A plant exosome-like nanovesicle, its preparation method and application

Abstract

The present invention relates to a plant exosome-like nanovesicle, a preparation method thereof and an application thereof, and relates to the technical fields of biotechnology and life science. The preparation method comprises the following steps: mixing and crushing a plant source and a buffer solution, then adding an enzyme for enzymatic hydrolysis to obtain a plant source juice; performing first centrifugal separation on the plant source juice to obtain a supernatant; performing second centrifugal separation on the supernatant, and then washing the obtained precipitate to obtain the plant exosome-like nanovesicle. The present invention combines an enzymatic hydrolysis method with an ultracentrifugation method to successfully prepare Rosa roxburghii / Artemisia sphaerocephala Krasch seeds / Morus alba leaves / Gynostemma pentaphyllum ELNs, which have the characteristics of simple process, safety, environmental protection, and good biological activity of the obtained ELNs. At the same time, in-depth research shows that the obtained plant exosome-like nanovesicles contribute to improving the repair of intestinal mucosal barrier injury, providing important data support for the development of products for restoring intestinal mucosal barrier function.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and life science technology, and in particular to a plant exosome-like nanovesicle and a preparation method and application thereof. Background Art

[0002] The intestine is the most important and crucial digestive, absorptive, and secretory organ in the human body, and is also the largest immune organ in the human body, serving as the first line of defense against toxins and harmful substances. Its mucosal barrier can prevent harmful substances in the intestine, such as bacteria, toxins, and food-derived antigens, from passing through the intestinal mucosa and entering other tissues, organs, and the blood circulation in the human body. Various reasons can lead to damage to the intestinal mucosal barrier function. When the intestinal barrier function is impaired, intestinal mucosal atrophy and increased intestinal permeability will result in the translocation of intestinal bacteria and toxins and the massive release of pro-inflammatory factors, leading to systemic inflammatory response syndrome and further developing into multiple organ dysfunction, such as Crohn's disease, bacterial enteritis, as well as alcoholic liver, fatty liver, pancreatitis, etc. Colitis is an inflammatory disease with a relatively high incidence caused by the impairment of the intestinal mucosal barrier function. The main clinical manifestations include diarrhea, abdominal pain, mucus stools and purulent bloody stools, tenesmus, and even constipation, with inability to defecate for several days; often accompanied by weight loss and fatigue, and it recurs frequently. With the increasing incidence of colitis, the disease course is long, and there is a risk of concurrent colon cancer, so it has received more and more attention.

[0003] Extracellular vesicles (EVs) are lipid bilayer nanoparticles released from cells into the extracellular space, including exosomes, exosome-like nanoparticles, and outer membrane vesicles, etc. EVs play an important role in the transport of biomolecules (such as proteins, lipids, etc.) and nucleic acids (including mRNA) in the human body. Due to their inherent biocompatibility, ability to cross physiological barriers, and low immunogenicity, and they can be specifically targeted and absorbed by tissues, they have now become a biological carrier with great potential. Plant exosome-like nanovesicles are nanoscale vesicles secreted by plant cells, with a wide range of sources and rich in bioactive substances, and play a prominent role in cell-to-cell communication and information transfer, maintaining homeostasis in the body, and treating diseases. Existing studies have shown that plant exosome-like nanovesicles can protect the intestinal barrier by participating in cell-to-cell communication involving DNA, mRNA, miRNA, proteins, lipids, and substances, improving the inflammatory response of intestinal cells, maintaining tight junction complexes, and regulating the inflammatory response, which is helpful for intestinal homeostasis.

[0004] Some herbal plants, such as Rosa roxburghii Tratt, Artemisia desertorum Spreng, Morus alba L., and Gynostemma pentaphyllum (Thunb.) Makino, are rich in flavonoids, alkaloids, phytosterols, dietary fiber, and various trace elements, and have high medicinal and health care values, and are often used for reducing blood lipids, anti-tumor, anti-inflammatory, antioxidant, etc. The exosome-like nanoparticles (ELNs) extracted from these plants not only have corresponding pharmacological effects, but also have the unique morphological and compositional characteristics of nanocarriers. Research shows that plant exosome-like nanovesicles reach the small intestine through the vesicle transport mechanism and are absorbed by the intestine, and are specifically phagocytosed by target cells, achieving anti-inflammatory, antioxidant, and mucosal repair effects, thus achieving the effect of alleviating inflammation, having functions such as maintaining intestinal homeostasis, and not causing problems such as specific immunity, and are expected to be used for the prevention and treatment of colitis. It can be achieved by means of oral preparations, injection needles, and transdermal drug delivery, etc.

[0005] So far, ELNs derived from grapes, grapefruits, ginger, kudzu roots, and ginseng have been reported to be used for the treatment of colitis due to their unique transport characteristics and biological functions. However, there is currently no relevant research report on the application of ELNs derived from Rosa roxburghii Tratt, Artemisia desertorum Spreng, Morus alba L., and Gynostemma pentaphyllum (Thunb.) Makino in improving intestinal mucosal barrier damage. Summary of the Invention

[0006] To solve the above problems, the present invention provides a plant exosome-like nanoparticle and its preparation method and application.

[0007] In the first aspect, the present invention provides a preparation method of a plant exosome-like nanoparticle, and the preparation method includes the following steps:

[0008] Mix and crush the plant source and the buffer solution, and then add an enzyme for enzymatic hydrolysis to obtain a plant source juice;

[0009] Perform a first centrifugal separation on the plant source juice to obtain a supernatant;

[0010] Perform a second centrifugal separation on the supernatant, and then wash the obtained precipitate to obtain the plant exosome-like nanoparticle;

[0011] Wherein, the plant source includes at least one of Rosa roxburghii Tratt, Artemisia desertorum Spreng seeds, Morus alba L., and Gynostemma pentaphyllum (Thunb.) Makino.

[0012] Furthermore, the weight ratio of the plant source to the buffer solution is 2:1; the buffer solution includes a PBS buffer solution, and the pH of the PBS buffer solution is 6.0.

[0013] Furthermore, calculated by weight parts, the addition amount of the enzyme accounts for 1-3 wt% of the total weight of the plant source and the buffer solution, and the enzyme includes cellulase.

[0014] Furthermore, the working condition parameters of the enzymatic hydrolysis include: the enzymatic hydrolysis temperature is 40 - 60°C, and the enzymatic hydrolysis time is 30 - 120 min.

[0015] Furthermore, the working condition parameters of the first centrifugal separation include: the centrifugal force is 1200g - 10000g, the centrifugal time is 20 - 100 min, and the centrifugal temperature is 4°C.

[0016] Furthermore, the step of subjecting the plant-derived juice to the first centrifugal separation to obtain the supernatant includes the following process:

[0017] At a temperature of 4°C, the plant-derived juice is centrifuged successively at a centrifugal force of 1200g for 20 min, at a centrifugal force of 3000g for 20 min, and at a centrifugal force of 10000g for 60 min to obtain the supernatant.

[0018] Furthermore, the working condition parameters of the second centrifugal separation include: the centrifugal force is 100000g, the centrifugal time is 120 min, and the centrifugal temperature is 4°C.

[0019] Furthermore, the preparation method further includes: subjecting the plant exosome-like nanovesicles to vacuum freeze-drying to prepare freeze-dried powder.

[0020] In a second aspect, the present invention provides a plant exosome-like nanovesicle, which is prepared by using the preparation method according to any one of the first aspect.

[0021] In a third aspect, the present invention provides the application of the plant exosome-like nanovesicle according to any one of the second aspect in the preparation of a product for improving the repair of intestinal mucosal barrier damage.

[0022] The above technical solutions provided by the embodiments of the present invention have at least the following advantages compared with the prior art:

[0023] The embodiments of the present invention provide a preparation method of plant exosome-like nanovesicles. The present invention combines an enzymatic hydrolysis method with an ultracentrifugation method to successfully prepare Rosa roxburghii / Artemisia sphaerocephala Krasch. / Mulberry leaf / Gynostemma pentaphyllum ELNs, which have the characteristics of simple process, safety and environmental protection, and good biological activity of the obtained ELNs. At the same time, in-depth research shows that the obtained plant exosome-like nanovesicles contribute to improving the repair of intestinal mucosal barrier damage, providing important data support for the development of products for restoring intestinal mucosal barrier function. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings herein are incorporated into the specification and constitute a part of this specification, showing embodiments consistent with the present invention and used together with the specification to explain the principles of the present invention.

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0026] Figure 1 It is a schematic flowchart of a preparation method of plant exosome-like nanovesicles provided by an embodiment of the present invention.

[0027] Figure 2 It is the test result of the storage stability of the sample obtained in Example 3 in the test example of the present invention for plant exosome-like nanovesicles at 4°C.

[0028] Figure 3 It is the test result of the storage stability of the sample obtained in Example 3 in the test example of the present invention for plant exosome-like nanovesicles at -20°C.

[0029] Figure 4 It is the test result of the antioxidant property of the sample obtained in Example 3 in the test example of the present invention for plant exosome-like nanovesicles by the FRAP method.

[0030] Figure 5 It is the test result of the antioxidant property of the sample obtained in Example 3 in the test example of the present invention for plant exosome-like nanovesicles by the DPPH method.

[0031] Figure 6 It is the test result of the digestion stability of the sample obtained in Example 3 in the test example of the present invention in the gastric juice and intestinal juice environments.

[0032] Figure 7 It is the test result of the colon length and appearance characteristics in the acute colitis mouse experiment of the sample obtained in Example 3 in the test example of the present invention.

[0033] Figure 8 It is the test result of the staining of colon tissue sections in the acute colitis mouse experiment of the sample obtained in Example 3 in the test example of the present invention.

[0034] Figure 9 It is the test result of the levels of inflammatory factors in the serum in the acute colitis mouse experiment of the sample obtained in Example 3 in the test example of the present invention.

[0035] Figure 10 It is the test result of the levels of intestinal tight junction proteins in the serum in the acute colitis mouse experiment of the sample obtained in Example 3 in the test example of the present invention.

[0036] Figure 11 It is the test result of in vivo fluorescence imaging and gastrointestinal imaging of the sample obtained in Example 3 in the test example of the present invention.

[0037] Figure 12 It is a technical concept flow chart of the preparation method of plant exosome-like nanovesicles provided by the present invention. Detailed implementation manners

[0038] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0039] Unless otherwise specifically stated, all kinds of raw materials, reagents, instruments and equipment used in the present invention can be obtained through market purchase or can be prepared by existing methods.

[0040] As an emerging biological nanomaterial, plant exosome-like nanovesicles show extraordinary potential in the biomedical field, especially in drug delivery, disease treatment and other aspects. However, the current related technologies such as its separation and extraction still face a series of challenges and deficiencies, such as complex operation and high cost, production limitation, environmental sensitivity, unstable delivery efficiency, etc., and the safety evaluation of its related products is insufficient, lacking unified standards and specifications. Specifically:

[0041] (1) Complex operation and high cost: Existing extraction methods, such as ultracentrifugation combined with sucrose density gradient centrifugation, can effectively purify, but the operation is cumbersome, time-consuming, and requires high equipment requirements, directly resulting in high extraction costs and limiting the possibility of its large-scale commercial application.

[0042] (2) Production limitation: The current technology has not achieved the efficient large-scale production of plant exosome-like nanovesicles. Although it can meet the clinical and scientific research needs, it is limited by production in its large-scale commercial production application.

[0043] (3) Environmental sensitivity: The stability of plant exosome-like nanovesicles is easily affected by external environmental factors, such as temperature, pH value, storage conditions, etc., which may lead to a decrease or inactivation of its biological activity. How to achieve long-term effective preservation while ensuring its biological activity is a difficult problem currently faced and continues to be overcome.

[0044] (4) Unstable delivery efficiency: The delivery efficiency of plant exosome-like nanovesicles in vivo is affected by various factors, such as blood circulation, tissue barriers, etc., and targeted delivery and improved delivery efficiency need to be achieved through further modification or optimization. Moreover, plant exosome-like nanovesicles from different plant sources differ in composition and properties, and it is difficult to uniformly control their extraction efficiency and purity.

[0045] In view of the existing problems, such as Figure 12 As shown, the primary object of the present invention is to provide a preparation method for four plant-derived exosome-like nanovesicles of Rosa roxburghii Tratt, Artemisia sphaerocephala Krasch seeds, mulberry leaves, and Gynostemma pentaphyllum, which can quickly and efficiently obtain the four plant-derived exosome-like nanovesicles. On the other hand: The present invention also provides the application of the above four plant-derived exosome-like nanovesicles in improving intestinal mucosal barrier injury, providing important data support for the development of oral products and drugs for the restoration of intestinal mucosal barrier function. The technical solutions provided by the present invention are as follows:

[0046] In the first aspect, the present invention provides a preparation method for plant exosome-like nanovesicles, as Figure 1 shown, the preparation method includes the following steps:

[0047] Mix and break a plant source and a buffer solution, and then add an enzyme for enzymatic hydrolysis to obtain a plant source juice;

[0048] Perform a first centrifugal separation on the plant source juice to obtain a supernatant;

[0049] Perform a second centrifugal separation on the supernatant, and then wash the obtained precipitate to obtain the plant exosome-like nanovesicles;

[0050] Wherein, the plant source includes at least one of Rosa roxburghii Tratt, Artemisia sphaerocephala Krasch seeds, mulberry leaves, and Gynostemma pentaphyllum.

[0051] The embodiments of the present invention provide a preparation method for plant exosome-like nanovesicles. The present invention has conducted in-depth research on the preparation method of Rosa roxburghii Tratt, Artemisia sphaerocephala Krasch, mulberry leaves, and Gynostemma pentaphyllum ELNs and their application in improving intestinal mucosal barrier injury, and proved that the four plant exosome-like nanovesicles obtained by this method are helpful for improving the repair of intestinal mucosal barrier injury and have practical application value.

[0052] The present invention uses an enzymatic hydrolysis and ultrafiltration method to prepare Rosa roxburghii Tratt / Artemisia sphaerocephala Krasch seeds / mulberry leaves / Gynostemma pentaphyllum ELNs. Not only is the operation process simple, safe, and environmentally friendly, effectively avoiding the pollution and damage that may be brought by traditional extraction methods, but also the extraction rate and purity of plant exosome-like nanovesicles are greatly improved. Moreover, the plant exosome-like nanovesicles prepared by this method are green, natural, have low immunogenicity, and stable structure, showing good antioxidant properties and biological activities.

[0053] Meanwhile, the plant-derived exosome-like nanovesicles were freeze-dried using vacuum freeze-drying technology to obtain freeze-dried powder of plant-derived exosome-like nanovesicles. The freeze-drying technology greatly improved the long-term storage stability of exosomes. The prepared exosome-like nanovesicles from Rosa roxburghii tratt and Morus alba L. have good storage stability, antioxidant activity and digestive stability, and the colon length of mice, the levels of inflammatory factors in serum and the levels of intestinal tight junction proteins in serum have all been improved to a certain extent, and it is expected to be used for the prevention and treatment of colitis.

[0054] In some specific embodiments, the weight ratio of the plant source to the buffer solution is 2:1; the buffer solution includes PBS buffer solution, and the pH of the PBS buffer solution is 6.0.

[0055] In some specific embodiments, by weight, the addition amount of the enzyme accounts for 1-3 wt% of the total weight of the plant source and the buffer solution, and the enzyme includes cellulase.

[0056] In some specific embodiments, the working condition parameters of the enzymatic hydrolysis include: the enzymatic hydrolysis temperature is 40-60 °C, and the enzymatic hydrolysis time is 30-120 min.

[0057] In some specific embodiments, the working condition parameters of the first centrifugal separation include: the centrifugal force is 1200g-10000g, the centrifugal time is 20-100 min, and the centrifugal temperature is 4 °C.

[0058] In some specific embodiments, the step of performing the first centrifugal separation on the plant source juice to obtain the supernatant includes the following process:

[0059] The plant source juice was centrifuged at 4 °C for 20 min at a centrifugal force of 1200g, 20 min at a centrifugal force of 3000g, and 60 min at a centrifugal force of 10000g in sequence to obtain the supernatant.

[0060] In some specific embodiments, the working condition parameters of the second centrifugal separation include: the centrifugal force is 100000g, the centrifugal time is 120 min, and the centrifugal temperature is 4 °C.

[0061] In some specific embodiments, the preparation method further includes: performing vacuum freeze-drying on the plant exosome-like nanovesicles to make freeze-dried powder.

[0062] In some specific embodiments, the preparation method of the plant exosome-like nanovesicles may specifically include:

[0063] Step 1: Put Rosa roxburghii / Artemisia sphaerocephala seeds / Mulberry leaves / Gynostemma pentaphyllum and PBS buffer into a crusher. After crushing, obtain Rosa roxburghii / Artemisia sphaerocephala seeds / Mulberry leaves / Gynostemma pentaphyllum juice. Add 2 wt% cellulase to the Rosa roxburghii / Artemisia sphaerocephala seeds / Mulberry leaves / Gynostemma pentaphyllum juice at room temperature in proportion and stir evenly.

[0064] Step 2: Centrifuge the liquid obtained in Step 1 at low speed several times. After each centrifugation, take the supernatant and centrifuge it again.

[0065] Step 3: Ultracentrifuge the supernatant obtained after centrifuging several times in Step 2. Take the precipitate and resuspend it with PBS buffer to obtain Rosa roxburghii / Artemisia sphaerocephala seeds / Mulberry leaves / Gynostemma pentaphyllum ELNs.

[0066] Step 4: Vacuum freeze-dry the phytogenic exosome-like nanovesicles obtained in Step 3 to obtain freeze-dried powder of Rosa roxburghii / Artemisia sphaerocephala seeds / Mulberry leaves / Gynostemma pentaphyllum ELNs.

[0067] Among them, the temperature of the centrifugation is 4°C. The number of times of repeated centrifugation in Step 2 is 3 times. The first centrifugation condition is 1200 g, 20 min; the second centrifugation condition is 3000 g, 20 min; the third centrifugation condition is 10000 g, 60 min. The removal effect of large particles, fine fragments and some impurities is the best under this centrifugation condition. The ultracentrifugation condition in Step 3 is 100000 g, 120 min. The purification effect is the best under this centrifugation condition.

[0068] In the second aspect, based on the same inventive concept, the present invention provides a phytogenic exosome-like nanovesicle, and the phytogenic exosome-like nanovesicle is prepared by the preparation method described in any item of the first aspect.

[0069] It should be noted that for each component involved in the phytogenic exosome-like nanovesicle and its preparation method provided in the embodiments of the present invention, if there is no special limitation or specific description, commercially available products can be used; at the same time, for the steps and parameters involved in the preparation method, if there is no special limitation or specific description, they can be carried out according to the existing process.

[0070] In the third aspect, based on the same inventive concept, the present invention provides the application of the phytogenic exosome-like nanovesicle described in any item of the second aspect in the preparation of products for improving intestinal mucosal barrier injury repair.

[0071] To sum up, the present invention provides the application of the preparation methods of four phytogenic exosome-like nanovesicles in mucosal barrier repair. Compared with the prior art, it has the following advantages:

[0072] (1) The present invention uses an enzymatic hydrolysis method combined with an ultracentrifugation method to successfully prepare Rosa roxburghii / Artemisia sphaerocephala Krasch. / Mulberry leaf / Gynostemma pentaphyllum ELNs. This method has a simple operation process, is safe, environmentally friendly, and effectively avoids the pollution and damage that may be caused by traditional extraction methods, greatly improving the extraction rate and purity of plant exosome-like nanovesicles. The ELNs prepared by this method have the characteristics of being green, natural, and having low immunogenicity. Their structure is stable, showing good antioxidant performance and biological activity.

[0073] (2) In addition, as natural nanoformulations, the above four plant exosome-like nanovesicles have the morphology and characteristics of nano-carriers and can be used as low-toxic nano-carriers to achieve targeted delivery of functional biological macromolecules (such as nucleic acids, therapeutic proteins, etc.). They are also superior to other nano-carriers in terms of in vivo distribution and stability, which is conducive to extending their effective action time in vivo; their rich components such as bioactive lipids, proteins, and RNAs can directly act on damaged mucosal cells to promote cell repair and regeneration; in mucosal barrier repair, this means that they can carry therapeutic substances to accurately reach the damaged mucosal site, improve the treatment effect and reduce the potential impact on other healthy tissues.

[0074] (3) The freeze-dried powder of the plant exosome-like nanovesicles prepared by the present invention has a fluffy appearance and compact powder, can be quickly redissolved, and the solution after redissolution is clear and transparent. Moreover, the properties such as the particle size and morphology of the plant exosome-like nanovesicles after redissolution have not changed significantly, which has important value for the long-term stability of exosomes.

[0075] The following further elaborates the present invention in combination with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually determined according to national standards. If there is no corresponding national standard, they are carried out according to general international standards, conventional conditions, or the conditions recommended by the manufacturer.

[0076] Example 1

[0077] This example provides a plant exosome-like nanovesicle and its preparation method. The preparation method includes the following steps:

[0078] (1) Put the clean plant source of Rosa roxburghii and PBS buffer solution with a pH of 6.0 into a crusher according to a weight ratio of 2:1 to obtain plant source juice;

[0079] (2) Centrifuge the plant source juice obtained in step 1 at a low speed several times at 4°C, and take the supernatant and centrifuge it again; among them, repeat 3 times, and the centrifugation conditions for the 3 times are 1200g, 20 min; 3000g, 20 min; 10000g, 60 min;

[0080] (3) Centrifuge the supernatant obtained after repeating step 2 three times at 4 °C at 100,000 g for 120 min, collect the precipitate, wash it three times with PBS buffer and resuspend it to obtain Rosa roxburghii Tratt ELNs, and store it in a refrigerator at -80 °C for later use;

[0081] (4) Vacuum freeze-dry the phytogenic exosome-like nanovesicles obtained in step 3. The working condition parameters of vacuum freeze-drying include: vacuum degree of 41.3 Pa, freezing temperature of 56.6 °C, and freezing time of 48 h, and the lyophilized powder can be obtained.

[0082] On the basis of using Rosa roxburghii Tratt as the plant source to prepare Rosa roxburghii Tratt exosome-like nanovesicles in Example 1 above, Artemisia sphaerocephala Krasch seeds, mulberry leaves, and Gynostemma pentaphyllum were used as plant sources to prepare Artemisia sphaerocephala Krasch seed exosome-like nanovesicles, mulberry leaf exosome-like nanovesicles, and Gynostemma pentaphyllum exosome-like nanovesicles respectively.

[0083] Example 2

[0084] This example provides a plant exosome-like nanovesicle and a preparation method thereof. The preparation method includes the following steps:

[0085] (1) Put the clean plant source Rosa roxburghii Tratt and PBS buffer with a pH of 6.0 into a crusher at a weight ratio of 2:1, add 1% cellulase according to the ratio at room temperature, and stir evenly to obtain a plant source juice;

[0086] (2) Centrifuge the plant source juice obtained in step 1 at a low speed several times at 4 °C, and take the supernatant and centrifuge it again; among them, repeat 3 times, and the centrifugation conditions for the 3 times are 1200 g, 20 min; 3000 g, 20 min; 10000 g, 60 min;

[0087] (3) Centrifuge the supernatant obtained after repeating step 2 three times at 4 °C at 100,000 g for 120 min, collect the precipitate, wash it three times with PBS buffer and resuspend it to obtain Rosa roxburghii Tratt ELNs, and store it in a refrigerator at -80 °C for later use;

[0088] (4) Vacuum freeze-dry the phytogenic exosome-like nanovesicles obtained in step 3. The working condition parameters of vacuum freeze-drying include: vacuum degree of 41.3 Pa, freezing temperature of 56.6 °C, and freezing time of 48 h, and the lyophilized powder can be obtained.

[0089] On the basis of using Rosa roxburghii Tratt as the plant source to prepare the freeze-dried powder of Rosa roxburghii Tratt exosome-like nanovesicles in Example 2 above, Artemisia sphaerocephala Krasch seeds, mulberry leaves, and Gynostemma pentaphyllum were used as plant sources to prepare the freeze-dried powder of Artemisia sphaerocephala Krasch seed exosome-like nanovesicles, the freeze-dried powder of mulberry leaf exosome-like nanovesicles, and the freeze-dried powder of Gynostemma pentaphyllum exosome-like nanovesicles respectively.

[0090] Example 3

[0091] This example provides a plant exosome-like nanovesicle and a preparation method thereof. The preparation method includes the following steps:

[0092] (1) Put clean Rosa roxburghii Tratt from plant source and PBS buffer solution with pH 6.0 into a crusher at a weight ratio of 2:1 for crushing. Then add 2% cellulase at room temperature in proportion and stir evenly to obtain plant source juice;

[0093] (2) Centrifuge the plant source juice obtained in step 1 at a low speed several times at 4 °C, and take the supernatant for further centrifugation. Among them, repeat 3 times, and the 3 centrifugation conditions are 1200g, 20 min; 3000g, 20 min; 10000g, 60 min respectively;

[0094] (3) Centrifuge the supernatant obtained by repeating the centrifugation in step 2 3 times at 100000g for 120 min at 4 °C, collect the precipitate, wash it three times with PBS buffer solution and then resuspend it to obtain Rosa roxburghii Tratt ELNs, and place it in a refrigerator at -80 °C for standby;

[0095] (4) Vacuum freeze-dry the plant-derived exosome-like nanovesicles obtained in step 3. The working condition parameters of vacuum freeze-drying include: vacuum degree is 41.3 Pa, freezing temperature is 56.6 °C, and freezing time is 48 h, and then the freeze-dried powder can be obtained.

[0096] On the basis of preparing the freeze-dried powder of Rosa roxburghii Tratt exosome-like nanovesicles with Rosa roxburghii Tratt as the plant source in the above Example 3, the freeze-dried powder of Artemisia sphaerocephala Krasch exosome-like nanovesicles, the freeze-dried powder of Morus alba L. exosome-like nanovesicles and the freeze-dried powder of Gynostemma pentaphyllum (Thunb.) Makino exosome-like nanovesicles are prepared with Artemisia sphaerocephala Krasch, Morus alba L. and Gynostemma pentaphyllum (Thunb.) Makino as the plant sources respectively.

[0097] Example 4

[0098] This example provides a plant exosome-like nanovesicle and a preparation method thereof. The preparation method includes the following steps:

[0099] (1) Put clean Rosa roxburghii Tratt from plant source and PBS buffer solution with pH 6.0 into a crusher at a weight ratio of 2:1 for crushing. Then add 3% cellulase at room temperature in proportion and stir evenly to obtain plant source juice;

[0100] (2) Centrifuge the plant source juice obtained in step 1 at a low speed several times at 4 °C, and take the supernatant for further centrifugation. Among them, repeat 3 times, and the 3 centrifugation conditions are 1200g, 20 min; 3000g, 20 min; 10000g, 60 min respectively;

[0101] (3) Centrifuge the supernatant obtained after repeating step 2 three times at 4°C at 100,000 g for 120 min, collect the precipitate, wash it three times with PBS buffer and resuspend it to obtain Rosa roxburghii Tratt ELNs, and place it in a refrigerator at -80°C for standby;

[0102] (4) Vacuum freeze-dry the plant-derived exosome-like nanovesicles obtained in step 3. The working condition parameters of vacuum freeze-drying include: vacuum degree of 41.3 Pa, freezing temperature of 56.6°C, and freezing time of 48 h to obtain the freeze-dried powder.

[0103] On the basis of obtaining the freeze-dried powder of Rosa roxburghii Tratt exosome-like nanovesicles using Rosa roxburghii Tratt as the plant source in Example 4 above, the freeze-dried powder of Artemisia sphaerocephala Krasch exosome-like nanovesicles, the freeze-dried powder of mulberry leaf exosome-like nanovesicles, and the freeze-dried powder of Gynostemma pentaphyllum exosome-like nanovesicles are prepared using Artemisia sphaerocephala Krasch, mulberry leaf, and Gynostemma pentaphyllum as the plant sources respectively.

[0104] Taking Rosa roxburghii Tratt exosome-like nanovesicles as an example, use a BCA protein quantification kit (Beyotime Biotechnology Co., Ltd.) to measure the protein concentration of the Rosa roxburghii Tratt exosome-like nanovesicles obtained in the above examples. The test results of exosome-like nanovesicles in the above examples are shown in Table 1 below.

[0105] Table 1

[0106] Result Protein concentration (mg / mL) Extraction rate (%) Example 1 5.12 0.034 Example 2 5.53 0.037 Example 3 7.16 0.048 Example 4 7.53 0.050

[0107] Example 5

[0108] On the basis of Example 4 above, taking Rosa roxburghii Tratt exosome-like nanovesicles as an example, using the single-factor variable method (that is, only adjusting a certain key condition parameter in Example 4, and the rest of the steps and conditions are the same as in Example 4), a screening experiment is carried out on the reaction condition parameters in each preparation step of Example 4. The specific condition parameters of each single variable and the protein concentration and extraction rate in the obtained Rosa roxburghii Tratt exosome-like nanovesicles are shown in Table 2.

[0109] Table 2

[0110]

[0111] Test Example

[0112] Take an appropriate amount of the four plant exosome-like nanovesicles obtained in Example 3 above, and use PBS buffer to dilute them to an appropriate concentration. In this example, performance test analysis is carried out on the four samples of Rosa roxburghii Tratt / Artemisia sphaerocephala Krasch / mulberry leaf / Gynostemma pentaphyllum exosome-like nanovesicles obtained in Example 3 above.

[0113] 1. Characterization analysis of exosome-like nanovesicles

[0114] (1) Storage stability

[0115] Appropriately absorb exosome-like nanovesicles of Rosa roxburghii / Artemisia sphaerocephala Krasch. / Mulberry leaf / Gynostemma pentaphyllum respectively, dilute them with PBS buffer and dispense them into test tubes. Store them at 4 °C and -20 °C for 12 d respectively, and regularly (0, 4, 8, 12 d) use a NanoZS90 particle size analyzer to measure the particle size of each sample in automatic mode to evaluate its storage stability. As Figure 2 and Figure 3 shown, the particle sizes of the four freshly prepared exosome-like nanovesicles are not significantly different, all less than 200 nm. Further storage observation found that under the storage condition of 4 °C ( Figure 2 ), the particle sizes of the four nanovesicles all showed an increasing trend to varying degrees over time, indicating that their stability gradually decreased. In contrast, when stored at -20 °C ( Figure 3 ), the increase in the particle sizes of the four exosome-like nanovesicles showed an obvious delaying trend. In addition, under the storage condition of -20 °C ( Figure 3 ), within 8 d, its particle size remained basically stable, showing relatively high storage stability. As the storage time was extended to 12 d, a significant increasing trend in particle size began to appear. To sum up, when the four exosome-like nanovesicles are stored at -20 °C, good storage stability can be maintained for at least 8 d.

[0116] (2) Antioxidant property

[0117] The FRAP (ferric reducing ability of plasma) method and DPPH (1,1-diphenyl-2-picrylhydrazyl) method were used to evaluate the antioxidant properties of exosome-like nanovesicles from four different sources, as Figure 4 and Figure 5 shown. It can be seen that the FRAP values and DPPH scavenging rates of exosome-like nanovesicles from four different sources all showed a significant upward trend with the increase in protein concentration. The larger the FRAP value and DPPH scavenging rate, the stronger its antioxidant property. Specifically, at the same protein concentration, the exosome-like nanovesicles of Gynostemma pentaphyllum had the highest FRAP value and DPPH scavenging rate, and its antioxidant property was the strongest. Followed by mulberry leaf, Rosa roxburghii, Artemisia sphaerocephala Krasch. in turn.

[0118] (3) Digestive stability

[0119] First, simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared. Subsequently, Rosa roxburghii / Artemisia sphaerocephala / Morus alba / Gynostemma pentaphyllum ELNs were diluted with PBS buffer respectively. Next, 0.5 mL of each diluted liquid was added to 5 mL of pre-prepared simulated gastric fluid. In a constant temperature shaker at 37 °C, it was left standing for 2 h. After the predetermined time, 1.5 mL was taken from each sample in each group and its particle size distribution was immediately measured to evaluate its stability in the simulated gastric environment. After completing the simulated gastric digestion stage, the simulated gastric fluid of each group was replaced with simulated intestinal fluid to continue the simulation of the intestinal digestion process. The samples were again placed in a constant temperature shaker at 37 °C and left standing for 2 h. After the time, 1.5 mL was also taken from each sample in each group and its particle size distribution was immediately measured. As Figure 6 shown, through this series of in vitro simulated digestion experiments, it can be clearly observed that the particle size of Rosa roxburghii / Artemisia sphaerocephala / Morus alba / Gynostemma pentaphyllum ELNs did not change significantly after undergoing simulated digestion in the stomach and intestine. This result indicates that Rosa roxburghii / Artemisia sphaerocephala / Morus alba / Gynostemma pentaphyllum ELNs exhibit good stability in the gastrointestinal environment, providing strong support for their application as potential drug delivery systems.

[0120] 2. Experiment on mice with acute colitis

[0121] (1) Establishment and grouping of mice models with acute colitis

[0122] Before the experiment, 42 SPF-grade Balb / c mice aged 6 - 8 weeks were adaptively fed for one week and randomly divided into 6 groups (n = 7 in each group): blank control group (Control), model group (DSS), Rosa roxburghii exosome-like nanovesicles (DSS + R-ELNs), Artemisia sphaerocephala exosome-like nanovesicles (DSS + A-ELNs), Morus alba exosome-like nanovesicles (DSS + M-ELNs), and Gynostemma pentaphyllum exosome-like nanovesicles (DSS + G-ELNs). No treatment was done to the 6 groups in terms of diet. Except for the control group, 3% DSS was added to the drinking water of the other 5 groups to induce colitis in mice. The four groups of plant exosome-like nanovesicles were given intragastric administration once a day, with a dosage of 10 mg / kg, for 14 consecutive days; all mice were sacrificed 24 hours after the administration on the 14th day.

[0123] (2) Colon length and appearance characteristics

[0124] The mice were sacrificed 24 hours after the administration on the 14th day. During the experiment, obvious colitis symptoms such as weight loss, loose stools, and bloody stools were observed in the mice. As Figure 7As shown in the figure, by observing the macroscopic state of the colon, it was found that the colon of the mice in the DSS group was significantly shorter than that of the normal mouse group. After treatment with Rosa roxburghii / Artemisia sphaerocephala / Kyoto University / ELNs, the shortening of the colon in the mice was improved, and the colon length increased slightly. Among them, the DSS+M-ELNs group and the DSS+G-ELNs group were closer to the normal state.

[0125] (3) Staining of colon tissue sections

[0126] The results of histopathological analysis showed ( Figure 8 ), in the normal group, the mucosa and crypts of the colon tissue were intact, the submucosa and peripheral muscle layers were thinner, there was no inflammatory infiltration, and no obvious pathological changes were seen. In the DSS group, the crypts were severely damaged and there was inflammatory cell infiltration. After treatment with 4 kinds of PELNs, the situation was improved, the number of crypts increased, the structure was more complete, and the edema and inflammatory cell infiltration decreased. In addition, compared with the other two groups, the Rosa roxburghii ELNs group and the Morus alba ELNs group could significantly improve and reduce the histological score of the colon.

[0127] (4) Levels of inflammatory factors in serum

[0128] As Figure 9 shown, compared with the control group, the levels of IL-1β, IL-6 and TNF-α in the serum of the mice induced by DSS were significantly increased. The ELNs group could effectively reduce the levels of each inflammatory factor, especially the Rosa roxburghii ELNs group and the Morus alba ELNs group had more significant effects.

[0129] (5) Levels of intestinal tight junction proteins in serum

[0130] Tight junctions are the main determinants for maintaining mucosal permeability of intestinal barrier function. Among them, Claudin-1, ZO-1 and Occludin play important roles and are widely used as markers of intestinal integrity. As Figure 10 shown, compared with the control group, the TJ proteins (Claudin-1, ZO-1 and Occludin) induced by DSS decreased significantly. The levels of TJ proteins in the 4 kinds of PELNs groups were significantly higher than those in the DSS group, and the Rosa roxburghii ELNs group and the Morus alba ELNs group were more significant than the other sample groups.

[0131] (6) In vivo fluorescence imaging and gastrointestinal imaging

[0132] Cy5.5-labeled exosome-like nanovesicles: Add 10 mM Cy5.5 dye to the plant exosome-like nanovesicles at a concentration of 2 mg / mL, and gently shake and incubate at room temperature for 60 min. The above experiments were carried out under light-proof conditions.

[0133] In vivo fluorescence imaging: C57BL / 6 mice were gavaged with Cy5.5-labeled plant exosome-like nanovesicles at a dose of 200 μL per mouse. The mice were anesthetized at 0.5, 1, 2, 4, 6, 8, and 10 h respectively for in vivo imaging.

[0134] Gastrointestinal imaging: The mice were sacrificed and the gastrointestinal tracts were removed for imaging. To further study the distribution of plant exosome-like nanovesicles in the gastrointestinal tract, ex vivo fluorescence imaging of the gastrointestinal tissues of the mice was performed at 0.5, 1, 2, 4, 6, 8, and 10 h.

[0135] As Figure 11 shown, in in vivo fluorescence imaging, clear fluorescence signals were detected in the upper abdomen of the mice 0.5 h after oral administration, and then gradually spread to the lower abdomen. The in vivo fluorescence signals were very weak after 10 h. In gastrointestinal imaging, at 0.5 h, plant exosome-like nanovesicles covered the stomach, duodenum, and jejunum. At 2 h, they were mainly in the ileum. After 4 h, there was almost no fluorescence signal in the upper half of the intestine, and there were strong fluorescence signals in the cecum and colon. After 8 h, the fluorescence signals in the whole intestine decreased sharply. At 10 h, only partial fluorescence remained in the stomach and the end of the intestine, indicating that most of the plant exosome-like nanovesicles were metabolized.

[0136] Various embodiments of the present invention may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be construed as a rigid limitation on the scope of the present invention; therefore, it should be considered that the described range description has specifically disclosed all possible sub-ranges and single values within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5, and 6, and this applies regardless of the range. Additionally, whenever a numerical range is indicated herein, it means including any cited number (fraction or integer) within the indicated range.

[0137] The above are only specific embodiments of the present invention, enabling those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather will conform to the broadest scope consistent with the principles and novel features claimed herein.

Claims

1. Use of plant exosome-like nanovesicles in the preparation of a drug for improving acute colitis, characterized in that: The method for preparing the plant exosome-like nanovesicles comprises the following steps: The plant source and the buffer solution are mixed and crushed, and then enzymes are added for enzymolysis to obtain plant source juice; Performing a first centrifugal separation on the plant-derived juice to obtain a supernatant; The supernatant is subjected to a second centrifugal separation, and the obtained precipitate is then washed to obtain the plant exosome-like nanovesicles; Wherein, the plant source is at least one of roxburghii, wormwood seeds, mulberry leaves and gynostemma pentaphyllum; The weight ratio of the plant source to the buffer is 2:1; the buffer comprises a PBS buffer, and the pH of the PBS buffer is 6.0; In terms of weight, the added amount of the enzyme accounts for 1-3wt% of the total weight of the plant source and the buffer solution, and the enzyme is cellulase.

2. The use according to claim 1, characterized in that: The working condition parameters of the enzymatic hydrolysis include: the enzymatic hydrolysis temperature is 40-60° C., and the enzymatic hydrolysis time is 30-120 min.

3. The use according to claim 1, characterized in that: The working condition parameters of the first centrifugal separation include: centrifugal force of 1200g to 10000g, centrifugal time of 20 to 100min, and centrifugal temperature of 4°C.

4. The use according to claim 1, characterized in that: The step of subjecting the plant-derived juice to a first centrifugal separation to obtain a supernatant comprises the following process: The plant-derived juice was centrifuged at 4° C. for 20 min at a centrifugal force of 1200 g, for 20 min at a centrifugal force of 3000 g, and for 60 min at a centrifugal force of 10000 g to obtain a supernatant.

5. The use according to claim 1, characterized in that: The working condition parameters of the second centrifugal separation include: centrifugal force of 100000g, centrifugal time of 120min, and centrifugal temperature of 4°C.

6. The use according to any one of claims 1 to 5, characterized in that: The method for preparing the plant exosome-like nanovesicles further includes: vacuum freeze-drying the plant exosome-like nanovesicles to prepare freeze-dried powder.

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