Composition containing rosemary exosome vesicles and application of composition in anti-inflammation

Abstract

The invention relates to the technical field of food, medicine and health care products, and discloses a composition containing rosemary exosome vesicles and application of the composition in anti-inflammatory, the composition is formed by mixing plant cell-derived exosome vesicles and functional active ingredients, the composition comprises the following components in percentage by mass: 45-55% of rosemary exosome and 45-55% of turmeric exosome; the preparation method of the composition containing the rosemary exosome vesicles comprises the following steps: S1, taking a certain amount of fresh rosemary and turmeric plants, removing root hairs and impurities, washing the fresh plants with domestic drinking water, washing the fresh plants with purified water for three times, and finally washing the fresh plants with ultrapure water for three times for 3-5 minutes each time; the product can carry various bioactive molecules, transfer cell signals in a transboundary manner, participate in anti-inflammation, inhibit tumor cell proliferation and promote normal tissue repair, has an anti-inflammatory effect, inhibits release of inflammatory factors, improves the immune function of a body and adjusts the immune balance state, and can play a role in neovascularization diseases of the body.

Description

Technical Field

[0001] The present invention relates to the technical field of food and medical health products, and in particular to a rosemary exosome vesicle composition and an application thereof in anti-inflammatory treatment. Background Art

[0002] Rosemary is a precious natural spice plant with a calming, refreshing and brain-awakening effect. It has a certain effect on indigestion and stomachache. It can also be used to treat insomnia, palpitations, headaches, indigestion and many other diseases. In addition, studies have found that rosemary also has the effects of strengthening the heart, promoting metabolism, and promoting blood circulation in peripheral blood vessels. Turmeric comes from the dried rhizomes of the plant turmeric, and is on the list of traditional Chinese medicines that can be used as health foods announced by the Ministry of Health. Turmeric contains a variety of chemical components, such as curcuminoids and sesquiterpenoids: curcuminone, etc. Recent studies have shown that curcumin has a variety of biological activities, such as anti-cancer, anti-inflammatory, antioxidant, anti-atherosclerotic, anti-diabetic, etc., and has health benefits in protecting the liver and stomach, lowering blood lipids, and enhancing immunity. Exosomes are vesicle structures produced by cells, carrying maternal cell nucleic acid, proteins, phospholipids and other components. Exosomes have a lipid structure similar to that of cell membranes, and can easily pass through various barriers in the body. They can perform important functions such as intercellular information flow and material transport, and have the functions of inhibiting inflammation, immune regulation, angiogenesis, and participating in tissue repair. Relevant experiments have shown that after plant-derived exosomes enter the body of mammals, they participate in related signal pathways and can complete cross-border regulation, playing a therapeutic and health-care function similar to that of the original plant.

[0003] Intestinal inflammatory diseases are a type of disease characterized by intestinal mucosal inflammatory reactions, such as Crohn's disease and ulcerative colitis. Currently, the treatments for this type of disease mainly include drug therapy, nutritional support, and biological agent therapy. However, existing treatments have certain limitations, such as large drug side effects, unstable treatment effects, and easy development of drug resistance. Summary of the invention

[0004] The purpose of the present invention is to provide a rosemary exosome vesicle composition and its application in anti-inflammatory treatment, so as to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a rosemary exosome vesicle composition, wherein the mass percentage of each component of the composition includes 45-55% of rosemary exosomes and 45-55% of turmeric exosomes;

[0006] The preparation method of the rosemary exosome vesicle composition comprises the following steps:

[0007] S1: Take a certain amount of fresh rosemary and turmeric plants, remove the roots and 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;

[0008] S2: Wash the fresh rosemary and turmeric plants and cut them into pieces, mix and crush them in juicers respectively, squeeze them three times in a row, filter and take out the juice to obtain the original juice of rosemary and turmeric plants respectively;

[0009] S3: The rosemary and turmeric plant juices were subjected to differential centrifugation at 4°C, with the first centrifugal force of 1000g for 10 minutes to obtain supernatant A; the supernatant A was then centrifuged at 3000g for 20 minutes to obtain supernatant B, and the supernatant B was then centrifuged at 10000g for 40 minutes to remove impurities to obtain supernatant C; 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 rosemary exosome extract and turmeric exosome extract;

[0010] S4: The rosemary exosome extract and the turmeric exosome extract obtained in step S3 are mixed at a volume ratio of 1:1 and set aside.

[0011] The invention discloses an application of a rosemary exosome vesicle composition in anti-inflammatory treatment. The application relates to a rosemary exosome vesicle composition. The rosemary exosome vesicle mixture has anti-inflammatory effect, inhibits the release of inflammatory factors, improves the body's immune function, and regulates the immune balance state.

[0012] Preferably, the rosemary exosome vesicle mixture can inhibit the proliferation of umbilical vein endothelial cells (HUVEC), and can play a role in neovascular diseases in the body.

[0013] The rosemary exosome vesicle composition is passed through a nanoparticle tracking analyzer to determine the particle size distribution and density of the plant exosome particles. The purity is preliminarily judged based on the peak distribution, and the fewer the peaks, the higher the purity. The yield of exosome particles is judged by the number of exosomes per ml, and the larger the number, the higher the yield.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1. The rosemary exosome vesicle composition of the present invention provides a stable source of exosome composition for intestinal anti-inflammatory treatment. The therapeutic effect of exosomes is more stable. The exosome vesicles of this plant can carry a variety of bioactive molecules, transmit cell signals, participate in anti-inflammation and tissue repair. The product has been tested and the exosomes have an excellent effect.

[0016] 2. The rosemary exosome vesicle composition of the present invention can regulate inflammatory response, inhibit or reduce the expression of inflammatory factors, and increase the secretion of anti-inflammatory factors, thereby achieving a good anti-inflammatory effect.

[0017] 3. The rosemary exosome vesicle composition of the present invention can inhibit macrophage apoptosis, increase immune function, and regulate immune status.

[0018] 4. The rosemary exosome vesicle composition of the present invention inhibits the increase of umbilical vein endothelial cells, has an antagonistic effect on new angiogenesis, and can exert an anti-tumor effect or abnormal vascular proliferative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the IL-12 protein expression level of the present invention.

[0020] Figure 2 This is a schematic diagram of the IL-1β protein expression level of the present invention.

[0021] Figure 3 This is a schematic diagram of the IL-10 protein expression level of the present invention.

[0022] Figure 4 This is a comparison chart of the effects of the rosemary extracellular vesicles of the present invention on the apoptosis of macrophages RAW.

[0023] Figure 5 Schematic diagram of the effect of the rosemary extracellular vesicles of the present invention on the apoptosis of macrophage RAW.

[0024] Figure 6 Schematic diagram of the effect of the rosemary extracellular vesicles of the present invention on the proliferation of umbilical vein endothelial cells HUVEC. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] See also Figures 1 to 6 , the present invention provides a technical solution: a rosemary exosome vesicle composition, wherein the mass percentage of each component of the composition includes 45-55% of rosemary exosomes and 45-55% of turmeric exosomes;

[0027] The preparation method of the rosemary exosome vesicle composition comprises the following steps:

[0028] S1: Take a certain amount of fresh rosemary and turmeric plants, remove the roots and 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;

[0029] S2: Wash the fresh rosemary and turmeric plants and cut them into pieces, mix and crush them in juicers respectively, squeeze them three times in a row, filter and take out the juice to obtain the original juice of rosemary and turmeric plants respectively;

[0030] S3: The rosemary and turmeric plant juices were subjected to differential centrifugation at 4°C, with the first centrifugal force of 1000g for 10 minutes to obtain supernatant A; the supernatant A was then centrifuged at 3000g for 20 minutes to obtain supernatant B, and the supernatant B was then centrifuged at 10000g for 40 minutes to remove impurities to obtain supernatant C; 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 rosemary exosome extract and turmeric exosome extract;

[0031] S4: The rosemary exosome extract and the turmeric exosome extract obtained in step S3 are mixed at a volume ratio of 1:1 and set aside.

[0032] Example 1: Detection of the anti-inflammatory function of rosemary extracellular vesicle composition

[0033] After 24 hours of culture, the revived RAW264.7 cells were centrifuged at 400 g for 10 minutes and the supernatant was discarded. The cell concentration was adjusted to 1×10 5 / mL with DMEM containing 10% FBS.

[0034] RAW264.7 cells in the logarithmic growth phase were seeded in a 96-well plate at 1×10 4 / well and placed in a 37° C. 5% CO 2 cell culture incubator overnight.

[0035] The next day, the old culture medium of each well was discarded, and LPS was added to a final concentration of 1ug / ml. After 12h, 40ug / ml of the prepared rosemary exosome vesicles were added according to Table 1. Two replicate wells were set up in each group, and a cell control group and an LPS positive control group were also set up.

[0036] Place in a 37°C 5% CO2 cell culture incubator and continue to culture for 24h and 48h, and take 80uL of cell supernatant at each time point. Centrifuge at 300xg for 10min at 4°C and collect the cell supernatant. According to the instructions of the multi-factor detection kit (BIO-RAD, 171304090M), the protein content of pro-inflammatory factors IL-1β, IFN-gamma, IL-12 and anti-inflammatory factor IL-10 in the supernatant was detected on the Luminex instrument platform.

[0037] The experimental groups are shown in Table 1 below:

[0038] Grouping Sample name Use concentration (ug / ml) 1 Cell control group 0 2 Positive control group 1ug / ml LPS 3 Drug group 1ug / ml LPS+40ug / ml M-EVs

[0039] The experimental results are as attached Figure 1 , Attachment Figure 2 , Attachment Figure 3 : In the LPS-induced inflammatory response, the rosemary extracellular vesicle composition can significantly inhibit the protein levels of pro-inflammatory factors IL-1β and IL-12, and increase the expression level of anti-inflammatory factor IL-10, which is significantly different from the LPS positive control group. Adding 40ug / ml of rosemary extracellular vesicles has a good anti-inflammatory effect.

[0040] Example 2: Detection of the antagonism of macrophage apoptosis by the rosemary-containing extracellular vesicle composition

[0041] After 24 hours of culture, the revived RAW264.7 cells were centrifuged at 400 g for 10 minutes, the supernatant was discarded, and the cell concentration was adjusted to 1×10 5 / mL with DMEM containing 10% FBS.

[0042] RAW264.7 cells in the logarithmic growth phase were seeded in a six-well plate at 1×105 / well and placed in a 37°C, 5% CO2 cell culture incubator overnight.

[0043] The next day, the old culture medium of each well was discarded, and LPS was added to a final concentration of 1ug / ml. After 12 hours, 40ug / ml of the prepared rosemary exosome vesicles were added according to Table 1, and the cells were placed in a 37°C, 5% CO2 cell culture incubator for 48h.

[0044] The supernatant was discarded, the cells were washed once with room temperature PBS, trypsin was added for digestion for 3 minutes, and then DMEM containing 10% FBS was added to terminate the digestion.

[0045] Centrifuge at 300xg for 10 min at 4°C to collect the cells. Remove the supernatant, wash twice, and resuspend with 500μL 1× Binding Buffer. Then add 5μL Annexin V-FITC and 10μL PI dye to each tube, mix well, incubate at room temperature in the dark for 5 minutes, and detect on a flow cytometer.

[0046] The experimental results are as attached Figure 4 , Attachment Figure 5 :LPS continuously stimulates macrophages to produce a large number of inflammatory factors, lysosomal enzymes, NO and other bioactive substances, which eventually cause cell damage and apoptosis. Rosemary exosome vesicles inhibit the release of inflammatory factors and reduce the number of apoptotic macrophages.

[0047] Example 3: Effect of rosemary-containing extracellular vesicle composition on inhibiting the proliferation of HUVEC

[0048] The HUVEC cell line was revived, centrifuged at 400 g for 10 min, the supernatant was discarded, and the cell concentration was adjusted to 5×10 4 cells / mL with 10% FBS ECM culture medium.

[0049] 100 μL was inoculated into a 96-well plate, with 5,000 cells per well, and cultured in a 37°C 5% CO2 incubator for 12 h.

[0050] After the cells adhered to the wall, 40ug / ml rosemary exosome vesicles were added, with 2 replicates in each group, and a cell control group was set up at the same time, and placed in a 37°C, 5% CO2 cell culture incubator for 72 hours. 10μL CCK8 solution was added to each well, incubated at 37°C in the dark for 2 hours, and the OD450nm absorbance was detected on an ELISA reader.

[0051] The experimental groups refer to Table 2 below:

[0052] Grouping Sample name Use concentration (ug / ml) 1 Cell control group 0 2 Single drug group 40ug / ml Rosemary Extract 3 Single drug group 40ug / ml Turmeric Extract 4 Combined drug group 40ug / ml Mix-EVs

[0053] The results are attached Figure 6 : Compared with the cell control group, both rosemary exosomes and turmeric exosomes can inhibit the proliferation of HUVECs, and the extracellular vesicles containing both substances have the most obvious inhibitory effect.

[0054] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A rosemary exosome vesicle composition, characterized in that: The mass percentages of the components of the composition include 45-55% of rosemary exosomes and 45-55% of turmeric exosomes; The preparation method of the rosemary exosome vesicle composition comprises the following steps: S1: Take a certain amount of fresh rosemary and turmeric plants, remove the roots and 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: Wash the fresh rosemary and turmeric plants and cut them into pieces, mix and crush them in juicers respectively, squeeze them three times in a row, filter and take out the juice to obtain the original juice of rosemary and turmeric plants respectively; S3: The rosemary and turmeric plant juices were subjected to differential centrifugation at 4°C, with the first centrifugal force of 1000g for 10 minutes to obtain supernatant A; the supernatant A was then centrifuged at 3000g for 20 minutes to obtain supernatant B, and the supernatant B was then centrifuged at 10000g for 40 minutes to remove impurities to obtain supernatant C; 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 rosemary exosome extract and turmeric exosome extract; S4: The rosemary exosome extract and the turmeric exosome extract obtained in step S3 are mixed at a volume ratio of 1:1 and set aside.

2. The use of a rosemary exosome vesicle composition in anti-inflammatory treatment, characterized in that: The application is to the rosemary exosome vesicle composition as described in claim 1. The rosemary exosome vesicle mixture has anti-inflammatory effects, inhibits the release of inflammatory factors, improves the body's immune function, and regulates the immune balance state.

3. The use of the rosemary exosome vesicle composition in anti-inflammatory treatment according to claim 2, characterized in that: The rosemary exosome vesicle mixture can inhibit the proliferation of umbilical vein endothelial cells (HUVECs), and can play a role in neovascular diseases in the body.

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