A method, kit, and system for the fractionated preparation of fresh pharmaceutical raw materials

Abstract

This invention discloses a method, assembly kit, and system for graded preparation of fresh medicinal raw materials, belonging to the field of natural product processing and biotechnology. The method first pre-treats the fresh raw materials by crushing, homogenizing, or enzymatically hydrolyzing the cell walls. Then, a liquid component rich in extracellular vesicles is obtained as the first component through low-shear solid-liquid separation. The residue obtained after separation is then mechanically pressed to extract juice, which is the second component. The solid residue obtained after mechanical pressing is then subjected to thermal extraction with water to obtain the extract as the third component. This establishes a core preparation sequence of gentle vesicle separation, followed by pressing to extract juice, and finally thermal extraction. This not only maximizes the preservation of the original active components from the nanoscale to the macroscale in the raw materials but also generates transformation products unique to traditional methods such as decoction through the final step, achieving a balance between preserving the original components and creating thermally transformed components.

Description

Technical Field

[0001] This invention relates to the fields of natural product processing and biotechnology, specifically to a method for graded preparation of fresh medicinal raw materials, a combination kit obtained by the method, and a system for implementing the method. Background Technology

[0002] Extracellular vesicles derived from plants and fungi show great promise as novel natural nanocarriers in the pharmaceutical and functional food fields. Existing fresh herb processing techniques, such as traditional juicing or decoction, typically only extract some of the active components from plant materials, leading to a systematic loss of the raw material's chemical information. For example, mechanical pressing is considered a destructive step for extracellular vesicles, while high-temperature decoction inactivates heat-sensitive active ingredients. Furthermore, existing technologies are mostly designed for industrial-scale mass production, lacking standardized operating procedures suitable for on-site preparation in clinics, pharmacies, and other similar settings, and failing to adequately consider the compatibility issues of the prepared components in subsequent preservation and compounding applications.

[0003] Therefore, there is an urgent need in this field for a technical solution that can achieve a stepwise extraction from mild to strong based on the differences in the physicochemical stability of different active components, thereby maximizing the preservation of the full spectrum of active components of fresh raw materials from the nanovesicle level, cell fluid level to cell wall matrix level. Summary of the Invention

[0004] In view of this, and to address the shortcomings of existing technologies, this invention aims to provide a method for graded preparation of fresh medicinal raw materials, as well as a combination kit obtained by this method and a system for implementing the method. This method is particularly suitable for on-site preparation of fresh medicinal raw materials and subsequent cold chain distribution scenarios. Based on the stability differences of different active components, this method designs a technically necessary preparation sequence, which not only maximizes the retention of native active components at different stability levels in fresh raw materials, from nanovesicles and cell sap to the cell wall matrix, but also generates decoction-specific transformation products through the final thermal extraction step. This achieves both the retention of native components and the generation of thermally transformed components, providing a high-quality component basis for subsequent short-term preservation and personalized compounding.

[0005] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for graded preparation of fresh medicinal raw materials, comprising the following steps: a) Preparation steps of the first component: After pretreatment of fresh raw materials by crushing, homogenizing or enzymatically hydrolyzing the cell walls, a liquid component rich in extracellular vesicles is obtained as the first component through low-shear solid-liquid separation. b) Second component preparation step: The residue obtained after separation in step a) is mechanically pressed to extract juice, and the pressed juice is used as the second component; c) Preparation of the third component: The solid residue obtained after mechanical pressing and juice extraction in step b) is subjected to thermal extraction with water to obtain the extract as the third component; Step b) must not begin before step a) is completed, and step c) must not begin before step b) is completed.

[0006] As a further aspect of the present invention, when the fresh raw material enters the preparation of the first component, it satisfies a moisture content ≥70wt% and meets any of the following conditions: (1) The preparation process begins within 24 hours of harvesting; (2) After harvesting, freeze at -80℃ and thaw at 4℃ before use; (3) Although it exceeds 24 hours after harvest, the extracellular vesicle recovery rate is not less than 85%, the rupture rate is not higher than 5%, and the water content is still ≥70wt%; Furthermore, the fresh raw materials have not undergone dehydration heat treatment or freeze-drying treatment at temperatures above 50°C.

[0007] As a further aspect of the present invention, the low-shear solid-liquid separation method in step a) ensures that the rupture rate of extracellular vesicles in the obtained first component is ≤5% and the recovery rate is ≥85%; the rupture rate is determined by the SYTOX Green nucleic acid dye method and the recovery rate is determined by a nanoparticle tracking analyzer.

[0008] As a further aspect of the present invention, the enzymatic cell wall pretreatment in step a) includes enzymatic hydrolysis using one or more of cellulase, pectinase, and hemicellulase. After enzymatic hydrolysis, there is no need for separate high-temperature inactivation; the reaction is terminated by immediately performing subsequent low-temperature, low-shear solid-liquid separation.

[0009] As a further aspect of the present invention, the low-shear solid-liquid separation method is selected from any of the following: (i) 6-10 layers, 18-22 mesh / cm 2 Medical degreased gauze filtration, wherein the gauze is placed at an angle of 30°-60° and driven by the gravity of the material itself, and each 100mL of raw material liquid is filtered within 3-5 minutes; (ii) Tangential flow filtration with a molecular weight cutoff of 300-500 kDa and a transmembrane pressure ≤0.3 bar; (iii) Graded low-speed centrifugation combined with 50-200μm sieve filtration.

[0010] As a further aspect of the present invention, the parameters for the graded low-speed centrifugation are as follows: at 4°C, centrifugation is performed sequentially at 300-500g for 10-15 minutes to remove cell debris, centrifugation at 1000-3000g for 15-20 minutes to remove larger vesicles, and centrifugation at 5000-10000g for 20-30 minutes to enrich the target vesicles.

[0011] As a further embodiment of the present invention, the mechanical pressing and juice extraction in step b) is carried out under low temperature conditions, with a pressing pressure of 0.1-1.0 MPa and a pressing time of 30 seconds to 5 minutes.

[0012] As a further embodiment of the present invention, the temperature of the thermal extraction in step c) is 80-100℃, the time is 30-90 minutes, and the mass-volume ratio of the solid residue to water is 1:5 to 1:15 g:mL.

[0013] As a further aspect of the present invention, after step a) is completed, the time T0 at which the preparation of the first component is completed is recorded, and step b) is completed within 30 minutes from T0.

[0014] As a further aspect of the present invention, the average particle size of the extracellular vesicles in the first component obtained in step a) is 50-200 nm, and the polydispersity index (PDI) is <0.3.

[0015] Secondly, the present invention provides a method for graded preparation of fresh Chinese medicinal materials and / or fresh medicinal fungal materials, comprising the following steps that must be strictly performed in sequence: a) First component preparation steps: After pretreatment of fresh raw materials by crushing, homogenizing or enzymatically hydrolyzing cell walls, a liquid component rich in extracellular vesicles is obtained as the first component through low-shear solid-liquid separation. b) Second component preparation step: The residue obtained after separation in step a) is mechanically pressed to extract juice, and the pressed juice is used as the second component. In this process, the solid residue after step b) is discarded or used for other purposes, and the thermal extraction step is not performed.

[0016] Thirdly, the present invention provides a fresh herb combination kit comprising the following three components, which are individually packaged and provided in an associated manner: First component container: contains a liquid component rich in extracellular vesicles prepared by a graded preparation method of a fresh medicinal raw material; Second component container: contains pressed juice components obtained from a graded preparation method of a fresh medicinal raw material; The third component container contains a hot extract component prepared by a graded preparation method of a fresh medicinal raw material; And a supporting information carrier, wherein the information carrier records at least the preparation completion time T0 of the first component, and the container of the first component is provided with a time indication unit associated with T0.

[0017] Fourthly, the present invention provides an on-site preparation system for implementing a graded preparation method for fresh medicinal raw materials, comprising, in sequence: a raw material pretreatment unit, a low-shear solid-liquid separation unit, a mechanical pressing and juice extraction unit, and optionally a thermal extraction unit.

[0018] Compared with the prior art, the method, kit, and system for graded preparation of fresh medicinal raw materials of the present invention have the following beneficial effects: This invention establishes a core preparation sequence of first gently separating vesicles, then pressing to extract juice, and finally thermal extraction. This sequence is the key technology for achieving a significant synergistic effect among the three components. It not only preserves the original active components from the nanoscale to the macroscale in the raw materials to the greatest extent, but also generates transformation products unique to traditional methods such as decoction in the final step, achieving both the preservation of original components and the creation of thermally transformed components. Reversing the sequence will lead to severe damage to the extracellular vesicle structure and complete loss of the synergistic effect.

[0019] Based on the stability differences of different active components under this preparation sequence, this invention first protects the fragile extracellular vesicles in the gentlest way, then obtains the active substances in the cell sap at medium and low temperatures, and finally obtains the transformation product through thermal extraction. This achieves the maximum retention of active components from the nanoscale to the macroscale and the generation of transformation products. It is applicable not only to different kinds of plant raw materials, but also to fungal raw materials.

[0020] To more clearly illustrate the structural features and effects of the present invention, the present invention will be described in detail below with reference to specific embodiments. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0022] The present invention provides a method for graded preparation of fresh medicinal raw materials, the core of which lies in strictly performing the following three steps in sequence, which can grade and obtain three active components with synergistic effects from fresh medicinal raw materials.

[0023] Step a) Preparation of the first component (rich in extracellular vesicle components): 1. Raw material preparation: Select fresh medicinal raw materials that meet the requirements, such as fresh ginseng, fresh Ganoderma lucidum, or fresh ginger. The moisture content of the raw materials should be ≥70 wt%, and they should not have undergone dehydration heat treatment above 50℃ or freeze-drying. The raw materials can be fresh materials processed within 24 hours after harvesting, or materials that have been quick-frozen at -80℃ and then thawed at 4℃.

[0024] 2. Pretreatment: The raw materials are broken down or homogenized to release intracellular substances. Enzymatic cell wall pretreatment can also be used. A preferred method is to place the raw materials in pre-cooled PBS (phosphate-buffered saline, pH 7.4, isotonic with the human physiological environment) and add one or more of cellulase, pectinase, and hemicellulase. For example, at 25°C and pH 5.5, 0.5% (w / v) pectinase and 0.3% (w / v) cellulase are added, and the mixture is gently stirred for 60 minutes. After enzymatic hydrolysis, a separate high-temperature inactivation step is unnecessary. The enzymatic reaction can be effectively terminated by immediately performing a subsequent low-temperature (4-10°C) low-shear solid-liquid separation operation, avoiding damage to the vesicles from high temperatures.

[0025] 3. Low-shear solid-liquid separation: The pretreated slurry is subjected to solid-liquid separation to obtain a liquid rich in extracellular vesicles (EVs). The key to this step is low shear force to ensure the structural integrity of the EVs. After separation, the rupture rate of EVs in the first fraction should be ≤5%, and the recovery rate should be ≥85% (rupture rate was determined using the SYTOX Green nucleic acid dye method, and recovery rate was determined using a nanoparticle tracking analyzer (NTA)).

[0026] Depending on the characteristics of the raw materials and the scale of production, one of the following low-shear separation methods can be selected: (1) Gravity gauze filtration (preferably on-site / small batch preparation): suitable for plant-based raw materials with high fiber content and relatively low slurry viscosity (such as ginseng and ginger). Pour the homogenate into 6-10 layers of 18-22 mesh gauze. 2 The gauze is placed at a 30°-60° angle on medical degreased gauze, and filtration is driven solely by the gravity of the material itself. The filtration speed is controlled to be approximately 3-5 minutes per 100mL of raw material solution. This method is gentle and the equipment is simple.

[0027] (2) Tangential flow filtration (preferably for large-scale production or special raw materials): suitable for fungal raw materials containing fine, hard particles (such as chitin fragments from Ganoderma lucidum) that easily clog filter media, or for scenarios requiring continuous production. Use a filter membrane with a molecular weight cutoff of 300-500 kDa and control the transmembrane pressure to ≤0.3 bar. This method effectively prevents membrane clogging and achieves gentle and efficient separation.

[0028] (3) Graded low-speed centrifugation combined with filtration (preferably research-grade purification): suitable for situations requiring further purification or analysis of vesicle subpopulations. At 4°C, the homogenate supernatant is subjected to the following sequential steps: centrifugation at 300-500g for 10-15 minutes to remove cell debris; centrifugation at 1,000-3,000g for 15-20 minutes to remove larger vesicles; and centrifugation at 5,000-10,000g for 20-30 minutes to enrich the target vesicles. The supernatant after centrifugation can be filtered through a 50-200μm sieve to remove residual large particles.

[0029] 4. Recording and Temporary Storage: The exact moment of completion of the first fraction (EVs enrichment solution) should be recorded immediately and marked as T0. This fraction should be temporarily stored at 4°C and proceeded to subsequent steps as soon as possible.

[0030] Step b) Preparation of the second component (pressed juice component): 1. Raw material transfer: Transfer the solid residue (non-filtrate) remaining after the low-shear separation in step a) to the pressing device.

[0031] 2. Mechanical pressing: The residue is mechanically pressed under low-temperature conditions (4-15℃). The pressing pressure is controlled at 0.1-1.0 MPa, and the pressing time lasts from 30 seconds to 5 minutes. This step aims to extract the juice from the cells through mechanical pressure, while avoiding the inactivation of heat-sensitive components (such as polyphenols and vitamins) caused by high temperatures. Any form of pressing of the raw material before step a) is strictly prohibited, as even light pre-pressing will severely impair the recovery rate and integrity of subsequent vesicles.

[0032] 3. Collection: Collect the pressed juice as the second component. Step b) must be initiated and completed within 30 minutes from time T0 to ensure a significant synergistic effect with the first component (combination index CI < 0.85). Delays exceeding 30 minutes will significantly weaken or even eliminate the synergistic effect.

[0033] Step c) Preparation of the third component (thermal extract component): 1. Raw material transfer: Transfer the solid residue remaining after mechanical pressing and juice extraction in step b) to a heating container.

[0034] 2. Thermal extraction: Add water to the residue, maintaining a solid-liquid ratio (residue mass: water volume) of 1:5 to 1:15 (g:mL). Heat and extract at 80-100℃ for 30-90 minutes. Extraction can be performed 1-2 times; combine the extracts.

[0035] 3. Collection: Filtration, the resulting filtrate is the third component. This step uses high temperature to induce the dissolution of components in the cell wall matrix and may cause transformation, generating active products unique to decoction.

[0036] This invention provides a fresh herbal combination kit, comprising three components prepared by the above-described method, which can be prepared into a combination kit that is easy to store, transport, and use. The kit includes three independently packaged containers, respectively containing the first component (EVs enriched solution), the second component (pressed juice), and the third component (hot extract). The kit also includes an information carrier (such as a label, instruction manual, or electronic chip) that records at least the preparation completion time T0 of the first component. This is crucial information to ensure the timeliness of subsequent compounding. The kit also includes a time indicator unit; a time indicator unit (such as a color-changing label with time scale) associated with T0 can be set on the first component container to visually indicate the remaining effective usage time of the component, ensuring compounding is performed within the effective time window.

[0037] To facilitate the implementation of the method of this invention in clinics, pharmacies, and other on-site settings, this invention also includes an on-site preparation system. This system comprises, in sequence: Raw material pretreatment unit: used for pretreatment of fresh raw materials by washing, crushing, homogenizing or enzymatic hydrolysis.

[0038] Low-shear solid-liquid separation unit: Depending on the scenario configuration, a gravity filtration device, a small tangential flow filtration system, or a benchtop centrifuge can be selected to perform step a).

[0039] Mechanical pressing and juice extraction unit: used for low-temperature pressing of the residue from step a), performing step b).

[0040] In this embodiment, the on-site preparation system further includes a thermal extraction unit, which can be an electric decoction device, used to heat and extract the residue from step b) to perform step c).

[0041] It should be noted that the preparation of this invention must strictly follow the order of steps a) → b) → c). Any reversal, such as step c) before step a) or the insertion of a destructive step, such as pressing before step a), will result in a large number of extracellular vesicles ruptured and the loss of synergistic effect, leading to a decrease in recovery rate, an increase in rupture rate, or a CI value >1 indicating antagonism.

[0042] The present invention provides a method for grading and preparing fresh medicinal raw materials applicable to fresh Chinese herbal medicine raw materials and / or fresh medicinal fungal raw materials. The fresh Chinese herbal medicine raw materials and / or fresh medicinal fungal raw materials refer to those with a moisture content ≥70 wt%, determined by the drying method according to General Chapter 0832 of the 2020 edition of the Chinese Pharmacopoeia, and meeting one of the following conditions when entering the preparation process: (1) The preparation process begins within 24 hours of harvesting; (2) After harvesting, freeze at -80℃ and thaw at 4℃ before use; (3) Although the above time is exceeded, the extracellular vesicle recovery rate is not less than 85%, the rupture rate is not higher than 5%, and the water content is still ≥70wt%.

[0043] Furthermore, the raw materials have not undergone dehydration heat treatment at temperatures above 50°C that would cause irreversible damage to the cell structure, nor have they undergone freeze-drying.

[0044] Step a) After pretreating the fresh raw material by crushing, homogenizing, or enzymatically hydrolyzing the cell wall, a liquid component rich in extracellular vesicles is obtained as the first component through low-shear solid-liquid separation. Here, crushing, homogenizing, or enzymatically hydrolyzing the cell wall refers to various optional methods for cell disruption of the fresh raw material. When enzymatically hydrolyzing the cell wall is used, the enzymatic process itself can partially degrade the cell wall, which is beneficial for the subsequent release of extracellular vesicles. To further improve the release efficiency of extracellular vesicles, homogenization can be optionally performed after enzymatic hydrolysis. This homogenization is a routine optimization operation for those skilled in the art and does not affect the definition of the core timing described in this invention.

[0045] The low-shear solid-liquid separation method refers to a solid-liquid separation method that maintains the structural integrity of extracellular vesicles. In this invention, the low-shear solid-liquid separation method ensures that the rupture rate of extracellular vesicles in the obtained first fraction is ≤5% and the recovery rate is ≥85%. The rupture rate is determined using the SYTOX Green nucleic acid dye method, specifically: SYTOX Green dye is added to the sample to a final concentration of 1 μM, incubated in the dark for 5 minutes, and then the fluorescence intensity is detected using a fluorescence microplate reader (excitation wavelength 488 nm / emission wavelength 525 nm). The fluorescence intensity after the sample is completely ruptured using Triton X-100 (final concentration 1%) is used as a 100% rupture control, and the fluorescence intensity of the untreated fresh sample is used as the background value to calculate the rupture rate. The recovery rate is determined using a nanoparticle tracking analyzer (NTA) and is calculated as the ratio of the total number of particles in the separated sample to the theoretical total number of particles in the pretreated homogenate.

[0046] In step b), the residue obtained after separation in step a) is mechanically pressed to extract juice, and the pressed juice is used as the second component. Mechanical pressing refers to a juice extraction process primarily driven by mechanical pressure to release the liquid phase from the material, excluding extraction using organic solvents. During the preparation process, staged low-speed centrifugation refers to differential centrifugation with a centrifugal acceleration ≤10,000g to avoid vesicle rupture due to high shear force. In step c), the solid residue obtained after mechanical pressing in step b) is thermally extracted with water to obtain the extract as the third component; step b) must not begin before step a) is completed, and step c) must not begin before step b) is completed.

[0047] In a preferred embodiment of the present invention, the first component, the second component, and the third component are derived from the same pharmaceutical raw material variety or the same harvesting batch to improve the consistency among the components. However, the present invention is not limited thereto. In at least one validated raw material model, raw materials from different batches can also yield a product combination with synergistic effects, as long as the timing described in the present invention is followed.

[0048] In a preferred embodiment of the present invention, the preparation completion time T0 is recorded after step a) is completed. Step b) is preferably completed within 30 minutes from T0. The inventors have found that within this efficacious window, the antioxidant activity of polyphenols in the second component can be preserved to the greatest extent, and a synergistic effect can be formed with the first component.

[0049] In a preferred embodiment of the present invention, the average particle size of the extracellular vesicles in the first component obtained in step a) is 50-200 nm, and the polydispersity index (PDI) is <0.3.

[0050] As a preferred embodiment particularly suitable for on-site scenarios, the low-shear solid-liquid separation method involves filtration using 6-10 layers of 18-22 mesh / cm² medical degreased gauze. The gauze is placed at a 30°-60° angle, driven by the material's own gravity, and filtration is completed within 3-5 minutes for every 100 mL of raw material. The inventors have found that this method achieves solid-liquid separation under mild conditions, effectively protecting the integrity of extracellular vesicle structures. In addition, tangential flow filtration with a molecular weight cutoff of 300-500 kDa and a transmembrane pressure ≤0.3 bar, or staged low-speed centrifugation combined with 50-200 μm sieve filtration, are also optional embodiments of this invention.

[0051] This invention also provides a fresh herb combination kit, comprising independently packaged first component containers, second component containers, and third component containers, as well as a matching information carrier. The information carrier records at least the preparation completion time T0. Preferably, the first component container is provided with a time indicator unit associated with T0 to prompt the operator to adhere to the time window in subsequent storage or compounding steps.

[0052] The method of this invention is applicable to a variety of fresh medicinal plants (such as ginseng and ginger) and fungi (such as Ganoderma lucidum). Depending on the physical characteristics of different raw materials (such as the fact that Ganoderma lucidum slurry contains fine chitin and is prone to clogging), a low-shear separation method can be flexibly selected (such as tangential flow filtration being preferred for Ganoderma lucidum).

[0053] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0054] First, the preparation of experimental materials and testing methods: Extracellular vesicle particle size and concentration: determined using a nanoparticle tracking analyzer (NTA) model ZetaViewPMX-120.

[0055] Rupture rate: Determined using the SYTOX Green nucleic acid dye method. Specific procedure: Add 100 μL of sample to a 96-well plate, add SYTOX Green dye to a final concentration of 1 μM, incubate in the dark for 5 minutes, and detect fluorescence intensity using a microplate reader (excitation wavelength 488 nm, emission wavelength 525 nm). Add Triton X-100 to the same sample to a final concentration of 1%, mix thoroughly to ensure complete vesicle rupture, and detect fluorescence intensity again as a 100% rupture control. Rupture rate (%) = (sample fluorescence intensity - background fluorescence intensity) / (fluorescence intensity after complete rupture - background fluorescence intensity) × 100%.

[0056] Recovery rate: determined using NTA. Take the pretreated homogenate and measure the particle concentration C1 and volume V1. Calculate the total number of particles N1 = C1 × V1. Take the first component obtained after separation in step a), and measure the particle concentration C2 and volume V2. Calculate the total number of particles N2 = C2 × V2. Recovery rate (%) = N2 / N1 × 100%.

[0057] Synergistic effect evaluation: Human skin fibroblasts (HSF) and human colon cancer cells (Caco-2) were used as models, and the MTT assay was used to determine the proliferative activity of the samples. HSF, encompassing skin, connective tissue, and stromal cells, is involved in tissue repair, anti-aging, and regenerative medicine, and can characterize the regenerative capacity of peripheral tissues. Caco-2 cells represent the intestinal epithelial barrier and absorptive cells, involved in intestinal health, absorptive function, and intestinal barrier repair, and characterize the health status of the intestinal system. The combination index (CI) was calculated using the Chou-Talalay method, with CI < 1 indicating synergy, CI = 1 indicating additive effects, and CI > 1 indicating antagonism. All experiments were repeated three times, and data are expressed as mean ± standard deviation.

[0058] Total polyphenol content determination: The Folin-Ciocalteu method was used, with gallic acid as the standard.

[0059] Example 1 This embodiment provides a method for preparing graded fresh ginseng, the preparation steps of which are as follows: Raw material: 50g of fresh ginseng, processed within 4 hours after harvesting, with a moisture content of 78%.

[0060] Step a): After chopping, add 3 times the volume of pre-cooled PBS (Phosphate-Buffered Saline) to homogenize. Centrifuge the homogenate at 500g for 10 minutes and then at 3000g for 15 minutes to remove large particles. Filter the supernatant through 8 layers of 20-mesh / cm² medical degreased gauze at a 45° angle using gravity to obtain 25 mL of the first fraction. Record T0. Analysis showed: average particle size 105.3 nm, PDI 0.21, concentration 2.1 × 10⁻⁶. 10 Particles / mL, recovery rate 93%, breakage rate 2%.

[0061] Step b): Place the residue filtered in step a) into a pressing device and press at 0.05 MPa for 30 seconds to obtain 20 mL of the second component, followed by T0+18 minutes. Analysis showed that the total polyphenol content was 2.8 mg / mL.

[0062] Step c): Add 400mL of water to the pressed residue, simmer over low heat for 35 minutes, filter to obtain 150mL of the third component, and simmer for T0+63 minutes.

[0063] Synergistic effect: The three components were mixed in a volume ratio of 1:3:6 and their activity was evaluated using human skin fibroblasts (HSF) and human colon cancer cells (Caco-2) as models. The combination index (CI) was calculated using the Chou-Talalay method. The CI values ​​were 0.65 and 0.71 in the two cell lines, respectively, both showing a significant synergistic effect.

[0064] Example 2 To verify the decisive role of preparation timing in the synergistic effect, this embodiment conducts time-series synergistic verification across batches of raw materials, and makes the following comparisons: Group A (same batch, ascending order): 50g of fresh ginseng from the same batch, prepared according to the method in Example 1, CI=0.65.

[0065] Group B (cross-batch ascending sequence): The first component comes from batch 1 (harvested on the day of harvest, moisture content 78%), the second component comes from batch 2 (24 hours after harvest, stored at 4℃, moisture content 72%), and the third component comes from batch 3 (48 hours after harvest, stored at 4℃, moisture content 70%), strictly following the sequence of this invention, CI=0.72.

[0066] Group C (same batch, reverse order): 50g of fresh ginseng from the same batch, first boiled in water for 30 minutes, cooled and then extracted for EVs, CI=1.35.

[0067] Conclusion: The preparation sequence is the key technology for achieving synergistic effects, while batch consistency is a preferred condition rather than a necessary limitation. Even when there are differences in the moisture content of the raw materials (72%-78%), significant synergistic effects (CI<0.8) can still be obtained as long as the preparation sequence of this invention is followed.

[0068] Example 3 This embodiment verifies the critical value of the aging window. After the preparation of the first component, step b) is started and the preparation of the second component is completed after different delay times. Each group starts step b) at the corresponding delayed time point and completes the preparation of the second component under the same pressing conditions. After the three components are jointly evaluated, the CI values ​​are shown in Table 1. Table 1 CI Value Detection Table

[0069] The results showed that 30 minutes was the critical time boundary for ensuring a significant synergistic effect (CI<0.85), the synergistic effect weakened to near the additive level at 45 minutes, and turned into antagonism at 60 minutes.

[0070] Example 4 This embodiment verifies the timing violation of the EV extraction process after hot extraction.

[0071] The control group was treated in the order of Example 1; the non-compliant group was first boiled in water for 30 minutes, cooled, and then EVs were extracted, as shown in Table 2. Table 2 Timing Violation Verification Table

[0072] Conclusion: Reversal of the time sequence leads to the destruction of vesicle structure and complete loss of synergistic effect.

[0073] Example 5 This embodiment verifies the effect of the gauze filtration parameter range on the quality of the first component.

[0074] With other conditions fixed, the effect of gauze filtration parameters on the quality of the first component was investigated, as shown in Table 3: Table 3. Influence of Gauze Filtration Parameter Range on the Quality of the First Component

[0075] The results show that the optimal mass of the first component can be obtained within the preferred parameter range of the present invention (6-10 layers, 30°-60°, gravity drive, 3-5 minutes / 100mL).

[0076] Example 6 This embodiment provides a method for preparing fresh Ganoderma lucidum through grading, using fresh Ganoderma lucidum as a representative of fungi. The steps are as follows: Raw material: 30g of fresh Ganoderma lucidum, processed within 24 hours after harvesting, with a moisture content of 82%.

[0077] Step a): After homogenization, tangential flow filtration was performed (molecular weight cutoff 300 kDa, transmembrane pressure 0.2 bar) to obtain 15 mL of the first fraction. The average particle size was 125.3 nm, PDI 0.24, recovery rate 89%, and breakage rate 3%. T0 was recorded.

[0078] Step b): Press the residue to obtain 12 mL of the second component, T0+15 minutes.

[0079] Step c): Add 300mL of water to the residue and decoct for 30 minutes to obtain 100mL of the third component, T0+55 minutes.

[0080] Synergistic effect: three-component mixture, CI=0.68.

[0081] Because the mycelia and fruiting bodies of Ganoderma lucidum produce a large number of fine chitin fragments after homogenization, this embodiment uses tangential flow filtration instead of the 8-layer gauze filtration of Example 1 to avoid clogging. This avoids the problem of chitin fragments clogging the gauze mesh, preventing filtration from being completed and causing blockage. Tangential flow filtration allows the liquid to flow tangentially at high speed across the membrane surface. Small molecules (including vesicles) permeate vertically through the membrane under pressure, while large molecules and particles (cell debris, chitin) are trapped and carried away by the liquid flow, preventing accumulation on the membrane surface. Moreover, the continuous high-speed liquid flow washing the membrane surface also effectively prevents the accumulation and blockage of fine chitin fragments. By selecting a membrane pore size with a molecular weight cutoff of 300-500 kDa, vesicles and chitin fragments can be efficiently separated under relatively low and controllable pressure, while ensuring a low vesicle breakage rate and a high recovery rate. In this embodiment, the low breakage rate is 3%, and the high recovery rate can reach 89%.

[0082] Example 7 This embodiment provides a method for grading and preparing fresh ginger, using fresh ginger as a representative of other plant types. The steps are as follows: Ingredients: 50g fresh ginger, processed within 12 hours of harvesting, with a moisture content of 85%.

[0083] Step a): After homogenization, the sample was gravity filtered through 8 layers of gauze to obtain 20 mL of the first fraction. The average particle size was 95.6 nm, PDI 0.25, recovery rate 87%, and breakage rate 4%. T0 was recorded.

[0084] Step b): Press the residue to obtain 18 mL of the second component, T0+20 minutes.

[0085] Step c): Add 350mL of water to the residue and decoct for 30 minutes to obtain 120mL of the third component, T0+70 minutes.

[0086] Synergistic effect: three-component mixture, CI=0.75.

[0087] Example 8 This embodiment avoids the impact of path verification method one: firstly, lightly press the vesicles to reduce their quality.

[0088] To verify the destructiveness of any form of pressing prior to step a), the following comparison is made: Group A (the present invention): Fresh ginseng was homogenized directly according to the method of Example 1, and then EVs were separated.

[0089] Group B (Avoidance Attempt): Take fresh ginseng from the same batch, first press it lightly at 0.02 MPa for 5 seconds, collect a small amount of juice and discard it, then homogenize and separate the pressed ginseng into EVs according to step a) of Example 1.

[0090] The recovery rate and rupture rate of EVs in the first component were tested in two groups. The results showed that the recovery rate of group A was 93% and the rupture rate was 2%; the recovery rate of group B dropped sharply to 61% and the rupture rate rose to 19%. The results indicate that any mechanical pressing force applied to the raw material before low-shear solid-liquid separation will cause a large number of EVs to rupture due to damage to the cell structure, which cannot meet the quality requirements of the first component of this invention.

[0091] Example 9 This embodiment demonstrates the second method for bypassing the verification path: the application and synergistic effect verification of enzymatic pretreatment.

[0092] To verify the applicability of the enzymatic hydrolysis method in the timing sequence of this invention, the following experiments were conducted: Take 50g of fresh ginseng, add PBS buffer containing 0.5% pectinase and 0.3% cellulase, and gently stir at 25℃ for 60 minutes for enzymatic hydrolysis. After enzymatic hydrolysis, homogenize, centrifuge at low speed, and filter through gauze according to the same method as in Example 1 to obtain the first component. The EVs showed an average particle size of 110.2nm, a PDI of 0.23, a recovery rate of 88%, and a fragmentation rate of 4%. Subsequent steps b) and c) were the same as in Example 1. After mixing the three components, the CI was 0.68 using HSF cell detection.

[0093] This embodiment demonstrates that enzymatic hydrolysis, as a method of pretreatment for crushing (homogenization after enzymatic hydrolysis), is also applicable to the method of the present invention, and the final product still has a significant synergistic effect (CI<1).

[0094] Example 10 This embodiment compares the synergistic effects of compound co-extraction and single-component fractionation.

[0095] To investigate the impact of different compatibility methods on synergistic effects, the following comparisons were made: Group A (compound co-extraction): 30g of fresh ginseng and 20g of fresh ginger were mixed and prepared using the three-step method described in Example 1. The particle size distribution and concentration of the mixed EVs were measured and compared with those of single-component EVs.

[0096] Group B (single-component extraction followed by mixing): Take 30g of fresh ginseng and 20g of fresh ginger, and prepare them independently according to the method in Example 1 to obtain ginseng EVs, ginseng juice, ginseng decoction, and ginger EVs, ginger juice, and ginger decoction. Then, mix the three components of ginseng with the three components of ginger in a ratio of 3:2.

[0097] The concentration index (CI) values ​​of groups A and B on HSF cells were measured. The results showed that group A (compound co-extraction) had a CI of 0.88, while group B (single-component extraction followed by mixing) had a CI of 0.69. These results indicate that although the compound co-extraction still exhibits some synergistic effect, its effect is significantly lower than that of the combination of single-component preparation followed by mixing. This provides strong experimental support for the preferred "single-component origin preparation - clinic terminal compounding" business model of this invention.

[0098] In application, the pretreatment method of this invention can be flexibly selected according to different application scenarios. For on-site preparation scenarios requiring rapid processing, such as clinics and pharmacies, the physical crushing combined with gauze filtration method described in Example 1 is preferred, as it is simple and quick to operate. For centralized production scenarios, the enzymatic hydrolysis method described in Example 9 can be used, although the processing time is longer, it is more suitable for large-scale production.

[0099] It should be noted that although Example 3 shows that the synergy index (CI) value is still less than 1 (0.95) after a delay of 45 minutes, indicating that a weak synergistic effect still exists, from the perspective of quality control in industrial production, to ensure that "significant synergy" (CI < 0.85) can be stably achieved across different batches and different raw material varieties, those skilled in the art have determined 30 minutes as the critical time window. Beyond 30 minutes, the stability and significance of the synergistic effect will be difficult to guarantee. Data from Example 6 (fresh Ganoderma lucidum CI = 0.68) and Example 7 (fresh ginger CI = 0.75) further demonstrate that, under the timing of this invention, different raw materials can obtain CI values ​​far below 0.85 within a 30-minute window, proving the robustness of this window across different varieties.

[0100] It should be noted that the core of the method described in this invention lies in the timing control of steps a), b), and c). Any unnecessary processing steps inserted before, during, or after the invention (e.g., but not limited to, ultrasound-assisted extraction, microwave pretreatment, pulsed electric field treatment, low-temperature plasma treatment, etc.) fall within the scope of protection of this invention, as long as they still include the three core steps defined in this invention and do not change their order. Even if the inserted steps may have a certain impact on the yield or activity of a certain component, as long as the final product still contains the component obtained through the three core steps and the timing relationship of the three steps is not changed, it constitutes an implementation of the technical solution of this invention. Examples 8 and 9 have demonstrated that any application of mechanical pressing force to the raw material or a change in the pretreatment method before step a) will lead to a significant decrease in the quality of extracellular vesicles, and maintaining the core timing described in this invention is the key to achieving the synergistic effect.

[0101] It should be noted that the use of the word "comprising" in the method of this invention is used to limit the scope of protection of this invention. The core of the fresh medicinal raw material graded preparation method of this invention lies in the timing control of the three preparation steps of the first component, the second component, and the third component. At the same time, the specification has clearly stated the principle of infringement determination that any inserted step does not change the core timing of this invention.

[0102] This invention utilizes a sequence of vesicle extraction, followed by juice extraction and then thermal extraction. Examples demonstrate that this sequence is crucial for achieving the synergistic effect of multiple components. Example 4 shows that reversing the sequence leads to a sharp drop in vesicle recovery rate from 93% to 42% and a deterioration in the CI value from 0.65 to 1.35, proving the technical necessity of this sequence. This invention includes a three-step fractional preparation method for the first, second, and third components, a two-step fractional preparation method for the first and second components, as well as a combination kit and on-site preparation system. This provides multi-layered protection from the core method to commercial applications. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of this invention, or "two-step circumvention" strategies may be employed. The scope of this invention is defined by the appended claims and their equivalents.

[0103] The present invention demonstrates through the above embodiments that cross-batch raw materials still exhibit a synergistic effect under the correct timing (CI=0.72), while the same batch of raw materials loses its synergy under the incorrect timing (CI=1.35), strongly proving the technical necessity of the preparation timing itself. Example 2 further clarifies that even when there are differences in the moisture content of the raw materials (72%-78%), a significant synergistic effect can still be obtained as long as the timing of the present invention is followed. Example 10 demonstrates that the preferred single-component fractionation followed by mixing mode of the present invention is significantly superior to compound co-extraction in terms of synergistic effect, providing data support for the legitimacy of the business model.

[0104] The above embodiments identify 30 minutes as the critical time window and clarify the quality indicators (fracture rate ≤5%, recovery rate ≥85%) and optimal parameter range for the low-shear separation method, providing operable and detectable specific boundaries for technology implementation. Furthermore, the specifications clearly define the detection methods for fracture rate and recovery rate, ensuring the repeatability of the technical solution and the operability of infringement determination. Examples 6 and 7 further demonstrate that the 30-minute window is robust across different raw material varieties, with CI = 0.68 for fresh Ganoderma lucidum and CI = 0.75 for fresh ginger.

[0105] The timing control of this invention not only maximizes the preservation of native extracellular vesicles and heat-sensitive small molecule active substances in the raw materials, but also generates a unique transformation product of decoction through the third-step thermal extraction, providing a more complete spectrum of active components than any single processing method. The preferred on-site preparation method and clearly defined time window make this method suitable not only for on-site preparation in clinics, but also for providing high-quality starting materials for cryopreservation of vesicles and juice, perfectly supporting a new business model of on-site preparation, cold chain distribution, and end-user compounding. The first component prepared by this method can be directly used in the cryopreservation method for vesicles, the second component can be directly used in the cryopreservation method for pressed juice, and all three components maintain excellent compounding compatibility after preservation.

[0106] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A method for graded preparation of fresh medicinal raw materials, characterized in that, Includes the following steps: The first component preparation steps are as follows: After pretreatment of fresh raw materials by crushing, homogenizing or enzymatically hydrolyzing the cell walls, a liquid component rich in extracellular vesicles is obtained by low-shear solid-liquid separation as the first component. Second component preparation step: The residue obtained after separation in the first component preparation step is mechanically pressed to extract juice, and the pressed juice is used as the second component. Third component preparation step: The solid residue obtained after mechanical pressing and juice extraction in the second component preparation step is subjected to thermal extraction with water to obtain the extract as the third component; The second component preparation step must not begin before the first component preparation step is completed, and the third component preparation step must not begin before the second component preparation step is completed.

2. The method for graded preparation of fresh medicinal raw materials according to claim 1, characterized in that, When the fresh raw material enters the preparation of the first component, it meets the following conditions: moisture content ≥ 70 wt% and satisfies any one of the following conditions: The preparation process begins within 24 hours of harvesting. After harvesting, freeze at -80℃ and thaw at 4℃ before use; Although it was more than 24 hours after harvest, the fresh raw material was tested and found to have an extracellular vesicle recovery rate of no less than 85%, a rupture rate of no more than 5%, and a moisture content of ≥70wt%. Furthermore, the fresh raw materials have not undergone dehydration heat treatment or freeze-drying treatment at temperatures above 50°C.

3. The method for graded preparation of fresh medicinal raw materials according to claim 2, characterized in that, In the first component preparation step, the low-shear solid-liquid separation method ensures that the rupture rate of extracellular vesicles in the obtained first component is ≤5% and the recovery rate is ≥85%; the rupture rate is determined by the SYTOX Green nucleic acid dye method and the recovery rate is determined by a nanoparticle tracking analyzer.

4. The method for graded preparation of fresh medicinal raw materials according to claim 3, characterized in that, The low-shear solid-liquid separation method is selected from any of the following: 6-10 layers, 18-22 mesh / cm 2 Medical degreased gauze filtration, wherein the gauze is placed at an angle of 30°-60° and driven by the gravity of the material itself, and each 100mL of raw material liquid is filtered within 3-5 minutes; Tangential flow filtration with a molecular weight cutoff of 300-500 kDa, and a transmembrane pressure ≤0.3 bar; Graded low-speed centrifugation combined with 50-200μm sieve filtration.

5. The method for graded preparation of fresh medicinal raw materials according to claim 4, characterized in that, The parameters for the graded low-speed centrifugation are as follows: at 4°C, centrifuge at 300-500g for 10-15 minutes to remove cell debris, centrifuge at 1000-3000g for 15-20 minutes to remove larger vesicles, and centrifuge at 5000-10000g for 20-30 minutes to enrich the target vesicles.

6. The method for graded preparation of fresh medicinal raw materials according to claim 1, characterized in that, After the preparation of the first component is completed, the time T0 when the preparation of the first component is completed is recorded, and the preparation of the second component is completed within 30 minutes from T0.

7. The method for graded preparation of fresh medicinal raw materials according to claim 1, characterized in that, The average particle size of extracellular vesicles in the first component is 50-200 nm, and the polydispersity index (PDI) is <0.

3.

8. A method for graded preparation of fresh medicinal raw materials, characterized in that, This includes the following steps that must be performed in strict order: a) Preparation steps of the first component: After pretreatment of fresh raw materials by crushing, homogenizing or enzymatically hydrolyzing the cell walls, a liquid component rich in extracellular vesicles is obtained as the first component through low-shear solid-liquid separation; b) Preparation of the second component: The residue obtained after separation in step a) is mechanically pressed to extract juice, and the pressed juice is used as the second component; In this process, the solid residue after step b) is discarded or used for other purposes, and the thermal extraction step is not performed.

9. A fresh herb combination kit, characterized in that, It contains the following three components, which are individually packaged and provided in an associated form: First component container: contains a liquid component rich in extracellular vesicles prepared by the method for graded preparation of fresh medicinal raw materials as described in any one of claims 1-7; The second component container contains the pressed juice component obtained by the graded preparation method of fresh medicinal raw materials as described in any one of claims 1-7; The third component container contains a hot extract component prepared by the graded preparation method of fresh medicinal raw materials as described in any one of claims 1-7; And a supporting information carrier, wherein the information carrier records at least the preparation completion time T0 of the first component, and the container of the first component is provided with a time indication unit associated with T0.

10. A field preparation system for implementing the graded preparation method for fresh medicinal raw materials as described in any one of claims 1-7, characterized in that, It includes, in sequence, a raw material pretreatment unit, a low-shear solid-liquid separation unit, a mechanical pressing and juice extraction unit, and optionally a thermal extraction unit.

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