Momordica grosvenori extracellular vesicles and application thereof in intestinal inflammation

Abstract

The invention relates to the field of health care products, and particularly provides fructus momordicae extracellular vesicles and application thereof in intestinal inflammation, the fructus momordicae extracellular vesicles comprise, by mass, 75-80% of fructus momordicae, 8-10% of a fructus momordicae fermentation broth, and 8-12% of a fructus momordicae extract; the preparation method of the siraitia grosvenorii extracellular vesicles comprises the following steps: S1, taking a certain amount of fresh siraitia grosvenorii plants, removing impurities, washing the fresh siraitia grosvenorii plants with domestic drinking water, washing the fresh siraitia grosvenorii plants with purified water for three times, and finally washing the fresh siraitia grosvenorii plants with ultrapure water for three times, s2, cutting cleaned fresh siraitia grosvenorii plants into blocks, respectively putting the blocks into a juicer for mixing and crushing, continuously squeezing for three times, filtering, and taking juice to obtain siraitia grosvenorii plant normal juice; experimental results of anti-inflammatory and intestinal cell proliferation promoting tests show that the extracellular vesicles of momordica grosvenori can promote proliferation of intestinal mucosa crypt cells, antagonize release of inflammatory factors induced by macrophages and increase generation of anti-inflammatory factors, so that the anti-inflammatory effect is achieved.

Description

Technical Field

[0001] This invention relates to the technical field of health products, specifically to a type of monk fruit extracellular vesicle and its application in intestinal inflammation. Background Technology

[0002] Monk fruit (Siraitia grosvenorii) is a climbing herbaceous plant belonging to the genus Siraitia in the family Cucurbitaceae. According to the *Lingnan Caiyao Lu* (Records of Medicinal Herbs from Lingnan), monk fruit is cool in nature and sweet in taste, entering the lung and large intestine meridians. It has the effects of clearing the lungs and relieving sore throat, resolving phlegm and relieving cough, and moistening the intestines and promoting bowel movements. It is mainly used to treat cough, asthma, sore throat, constipation, and other ailments. The extracellular vesicles of monk fruit contain abundant bioactive substances, lipids, and nucleic acids, which can quickly open cell membrane channels, be absorbed by cells, have minimal irritation to the gastrointestinal tract, and exert therapeutic effects, thus possessing excellent market prospects.

[0003] While traditional anti-inflammatory drugs can reduce inflammation in the treatment of inflammatory bowel diseases, their effects on intestinal cell proliferation and repair are often limited. Furthermore, long-term use of these drugs may lead to adverse reactions and drug resistance. Although natural plant extracts such as monk fruit have been proven to have anti-inflammatory effects, their application in the treatment of inflammatory bowel diseases is not widespread due to issues such as the purity, stability, bioavailability, and delivery methods of the extracts. Summary of the Invention

[0004] The purpose of this invention is to provide a monk fruit extracellular vesicle and its application in intestinal inflammation, in order to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a monk fruit extracellular vesicle, wherein the monk fruit extracellular vesicle comprises, by weight percentage, 75-80% monk fruit, 8-10% monk fruit fermentation broth, and 8-12% monk fruit extract;

[0006] The method for preparing the extracellular vesicles of monk fruit includes the following steps:

[0007] S1: Take a certain amount of fresh monk fruit plants, remove impurities, rinse with drinking water, then rinse three times with purified water, and finally rinse three times with ultrapure water, each rinse lasting 3-5 minutes.

[0008] S2: Cut the washed fresh monk fruit plant into pieces, put them into a juicer and mix and grind them. After juicing three times, filter and collect the juice to obtain the original monk fruit plant juice.

[0009] S3: Centrifuge the original juice of monk fruit at 4℃ using differential speed. The first centrifugation is performed at 1000g for 10 min to obtain supernatant A. Then, centrifuge supernatant A at 3000g for 20 min to obtain supernatant B. Then, centrifuge supernatant B at 10000g for 40 min to remove impurities and obtain supernatant C. Finally, centrifuge supernatant C at 150000g for 120 min, collect the precipitate, and wash it three times with phosphate buffer to obtain the extracellular vesicles of monk fruit.

[0010] S4: Set aside the extracellular vesicles of monk fruit obtained in step S3.

[0011] Preferably, the extracellular vesicle size distribution of the monk fruit is between 30-150 nm.

[0012] The application of monk fruit extracellular vesicles in intestinal inflammation, the application of monk fruit extracellular vesicles in drugs for treating inflammatory responses.

[0013] Preferably, the application of the monk fruit in the preparation of a treatment for intestinal injury diseases.

[0014] Preferably, the use of the monk fruit extracellular vesicles in the treatment of enteritis or in the suppression of inflammatory responses of immune cells.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] This invention provides an anti-inflammatory extracellular vesicle from monk fruit, which is easy to extract. Experiments on its anti-inflammatory and intestinal cell proliferation-promoting effects show that the extracellular vesicles from monk fruit can promote the proliferation of intestinal mucosal crypt cells, antagonize the release of inflammatory factors induced by macrophages, and increase the production of anti-inflammatory factors, thereby producing an anti-inflammatory effect. Attached Figure Description

[0017] Figure 1 This is a characterization diagram of extracellular vesicles in Luo Han Guo (Siraitia grosvenorii) observed by transmission electron microscopy according to the present invention.

[0018] Figure 2 This is a diagram showing the particle size distribution of extracellular vesicles in monk fruit according to the present invention.

[0019] Figure 3 This is a schematic diagram illustrating how the extracellular vesicles of monk fruit can promote the proliferation of small intestinal crypt epithelial cells, according to the present invention.

[0020] Figure 4 This is a schematic diagram illustrating how the extracellular vesicles of monk fruit can significantly inhibit the protein level of the pro-inflammatory factor IL-1β, as described in this invention.

[0021] Figure 5 This is a schematic diagram illustrating how the extracellular vesicles of monk fruit can significantly inhibit the protein level of the pro-inflammatory factor IFN-gamma, as described in this invention.

[0022] Figure 6 This is a schematic diagram illustrating how the extracellular vesicles of monk fruit can significantly inhibit the protein level of the pro-inflammatory factor IL-12, as described in this invention.

[0023] Figure 7 This is a schematic diagram illustrating how the extracellular vesicles of monk fruit can significantly increase the protein level of the anti-inflammatory factor IL-10, as described in this invention. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Please see Figures 1 to 7 The present invention provides a technical solution: a monk fruit extracellular vesicle, wherein the monk fruit extracellular vesicle comprises, by weight percentage, 75-80% monk fruit, 8-10% monk fruit fermentation broth, and 8-12% monk fruit extract.

[0026] The preparation method of extracellular vesicles from monk fruit includes the following steps:

[0027] S1: Take a certain amount of fresh monk fruit plants, remove impurities, rinse with drinking water, then rinse three times with purified water, and finally rinse three times with ultrapure water, each rinse lasting 3-5 minutes.

[0028] S2: Cut the washed fresh monk fruit plant into pieces, put them into a juicer and mix and grind them. After juicing three times, filter and collect the juice to obtain the original monk fruit plant juice.

[0029] S3: Centrifuge the original juice of monk fruit at 4℃ using differential speed. The first centrifugation is performed at 1000g for 10 min to obtain supernatant A. Then, centrifuge supernatant A at 3000g for 20 min to obtain supernatant B. Then, centrifuge supernatant B at 10000g for 40 min to remove impurities and obtain supernatant C. Finally, centrifuge supernatant C at 150000g for 120 min, collect the precipitate, and wash it three times with phosphate buffer to obtain the extracellular vesicles of monk fruit.

[0030] S4: Set aside the extracellular vesicles of monk fruit obtained in step S3.

[0031] Example 1: Morphological observation of extracellular vesicles in Luo Han Guo (Siraitia grosvenorii) cells using transmission electron microscopy:

[0032] First, the grid was hydrophilically treated using a glow discharge apparatus to facilitate sample adsorption onto the carbon film. Then, the grid was held by its edge with self-locking tweezers and placed on a petri dish to suspend it. 10 μL of sample was then dropped onto the grid and allowed to stand for 5 minutes.

[0033] After blotting away excess liquid with lens paper, add 1 drop of ultrapure water to the screen, wipe it off after 10 seconds, and repeat 3 times to remove salt components.

[0034] Then, 10 μL of phosphotungstic acid was added to the screen, stained for 1 minute, and then wiped off with lens paper.

[0035] The carrier screen was dried under a baking lamp and then examined using a 120kV transmission electron microscope.

[0036] Example 3: Nanoparticle tracking analysis of extracellular vesicle size in monk fruit

[0037] Open the ZetaView software and warm it up.

[0038] Use a syringe to inject ultrapure water into the chamber for cleaning. After the instrument passes the self-test, inject a standard sample of 100nm microspheres into the chamber for calibration. After calibration, clean the chamber again with ultrapure water and prepare for sample loading.

[0039] Select autofocus, and start the detection after focusing is complete to obtain the concentration and particle size distribution of exosomes in the sample.

[0040] As attached Figure 2 The results showed that the extracellular vesicle size distribution of monk fruit ranged from 30 to 150 nm.

[0041] Example 3: Extracellular vesicles of monk fruit can promote the proliferation of small intestinal crypt epithelial cells.

[0042] Resuscitate IEC-6 cells, centrifuge at 400g for 10 min, and discard the supernatant; adjust the concentration of IEC-6 cells to 1×10⁵ / mL with DMEM containing 10% FBS.

[0043] IEC-6 cells in the logarithmic growth phase were seeded into 96-well plates at a density of 10,000 cells / well, for a total volume of 200 μL, and then incubated statically in a 37°C, 5% CO2 cell culture incubator.

[0044] After the cells adhered overnight, 40 μg / mL of monk fruit filtrate and monk fruit extracellular vesicles were added, and the cells were incubated for 48 h. Each group had two replicates, and a cell control group and a culture medium blank group were also included. After culturing for another 48 hours, 20 μL of CCK8 reagent was added, and the cells were incubated at 37℃ in the dark for 0.5 h–1 h. The absorbance of each well was measured at 450 nm using a microplate reader.

[0045] Calculate the cell proliferation percentage using the following formula: Cell viability percentage = [(AC) / (BC)] × 100% (A is the absorbance value of the experimental group; B is the absorbance value of the control group; C is the absorbance value of the blank group) (containing only culture medium and CCK8 reagent)

[0046] Appendix Figure 3 The results showed that the extracellular vesicles of monk fruit significantly promoted the proliferation of small intestinal crypt epithelial cells, with a significant difference compared to the cell control group; although the monk fruit filtrate also promoted the proliferation of small intestinal crypt epithelial cells, there was no difference compared to the cell control group.

[0047] Example 4: Detection of the anti-inflammatory function of extracellular vesicles of monk fruit

[0048] After reviving and culturing the RAW264.7 cell line for 24 hours, centrifuge at 400g for 10 minutes and discard the supernatant; adjust the cell concentration to 1×10⁵ / mL with DMEM containing 10% FBS.

[0049] RAW264.7 cells in the logarithmic growth phase were seeded into 96-well plates at 1×10⁴ cells / well and incubated overnight at 37°C in a 5% CO₂ cell culture incubator.

[0050] The next day, discard the old culture medium in each well, add LPS to a final concentration of 1ug / ml, and 12 hours later add the prepared 30ug / ml monk fruit filtrate or 30ug / ml monk fruit extracellular vesicles according to Table 1. Two replicates were set up for each group, and a cell control group and an LPS positive control group were also set up.

[0051] The cells were cultured in a 37℃ 5% CO2 cell culture incubator for 24h and 48h, and 80uL of cell supernatant was collected at each time point.

[0052] Cells were centrifuged at 300xg for 10 min at 4°C, and the cell supernatant was collected. Following the instructions of the multifactor assay kit (BIO-RAD, 171304090M), the protein levels of pro-inflammatory factors IL-1β, IFN-gamma, IL-12, and anti-inflammatory factor IL-10 in the supernatant were measured using a Luminex instrument platform.

[0053] The experimental group is shown in Table 1 below:

[0054] Grouping Sample Name Use concentration (ug / ml) 1 Cell control group 0 2 Positive control group 1ug / ml LPS 3 Drug administration group 1ug / ml LPS+30ug / ml Siraitia-filtrate 4 Drug administration group 1ug / ml LPS+30ug / ml Siraitia-EVs

[0055] The experimental results are attached. Figure 4 Appendix Figure 5 Appendix Figure 6 Appendix Figure 7In LPS-induced inflammatory responses, extracellular vesicles of *Siraitia grosvenorii* significantly inhibited the protein levels of pro-inflammatory cytokines IL-1β, IFN-gamma, and IL-12, while increasing the expression level of the anti-inflammatory cytokine IL-10, showing a significant difference compared to the LPS-positive control group. *Siraitia grosvenorii* filtrate also decreased the expression levels of IL-1β, IFN-gamma, and IL-12, and slightly increased the expression level of the anti-inflammatory cytokine IL-10, but without a significant difference compared to the LPS-positive control group. Adding *Siraitia grosvenorii* extracellular vesicles at a protein concentration of 30 μg / ml demonstrated a good anti-inflammatory effect.

[0056] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A Momordica grosvenori extracellular vesicle, characterized in that: The monk fruit extracellular vesicle comprises, by mass percentage, 75-80% of monk fruit, 8-10% of monk fruit fermentation liquid, and 8-12% of monk fruit extract; The method for preparing the extracellular vesicles of Momordica grosvenori comprises the following steps: S1: Take a certain amount of fresh monk fruit plants, remove impurities, rinse with drinking water, then wash three times with purified water, and finally wash three times with ultrapure water, each washing time is 3-5 minutes; S2: Cut the washed fresh Momordica grosvenori plants into pieces, put them into a juicer for mixing and crushing, squeeze them three times in a row, filter and take out the juice to obtain the Momordica grosvenori plant juice; S3: The monk fruit juice was subjected to differential centrifugation at 4°C, with the first centrifugal force of 1000g for 10 minutes to obtain supernatant A; then the supernatant A was centrifuged at 3000g for 20 minutes to obtain supernatant B, and then the supernatant B was centrifuged at 10000g for 40 minutes to remove impurities to obtain supernatant C, and finally the supernatant C was ultra-centrifuged at 150000g for 120 minutes, the precipitate was taken, and washed three times with phosphate buffer to obtain monk fruit extracellular vesicles; S4: The extracellular vesicles of Momordica grosvenori obtained in step S3 are set aside.

2. The extracellular vesicle of Momordica grosvenori according to claim 1, characterized in that: The particle size distribution of the monk fruit extracellular vesicles is between 30-150 nm.

3. An application of Momordica grosvenori extracellular vesicles in intestinal inflammation, characterized in that: The application is the monk fruit extracellular vesicles as described in any one of claims 1-2, and the application of the monk fruit extracellular vesicles in drugs for treating inflammatory reactions.

4. The use of a Momordica grosvenori extracellular vesicle in intestinal inflammation according to claim 3, characterized in that: The application of the monk fruit cells in the external preparation for treating intestinal damage diseases.

5. The extracellular vesicle of Momordica grosvenori and its use in intestinal inflammation according to claim 3, characterized in that: The application of the monk fruit extracellular vesicles in treating enteritis or inhibiting the inflammatory response of immune cells.

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