an extremely high yield per gram of dry weight) indicates that this method can obtain high concentrations of PELNs particles; DLS shows that the Zeta potentials of all PELNs are significantly negative (-20 mV to -45 mV), indicating that the vesicle system has good colloidal stability in solution and is not prone to aggregation (see Table 1 for examples and...). Figure 2 Example groups 1-6).

[0072] Comparative control group (traditional methods applied to dried medicinal materials): NTA analysis showed that the particle number concentration of dandelion treated with traditional methods was only 10. 8 The particle size distribution is on the order of particles / mL (2-3 orders of magnitude lower than the method of this invention), and the particle size distribution is disordered with extremely low protein concentration (see Table 1 for comparative examples and...). Figure 2 (Comparative group). The data confirms that traditional methods have extremely low extraction efficiency for dried medicinal materials.

[0073] 3. Conclusion In summary, this embodiment powerfully demonstrates that the conventional "rehydration + physical disruption + single enzymatic hydrolysis" method (comparative example) suffers from severe yield bottlenecks and structural damage when processing dried Chinese herbal medicine pieces, failing to yield qualified samples. However, the "cold permeation-warm degradation dual-temperature-level enzymatic hydrolysis + critical low-energy release + sequential centrifugation" strategy proposed in this invention successfully overcomes the mechanical barrier of the cell walls of dried medicinal materials, effectively solving the limitations of existing technologies and achieving efficient and non-destructive extraction of PELNs. The yield is more than 100 times higher than traditional methods, while maintaining good structural integrity. This method has good universality for various sources and textures of Chinese herbal medicine pieces, including leaves (dandelion, green tea), fruits (Vitex trifolia), rhizomes (rhubarb, Coptis chinensis), and bulbs (lily), laying a solid technical foundation for the large-scale preparation and application of Chinese herbal medicine exosomes.

[0074]

[0075] In summary, this invention, targeting the special matrix of dried Chinese herbal medicine slices, has achieved the following significant technical advancements and beneficial effects: 1. Achieved non-destructive extraction of dried medicinal materials (core advantage): Most existing technologies are based on the logic of physically destroying tissue to facilitate enzymatic hydrolysis, which leads to a large number of broken vesicles in dried medicinal materials. This invention creatively proposes a reverse process of softening followed by peeling. In particular, it establishes a critical low-energy release window of 6000-8000 rpm, completely avoiding the high-frequency shearing and thermal damage caused by physical grinding, and achieving the retention of more than 90% of the perfect cup-shaped double-layer membrane structure of the vesicles under transmission electron microscopy.

[0076] 2. Employing a dual-temperature-stage process of "cold permeation-warm degradation" overcomes the bottleneck in extraction rate: This invention does not directly add enzymes at the optimal enzyme activity temperature. Instead, it utilizes temperature sensitivity, first using a 4°C cold penetration method to allow the complex enzyme to uniformly penetrate the dehydrated tissue, and then using a 40°C heating method to achieve three-dimensional degradation from the inside out. This completely overcomes the gel-blocking effect caused by the rapid degradation of epidermal polysaccharides in dried medicinal materials, resulting in an exponential increase in vesicle extraction rate (reaching 10). 11 (particles / g level), far exceeding existing technologies.

[0077] 3. A specific "osmotic pressure-dual enzyme" buffer system was constructed, which solved the rehydration bursting problem: This invention specifically adds 0.4 M mannitol to construct an osmotic pressure buffer system for the rehydration of dried cells, effectively preventing vesicle rupture caused by the instantaneous osmotic pressure shock during rehydration. Simultaneously, the specific combination of cellulase and dissociative enzyme successfully breaks down the complex cross-linked lignified structure generated during the processing of traditional Chinese medicine.

[0078] 4. The sample has high purity and retains its biological activity intact: The gentle release process avoids the excessive dissolution of intracellular starch, pigments, and secondary metabolites, greatly reducing the co-precipitation of impurities and vesicles. The resulting PELNs have a clean background and stable potential, maximizing the preservation of natural bioactive components derived from traditional Chinese medicine, making them suitable for the subsequent development of drug delivery carriers.

[0079] 5. The process is stable and has the potential for large-scale production: This invention establishes a standardized process (rehydration-enzymatic hydrolysis-homogenization-centrifugation) with clear parameters and strong controllability, avoiding the randomness of operations such as liquid nitrogen grinding, with good batch repeatability, and the required equipment (constant temperature shaker, homogenizer) are all conventional equipment, which is easy to transform into industrial production.

[0080] The technical means disclosed in this invention are not limited to those disclosed in the above embodiments, but also include technical solutions composed of any combination of the above technical features. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications are also considered within the scope of protection of this invention.

Claims

1. A method for non-destructive extraction of exosome-like nanovesicles from dried Chinese medicinal herbs, characterized in that, Includes the following steps: The dried Chinese herbal medicine slices were mixed with an extraction buffer containing cell wall degrading enzymes and osmotic pressure stabilizers, and then incubated for the first time at a temperature below the optimal operating temperature of the enzymes to allow the enzymes to penetrate into the slices. The infiltrated mixture was heated to the optimal operating temperature of the enzyme for a second incubation to degrade the cell wall; Mechanical energy below the exosome-like nanovesicle membrane rupture threshold was applied to the degraded mixture to release the vesicles; Exosome-like nanovesicles were isolated from the release products.

2. The method according to claim 1, characterized in that, The osmotic pressure stabilizer is selected from mannitol or sucrose, and its concentration in the extraction buffer is 0.4 M to 0.6 M.

3. The method according to claim 1, characterized in that, The cell wall degrading enzymes include cellulase and dissociation enzymes.

4. The method according to claim 1, characterized in that, The mechanical energy is provided by a tissue homogenizer with a homogenization speed of 6000 rpm to 8000 rpm.

5. The method according to claim 1, characterized in that, The separation includes differential centrifugation and ultracentrifugation.

6. A dried traditional Chinese medicine decoction piece with exosome-like nanovesicles prepared by the method described in any one of claims 1 to 5.

7. The application of the dried traditional Chinese medicine decoction pieces exosome-like nanovesicles according to claim 6 in the preparation of products that improve intestinal mucosal barrier damage, characterized in that, The dried Chinese medicinal herbs are selected from at least one of the following: dandelion, green tea, vitex fruit, rhubarb, coptis, and lily.

8. The application according to claim 7, characterized in that, The product is a medicine or a functional food.

9. A pharmaceutical composition for improving intestinal mucosal barrier damage, characterized in that, The product comprises an effective amount of the dried Chinese herbal medicine slices described in claim 6, and a pharmaceutically acceptable carrier.

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