Extraction method and application of plant exosome

Abstract

The invention discloses a plant exosome extraction method and application thereof, and belongs to the technical field of exosome extraction. The extraction method of the plant exosome comprises the following steps: S1, mixing a plant sample with a buffer solution, performing juicing treatment, and filtering to obtain a crude extract; s2, carrying out continuous flow centrifugal treatment on the crude extract to obtain a refined extract; s3, carrying out concentration treatment on the refined extract to obtain a concentrated solution; and S4, carrying out microfluidic purification treatment on the concentrated solution to obtain the plant exosome. According to the method, the exosome of the plant sample is extracted by adopting a treatment method combining juicing treatment, continuous flow centrifugal treatment, concentration treatment and microfluidic purification treatment, so that large-volume, high-throughput, high-efficiency and low-cost exosome extraction and separation of the plant sample are realized; in addition, it is found for the first time that the panax notoginseng source exosome not only can effectively inhibit expression of inflammatory factors, but also has a good effect on psoriasis treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of exosome extraction, and specifically relates to a method for extracting plant exosomes and an application thereof. Background Art

[0002] Plant exosomes are nano-scale vesicles secreted by plant cells, which have important biological functions and application prospects. Common methods for extracting plant exosomes include differential centrifugation, density gradient centrifugation, ultrafiltration and immunocapture. Among them, differential centrifugation removes impurities by gradually increasing the centrifugal force, and finally precipitates exosomes under high centrifugal force; density gradient centrifugation is based on differential centrifugation, and the exosome suspension is loaded on a pre-prepared density gradient medium, and the exosomes are further purified by ultrahigh-speed centrifugation. Ultrafiltration utilizes the selective permeability characteristics of ultrafiltration membranes to filter out macromolecules and impurities under a certain pressure, while retaining exosomes. The immunocapture method is based on specific markers on the surface of exosomes, and exosomes are captured by antibodies that specifically bind to the markers. However, all of the above methods have problems such as low purity and yield, and complex operation. It is still necessary to further optimize the extraction method to improve the purity and yield of plant exosomes.

[0003] Psoriasis is a chronic, relapsing, immune-mediated skin disease whose treatment goals are to control symptoms, prolong remission, and improve the quality of life of patients. Common treatment drugs include topical medications, systemic treatments, and biologics. Topical medications such as glucocorticoids, vitamin D3 analogs, and tacrolimus relieve symptoms mainly by anti-inflammatory and inhibiting keratinocyte proliferation. Systemic treatment drugs include methotrexate, cyclosporine, and acitretin, which work by regulating immune responses and inhibiting cell proliferation. In recent years, biologics have made significant progress in the treatment of psoriasis, including tumor necrosis factor (TNF) inhibitors and interleukin (IL) inhibitors, such as adalimumab, etanercept, secukinumab, and ustekinumab, which control inflammatory responses and skin symptoms by targeting specific immune pathways. Overall, these drugs have improved the condition of psoriasis patients to varying degrees. However, it is necessary to further study and develop new treatment strategies to address the limitations of existing treatments and improve the quality of life of patients.

[0004] Currently, there are no reports on the use of Panax notoginseng-derived exosomes for the prevention and / or treatment of psoriasis. Summary of the invention

[0005] The purpose of the present invention is to provide a method for extracting plant exosomes and its application. The present invention adopts a specific separation and extraction method to achieve large-volume, high-throughput, high-efficiency, and low-cost separation of exosomes from plant samples; further, the present invention uses Panax notoginseng-derived exosomes for the treatment of psoriasis for the first time, and the Panax notoginseng-derived exosomes can not only effectively inhibit the expression of inflammatory factors, but also have a good effect on the treatment of psoriasis.

[0006] In a first aspect, the present invention provides a method for extracting plant exosomes, comprising the following steps: S1, mixing a plant sample with a buffer solution and then juicing the mixture, and filtering the mixture to obtain a crude extract; S2, subjecting the crude extract to continuous flow centrifugation to obtain a refined extract; S3, concentrating the refined extract to obtain a concentrated solution; S4, subjecting the concentrated solution to microfluidic purification to obtain plant exosomes.

[0007] In the present invention, the inventors have found that by combining juicing, continuous flow centrifugation, concentration and microfluidic purification, large-volume, high-throughput, high-efficiency and low-cost exosome extraction and separation of plant samples can be achieved. Therefore, this extraction method has good application prospects in the extraction of plant exosomes.

[0008] In some embodiments, in step S1, the plant sample includes a Panax notoginseng sample, and the buffer includes a PBS buffer.

[0009] In the present invention, the plant samples can be conventionally selected according to actual use needs. For example, preferably, the plant samples in the present invention include Panax notoginseng samples; further, the buffer solution can be conventionally selected according to actual use needs. For example, preferably, the buffer solution in the present invention includes PBS buffer solution.

[0010] In some embodiments, the mass volume ratio of the plant sample to the buffer is (400-600 g):(1-3 L), for example, 400 g:1 L, 400 g:2 L, 400 g:3 L, 500 g:1 L, 500 g:2 L, 500 g:3 L, 600 g:1 L, 600 g:2 L, 600 g:3 L or other ratios within this range.

[0011] In some preferred embodiments, the filtering step specifically comprises: filtering using gauze.

[0012] In some embodiments, in step S2, the continuous flow centrifugation step specifically includes: centrifuging for 20-40 min, for example, 20 min, 25 min, 30 min, 35 min, 40 min or other values ​​within the range, at a temperature of 0-4°C (for example, 0°C, 1°C, 2°C, 3°C, 4°C or other values ​​within the range) and a centrifugal force of 10000-15000 g (for example, 10000 g, 11000 g, 12000 g, 13000 g, 14000 g, 15000 g or other values ​​within the range).

[0013] In some preferred embodiments, in step S2, the continuous flow centrifugation step specifically comprises: centrifuging for 30 minutes at a temperature of 4°C and a centrifugal force of 10,000 g.

[0014] In the present invention, during the continuous flow centrifugation process, under the action of centrifugal force, large impurities (such as cell debris, protein aggregates, etc.) will accumulate on the wall of the centrifugal chamber to form precipitates, while smaller particles such as exosomes will dissolve in the supernatant. Compared with traditional batch centrifugation, continuous flow centrifugation has the advantages of large processing capacity, high separation efficiency, no need for balancing to reduce sample processing time and reduce rotor wear.

[0015] In some embodiments, in step S3, the concentration treatment includes at least one of membrane concentration treatment and evaporation concentration treatment.

[0016] In the present invention, the concentration treatment can be conventionally selected according to actual use needs. For example, preferably, the concentration treatment includes at least one of membrane concentration treatment and evaporation concentration treatment.

[0017] In some preferred embodiments, the concentration step specifically comprises: using a 100-300KD membrane to concentrate the extract.

[0018] In the present invention, the molecular sieve principle is used to physically concentrate the extract to reduce the loss of exosomes (vesicles).

[0019] In some embodiments, in step S4, before the concentrate is subjected to microfluidic purification, the step of mixing the concentrate is also included.

[0020] In the present invention, the concentrated solution is mixed so that the concentrated solution is dispersed more evenly, which facilitates the subsequent efficient purification of the concentrated solution to obtain exosomes with higher purity and concentration.

[0021] In some embodiments, the microfluidic purification step specifically includes: passing the mixed concentrated solution into a microfluidic chip, and then recovering the plant exosomes from the recovery end of the microfluidic chip.

[0022] In the present invention, after the concentrated liquid enters the chip, the single-chip processing efficiency is as high as 100mL / h. A multi-stage stacked microfluidic chip is adopted, and the channels within the chip are utilized to achieve precise manipulation of samples and efficient purification and separation, thereby obtaining exosomes with higher purity and concentration, greatly saving time costs.

[0023] In a second aspect, the present invention provides the use of plant exosomes extracted by any of the above extraction methods in the preparation of drugs for preventing and / or treating psoriasis.

[0024] In some embodiments, psoriasis is caused by inflammation of the skin.

[0025] In the present invention, the inventors discovered for the first time that exosomes derived from Panax notoginseng can not only effectively inhibit the expression of inflammatory factors, but also have a good effect on the treatment of psoriasis.

[0026] In a third aspect, the present invention provides a pharmaceutical composition comprising plant exosomes extracted by any of the extraction methods described above.

[0027] In some embodiments, a pharmaceutically acceptable carrier is also included.

[0028] In the present invention, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Exemplarily, pharmaceutically acceptable carriers include buffers, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents that are compatible with drug administration. Each carrier must be "acceptable", meaning compatible with the other ingredients of the composition and harmless to the patient.

[0029] The beneficial effects of the present invention are as follows: Different from the prior art, the present invention uses a processing method combining juicing, continuous flow centrifugation, concentration and microfluidic purification to extract exosomes from plant samples, achieving large-volume, high-throughput, high-efficiency and low-cost exosome extraction and separation from plant samples; in addition, the present invention discovers for the first time that exosomes from Panax notoginseng can not only effectively inhibit the expression of inflammatory factors, but also have a good effect on the treatment of psoriasis. Therefore, the extraction method of the present invention has a good application prospect in the extraction of plant exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The following are the results of the identification of Panax notoginseng exosomes extracted from Example 1 of the present invention, wherein (A) is the result of the NTA test, and (B) is the transmission electron microscopy image; Figure 2 The following are the results of the identification of Panax notoginseng exosomes extracted in Comparative Example 1 of the present invention, wherein (A) is the NTA test result diagram, and (B) is the transmission electron microscopy diagram; Figure 3A The morphological diagram of mouse skin tissue in different treatment groups in the application test of the present invention; Figure 3B This is a diagram showing the staining results of mouse skin tissue in different treatment groups in the application test of the present invention; Figure 4A This is a result diagram of differentially expressed genes in mouse skin tissue after exosome treatment in the application test of the present invention; Figure 4B This is a diagram showing the results of KEGG enrichment analysis in mouse skin tissue after exosome treatment in the application test of the present invention; Figure 4C This is a graph showing the changes in inflammatory factors in mouse skin tissue after treatment with exosomes in the application test of the present invention; Figure 5 The relative expression results of inflammatory factors in mouse skin tissue in each treatment group in the application test of the present invention are shown in Figure 1, wherein (A) is the relative expression result of CCL7, (B) is the relative expression result of CXCL10, and (C) is the relative expression result of IL-1β; Figure 6 The results of the content of inflammatory factors in the skin tissue of mice in each treatment group in the application test of the present invention are shown in Figure 1, wherein (D) is the result of the content of CCL7, (E) is the result of the content of CXCL10, and (F) is the result of the content of IL-1β; Figure 7 Schematic diagram of the structure of the microfluidic chip in the present invention; The reference numerals have the following meanings: 1: injection hole; 2: filtering microchannel; 3: filter membrane; 4: filter membrane support portion; 5: sample outlet; 6: waste liquid pool. DETAILED DESCRIPTION

[0031] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The experimental methods without specific conditions in the examples are usually carried out according to conventional conditions and conditions described in the manual, or according to conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all available from commercial channels unless otherwise specified.

[0033] See also Figure 7, which is a schematic diagram of the structure of the microfluidic chip in the present invention. The concentrated liquid is passed into the sampling hole 1 and enters the filtering microchannel 2. The filtering microchannel 2 is etched on the filter membrane support part 4. The filter membrane 3 is arranged on the filtering microchannel 2. After the concentrated liquid is purified by the filter membrane 3, it flows out from the sample outlet 5 and is collected, and the waste liquid enters the waste liquid pool 6.

[0034] Among them, the pore size of the filter membrane 3 is 100-300KD.

[0035] Example 1 A method for extracting Panax notoginseng exosomes comprises the following steps: S1. After cleaning the Panax notoginseng sample, take 500 g of clean Panax notoginseng sample and 2 L of PBS buffer solution and add them into a juicer to mix them. After mixing evenly, squeeze the juice and filter through gauze to obtain a crude extract. S2, passing the crude extract obtained in step S1 into a continuous flow centrifuge, and centrifuging for 30 minutes at a temperature of 4° C. and a centrifugal force of 10,000 g to obtain a refined extract; S3, passing the refined extract obtained in step S2 into a membrane concentration device (membrane pore size of 100KD) for concentration treatment to obtain a concentrated solution; S4. The concentrated solution obtained in step S3 is passed into a mixing pool for thorough mixing, and then the mixed concentrated solution is passed into a microfluidic chip, and then Panax notoginseng exosomes are recovered from the recovery end of the microfluidic chip.

[0036] Comparative Example 1 This comparative example provides a method for extracting Panax notoginseng exosomes, comprising the following steps: S1. After cleaning the Panax notoginseng sample, take 500 g of clean Panax notoginseng sample and 2 L of PBS buffer solution and add them into a juicer to mix them. After mixing evenly, squeeze the juice and filter through gauze to obtain a crude extract. S2, passing the crude extract obtained in step S1 into a continuous flow centrifuge, and centrifuging for 30 minutes at a temperature of 4° C. and a centrifugal force of 10,000 g to obtain a refined extract; S3. The extract obtained in step S2 is passed into a membrane concentration device (membrane pore size is 100KD) for concentration treatment to obtain Panax notoginseng exosomes.

[0037] Performance Testing The exosomes of Panax notoginseng extracted from Example 1 and Comparative Example 1 were subjected to NTA test and transmission electron microscopy observation, and the results were as follows: Figure 1 and 2 shown.

[0038] from Figure 1 and 2It can be seen from the figure that the particle size distribution of the Panax notoginseng exosomes extracted in Example 1 is uniform and the concentration is high. From the transmission electron microscopy image, it can be seen that the morphology of the Panax notoginseng exosomes is complete and there are no obvious impurities ( Figure 1 ); The particle size distribution of the Panax notoginseng exosomes extracted in Comparative Example 1 is uneven, and the concentration is low. From the transmission electron microscopy image, it can be seen that the morphology of the Panax notoginseng exosomes is incomplete and contains certain impurities ( Figure 2 ); The results show that the extraction method of the present invention can obtain Panax notoginseng exosomes with higher purity and concentration.

[0039] Application Testing In this application test, a mouse psoriasis model was first established, and then the exosomes (EVs) and extracts (EXT) extracted by the present invention were used to treat the psoriasis mice. After the treatment, the skin tissues of the mice were collected for relevant tests.

[0040] The preparation steps of the extract (EXT) are as follows: weigh the Panax notoginseng, then add 100g of Panax notoginseng to 500ml of PBS at a mass-to-volume ratio for juicing, centrifuge the juicing homogenate at 3000g for 30min, collect the supernatant, and obtain the extract (EXT).

[0041] Specifically, 8-week-old male wild-type Balb / c mice were first selected and divided into four groups: Group 1: normal group (Normal), Group 2: model group (Psoriasis), Group 3: model group + exosome treatment (Psoriasis+EV), Group 4: model group + extract treatment (Psoriasis+EXT). Among them, the backs of some mice were shaved and imiquimod (IMQ) ointment (Sichuan Mingxin Pharmaceutical, product number H20030128) was used to establish a psoriasis mouse model. 50 mg of imiquimod ointment was applied to the back skin of the mice every day, and the normal group of mice was applied with the same dose of vaseline ointment for 2 consecutive days; starting from the 3rd day, the third group of mice were applied with 100 μg / ml of Panax notoginseng exosomes, and the fourth group of mice were applied with 100 μg / ml of extracts; after 5 consecutive days of treatment, the skin conditions of the mice in each treatment group were observed, and on the 8th day, the mice were killed, blood was collected, and skin tissues were collected; one part of the mouse skin tissue was frozen and preserved, and the other part was fixed with 4% paraformaldehyde (Sinopharm, product number 80096618) and then observed by tissue staining. The results are as follows: Figure 3A and 3B shown.

[0042] from Figure 3A and 3BIt can be seen that compared with the mice in the normal group, the mice in the model group developed typical skin symptoms of psoriasis, and there were inflammatory cells in the skin tissue; after being treated with the exosomes and extracts extracted by the present invention, the skin symptoms of the psoriasis mice improved to a certain extent, and the inflammatory cells decreased; compared with the extracts, the exosomes had a better effect. The results show that compared with the extracts with low purity, the exosomes with higher purity extracted by the present invention have a better therapeutic effect on psoriasis mice.

[0043] Next, high-throughput sequencing technology was used to study the differentially expressed genes in the skin tissue of mice after treatment with exosomes.

[0044] Specifically, the RNA in mouse skin tissue was isolated and purified using the TRIzol (Invitrogen, Carlsbad, CA, USA) method according to the manufacturer's operating protocol; the quality and purity of the extracted RNA were then controlled using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA); finally, the integrity of the RNA was tested using Agilent 2100, with RIN number>7.0 as the qualified standard.

[0045] 5 μg of total RNA that passed the above quality control was taken, and the mRNA with PolyA (polyadenylic acid) was specifically captured by oligo(dT) magnetic beads through two rounds of purification. The captured mRNA was fragmented by divalent cations under high temperature conditions, and the fragmented RNA was synthesized into cDNA by reverse transcriptase; then E. coli DNA polymerase I and RNase H were used for double-strand synthesis to convert the composite double-stranded DNA and RNA into DNA double-stranded DNA, and DUTP was incorporated into the double-stranded DNA to fill the ends of the double-stranded DNA into blunt ends; then an A base was added to each end so that it could be connected to the adapter with a T base at the end, and then the fragment size was screened and purified by magnetic beads; the second strand was digested with UDG enzyme, and then PCR was performed to form a library with a fragment size of 300 bp±50 bp; finally, Illumina HiSeq 4000 (LC Bio, China) was used for double-end sequencing according to standard operations, with a read length of 150 bp, and the results were as follows: Figures 4A-4C shown.

[0046] from Figures 4A-4C It can be seen that the up-regulated genes in the model group were slightly higher than the down-regulated genes compared with the normal group, the up-regulated genes in the model group + exosome treatment were slightly higher than the down-regulated genes compared with the model group, and the up-regulated genes in the model group + exosome treatment were higher than the down-regulated genes ( Figure 4A ); KEGG enrichment analysis showed that the IL-17 signaling pathway was significantly different ( Figure 4B In addition, sequencing showed that the changes in inflammatory factors were quite different ( Figure 4C ). The above results showed that after exosome treatment, inflammatory factors in mouse skin tissues changed significantly.

[0047] Finally, qPCR was used to detect the relative expression levels of inflammatory factors in the skin tissue of mice in the above-mentioned treatment groups, and the ELISA method was used to detect the content of inflammatory factors.

[0048] Specifically, the operating steps for qPCR detection of the relative expression levels of inflammatory factors in the skin tissue of mice in each of the above treatment groups are as follows: First, RNA from tissues was extracted using the following method: 1) Add 50-100 mg of tissue / cells to Trizol, homogenize with a homogenizer, and centrifuge to remove the precipitate; 2) Add chloroform (200 μL chloroform per 1 mL Trizol), quickly invert and mix for about 15 seconds, and let stand at room temperature for 3 minutes; 3) Centrifuge at 4°C, 12,000 rpm for 10-15 min. The sample in the centrifuge tube is divided into three layers: organic phase (protein and polysaccharide), middle phase (DNA) and aqueous phase (RNA). RNA is mainly present in the upper aqueous phase. Transfer the aqueous phase to a new RNase-Free centrifuge tube. 4) Add an equal volume of isopropanol, mix well, and leave at room temperature for 20-30 minutes; 5) Centrifuge at 4°C, 12,000 rpm for 10 min. The white precipitate on the side and bottom of the tube is RNA. 6) Discard the supernatant and add 70% ethanol to the centrifuge tube to wash the precipitate (be careful not to blow the precipitate away). For every 1 mL of Trizol used, add at least 1 mL of 70% ethanol; 7) Centrifuge at 4°C, 5000 rpm for 3 min, remove the supernatant, and air-dry in a clean bench for about 2-3 min. Do not over-dry, otherwise it will be difficult to dissolve later. 8) Add an appropriate amount (about 30 to 100 μL) of DEPC water according to the amount of precipitation to fully dissolve the RNA; Then, the extracted RNA was reverse transcribed into cDNA using ReverTra Ace qPCR RT Kit (FSQ-101, 200T, Toyobo) and qPCR was performed. The internal reference gene β-actin was used as a control to calculate the relative expression levels of inflammatory factors CCL7, CXCL10, and IL-1β. The results are shown in Figure 5 shown.

[0049] Among them, the detection primer sequences of β-actin gene, inflammatory factors CCL7, CXCL10 and IL-1β are as follows: β-actin-F: 5'-CCGCTGCCTCTCCTCCTC-3' (SEQ ID NO: 1); β-actin-R: 5'-AACGCTCATGCCGATGGTG-3' (SEQ ID NO: 2); CCL7-F: 5'-AATGCATCCACATGCTGCTA-3' (SEQ ID NO: 3); CCL7-R: 5'-ATAGCCTCCTCGACCCACTT-3' (SEQ ID NO: 4); CXCL10-F: 5'-AAGTGCTGCCGTCATTTTCT-3' (SEQ ID NO: 5); CXCL10-R: 5'-GTGGCAATGATCTCAACACG-3' (SEQ ID NO: 6); IL-1β-F: 5'-GGGCCTCAAAGGAAAGAATC-3' (SEQ ID NO: 7); IL-1β-R: 5'-TACCAGTTGGGGAACTCTGC-3' (SEQ ID NO: 8).

[0050] from Figure 5 It can be seen that after treatment with the exosomes and extracts obtained by the present invention, the relative expression levels of inflammatory factors in the skin of psoriasis mice decreased, but after treatment with exosomes, the relative expression levels of inflammatory factors in the skin of psoriasis mice decreased significantly. The results show that the exosomes and extracts in the present invention can inhibit the expression of inflammatory factors, thereby treating psoriasis, and the exosomes with higher purity extracted by the present invention can more significantly inhibit the expression of inflammatory factors.

[0051] The steps for detecting the content of inflammatory factors using the ELISA method are as follows: 1) Set up standard wells, blank wells and sample wells respectively, add 100 μL of serially diluted standard (inflammatory factors CCL7, CXCL10 and IL-1β) to the standard wells, add 100 μL of standard & sample diluent to the blank wells, and add 100 μL of the sample to be tested to the remaining wells (where the sample to be tested is the supernatant after centrifugation of the blood collected in the previous step. It is recommended that all samples to be tested and standards be set up in duplicate wells during the test); cover the ELISA plate with a film and incubate at 37°C for 90 minutes; Tips: When adding samples, add the samples to the bottom of the ELISA plate, try not to touch the well wall, gently shake to mix, avoid bubbles, and the adding time should be controlled within 10 minutes; 2) Shake off the liquid in the wells without washing. Add 100 μL of biotinylated antibody working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 1 hour.

[0052] 3) Shake off the liquid in the wells and pat dry on clean absorbent paper. Add 350μL of washing solution to each well and soak for 1 minute. Absorb or shake off the liquid in the ELISA plate and pat dry. Repeat this washing step 3 times. After washing, proceed to the next step immediately and do not let the microplate dry. 4) Add 100 μL of enzyme conjugate working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 30 minutes; 5) Shake off all liquid in the wells and wash the plate 5 times, using the same method as step 3); 6) Add 90 μL of substrate solution (TMB) to each well, cover the ELISA plate with a film, and incubate at 37°C in the dark for about 15 minutes; Tip: shorten or extend the time as appropriate according to the actual color development situation, but it should not exceed 30 minutes. When a clear gradient appears in the standard well (clear blue gradient appears in the first 4 color development wells), it can be terminated. Turn on the ELISA reader 15 minutes in advance to preheat; 7) Add 50 μL of stop solution to each well to terminate the reaction. Tip: The order of adding the stop solution should be the same as that of adding the substrate solution. 8) Immediately measure the optical density (OD value) of each well using a microplate reader at a wavelength of 450 nm.

[0053] The standard curve equations were established using the standard samples, and then the contents of the samples (inflammatory factors CCL7, CXCL10, and IL-1β) were calculated. The results are as follows: Figure 6 shown.

[0054] from Figure 6 It can be seen that after treatment with the exosomes and extracts obtained by the present invention, the levels of inflammatory factors in the blood of psoriasis mice decreased, but after treatment with exosomes, the levels of inflammatory factors in the blood of psoriasis mice decreased significantly. The results show that the exosomes and extracts in the present invention can inhibit the expression of inflammatory factors, thereby treating psoriasis, and the exosomes with higher purity extracted by the present invention can more significantly reduce the content of inflammatory factors.

[0055] In summary, the present invention adopts a specific separation and extraction method to achieve large-volume, high-throughput, high-efficiency and low-cost separation of exosomes from plant samples; further, the present invention uses Panax notoginseng-derived exosomes for the treatment of psoriasis for the first time. The Panax notoginseng-derived exosomes can not only effectively inhibit the expression of inflammatory factors, but also have a good effect on the treatment of psoriasis.

[0056] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0057] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for extracting plant exosomes, characterized in that: The steps include: S1, mixing the plant sample with a buffer solution and then juicing the mixture, and filtering the mixture to obtain a crude extract; S2, subjecting the crude extract to continuous flow centrifugation to obtain a refined extract; S3, concentrating the extract to obtain a concentrated solution; S4. Performing microfluidic purification on the concentrated solution to obtain plant exosomes.

2. The method for extracting plant exosomes according to claim 1, characterized in that: In step S1, the plant sample includes a Panax notoginseng sample, and the buffer includes a PBS buffer.

3. The method for extracting plant exosomes according to claim 2, characterized in that: The mass volume ratio of the plant sample to the buffer solution is (400-600 g): (1-3 L).

4. The method for extracting plant exosomes according to claim 1, characterized in that: In step S2, the continuous flow centrifugation step specifically includes: centrifuging for 20-40 minutes at a temperature of 0-4°C and a centrifugal force of 10000-15000g.

5. The method for extracting plant exosomes according to claim 1, characterized in that: In step S3, the concentration step specifically includes: using a 100-300KD membrane to concentrate the extract; in step S4, before the concentrate is subjected to microfluidic purification, it also includes a step of mixing the concentrate.

6. The method for extracting plant exosomes according to claim 5, characterized in that: The microfluidic purification treatment step specifically includes: passing the mixed concentrated solution into a microfluidic chip, and then recovering the plant exosomes from the recovery end of the microfluidic chip.

7. Use of plant exosomes extracted by the extraction method according to any one of claims 1 to 6 in the preparation of drugs for preventing and / or treating psoriasis.

8. The use according to claim 7, characterized in that: Psoriasis is caused by inflammation of the skin.

9. A pharmaceutical composition, characterized in that The invention comprises plant exosomes extracted by the extraction method described in any one of claims 1 to 6.

10. The pharmaceutical composition according to claim 9, characterized in that Also included are pharmaceutically acceptable carriers.

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