Application of fissistigma oldhamii outer vesicles in preparation of medicine for treating inflammation

Abstract

The invention discloses application of fissistigma oldhamii outer vesicles in preparation of a medicine for treating inflammation, and belongs to the technical field of food and medical health care products. According to the application disclosed by the invention, the outer vesicles are separated and extracted from fissistigma oldhamii, and experimental verification shows that the fissistigma oldhamii outer vesicles can be used for remarkably inhibiting the proliferation of Raw 264.7 cells, and can be used for remarkably inhibiting the inflammatory response of the Raw 264.7 cells induced by LPS (Lipopolysaccharide). The fissistigma oldhamii outer vesicles provided by the invention are derived from plants, are non-toxic to healthy cells, show an obvious anti-inflammatory function and inhibit cancer cell proliferation, have small side effects, provide a new treatment means for inflammation treatment and cancer prevention, and have great significance in scientific, reasonable and sustainable development and utilization of fissistigma oldhamii resources.

Description

Technical Field

[0001] The present invention relates to the technical field of food and pharmaceutical health products, and particularly to the application of Fissistigma oldhamii exosomes in the preparation of drugs for treating inflammation. Background Art

[0002] The plant resources of Fissistigma are rich, with about 75 species globally, widely distributed in tropical and subtropical regions, and having extremely high medicinal value, with cardiovascular protection, anti-inflammatory and immunosuppressive, anti-tumor, anti-bronchial asthma, anti-tuberculosis, antioxidant, antibacterial, analgesic and other effects. As a traditional Chinese medicine, the stem of Fissistigma oldhamii is often used to treat arthritis and rheumatic bone pain, etc. Plant exosomes are a kind of natural active substances that have been widely concerned in recent years. They have similar structures and functions to animal exosomes, and promote cell communication by carrying and transporting bioactive molecules (including DNA, RNA, proteins and lipids), changing the physiological and pathological functions of recipient cells. Plant exosomes have the advantages of low toxicity and side effects and low immunogenicity, and have great potential for drug delivery. However, the current research on plant exosomes is still in its infancy, and the extraction and purification methods have become the biggest obstacles to the research. Moreover, there are significant differences in the structures and functions of different plant exosomes. There have been no reports on Fissistigma oldhamii exosomes so far. Summary of the Invention

[0003] The purpose of the present invention is to provide the application of Fissistigma oldhamii exosomes in the preparation of drugs for treating inflammation, so as to solve the problems existing in the above-mentioned prior art. Fissistigma oldhamii exosomes can significantly inhibit the inflammatory response produced by mouse mononuclear macrophage leukemia cells Raw264.7 induced by LPS, and have a significant anti-inflammatory effect, providing a new treatment means for inflammation treatment and cancer prevention.

[0004] To achieve the above purpose, the present invention provides the following scheme:

[0005] The present invention provides the application of Fissistigma oldhamii exosomes in the preparation of drugs for treating inflammation, and the Fissistigma oldhamii exosomes are isolated from Fissistigma oldhamii.

[0006] The present invention also provides the application of Fissistigma oldhamii exosomes in the preparation of drugs for inhibiting the proliferation of leukemia cells, and the Fissistigma oldhamii exosomes are isolated from Fissistigma oldhamii.

[0007] Preferably, the isolation includes the following steps:

[0008] Crush and filter the stem of Fissistigma oldhamii to obtain the original juice of Fissistigma oldhamii;

[0009] Extract the original juice of Fissistigma oldhamii by gradient centrifugation and collect the precipitate;

[0010] Resuspend the precipitate with sterile physiological saline, centrifuge, and collect the supernatant to obtain the Fissistigma oldhamii exosomes.

[0011] Preferably, the extraction by gradient centrifugation comprises the following steps:

[0012] Based on the total volume of the original Fissistigma oldhamii juice, add sodium bicarbonate with a mass - volume ratio of 3 - 10%, proline with a mass - volume ratio of 0.5% - 10%, and ethyl acetate with a mass - volume ratio of 0.5 - 5% to the original Fissistigma oldhamii juice. After standing at room temperature for 20 - 30 min, then centrifuge at 10°C and 6500g for 10 min to obtain supernatant A;

[0013] Centrifuge the supernatant A at 10°C and 10000g for 20 min to obtain supernatant B;

[0014] Mix supernatant B and extraction reagent A according to a volume ratio of 2:1, let it stand, and then centrifuge at 10°C and 12000g for 10 min to obtain supernatant C;

[0015] Mix supernatant C and extraction reagent B according to a volume ratio of 3:1, let it stand, and then centrifuge at 10°C and 13500g for 30 min. Discard the supernatant and collect the precipitate;

[0016] After resuspending the precipitate, centrifuge at 2000g for 10 min.

[0017] Preferably, the inflammation includes anti - LPS - induced inflammation in Raw 264.7 cells.

[0018] The present invention also provides a method for preparing Fissistigma oldhamii exosomes, comprising the following steps:

[0019] Crush and filter the stems of Fissistigma oldhamii to obtain the original Fissistigma oldhamii juice;

[0020] Extract the original Fissistigma oldhamii juice by gradient centrifugation and collect the precipitate;

[0021] Resuspend the precipitate with sterile normal saline, centrifuge, and collect the supernatant to obtain the Fissistigma oldhamii exosomes.

[0022] Preferably, the extraction by gradient centrifugation comprises the following steps:

[0023] Based on the total volume of the original Fissistigma oldhamii juice, add sodium bicarbonate with a mass - volume ratio of 3 - 10%, proline with a mass - volume ratio of 0.5% - 10%, and ethyl acetate with a mass - volume ratio of 0.5 - 5% to the original Fissistigma oldhamii juice. After standing at room temperature for 20 - 30 min, then centrifuge at 10°C and 6500g for 10 min to obtain supernatant A;

[0024] Centrifuge the supernatant A at 10°C and 10000g for 20 min to obtain supernatant B;

[0025] After mixing supernatant B and extraction reagent A at a volume ratio of 2:1, let it stand, and then centrifuge at 10°C and 12,000 g for 10 min to obtain supernatant C;

[0026] After mixing the supernatant C and extraction reagent B at a volume ratio of 3:1, let it stand, and then centrifuge at 10°C and 13,500 g for 30 min. Discard the supernatant and collect the precipitate.

[0027] Preferably, after resuspending the precipitate, centrifuge at 2,000 g for 10 min.

[0028] The present invention also provides a drug for treating inflammation or inhibiting the proliferation of leukemia cells, comprising the artabotrys hexapetalus exosomes prepared by the described method.

[0029] The present invention discloses the following technical effects:

[0030] (1) The artabotrys hexapetalus exosomes of the present invention provide a stable source of exosomes for inflammation treatment. The artabotrys hexapetalus exosomes can carry a variety of bioactive molecules, transmit signals to participate in anti-inflammation, and after experiments, the exosomes play an excellent role.

[0031] (2) As a plant-derived drug, the material artabotrys hexapetalus of the present invention shows obvious inflammation inhibitory ability and has little side effect, providing a new treatment means for inflammation treatment and cancer prevention, and is of great significance for the scientific, reasonable and sustainable development and utilization of artabotrys hexapetalus resources.

[0032] (3) The present invention is based on an in vitro inflammation model of LPS-induced Raw 264.7 cells. By detecting whether the inflammatory response generated by LPS-activated Raw 264.7 cells is inhibited by the intervention of artabotrys hexapetalus exosomes, the results show that artabotrys hexapetalus exosomes can inhibit the LPS-induced inflammatory level and restore the pro-inflammatory factor level to the basal state, providing a new technical idea for inhibiting the inflammatory response generated by LPS-induced Raw264.7 cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order 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 in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0034] Figure 1 It is the transmission electron microscope image of the artabotrys hexapetalus exosomes of the present invention;

[0035] Figure 2 It is the nano-particle size image of the artabotrys hexapetalus exosomes of the present invention;

[0036] Figure 3 This invention is for detecting the effect of Fissistigma oldhamii exosomes on the proliferation of Raw 264.7 cells by MTT assay;

[0037] Figure 4 This invention is for detecting the expression of inflammatory factors in the supernatant of LPS-induced Raw 264.7 cells treated with Fissistigma oldhamii exosomes by Elisa assay;

[0038] Figure 5 This invention is for detecting the expression of inflammation-related genes in LPS-induced Raw 264.7 cells treated with Fissistigma oldhamii exosomes by qPCR assay. Detailed implementation manners

[0039] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0040] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0041] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0042] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are also obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0043] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0044] Example 1: Preparation method of Fissistigma oldhamii exosomes

[0045] S1: Take a certain amount of fresh stems of *Fissistigma oldhamii*, place them in a water tank, rinse them thoroughly with domestic drinking water, and then rinse them with ultrapure water.

[0046] S2: Cut the washed stems of *Fissistigma oldhamii* into pieces, place them in a juicer for mixing and crushing, filter, and take the juice to obtain the original juice of *Fissistigma oldhamii*.

[0047] S3: Based on the total volume of the original juice of *Fissistigma oldhamii*, add sodium bicarbonate with a mass-volume ratio of 3 - 10% (g / L), proline with a mass-volume ratio of 0.5% - 10% (g / L), and ethyl acetate with a mass-volume ratio of 0.5 - 5% (g / L) to the original juice of *Fissistigma oldhamii*. Centrifuge at 6500 g for 10 min at 10°C to obtain supernatant A; centrifuge supernatant A at 10000 g for 20 min at 10°C to obtain supernatant B; add extraction reagent A according to the volume ratio of supernatant B:extraction reagent A = 2:1, mix well, and let it stand at 4°C for 10 min. After standing, centrifuge at 12000 g for 10 min at 10°C to obtain supernatant C; add extraction reagent B according to the volume ratio of supernatant C:extraction reagent B = 3:1, mix well, and let it stand at 4°C for 1 h. After standing, centrifuge at 13500 g for 30 min at 10°C, discard the supernatant, and collect the precipitate.

[0048] S4: Resuspend the precipitate with sterile normal saline and centrifuge at 2000 g for 10 min to obtain supernatant D, which is the exosomes of *Fissistigma oldhamii*.

[0049] The above extraction reagent A and extraction reagent B respectively correspond to Isolation Reagent A and Isolation Reagent B in the plant tissue exosome extraction kit (product number: S309) sold by Shenzhen Shifangjie Technology Co., Ltd.

[0050] Example 2: Identification of exosomes of *Fissistigma oldhamii*

[0051] Perform transmission electron microscopy (TEM) detection on the extracted exosomes of *Fissistigma oldhamii*. The results are as Figure 1 shown. The results show that obvious cup-shaped bilayer vesicle structures can be observed under the transmission electron microscope.

[0052] Use a nanoparticle tracking analyzer (NTA) to measure the particle size distribution and density of the exosomes of *Fissistigma oldhamii*. Initially judge its purity based on the distribution of the peaks. The fewer the number of peaks, the higher the purity; judge the yield of the exosomes based on the number of exosomes per milliliter. The larger the number, the higher the yield. The results are as Figure 2 shown. The NTA results show that the average particle size of the isolated exosomes of *Fissistigma oldhamii* is 119.9 ± 10.2 nm, and the concentration is 9.49×10 10 particle / mL. It meets the particle size range of 30 - 150 nm for exosomes, and has high purity and high yield.

[0053] Example 3: Effect of Fissistigma oldhamii exosomes on the proliferation of Raw264.7 cells

[0054] 1. Experimental materials

[0055] Cells: Mouse monocyte macrophage leukemia cells Raw 264.7

[0056] Cell culture medium: DMEM basal medium (Servicebio) containing 10% fetal bovine serum (Sunview) and 1% penicillin-streptomycin mixture (Servicebio).

[0057] Reagents: MTT was purchased from Servicebio

[0058] 2. Experimental methods

[0059] 2.1 Grouping

[0060] Divided into blank group (0 μg / mL) and Fissistigma oldhamii exosome treatment groups (25, 50, 75, 100 μg / mL).

[0061] 2.2 Experimental procedures

[0062] Collect cells in the logarithmic phase, seed the cells into 96-well plates at a density of 1×10 4 / well. After the cells adhered, add the corresponding Fissistigma oldhamii exosomes and continue to incubate in the incubator for 24 h. Then add 15 μL of MTT solution to each well and continue to incubate in the cell incubator for 3 - 4 h (the reaction can be terminated when purple crystal substances appear at the bottom of the well plate). Aspirate the supernatant, try not to touch the cells, add 120 μL of DMSO dissolution solution to each well, and shake well in the dark on a shaker for 15 min. After the crystal substances are dissolved, measure the absorbance (OD value) at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0063] 2.3 Data processing

[0064] Statistical analysis was performed using Prism 6.0 (GraphPad) statistical software. One-way analysis of variance (One-way ANOVA) was used for comparison between multiple groups, and the results were expressed as mean ± standard deviation

[0065] 3. Experimental results

[0066] As Figure 3 shown, the results showed that each group of Fissistigma oldhamii exosomes significantly inhibited the proliferation of Raw264.7 cells. Subsequently, 25 μg / mL was selected for treatment.

[0067] Example 4: Effect of Fissistigma oldhamii exosomes on the inflammation of LPS-induced Raw264.7 cells

[0068] 1. Experimental materials

[0069] Cells: Mouse mononuclear macrophage leukemia cells Raw 264.7.

[0070] Cell culture medium: DMEM basal medium (Servicebio) containing 10% fetal bovine serum (Sunview) and a mixture of penicillin and streptomycin (Servicebio).

[0071] Reagents: TNF-α ELISA kit, IL-1β ELISA kit, IL-6 ELISA kit, all purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.; LPS was purchased from Macklin; RNA extraction kit and reverse transcription kit were purchased from Servicebio, and qPCR reagents were purchased from Sunview.

[0072] 2. Experimental methods

[0073] 2.1 Grouping

[0074] Divided into 3 groups, as shown in Table 1 specifically.

[0075] Table 1 Grouping

[0076] Group Treatment Control group No drug administration LPS group 1 μg / mL LPS Fissistigma oldhamii exosome group 1 μg / mL LPS + 25 μg / mL Fissistigma oldhamii exosomes

[0077] 2.2 Experimental procedures

[0078] Collect cells in the logarithmic growth phase, seed the cells at 1×10 5 / well into a 12-well plate and continue culturing in a cell culture incubator. The next day, add Fissistigma oldhamii exosomes according to the grouping and incubate for 24 h, then add LPS according to the grouping and continue culturing for 24 h. Collect the cell supernatant. Centrifuge at 13500 g for 10 min at 4°C, collect the cell supernatant, and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the pro-inflammatory factors TNF-α, IL-1β, and IL-6 in the supernatant according to the kit instructions. Collect the cells and detect NLRP3, Caspase1, TNF-α, IL-1β, and IL-6 in the cells according to the RNA extraction kit, reverse transcription kit, and qPCR detection instructions. The primer sequences are shown in Table 2.

[0079] Table 2 Primer sequences

[0080]

[0081] 2.3 Data processing

[0082] Statistical analysis was performed using Prism 6.0 (GraphPad) statistical software. One-way analysis of variance (One-way ANOVA) was used for comparison among multiple groups, and the results are expressed as mean ± standard deviation.

[0083] 3. Experimental Results

[0084] As Figure 4 and Figure 5 shown, after LPS treatment, the secretion level of pro-inflammatory factors in the cell supernatant and the mRNA expression level were significantly increased, while the exosomes of Fissistigma oldhamii could inhibit the LPS-induced inflammatory level and restore the pro-inflammatory factor level to the basal state. This indicates that the exosomes of Fissistigma oldhamii have anti-inflammatory effects, can inhibit the release of inflammatory factors, improve the immune function of the body, and regulate the immune balance state.

[0085] From the above embodiments, it can be seen that the present invention prepares exosomes of Fissistigma oldhamii and explores their role in inflammation, which not only provides a method for preparing exosomes of Fissistigma oldhamii, but also provides a new idea for inflammation research. Compared with animal exosomes, plant exosomes have higher safety and wider sources. The research results of the present invention will be beneficial to the scientific, comprehensive and sustainable development and utilization of Fissistigma oldhamii resources, and also provide certain support and help for the subsequent development of drugs for treating inflammation, such as new drugs for arthritis.

[0086] The above-described embodiments are only used to describe the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. The use of the extracellular vesicles of Glechoma longituba in preparing a drug for treating inflammation, characterized in that: The extracellular vesicles of the genus Cucurbitacin are separated from the genus Cucurbitacin.

2. The use of the extracellular vesicles of Glechoma longituba in the preparation of a drug for inhibiting the proliferation of leukemia cells, characterized in that: The extracellular vesicles of the genus Cucurbitacin are separated from the genus Cucurbitacin.

3. The use according to claim 1 or 2, characterized in that: The separation comprises the following steps: The stems of the cyperus rotundus are crushed and filtered to obtain the cyperus rotundus juice; Extracting the guava wood juice by gradient centrifugation and collecting the precipitate; The precipitate is resuspended with sterile physiological saline, centrifuged, and the supernatant is collected to obtain the extracellular vesicles of the Guarana oleracea.

4. The use according to claim 3, characterized in that The gradient centrifugation extraction comprises the following steps: According to the total volume of the Guafu Wood original juice, 3-10% sodium bicarbonate, 0.5%-10% proline and 0.5-5% ethyl acetate are added to the Guafu Wood original juice in a mass volume ratio, and after standing at room temperature for 20-30 minutes, centrifuge at 10°C and 6500g for 10 minutes to obtain supernatant A; The supernatant A is centrifuged at 10°C and 10000g for 20 min to obtain supernatant B; Supernatant B and extraction reagent A were mixed in a volume ratio of 2:1, allowed to stand, and then centrifuged at 10°C and 12000g for 10 min to obtain supernatant C; The supernatant C and the extraction reagent B were mixed in a volume ratio of 3:1, allowed to stand, and then centrifuged at 10°C and 13500g for 30 min, the supernatant was discarded, and the precipitate was collected; After the precipitate was resuspended, it was centrifuged at 2000 g for 10 min.

5. The use according to claim 1, characterized in that The inflammation includes anti-LPS-induced inflammation of Raw 264.7 cells.

6. A method for preparing extracellular vesicles of Glehnia littoralis, characterized in that: The following steps are involved: The stems of the cyperus rotundus are crushed and filtered to obtain the cyperus rotundus juice; Extracting the guava wood juice by gradient centrifugation and collecting the precipitate; The precipitate is resuspended with sterile physiological saline, centrifuged, and the supernatant is collected to obtain the extracellular vesicles of the Guarana oleracea.

7. The preparation method according to claim 6, characterized in that: The gradient centrifugation extraction comprises the following steps: According to the total volume of the Guafu Wood original juice, 3-10% sodium bicarbonate, 0.5%-10% proline and 0.5-5% ethyl acetate are added to the Guafu Wood original juice in a mass volume ratio, and after standing at room temperature for 20-30 minutes, centrifuge at 10°C and 6500g for 10 minutes to obtain supernatant A; The supernatant A is centrifuged at 10°C and 10000g for 20 min to obtain supernatant B; Supernatant B and extraction reagent A were mixed in a volume ratio of 2:1, allowed to stand, and then centrifuged at 10°C and 12000g for 10 min to obtain supernatant C; The supernatant C and the extraction reagent B were mixed in a volume ratio of 3:1, allowed to stand, and then centrifuged at 10°C and 13500g for 30 minutes, and the supernatant was discarded to collect the precipitate.

8. The preparation method according to claim 6, characterized in that: After the precipitate was resuspended, it was centrifuged at 2000 g for 10 min.

9. A drug for treating inflammation or inhibiting the proliferation of leukemia cells, characterized in that: The invention comprises extracellular vesicles of Citrus aurantium obtained by the method described in any one of claims 6 to 8.

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