Method for extracting extracellular vesicles from dried traditional Chinese medicine product

Abstract

The embodiment of the invention discloses a method for extracting extracellular vesicles from a traditional Chinese medicine dry product. The method comprises the following steps: taking a dry traditional Chinese medicine product as a raw material, and sequentially performing crushing, soaking, first-order enzymolysis, wall breaking, differential centrifugation, second-order enzymolysis, high-speed centrifugation, ultracentrifugation and ultrafiltration enrichment to obtain the extracellular vesicles. A two-step enzymolysis method is adopted in the enzymolysis process, impurities are removed to the maximum extent in a sequence difference enzymolysis mode, and the structure and function stability of the exosome are protected. According to the invention, the differential centrifugation and the enzymolysis method are alternately used, so that impurities with different sizes and different properties can be removed more thoroughly. According to the method, the cost is strictly controlled, the exosome purification efficiency is relatively high, the concentration of the obtained dry exosome is relatively high, and the operability is extremely high.

Description

Technical Field

[0001] The embodiments of the present invention relate to the technical field of extracellular vesicle preparation, and specifically to a method for extracting extracellular vesicles from dried traditional Chinese medicine. Background Art

[0002] Extracellular vesicles (EVs) are cellular products that are metabolized and transported through cells, containing a rich array of substances. These substances include nucleic acids, proteins, lipids, and various other molecular components. Some of these components have been shown to be unique to their specific organisms, making them of high research value and currently being considered as key mediators of intercellular communication. As a type of EV, plant EVs possess the unique targeting and safety characteristics of natural products, while also offering the advantages of low cost and ease of acquisition.

[0003] Traditional Chinese Medicine (TCM) is a medical science that has stood the test of thousands of years. TCM is used in a variety of ways, including fumigation, direct administration, and concentrated pills. TCM materials are also processed in numerous ways, enjoying widespread clinical application. The most important way to use TCM materials is by decocting them from dried materials into decoctions. Processing and decocting are also effective ways to concentrate the medicinal ingredients. Experiments have demonstrated that exosomes can be extracted from dried materials, suggesting that these highly stable exosomes may contain many of the components of TCM and hold great research value.

[0004] Current plant exosome extraction techniques largely follow the principles of animal exosomes. Fresh plant exosomes are typically extracted by disrupting their cell walls. However, the extraction results vary depending on the plant species, and even with the same method, personalized, combined, and cost-effective extraction methods are needed for different plant species. Medicinal plants hold immense research value in Traditional Chinese Medicine. While dried plants are typically used as fresh extracts, membrane-bound exosomes can be obtained during the extraction process. However, these extracts often contain significant amounts of impurities, hindering subsequent experiments and analysis. Key challenges remain: 1. Exosomes from dried plants contain significant impurities; 2. Conventional extraction methods for dried exosomes may yield lower concentrations than fresh exosomes; and 3. Impurities in dried exosomes are more challenging to handle than those in fresh plants. Because the preparation of dried exosomes differs from that of fresh plants, a more systematic and targeted extraction approach is required. Furthermore, while maintaining purity, increasing yield is crucial. Summary of the Invention

[0005] To address the current problems encountered in the extraction of plant exosomes, the present invention provides a method for extracting extracellular vesicles from dried traditional Chinese medicine. This method has high extraction purity and higher efficiency than traditional impurity removal methods. Compared with size exclusion and ion chromatography, the cost can be controlled lower. In terms of extraction effect, it has a complete and stable exosome structure and concentration.

[0006] In order to achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] A method for extracting extracellular vesicles from dried Chinese medicine products comprises the following steps: using the dried Chinese medicine products as raw materials, sequentially subjecting the raw materials to crushing, soaking, first-order enzymatic hydrolysis, cell wall breaking, differential centrifugation, second-order enzymatic hydrolysis, high-speed centrifugation, ultracentrifugation, and ultrafiltration enrichment to obtain the extracellular vesicles.

[0008] Furthermore, the particle size of the crushed medicinal material is 48 μm-150 μm.

[0009] Furthermore, the soaking conditions are as follows: the crushed medicinal materials are added to a phosphate buffer solution at a liquid-to-material ratio of 1:10-50 and soaked for 30 minutes.

[0010] Furthermore, based on 10g-50g of dried Chinese medicine, the conditions for the first enzymatic hydrolysis are: adding 0.15g-0.25g of cellulase with an enzyme activity of 3500u / g, and enzymatic hydrolysis at 25°C for 4-12h; then adding 0.01g-0.025g of lignin peroxidase with an enzyme activity of 100000u / g, and enzymatic hydrolysis at 37°C for 4-12h; finally, adding 0.5g-1g of cellobiase with an enzyme activity of 200u / g, and enzymatic hydrolysis at 37°C for 4-12h.

[0011] Furthermore, the conditions for the cell wall breaking are: cell wall breaking power is 1000W, rotation speed is 36000rpm, and temperature is maintained at 4°C.

[0012] Furthermore, the conditions of the differential centrifugation are: 500xg, 30 min; 5000xg, 40-60 min; 10000xg, 90-120 min.

[0013] Furthermore, based on 10g-50g of dried Chinese medicine, the conditions for the second enzymatic hydrolysis are: adding 0.01g of pectinase with an enzyme activity of 18000u / g, and incubating at 25-37°C for 2-4h; then adding 0.015g-0.025g of xylanase with an enzyme activity of 110000u / g, and incubating at 25-37°C for 2-4h; then adding 0.15g-0.25g of β-glucanase with an enzyme activity of 12000u / g, and incubating at 25-37°C for 2-4h; finally, adding 0.15g-0.25g of α-amylase with an enzyme activity of 10000u / g, and incubating at 25-37°C for 2-4h.

[0014] Furthermore, the conditions of the high-speed centrifugation are: 10,000 x g, 1-2 h.

[0015] Furthermore, the ultracentrifugation conditions are: 150,000×g, 1.5 h-2 h.

[0016] Furthermore, the molecular weight cut-off of the ultrafiltration membrane used in the ultrafiltration is 10-100 kDa.

[0017] The embodiments of the present invention have the following advantages:

[0018] (1) The present invention adopts the method of crushing the dry Chinese medicine to extract the exosomes from the dry product. Compared with other methods, the exosomes extracted by this method are more comprehensive and can better characterize the biological activity of the dry Chinese medicine, which is conducive to downstream multi-faceted verification.

[0019] (2) The present invention adopts a two-step enzymatic hydrolysis method during the enzymatic hydrolysis process, which removes impurities to the maximum extent through sequential differential enzymatic hydrolysis and protects the structural and functional stability of exosomes.

[0020] (3) The present invention uses differential centrifugation and enzymatic hydrolysis alternately to more thoroughly remove impurities of different sizes and properties.

[0021] (4) The present invention has strict cost control, high efficiency in purifying exosomes, high concentration of dry exosomes, and strong operability. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0023] Figure 1 The particle size distribution diagram (a) and transmission electron microscopy image (b) of the extracellular vesicles extracted in Example 1;

[0024] Figure 2 The particle size distribution diagram (a) and transmission electron microscopy image (b) of the rhubarb-derived extracellular vesicles prepared in Comparative Example 1;

[0025] Figure 3 (a) and (b) the particle size distribution of extracellular vesicles derived from Bupleurum chinense in Example 2;

[0026] Figure 4 The particle size distribution diagram (a) and transmission electron micrograph (b) of the extracellular vesicles obtained from the fresh extract of Bupleurum chinense in Comparative Example 2 are shown;

[0027] Figure 5 The particle size distribution diagram (a) and transmission electron micrograph (b) of the extracellular vesicles obtained from the extraction method of Bupleurum chinense with different liquid-to-solid ratios in Comparative Example 3 are shown;

[0028] Figure 6 The particle size distribution diagram (a) and transmission electron microscopy image (b) of extracellular vesicles obtained from Bupleurum chinense by ultrasonic nanofiltration in Comparative Example 4;

[0029] Figure 7 (a) and (b) the particle size distribution of extracellular vesicles derived from red peony root in Example 3;

[0030] Figure 8 The particle size distribution diagram (a) and transmission electron micrograph (b) of the extracellular vesicles derived from the whole herb of Scutellaria barbata in Example 4 are shown;

[0031] Figure 9 The particle size distribution diagram (a) and transmission electron micrograph (b) of the extracellular vesicles derived from the dry product of Compound Yinchen Wuling Powder in Example 5;

[0032] Figure 10 This is a transmission electron microscopic comparison of extracellular vesicles derived from the dry product of Compound Banxia Xiexin Decoction in Example 6 and Comparative Example 5;

[0033] Figure 11 This is a comparison of the particle size distribution of extracellular vesicles derived from dried Cnidium monnieri seeds in Example 7 and Comparative Example 6;

[0034] Figure 12 This is the particle size distribution diagram of the extracellular vesicles derived from the dried product of Radix Pseudostellariae in Example 8;

[0035] Figure 13 This is the particle size distribution diagram of the extracellular vesicles derived from the dried Codonopsis pilosula in comparative example 7;

[0036] Figure 14 This is the particle size distribution diagram of extracellular vesicles of dry Artemisia annua at different liquid-to-solid ratios in Example 9;

[0037] Figure 15 This is the particle size distribution diagram of the extracellular vesicles derived from the dry product of Smilax glabra in Example 10 and the extracellular vesicles derived from the dry product of Toosendan fruit in Example 11. DETAILED DESCRIPTION

[0038] The following describes the implementation of the present invention using specific embodiments. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. Obviously, the embodiments described are only a portion of the present invention, not all of it. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0039] In the following examples, unless otherwise specified, all methods are conventional methods; the reagents and materials described, unless otherwise specified, can be obtained from commercial sources.

[0040] Example 1

[0041] This example provides a method for extracting extracellular vesicles from dried rhubarb:

[0042] S1. Remove the rhubarb dust using a vacuum cleaner and electrostatic methods.

[0043] S2. Use a traditional Chinese medicine grinder to grind for 0.5 min three times in a row to make the output particle size about 100 μm.

[0044] S3, rhubarb powder 25g is added to phosphate buffer solution 500ml, after soaking 30min, add cellulase 0.15g (enzyme activity 3500u / g) and put into shaking table, be set to 100rpm, the time is 12h, and temperature is at 25 ℃, lignin catalase 0.01g (enzyme activity 100000u / g) and cellobiase 0.5g (enzyme activity 200u / g) put into shaking table, be set to 100rpm, respectively 4h, temperature is controlled at 37 ℃, rotating speed 100rpm.The soaking liquid obtained is utilized wall breaking machine to carry out broken wall process, and broken wall power is 1000w, and rotating speed is 36000rpm, and temperature remains on 4 ℃, and broken wall time is three minutes, runs three times.

[0045] S4. The wall-broken rhubarb extract was passed through a 300-mesh sieve and subjected to differential centrifugation at 500 x g for 30 min, 5000 x g for 40 min, and 10000 x g for 120 min to remove impurities with different sedimentation coefficients.

[0046] S5. Add pectinase 0.01g (enzyme activity 18000u / g), xylanase 0.015g-0.02g (enzyme activity 110000u / g), β-glucanase 0.15g-0.2g (enzyme activity 12000u / g), and α-amylase 0.15g (enzyme activity 10000u / g) to the obtained liquid in accordance with the type of extracted Chinese medicinal materials, incubate for 2h each, control the temperature at 30°C, and the rotation speed at 100rpm.

[0047] S6. The obtained liquid was centrifuged at 10,000 x g for 1 h and filtered through a 300-mesh filter.

[0048] S7. The liquid obtained in S6 was vacuum filtered and then enriched by ultracentrifugation. The ultracentrifugation conditions were 150,000 x g for 2 h.

[0049] S8. Resuspend the ultracentrifuged precipitate in phosphate buffer and ultrafilter it. The ultrafiltration conditions are: 50 ml outer tube, 15 ml inner tube, 100 kDa ultrafiltration tube, ultrafiltration process is 5000 x g, about 10 minutes, and the temperature is maintained at 4°C.

[0050] The rhubarb exosomes were characterized by nanoparticle size analysis, such as Figure 1 As shown, the average particle size of the particles in the extract was 110.4 nm, and the concentration was 2.0E+10 particles / mL. Transmission electron microscopy revealed a distinct saucer-like double-layer membrane structure in the extract.

[0051] Comparative Example 1

[0052] This comparative example provides a method for extracting extracellular vesicles from dried rhubarb, which is carried out according to the extraction method of fresh rhubarb:

[0053] S1. Soak the dried rhubarb in a phosphate solution at a ratio of 1:20 for 0.5 h.

[0054] S2. The rhubarb was subjected to a wall-breaking treatment 3 times for 3 minutes each time, with a wall-breaking power of 1000W, a rotation speed of 36000rpm, and a temperature maintained at 4°C.

[0055] S3. The broken medicinal material liquid was passed through 200-mesh and 300-mesh sieves respectively, and the extract was subjected to differential centrifugation at 500×g for 30 min, 5000×g for 40 min, and 10000×g for 120 min to remove impurities with different sedimentation coefficients.

[0056] S4. The liquid obtained from S4 was vacuum filtered and then enriched by ultracentrifugation at 150,000 x g for 1.5 to 2 hours. The obtained precipitate was resuspended in phosphate buffer.

[0057] The rhubarb extract extracted by traditional fresh product wall breaking method was characterized and analyzed by nanoparticle size analyzer, such as Figure 2 As shown in the results of nanoparticle size analysis, the average particle size of the extract was 111.7 nm, and the concentration was 1.3E+10 particles / mL. Under transmission electron microscopy, it was observed that the extract had a clear saucer-like double-layer membrane structure. Figure 2 (a)(b).

[0058] Compared with the two extraction methods, the exosomes of dried rhubarb extracted using this scheme are far superior to the second scheme in terms of concentration and concentration of particle size distribution range. The concentration of Example 1 is higher than that of Comparative Example 1, and the peak shape in the particle size distribution diagram is more concentrated, and the vesicle distribution and structural integrity under the field of view of the electron microscope image are better.

[0059] In summary, this invention scheme has better effect among the extraction schemes of dry rhubarb products.

[0060] Example 2

[0061] This example provides a method for extracting extracellular vesicles from dried Bupleurum chinense:

[0062] S1. Use a vacuum cleaner and electrostatic method to remove dust from the Bupleurum chinense.

[0063] S2. Use a traditional Chinese medicine grinder to crush the root for 40 seconds three times. Since dried Bupleurum chinense is mainly composed of roots, and dried Rhubarb is mainly composed of tubers, the crushing time should be increased.

[0064] S3, Radix Bupleuri powder 50g is added phosphate buffer solution 500ml, after soaking 30min, add cellulase 0.15g (enzyme 3500u / g alive), be set to 100rpm, 8h, temperature is controlled at 25 ℃, lignin catalase 0.01g (enzyme 100000u / g alive) and cellobiase 0.5g (enzyme 200u / g alive), be set to 100rpm, 8h respectively, temperature is controlled at 37 ℃, the soak liquor that obtains is carried out broken wall process, and the broken wall time is three minutes, operates three times.Because the Radix Bupleuri dry product fiber is more, therefore increase the enzymolysis treatment time for the first time.

[0065] S4. The liquid extracted from the broken Bupleurum chinense was passed through 200-mesh and 300-mesh sieves, respectively, and the extract was subjected to differential centrifugation at 500×g for 30 min, 5000×g for 40 min, and 10000×g for 120 min to remove impurities with different sedimentation coefficients.

[0066] S5. Add 0.01 g pectinase (enzyme activity 18,000 u / g), 0.02 g xylanase (110,000 u / g), 0.2 g β-glucanase (12,000 u / g), and 0.15 g α-amylase (10,000 u / g) to the obtained liquid according to the type of extracted Chinese medicinal materials, and incubate for 2 hours each. Control the temperature at 30°C and the rotation speed at 100 rpm.

[0067] S6. The obtained liquid was centrifuged at 10,000 x g for 1 h and filtered through a 300-mesh filter.

[0068] S7. The liquid obtained in S6 was vacuum filtered and then enriched by ultracentrifugation at 150,000 x g for 2 h.

[0069] S8. Resuspend the precipitate obtained by ultracentrifugation in phosphate buffer and perform ultrafiltration.

[0070] The extract of Bupleurum chinense was characterized by nanoparticle size analyzer. The results are as follows: Figure 3 As shown in Figure 2, the average particle size of the particles in the extract is 132nm and the concentration is 4.7E+11Particles / mL. Under transmission electron microscopy, it was observed that the extract had a clear saucer-like double-layer membrane structure and abundant aggregation. Figure 3 (a)(b).

[0071] Comparative Example 2

[0072] This comparative example provides a method for extracting extracellular vesicles from dried Bupleurum chinense, which is carried out according to the extraction method of fresh product:

[0073] S1. Soak 50 g of dried Bupleurum chinense in 50 ml of phosphate solution at a ratio of 1:10 for 0.5 h.

[0074] S2. Crush the wall of the dried Bupleurum chinense three times for 3 minutes each time.

[0075] S3. The wall-broken medicinal material liquid is subjected to differential centrifugation at 500×g for 30 min, 5000×g for 40 min, and 10000×g for 120 min.

[0076] S4. The broken medicinal material liquid was passed through 200-mesh and 300-mesh sieves respectively, and the extract was subjected to differential centrifugation at 500×g for 30 min, 5000×g for 40 min, and 10000×g for 2 h to remove impurities with different sedimentation coefficients.

[0077] S5. The liquid obtained in S4 was vacuum filtered and then enriched by ultracentrifugation at 150,000 x g for 2 h. The obtained precipitate was resuspended in phosphate buffer.

[0078] The Bupleurum chinense extract extracted by the traditional fresh product wall breaking method was characterized and analyzed by nanoparticle size analyzer.

[0079] The extract of Bupleurum chinense was characterized by nanoparticle size analyzer. The results are as follows Figure 4 As shown in Figure 2, the average particle size of the particles in the extract is 145.6 nm and the concentration is 4.0E+11 particles / mL. Under transmission electron microscopy, it was observed that the extract had a clear saucer-like double-layer membrane structure. Figure 4 (a)(b).

[0080] Comparative Example 3

[0081] This comparative example provides a method for extracting extracellular vesicles from dried Bupleurum chinense. The difference between the comparative example and Example 2 is that the liquid-to-solid ratio is 1:20, that is, in step S3, 50 g of Bupleurum chinense powder is added to 100 ml of phosphate buffer solution.

[0082] The extract of Bupleurum chinense was characterized by nanoparticle size analyzer. The results are as follows Figure 5 As shown, the average particle size of the particles in the extract was 126.1 nm, and the concentration was 7.2E+9 particles / mL. Under transmission electron microscopy, a clear saucer-like double-layer membrane structure was observed in the extract.

[0083] Extraction of Bupleurum chinense dry product with different liquid-to-mass ratios

[0084] S1. Use a vacuum cleaner and electrostatic method to remove dust from the Bupleurum chinense.

[0085] S2. Weigh Bupleurum chinense at a liquid-to-solid ratio of 1:20 and crush using a traditional Chinese medicine grinder for 40 seconds three times. Dried Bupleurum chinense is primarily root, while dried rhubarb is primarily tuber, so increase the crushing time.

[0086] S3, Radix Bupleuri powder 50g is added phosphate buffer solution 1000ml, after soaking 30min, add the cellulase 0.25g (enzyme 3500u / g alive) of configuration, 18h, temperature is set at 25 ℃, rotating speed 100rpm lignin peroxidase 0.01g (enzyme 100000u / g alive) and cellobiase 1g (enzyme 200u / g alive), 8h temperature is controlled at 37 ℃ respectively, rotating speed 100rpm is set the soak liquor that obtains is carried out broken wall process, broken wall time is three minutes, operates three times.Because Radix Bupleuri dry product fiber is more, therefore increase the enzymolysis treatment time for the first time.

[0087] S4. The liquid extracted from the broken Bupleurum chinense was passed through a 200-mesh sieve and a 300-mesh sieve, respectively, and the extract was subjected to differential centrifugation at 500 x g for 30 minutes, 5000 x g for 40 minutes, and 10000 x g for 2 hours to remove impurities with different sedimentation coefficients. Because the sedimentation coefficient of the impurity particles in Bupleurum chinense is high at a centrifugal force of 10000 x g, the centrifugation time was extended.

[0088] S5. Add 0.01 g pectinase (enzyme activity 18,000 u / g), 0.02 g xylanase (110,000 u / g), 0.2 g β-glucanase (12,000 u / g), and 0.15 g α-amylase (10,000 u / g) to the obtained liquid in accordance with the type of extracted Chinese medicinal materials, incubate for 2 h each, control the temperature at 30°C, and the rotation speed at 100 rpm.

[0089] S6. The obtained liquid was centrifuged at 10,000 x g for 1 h and filtered through a 300-mesh filter.

[0090] S7. The liquid obtained in S6 was vacuum filtered and then enriched by ultracentrifugation at 150,000 x g for 2 h.

[0091] S8. Resuspend the precipitate obtained by ultracentrifugation in phosphate buffer and perform ultrafiltration.

[0092] The extract of Bupleurum chinense was characterized by nanoparticle size analyzer. The results are as follows Figure 5 As shown in Figure 2, the average particle size of the particles in the extract is 126.1 nm and the concentration is 7.2E+9 particles / mL. Under transmission electron microscopy, it was observed that the extract had a clear saucer-like double-layer membrane structure.Figure 5 (a) (b).

[0093] Comparative Example 4

[0094] The present comparative example provides a method for extracting extracellular vesicles from dried Bupleurum chinense, which adopts ultrasonic nanofiltration method:

[0095] S1, soak the dried Bupleurum chinense in the phosphate solution at a ratio of 1:10 for 0.5 h.

[0096] S2, perform wall breaking treatment on the dried Bupleurum chinense for 3 times, 3 minutes each time.

[0097] S3, centrifuge the wall-broken medicinal material liquid at 500xg for 30 min, 5000xg for 40 min, and 10000xg for 120 min.

[0098] S4, pass the wall-broken medicinal material liquid through 200-mesh and 300-mesh screens, respectively, and centrifuge the extraction liquid at 500xg for 30 min, 5000xg for 40 min, and 10000xg for 2 h to remove impurities of different sedimentation coefficients.

[0099] S5, use exdous 600 to perform ultrasonic nanofiltration to extract exosomes from the liquid obtained in S4. The ultrasonic nanofiltration is based on negative pressure oscillation and chord wave oscillation technology, with a frequency of 5922 Hz and a time of 5 ms. Nanoparticle tracking analysis and electron microscopy are used to characterize the exosomes. Since the ultrasonic nanofiltration method can improve the purity of exosomes, it is compared with the previous scheme for reference.

[0100] The dandelion extract is characterized and analyzed by a nanoparticle size analyzer, and the results are shown in Figure 6 The average particle size of the particles in the extract is 163.6 nm, and the concentration is 5.8E+11 Particles / mL. Under transmission electron microscopy, the extract has a clear tea tray-like double-membrane structure, as shown in Figure 6 (a) (b).

[0101] Example 3

[0102] The present example provides a method for extracting extracellular vesicles from dried Radix Paeoniae Rubra:

[0103] S1, use a dust collector and electrostatic method to remove dust from Radix Paeoniae Rubra.

[0104] S2, dry the traditional Chinese medicinal material, weigh the material according to a liquid-to-material ratio of 1:20, and use a traditional Chinese medicine pulverizer to pulverize for 0.5 min, three times in succession.

[0105] S3. Add 25g of red peony root powder to 500ml of phosphate buffer solution, soak for 30min, then add 0.15g of the configured cellulase, for 12h, at 25°C, with a speed set to 100rpm, 0.01g of lignin peroxidase (enzyme activity 100000u / g) and 0.5g of cellobiase (enzyme activity 200u / g), set to 100rpm, for 4h respectively, and control the temperature at 37°C. The obtained soaking solution is subjected to wall-breaking treatment with a wall-breaking time of three minutes, and run three times.

[0106] S4. The broken red peony root extract was passed through a 300-mesh sieve and subjected to differential centrifugation at 500 x g for 30 minutes, 5000 x g for 60 minutes, and 10000 x g for 1.5 hours to remove impurities with different sedimentation coefficients. Because the sedimentation coefficient of impurity particles in red peony root is higher at 5000 x g, the centrifugation time was extended.

[0107] S5. Add 0.01 g pectinase (enzyme activity 18,000 u / g), 0.02 g xylanase (enzyme activity 110,000 u / g), 0.2 g β-glucanase (enzyme activity 12,000 u / g), and 0.15 g α-amylase (enzyme activity 10,000 u / g) to the obtained liquid according to the type of extracted Chinese medicinal materials, incubate for 2 hours each, control the temperature at 30°C, and the rotation speed at 100 rpm.

[0108] S6. The obtained liquid was centrifuged at 10,000 x g for 1 h and filtered through a 300-mesh filter.

[0109] S7. The liquid obtained in S6 was enriched by ultracentrifugation at 150,000 x g for 2 h.

[0110] S8. Resuspend the precipitate obtained by ultracentrifugation in phosphate buffer and perform ultrafiltration.

[0111] The extracellular vesicles of dried red peony root were characterized by nanoparticle size analyzer. The results are as follows: Figure 7 As shown in Figure 2, the average particle size of the particles in the extract is 150.1 nm and the concentration is 6.4E+10 particles / mL. Under transmission electron microscopy, it was observed that the extract had a clear saucer-like double-layer membrane structure. Figure 7 (a)(b). This extraction method yielded good results for dried red peony root.

[0112] Example 4

[0113] This embodiment provides a method for extracting extracellular vesicles from dried Scutellaria barbata:

[0114] S1. Remove the dust from the Scutellaria barbata using a vacuum cleaner and electrostatic methods.

[0115] S2. Weigh the dried Chinese medicinal materials according to the liquid-to-material ratio of 1:20, and crush them with a Chinese medicine grinder for 0.5 minutes three times in a row.

[0116] S3, 25g of Scutellaria barbata powder was added to 500ml of phosphate buffer solution and soaked for 30min. Then, 0.15g of cellulase (3500u / g enzyme activity) was added and placed in a shaking table, set to 100rpm, for 12h at 25°C. 0.01g of lignin peroxidase (100000u / g enzyme activity) and 0.5g of cellobiase (200u / g enzyme activity) were placed in a shaking table, set to 100rpm, for 4h respectively, with the temperature controlled at 37°C and a speed of 100rpm. The resulting soaking solution was subjected to a wall-breaking process with a wall-breaking time of three minutes and three runs.

[0117] S4. The liquid extracted from the broken Scutellaria barbata was passed through a 300-mesh sieve and subjected to differential centrifugation at 500 x g for 30 minutes, 5000 x g for 60 minutes, and 10000 x g for 1.5 hours to remove impurities with different sedimentation coefficients. Because the sedimentation coefficient of impurity particles in the Scutellaria barbata is higher at 5000 x g, the centrifugation time was extended.

[0118] S5. Add 0.01 g pectinase (enzyme activity 18,000 u / g), 0.02 g xylanase (enzyme activity 110,000 u / g), 0.2 g β-glucanase (enzyme activity 12,000 u / g), and 0.15 g α-amylase (enzyme activity 10,000 u / g) to the obtained liquid according to the type of extracted Chinese medicinal materials, incubate for 2 hours each, control the temperature at 30°C, and the rotation speed at 100 rpm.

[0119] S6. The obtained liquid was centrifuged at 10,000 x g for 1 h and filtered through a 300-mesh filter.

[0120] S7. The liquid obtained in S6 was enriched by ultracentrifugation at 150,000 x g for 2 h.

[0121] S8. Resuspend the precipitate obtained by ultracentrifugation in phosphate buffer and perform ultrafiltration.

[0122] The extract of Scutellaria barbata was characterized and analyzed by nanoparticle size analyzer. Figure 8 As shown in Figure 2, the average particle size of the particles in the extract is 123.9 nm and the concentration is 5.2E+10 particles / mL. Under transmission electron microscopy, it was observed that the extract had a clear saucer-like double-layer membrane structure. Figure 8 (a)(b).

[0123] Example 5

[0124] This example provides a method for extracting extracellular vesicles from the dried product of Yinchen Wuling San:

[0125] S1. The dry ingredients required for Yinchen Wuling Powder (the dosage of Yinchen, Atractylodes macrocephala, Poria cocos, Alisma orientalis, Cinnamomum cassia twig, and Polyporus umbellatus are formulated according to the dosage of Treatise on Febrile Diseases, i.e., 37.5 g of Yinchen and 18.8 g of Wuling Powder, respectively) are removed by vacuum cleaner and electrostatic method respectively.

[0126] S2. Weigh the dried Chinese medicinal materials according to the liquid-to-material ratio of 1:50, and grind them with a Chinese medicine grinder for 0.5 minutes three times in a row.

[0127] S3, 10g of Artemisia capillaris powder is added to 500ml of phosphate buffer solution, after soaking for 30min, 0.15g of cellulase (3500u / g of enzyme activity) is added and put into shaking table, be set to 100rpm, the time is 8h, the temperature is at 25 ℃, 0.01g of lignin peroxidase (100000u / g of enzyme activity) and 0.5g of cellobiase (200u / g of enzyme activity) are put into shaking table, be set to 100rpm, respectively 4h, temperature is controlled at 37 ℃, rotating speed 100rpm.The soaking liquid obtained is utilized wall breaking machine to carry out wall breaking process, wall breaking power is 1000w, rotating speed is 36000rpm, temperature remains at 4 ℃, wall breaking time is three minutes, run three times.

[0128] S4. Pass the broken Yinchen Wuling Powder extract through a 300-mesh sieve and perform differential centrifugation at 500 x g for 60 minutes, 5000 x g for 40 minutes, and 10000 x g for 1 hour to remove impurities with different sedimentation coefficients. Because impurity particles in Yinchen Wuling Powder are more concentrated at a centrifugal force between 500 and 1000 x g, the centrifugation time is extended.

[0129] S5. Add 0.01 g pectinase (enzyme activity 18,000 u / g), 0.02 g xylanase (110,000 u / g), 0.2 g β-glucanase (12,000 u / g), and 0.15 g α-amylase (10,000 u / g) to the obtained liquid in accordance with the type of extracted Chinese medicinal materials, incubate for 2 h each, control the temperature at 30°C, and the rotation speed at 100 rpm.

[0130] S6. The obtained liquid was centrifuged at 10,000 x g for 2 h and filtered through a 300-mesh filter.

[0131] S7. The liquid obtained from S6 was vacuum filtered and then enriched by ultracentrifugation at 150,000 x g for 1.5 h.

[0132] S8. Resuspend the precipitate obtained by ultracentrifugation in phosphate buffer and perform ultrafiltration.

[0133] According to ancient records, Yinchen Wuling Powder is directly crushed into dry products for use, and does not need to be made into a decoction. Therefore, its exosome extraction also follows this method, which can retain the active ingredients to the greatest extent. The extract of Yinchen Wuling Powder was characterized and analyzed by nanoparticle size analyzer, and the results are as follows Figure 9 As shown in Figure a, the average particle size of the extract is 123.9 nm, and the concentration is 5.2E+10 particles / mL. Transmission electron microscopy revealed that the exosomes in the extract, with significantly different particle sizes, had a distinct saucer-like double-layer membrane structure. This demonstrates that exosomes can also be extracted from traditional Chinese medicine compounds using this method, with fewer impurities. Figure 9 (a)(b).

[0134] Example 6

[0135] This example provides a method for extracting extracellular vesicles from the dried product of Banxia Xiexin Decoction (improved):

[0136] S1. Weigh 50 g of the dried ingredients needed for Banxia Xiexin Decoction (total amount: 65 g of washed Pinellia, 45 g of Scutellaria, 45 g of dried ginger, 45 g of ginseng, 45 g of roasted Licorice, 15 g of Coptis, and 12 split jujubes, according to a total amount of 50 g). 8.5 g of Pinellia, 6 g of Scutellaria, 6 g of dried ginger, 6 g of ginseng, 6 g of roasted Licorice, 2 g of Coptis, and 15.5 g of jujube (total 50 g) were weighed separately. Dust was removed using a vacuum cleaner and an electrostatic method.

[0137] S2. Weigh the dried Chinese medicinal materials according to the liquid-to-material ratio of 1:10, and crush them with a Chinese medicine grinder for 0.5 minutes three times in a row.

[0138] S3, 50g of Banxia Xiexin Decoction powder was added to 500ml of phosphate buffer solution, and after soaking for 30min, 0.15g of cellulase (3500u / g of enzyme activity) was added and put into a shaking table, which was set to 100rpm for 12h, and the temperature was at 25°C. 0.01g of lignin peroxidase (100000u / g of enzyme activity) and 0.5g of cellobiase (200u / g of enzyme activity) were put into a shaking table, which was set to 100rpm for 4h, respectively, and the temperature was controlled at 37°C and the speed was 100rpm. The soaking liquid obtained was subjected to wall breaking treatment using a wall breaking machine with a wall breaking power of 1000w, a rotating speed of 36000rpm, a temperature maintained at 4°C, a wall breaking time of three minutes, and three operations.

[0139] S4. The liquid extracted from the Banxia Xiexin Decoction after the cell wall was broken was passed through a 300-mesh sieve, and the extract was subjected to differential centrifugation at 500 x g for 60 min, 5000 x g for 40 min, and 10000 x g for 1 h to remove impurities with different sedimentation coefficients.

[0140] S5. Add 0.01 g pectinase (enzyme activity 18,000 u / g), 0.02 g xylanase (enzyme activity 18,000 u / g), 0.2 g β-glucanase (enzyme activity 12,000 u / g), and 0.15 g α-amylase (enzyme activity 10,000 u / g) to the obtained liquid according to the type of extracted Chinese medicinal materials, incubate for 2 hours each, control the temperature at 30°C, and the rotation speed at 100 rpm.

[0141] S6. The obtained liquid was centrifuged at 10,000 x g for 2 h and filtered through a 300-mesh filter.

[0142] S7. The liquid obtained from S6 was vacuum filtered and then enriched by ultracentrifugation at 150,000 x g for 1.5 h.

[0143] S8. Resuspend the precipitate obtained by ultracentrifugation in phosphate buffer and perform ultrafiltration.

[0144] The obtained liquid was characterized by nanoparticle tracking analyzer and transmission electron microscopy, respectively.

[0145] Comparative Example 5

[0146] This example provides a method for extracting extracellular vesicles from the dried product of Banxia Xiexin Decoction (improved):

[0147] S1. Remove 50g of the dry ingredients needed for Banxia Xiexin Decoction (a total of 65g of washed Pinellia, 45g of Scutellaria, 45g of dried ginger, 45g of ginseng, 45g of roasted Licorice, 15g of Coptis, and 12 jujubes (split)) by vacuuming and electrostatic methods respectively.

[0148] S2, dry Chinese medicinal material are weighed according to liquid-to-material ratio 1:10, after adding 1Lpbs and soaking 30min, boil 2h residue 500ml with earthenware pot, add cellulase 0.15g (enzyme activity 3500u / g) after being down to room temperature and put into shaking table, be set to 100rpm, the time is 12h, temperature is at 25 DEG C, lignin peroxidase 0.01g (enzyme activity 100000u / g) and cellobiase 0.5g (enzyme activity 200u / g) put into shaking table, be set to 100rpm, respectively 4h, temperature is controlled at 37 DEG C, rotating speed 100rpm.The soaking solution obtained is utilized wall breaking machine to carry out broken wall process, and broken wall power is 1000w, and rotating speed is 36000rpm, and temperature remains at 4 DEG C, and broken wall time is three minutes, runs three times.

[0149] S3. The liquid extracted from the Banxia Xiexin Decoction after the wall breaking was passed through a 300-mesh sieve, and the extract was subjected to differential centrifugation at 500 x g for 60 min, 5000 x g for 40 min, and 10000 x g for 1 h to remove impurities with different sedimentation coefficients.

[0150] S4. Add 0.01 g pectinase (enzyme activity 18,000 u / g), 0.02 g xylanase (enzyme activity 110,000 u / g), 0.2 g β-glucanase (enzyme activity 12,000 u / g), and 0.15 g α-amylase (10,000 u / g) to the obtained liquid according to the type of extracted Chinese medicinal materials, incubate for 2 hours each, control the temperature at 30°C, and the rotation speed at 100 rpm.

[0151] S5. The obtained liquid was centrifuged at 10,000 x g for 2 h and filtered through a 300-mesh filter.

[0152] S6. The liquid obtained from S5 was vacuum filtered and then enriched by ultracentrifugation at 150,000 x g for 1.5 h.

[0153] S7. Resuspend the precipitate obtained by ultracentrifugation in phosphate buffer and perform ultrafiltration.

[0154] The extraction method of the extracellular vesicles of the dried Banxia Xiexin Decoction is an extension of this method for extracting extracellular vesicles. Because Banxia Xiexin can be used as a decoction and can be used as pills and powders, some steps are optimized on this method to compare the two extraction methods. Based on this method, the extraction results have a concentration of 4.93E+11Particles / mL and 5.82E+11Particles / mL. The vesicle structure can still be seen after boiling, indicating that extracellular vesicles may play a role in the Chinese herbal decoction. Because the compound is a combination of multiple vesicles, nanoflow cytometry is used to characterize its particle size distribution. Transmission electron microscopy is also used to observe the extracellular vesicle structure. Figure 10 .

[0155] Example 7

[0156] This example provides a method for extracting extracellular vesicles from dried Celosia chinensis seeds:

[0157] S1. Remove the dust from the Amaranthus seeds using a vacuum cleaner and electrostatic methods.

[0158] S2. Weigh the Amaranthus seeds at a liquid-to-solid ratio of 1:50 and crush them using a traditional Chinese medicine grinder for 30 seconds three times. Dried Bupleurum chinense is primarily the root, while dried Rhubarb is primarily the tuber, so increase the crushing time.

[0159] S3, 10g of Celosia seed powder is added to 500ml of phosphate buffer solution, after soaking for 30min, 0.15g of cellulase (3500u / g of enzyme activity) is added and put into shaking table, be set to 100rpm, the time is 12h, the temperature is at 25 ℃, lignin peroxidase 0.01g (100000u / g of enzyme activity) and cellobiase 0.5g (200u / g of enzyme activity) are put into shaking table, be set to 100rpm, respectively 4h, temperature is controlled at 37 ℃, rotating speed 100rpm. The soaking liquid obtained is processed by wall breaking machine, wall breaking power is 1000w, rotating speed is 36000rpm, temperature is maintained at 4 ℃, wall breaking time is three minutes, run three times.

[0160] S4. The liquid extracted from the broken Celosia seeds was passed through 200-mesh and 300-mesh sieves, respectively, and the extract was subjected to differential centrifugation at 500×g for 30 min, 5000×g for 40 min, and 10000×g for 1 h to remove impurities with different sedimentation coefficients.

[0161] S5. Add 0.01g pectinase (enzyme activity 18000u / g), 0.025g xylanase (enzyme activity 110000u / g), 0.25g β-glucanase (enzyme activity 12000u / g), and 0.215g α-amylase (enzyme activity 10000u / g) to the obtained liquid according to the type of extracted Chinese medicinal materials, and incubate for 4h respectively, control the temperature at 30℃ and the speed at 100rpm. Because the seeds of Amaranthus contain more substances, the second enzymatic hydrolysis time is extended.

[0162] S6. The obtained liquid was centrifuged at 10,000 x g for 2 h and filtered through a 300-mesh filter. The centrifugation time was extended because the aggregation phenomenon after enzymatic hydrolysis was more serious.

[0163] S7. The liquid obtained in S6 was vacuum filtered and then enriched by ultracentrifugation at 150,000 x g for 2 h.

[0164] S8. Resuspend the precipitate obtained by ultracentrifugation in phosphate buffer and perform ultrafiltration.

[0165] The extract of Amaranthus chinensis seeds was characterized by a nanoparticle size analyzer. The average particle size of the particles in the extract was 159.9nm and the concentration was 5.9E+10 particles / mL. Under transmission electron microscopy, a clear saucer-like double-layer membrane structure was observed in the extract. Figure 11 (a).

[0166] Comparative Example 6

[0167] This comparative example provides a method for extracting extracellular vesicles from dried Atractylodes macrocephala seeds:

[0168] S1. Remove the dust from the Amaranthus seeds using a vacuum cleaner and electrostatic methods.

[0169] S2. Weigh the Celosia seeds according to the liquid-to-material ratio of 1:10, and crush them with a traditional Chinese medicine grinder for 30 seconds three times in a row.

[0170] S3. Add 50g of Celosia seed powder to 500ml of phosphate buffer solution, soak for 30min, then add 0.15g of the configured cellulase, for 12h, at a speed of 100rpm and a temperature of 25°C, 0.01g of lignin peroxidase, with an enzyme activity of 100,000u per g, and 0.5g of cellobiase, with an enzyme activity of 200u / g, set to 100rpm, for 8h respectively, and the temperature is controlled at 37°C. The obtained soaking liquid is subjected to a wall-breaking treatment with a wall-breaking time of three minutes, and run three times.

[0171] S4. The liquid extracted from the broken Celosia seeds was passed through 200-mesh and 300-mesh sieves, respectively, and the extract was subjected to differential centrifugation at 500×g for 30 min, 5000×g for 40 min, and 10000×g for 1 h to remove impurities with different sedimentation coefficients.

[0172] S5. Add 0.01 g pectinase (enzyme activity 18,000 u / g), 0.025 g xylanase (enzyme activity 110,000 u / g), 0.25 g β-glucanase (enzyme activity 12,000 u / g), and 0.25 g α-amylase (enzyme activity 10,000 u / g) to the resulting liquid in the order of the extracted Chinese medicinal materials. Incubate for 4 hours at 30°C and 100 rpm. Because Celosia chinensis contains a large amount of substances, the second enzymatic hydrolysis time is extended.

[0173] S6. The obtained liquid was centrifuged at 10,000 x g for 2 h and filtered through a 300-mesh filter. The centrifugation time was extended because the aggregation phenomenon after enzymatic hydrolysis was more serious.

[0174] S7. The liquid obtained in S6 was vacuum filtered and then enriched by ultracentrifugation at 150,000 x g for 2 h.

[0175] S8. Resuspend the precipitate obtained by ultracentrifugation in phosphate buffer and perform ultrafiltration.

[0176] The extracellular vesicles of C. aurantifolia seeds were characterized by nanoparticle size analyzer, and the results were as follows: Figure 11 As shown in Figure (a), the average particle size of the extract is 196.3 nm, and the concentration is 7.2E+10 particles / mL. Experiments have shown that different liquid-to-solid ratios significantly affect the extraction of exosomes. Depending on the extraction and measurement methods, a 1:10 liquid-to-solid ratio provides more accurate results.

[0177] Example 8

[0178] This example provides a method for extracting extracellular vesicles from dried Radix Pseudostellariae:

[0179] S1. Remove the dust from the Radix Pseudostellariae using a vacuum cleaner and electrostatic methods.

[0180] S2 and Pseudostellaria heterophylla were weighed according to a liquid-to-material ratio of 1:20, and crushed using a traditional Chinese medicine grinder for 30 seconds three times in a row.

[0181] S3, Radix Pseudostellariae powder 25g is added to phosphate buffer solution 500ml, after soaking 30min, add cellulase 0.15g (enzyme activity 3500u / g) and put into shaking table, be set to 100rpm, the time is 12h, temperature is at 25 ℃, lignin catalase 0.01g (enzyme activity 100000u / g) and cellobiase 0.5g (enzyme activity 200u / g) put into shaking table, be set to 100rpm, respectively 4h, temperature is controlled at 37 ℃, rotating speed 100rpm.The soak liquor obtained is utilized wall breaking machine to carry out broken wall process, broken wall power is 1000w, and rotating speed is 36000rpm, and temperature remains on 4 ℃, and broken wall time is three minutes, runs three times.

[0182] S4. The primary extract of the Pseudostellaria baicalensis with broken wall was passed through 200-mesh and 300-mesh sieves, respectively, and the extract was subjected to differential centrifugation at 500×g for 30 min, 5000×g for 40 min, and 10000×g for 1 h to remove impurities with different sedimentation coefficients.

[0183] S5. Add 0.01g pectinase (enzyme activity 18,000u / g), 0.02g xylanase (enzyme activity 110,000u / g), 0.2g β-glucanase (enzyme activity 12,000u / g), and 0.15g α-amylase (enzyme activity 10,000u / g) to the resulting liquid in the order of the extracted Chinese medicinal materials. Incubate for 4 hours at 30°C and 100 rpm. Because ginseng contains more substances, the second enzymatic hydrolysis time is extended.

[0184] S6. The obtained liquid was centrifuged at 10,000 x g for 2 h and filtered through a 300-mesh filter. The centrifugation time was extended because the aggregation phenomenon after enzymatic hydrolysis was more serious.

[0185] S7. The liquid obtained in S6 was vacuum filtered and then enriched by ultracentrifugation at 150,000 x g for 2 h.

[0186] S8. Resuspend the precipitate obtained by ultracentrifugation in phosphate buffer and perform ultrafiltration.

[0187] The extracellular vesicles of Radix Pseudostellariae were characterized by nanoparticle size analyzer. The results are as follows: Figure 12As shown, the average particle size of the particles in the extract is 136 nm and the concentration is 6.3E+10 particles / mL.

[0188] Comparative Example 7

[0189] This comparative example provides a method for extracting extracellular vesicles from dried Codonopsis pilosula:

[0190] S1. Remove the dust from the Codonopsis pilosula using a vacuum cleaner and electrostatic methods.

[0191] S2. Weigh Codonopsis pilosula according to the liquid-to-material ratio of 1:20, and crush it with a traditional Chinese medicine grinder for 40 seconds three times in a row.

[0192] S3. Add 25 g of Codonopsis pilosula powder to 500 ml of phosphate buffer solution and soak for 30 minutes. Then, add 0.15 g of the configured cellulase for 12 hours at 25°C and a speed of 100 rpm. Add 0.01 g of lignin peroxidase and 0.5 g of cellobiase for 4 hours respectively. The temperature is controlled at 37°C and the speed is 100 rpm. The obtained soaking liquid is subjected to a wall-breaking treatment with a wall-breaking time of three minutes and run three times.

[0193] S4. The liquid extracted from the broken Codonopsis pilosula was passed through 200-mesh and 300-mesh sieves, respectively, and the extract was subjected to differential centrifugation at 500×g for 30 min, 5000×g for 40 min, and 10000×g for 2 h to remove impurities with different sedimentation coefficients.

[0194] S5. Add 0.01 g pectinase (enzyme activity 18,000 u / g), 0.02 g xylanase (enzyme activity 110,000 u / g), 0.2 g β-glucanase (enzyme activity 12,000 u / g), and 0.15 g α-amylase (enzyme activity 10,000 u / g) to the obtained liquid according to the type of extracted Chinese medicinal materials, and incubate for 4 hours each. Control the temperature at 30°C and the rotation speed at 100 rpm.

[0195] S6. The obtained liquid was centrifuged at 10,000 x g for 1 h and filtered through a 300-mesh filter.

[0196] S7. The liquid obtained in S6 was vacuum filtered and then enriched by ultracentrifugation at 150,000 x g for 2 h.

[0197] S8. Resuspend the precipitate obtained by ultracentrifugation in phosphate buffer and perform ultrafiltration.

[0198] The extracellular vesicles of Codonopsis pilosula were characterized by nanoparticle size analyzer. The results are as follows: Figure 13 As shown, the average particle size of the particles in the extract is 123 nm and the concentration is 4.9E+8 particles / mL.

[0199] The experimental comparison proves that under the same conditions and the same liquid-material ratio, the extraction effect of medicinal materials of the same genus also has a large difference. It further proves that the extraction scheme of the outer vesicle in the dry medicinal materials needs to be customized.

[0200] Example 9

[0201] The present embodiment provides a method for extracting extracellular vesicles from dry Artemisia vulgaris:

[0202] S1, the dust in the dry Artemisia vulgaris is removed by using a dust collector and an electrostatic method. Because the dry Artemisia vulgaris has more attached impurities, the dust removal time is prolonged.

[0203] S2, the dry Artemisia vulgaris is weighed according to the liquid-material ratio of 1:10, 1:20 and 1:50, respectively, and is crushed by a traditional Chinese medicine crusher for 0.5 minutes, three times in succession.

[0204] S3, the dry Artemisia vulgaris is added into 500ml phosphate buffer solution according to the mass of 50g, 25g and 10g, respectively, and is soaked for 30min, then 0.15g of cellulase (enzyme activity 3500u / g) is added, and is put into a shaking bed, which is set to 100rpm, and the time is 12h, and the temperature is 25℃, and 0.01g of lignin peroxidase (enzyme activity 100000u / g) and 0.5g of cellobiase (enzyme activity 200u / g) are put into the shaking bed, which is set to 100rpm, and the time is 4h, respectively, and the temperature is controlled at 37℃, and the rotation speed is 100rpm. The obtained soaking liquid is treated by a cell wall breaking machine, the cell wall breaking power is 1000w, the rotation speed is 36000rpm, the temperature is maintained at 4℃, and the cell wall breaking time is three minutes, and the operation is three times. Because the Artemisia vulgaris is a whole grass, and the pectin content is high, the fiber content and hardness are lower than those of the rhizome and tuber dry products, so the first enzyme hydrolysis process is shortened, and the second enzyme hydrolysis process is prolonged.

[0205] S4, the liquid of the broken cell wall of the medicinal materials is passed through 200 mesh and 300 mesh sieves, respectively, and the extraction liquid is subjected to differential centrifugation at 500xg for 30min, 5000xg for 40min, 10000xg for 120min to remove impurities of different sedimentation coefficients.

[0206] S5, the obtained liquid is sequentially added with 0.01g of pectinase (enzyme activity 18000u / g), 0.02g of xylanase (enzyme activity 110000u / g), 0.2g of β-glucanase (enzyme activity 12000u / g) and 0.15g of α-amylase (enzyme activity 10000u / g), and each is incubated for 4h, and the temperature is controlled at 30℃, and the rotation speed is 100rpm.

[0207] S6, the obtained liquid is subjected to centrifugal treatment at 10000xg for 1h, and is passed through a 300 mesh filter screen.

[0208] S7. The liquid obtained in S6 was enriched by ultracentrifugation at 150,000 x g for 1.5 h.

[0209] S8. Resuspend the precipitates obtained by ultracentrifugation in phosphate buffer and perform ultrafiltration.

[0210] Three groups of extracellular vesicle extracts from dried Artemisia annua were characterized by nanoparticle size analysis. The concentrations were 1.8E+10 particles / mL, 1.5E+10 particles / mL, and 5.0E+9 particles / mL, respectively, with average particle sizes of 131.3 nm, 130.7 nm, and 126.8 nm. The results showed that the extraction concentrations of dried Artemisia annua remained similar at a liquid-to-solid ratio of 1:10-1:20, but decreased significantly at 1:50. However, the distribution of extracellular vesicles at this concentration was more uniform. Figure 14 (a)(b)(c).

[0211] Example 10

[0212] This example provides a method for extracting extracellular vesicles from dry Smilax glabra.

[0213] S1. Dust in the dried Smilax glabra product is removed by using a vacuum cleaner and electrostatic methods.

[0214] S2. Weigh the dried Chinese medicinal materials according to the liquid-to-material ratio of 1:5, and crush them with a Chinese medicine grinder for 0.5 minutes three times in a row.

[0215] S3, Rhizoma Smilacis Glabrae dry product is added to the phosphate buffer of 500ml according to quality 100g, soak liquor is phosphate buffer solution, after soaking 30min, adds cellulase 0.15g (enzyme 3500u / g alive) and puts into shaking table, is set to 100rpm, the time is 12h, temperature is at 25 DEG C, lignin peroxidase 0.01g (enzyme 100000u / g alive) and cellobiase 0.5g (enzyme 200u / g alive) put into shaking table, is set to 100rpm, respectively 4h, temperature is controlled at 37 DEG C, rotating speed 100rpm.The soak liquor obtained is utilized wall breaking machine to carry out broken wall process, and broken wall power is 1000w, and rotating speed is 36000rpm, and temperature remains at 4 DEG C, and broken wall time is three minutes, runs three times.

[0216] S4. The broken medicinal material liquid is passed through 200-mesh and 300-mesh sieves respectively, and the extract is subjected to differential centrifugation at 500×g for 30 min, 5000×g for 40 min, and 10000×g for 120 min to remove impurities with different sedimentation coefficients.

[0217] S5, the obtained liquid was added with pectinase 0.01g (enzyme activity 18000u / g), xylanase 0.02g (enzyme activity 110000u / g), β-glucanase 0.2g (enzyme activity 12000u / g), α-amylase 0.15g (enzyme activity 10000u / g) in sequence according to the type of Chinese medicinal materials extracted, and each was incubated for 4h, the temperature was controlled at 30°C and the speed was 100rpm. The polysaccharide content in Smilax glabra is relatively high, so the second enzymolysis time is extended.

[0218] S6. The obtained liquid was centrifuged at 10,000 x g for 1 h and filtered through a 300-mesh filter.

[0219] S7. The liquid obtained in S6 is vacuum filtered and then enriched by ultracentrifugation at 150,000 x g for 1.5 to 2 hours.

[0220] S8. Resuspend the precipitate obtained by ultracentrifugation in phosphate buffer and perform ultrafiltration.

[0221] The dry product of Smilax glabra was characterized by nanoparticle size analyzer. The results are as follows: Figure 15 As shown in (a), the average particle size of the particles in the extract is 144.7 nm and the concentration is 5.4E+10 particles / mL.

[0222] Example 11

[0223] This example provides a method for extracting extracellular vesicles from dried Toosendan fruit:

[0224] S1. The dust in the dried Toosendan fruit is removed by using a vacuum cleaner and electrostatic method.

[0225] S2. Weigh the dried Toosendan fruit according to the liquid-to-material ratio of 1:20, and grind it with a traditional Chinese medicine grinder for 0.5-2 minutes three times in a row. The grinding particle size should reach 48um-150um.

[0226] S3, Fructus Toosendan dry product 25g is added to the soaking liquid of 500ml, soaking liquid is a phosphate buffered solution, after soaking 30min, add cellulase 0.15g (enzyme 3500u / g active) and put into shaking table, be set to 100rpm, the time is 12h, temperature is at 25 ℃, lignin catalase 0.01g (enzyme 100000u / g active) and cellobiase 0.5g (enzyme 200u / g active) put into shaking table, be set to 100rpm, respectively 4h, temperature is controlled at 37 ℃, rotating speed 100rpm.The soaking liquid obtained is utilized wall breaking machine to carry out broken wall process, and broken wall power is 1000w, and rotating speed is 36000rpm, and temperature remains on 4 ℃, and the broken wall time is three minutes, runs three times.

[0227] S4. The broken medicinal material liquid is passed through 200-mesh and 300-mesh sieves respectively, and the extract is subjected to differential centrifugation at 500×g for 30 min, 5000×g for 40 min, and 10000×g for 120 min to remove impurities with different sedimentation coefficients.

[0228] S5. Add 0.01 g pectinase (enzyme activity 18,000 u / g), 0.02 g xylanase (enzyme activity 110,000 u / g), 0.2 g β-glucanase (enzyme activity 12,000 u / g), and 0.15 g α-amylase (enzyme activity 10,000 u / g) to the obtained liquid according to the type of extracted Chinese medicinal materials, incubate for 2 hours each, control the temperature at 30°C, and the rotation speed at 100 rpm.

[0229] S6. The obtained liquid was centrifuged at 10,000 x g for 1 h and filtered through a 300-mesh filter.

[0230] S7. The liquid obtained in S6 was enriched by ultracentrifugation at 150,000 x g for 1.5 h.

[0231] S8. Resuspend the precipitate obtained by ultracentrifugation in phosphate buffer and perform ultrafiltration.

[0232] The dried Toosendan fruit was characterized by a nanometer particle size analyzer. The results are as follows: Figure 15 As shown in b, the average particle size of the particles in the extract is 145.6 nm and the concentration is 1.5E+11 particles / mL.

[0233] The above results show that the extraction of dried Chinese medicine products through this scheme can avoid the appearance of a large number of impurities, and can extract a large number of plant extracellular vesicles in an efficient and low-cost manner, and this is the result of avoiding incomplete vesicle characterization in all parts of the dried products.

[0234] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for extracting extracellular vesicles from dried traditional Chinese medicine, characterized in that: The extracellular vesicles are obtained by taking dried traditional Chinese medicine as raw materials and sequentially undergoing crushing, soaking, first-order enzymatic hydrolysis, cell wall breaking, differential centrifugation, second-order enzymatic hydrolysis, high-speed centrifugation, ultracentrifugation and ultrafiltration enrichment.

2. The method for extracting extracellular vesicles from dried Chinese medicine according to claim 1, wherein The particle size of the crushed medicinal material is 48μm-150μm.

3. The method for extracting extracellular vesicles from dried Chinese medicine according to claim 1, wherein The soaking conditions are as follows: the crushed medicinal materials are added to a phosphate buffer solution at a liquid-to-material ratio of 1:10-50, and soaked for 30 minutes.

4. The method for extracting extracellular vesicles from dried Chinese medicine according to claim 1, characterized in that Based on 10g-50g of dried Chinese medicine, the conditions for the first enzymatic hydrolysis are: adding 0.15g-0.25g of cellulase with an enzyme activity of 3500u / g, and enzymatic hydrolysis at 25°C for 4-12h; then adding 0.01g-0.025g of lignin peroxidase with an enzyme activity of 100000u / g, and enzymatic hydrolysis at 37°C for 4-12h; finally, adding 0.5g-1g of cellobiase with an enzyme activity of 200u / g, and enzymatic hydrolysis at 37°C for 4-12h.

5. The method for extracting extracellular vesicles from dried traditional Chinese medicine according to claim 1, characterized in that: The conditions for the wall breaking are: the wall breaking power is 1000w, the rotation speed is 36000rpm, and the temperature is maintained at 4°C.

6. The method for extracting extracellular vesicles from dried traditional Chinese medicine according to claim 1, characterized in that: The conditions of the differential centrifugation are: 500×g, 30 min; 5000×g, 40-60 min; 10000×g, 90-120 min.

7. The method for extracting extracellular vesicles from dried traditional Chinese medicine according to claim 1, characterized in that: Based on 10g-50g of dried traditional Chinese medicine, the conditions for the second enzymatic hydrolysis are as follows: adding 0.01g of pectinase with an enzyme activity of 18000u / g and incubating at 25-37°C for 2-4h; then adding 0.015g-0.025g of xylanase with an enzyme activity of 110000u / g and incubating at 25-37°C for 2-4h; then adding 0.15g-0.25g of beta-glucanase with an enzyme activity of 12000u / g and incubating at 25-37°C for 2-4h; finally adding 0.15g-0.25g of alpha-amylase with an enzyme activity of 10000u / g and incubating at 25-37°C for 2-4h.

8. The method for extracting extracellular vesicles from dried traditional Chinese medicine according to claim 1, characterized in that: The conditions of the high-speed centrifugation are: 10,000×g, 1-2 h.

9. The method for extracting extracellular vesicles from dried traditional Chinese medicine according to claim 1, characterized in that: The ultracentrifugation conditions are: 150,000×g, 1.5 h-2 h.

10. The method for extracting extracellular vesicles from dried traditional Chinese medicine according to claim 1, characterized in that: The ultrafiltration membrane used in the ultrafiltration has a molecular weight cut-off of 10-100 kDa.

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