Vesicle composition as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to a vesicle composition as well as a preparation method and application thereof. The vesicle composition comprises lactobacillus vesicles and a free radical scavenging compound wrapped in the lactobacillus vesicles. According to the technical scheme, lactobacillus extracellular vesicles are extracted through an ultracentrifugation method, and astaxanthin and the vesicles are combined to form the vesicle composition. The oxidation resistance of the vesicle composition is analyzed through a free radical scavenging experiment, a fluorescence image of cells is collected by using a fluorescence microscope, and the influence of the vesicle composition on ROS change is observed. Results show that the vesicle composition effectively improves the oxidation resistance and stability of astaxanthin and has remarkable free radical scavenging capacity.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and in particular relates to a vesicle composition and a preparation method and application thereof. Background Art

[0002] Astaxanthin is a ketocarotenoid with strong antioxidant capacity. It can remove nitrogen dioxide, sulfide, disulfide, etc., reduce lipid peroxidation, and effectively inhibit lipid peroxidation caused by free radicals. In addition, astaxanthin is widely used in cosmetics, mainly due to its unique molecular structure and powerful physiological functions, including antioxidant and anti-aging, anti-inflammatory and soothing, and strengthening the skin barrier. However, astaxanthin is easily degraded during extraction, storage and processing, so it is necessary to improve the stability of astaxanthin.

[0003] With the application and promotion of nanotechnology, many new carrier systems have been developed, such as liposomes and nanovesicles. Lactobacillus, as a probiotic widely distributed in nature, can regulate the body's microecological balance, produce nutrients, etc. In recent years, skin microecological studies have shown that the balance of microbial flora on human skin is crucial to skin health. Lactobacillus is safer and more effective as a probiotic, and can improve skin health by regulating the balance of skin microbiota. Other bacterial vesicles may not be safely and effectively applied to the skin or body. Lactobacillus can secrete extracellular vesicles with a size of 20-200nm, which can be used as a carrier system to transport substances and are stable. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a vesicle composition and a preparation method and application thereof.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] A vesicle composition comprises lactobacillus vesicles and free radical scavenging compounds encapsulated in the lactobacillus vesicles.

[0007] The lactobacillus vesicles are prepared in the following manner: S11) inoculating lactobacillus into MRS culture medium and culturing at 37° C. to obtain lactobacillus fermentation liquid; S12) removing large particle impurities in the extract by low-speed centrifugation, and filtering by a filtering device to obtain a supernatant; S13) concentrating the collected supernatant by a concentrating device; S14) ultracentrifuging the filtrate after concentration to obtain a lactobacillus extracellular vesicle precipitate; S15) resuspending the obtained precipitate with a sterile PBS solution, and filtering by a filter membrane to obtain the final lactobacillus extracellular vesicles.

[0008] The lactobacillus is plant lactobacillus.

[0009] The rotation speed of the low-speed centrifugation in step S12) is 6000 rpm; steps S12)-S13) are performed under ice bath conditions; the ultracentrifugation condition of step S14) is ultracentrifugation at 100000 rpm for 2 hours; the filter membrane used for filtration in step S14) is 0.22 μm.

[0010] The free radical scavenging compound is astaxanthin.

[0011] The present invention also includes a method for preparing the vesicle composition, comprising the following steps: S21) dissolving PLGA and astaxanthin in dichloromethane, and dissolving by water bath ultrasound to obtain an organic phase; S22) adding the organic phase solution to a polyvinyl alcohol solution, and using an ultrasonic crusher to crush the organic phase to obtain an emulsion; S23) stirring at room temperature until the organic solvent is completely evaporated; centrifuging at low temperature and high speed to remove the supernatant, and resuspending the mixture with PBS to obtain a PLGA and astaxanthin composition; S24) mixing the PLGA and astaxanthin composition with lactobacillus vesicles in a centrifuge tube, performing water bath ultrasound, performing low temperature and high speed centrifugation to remove the supernatant, and resuspending the mixture with PBS to obtain a vesicle composition.

[0012] The conditions of low-temperature high-speed centrifugation in steps S23) and S24) are 14000 rpm and 40 min.

[0013] The mass ratio of PLGA to astaxanthin is (10-30):1; preferably 20:1; preferably, the molecular weight of PLGA is 17 kDa; and the concentration of polyvinyl alcohol is 1wt%.

[0014] In step 24), the ratio of the PLGA and astaxanthin composition to the lactobacillus vesicles is 1:(5-10).

[0015] The present invention also includes an application of the vesicle composition.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The technical solution of the present invention extracts lactobacillus extracellular vesicles by ultracentrifugation, and combines astaxanthin with the vesicles to form a vesicle composition. The antioxidant activity of the vesicle composition is analyzed by free radical scavenging experiments, and fluorescent images of cells are collected using a fluorescence microscope to observe the effect of the vesicle composition on ROS changes. The results show that the vesicle composition of the present invention effectively improves the antioxidant performance and stability of astaxanthin and has significant free radical scavenging ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is an electron micrograph of extracellular vesicles of Lactobacillus.

[0019] Figure 2 These are the results of the stability test under light conditions.

[0020] Figure 3 This is the DPPH free radical scavenging rate diagram.

[0021] Figure 4 Experimental results of induced oxidative stress model - fluorescence images. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and the best embodiments.

[0023] The preparation method of the vesicle composition includes the following specific steps:

[0024] 1) Preparation of lactobacillus vesicles: S11) Activate lactobacillus to obtain seed fermentation liquid, the strain number of plant lactobacillus is Lactobaci llus plantarum. The plant lactobacillus is purchased from Shanghai Keborui Biotechnology Co., Ltd. The seed fermentation liquid is inoculated into 2L MRS culture medium at an inoculum of 2%, and cultured for 24 hours at 37°C and 100rpm. S12) Treatment of fermentation liquid: ice bath the fermented bacterial liquid, divide it into 50ml centrifuge tubes after ice bathing, put it into a pre-cooled centrifuge, centrifuge at 6000rpm to remove large particle impurities in the extract, and take the supernatant after centrifugation for the next step of treatment; S13 add the centrifuged supernatant into a suction filtration device for suction filtration to obtain the filtrate, and store it in a low-temperature refrigerator. The specific steps of filtrate concentration are: put the filtered fermentation liquid into the concentration device, connect one end to the waste sample tube and the other end to the sample tube. The ultrafiltration membrane package can filter the macromolecular substances to increase the concentration, and finally obtain the concentrated filtrate; all the above operations are carried out under ice bath conditions. S14) Separation of extracellular vesicles: ice bath the concentrated filtrate for 1 hour and then package it. Weigh the concentrated filtrate in an ultracentrifuge tube on a precision balance and perform ultracentrifugation at 100,000 rpm for 2 hours. The precipitate obtained is the extracellular vesicles of Lactobacillus. S15) Resuspend the obtained precipitate with a small amount of sterile PBS solution, mix the resuspended liquids in multiple tubes, and filter with a 0.22μm filter membrane to obtain all the final extracellular vesicles of Lactobacillus such as Figure 1 shown.

[0025] 2) Preparation of vesicle composition;

[0026] S21) Dissolve 10 mg of PLGA (Mw = 17 kDa, polylactic acid-glycolic acid copolymer, Shanghai McLin) and 0.5 mg of astaxanthin in 1 ml of dichloromethane, and dissolve in water bath ultrasound to obtain an organic phase. S22) Slowly add the organic phase to 10 ml of a 1 wt% polyvinyl alcohol (degree of alcoholysis: 87.0-89.0 (mol / mol)) aqueous solution being stirred, and use an ultrasonic crusher to crush to obtain an emulsion. S23) Use a magnetic stirrer at room temperature at 400 rpm to stir until the organic solvent evaporates. Use a high-speed centrifuge to centrifuge at 14000 rpm (40 min) at 4°C to remove the supernatant, and resuspend in PBS to obtain a PLGA and astaxanthin composition. S24) taking PLGA and astaxanthin compositions in a ratio of 1:5 and 1:10 respectively and mixing them with lactobacillus vesicles in a centrifuge tube, ultrasonicating in a water bath at maximum power for 5 min, removing the supernatant at 14000 rpm (40 min), and resuspending in PBS to obtain a vesicle composition.

[0027] Test the results:

[0028] 1. Stability test results

[0029] The prepared vesicle composition and astaxanthin solution were dissolved in equal amounts of oil phase. The color changes of the vesicle composition and astaxanthin solution were observed under light for 7 days. Figure 2 As shown, the results showed that there was no obvious change in the discoloration of the vesicle composition, while the solution of the group in which astaxanthin was added alone had a change in discoloration.

[0030] 2. Determination of encapsulation efficiency of vesicle composition: Accurately weigh an appropriate amount of freeze-dried vesicle composition, add an appropriate amount of dichloromethane: ethanol (1:10) solution to break the emulsion, ultrasonicate for 3 minutes, vortex for 3 minutes, and then adjust the volume of the above solution. The absorbance of the solution is detected by an enzyme marker at a wavelength of 480nm. According to the AST standard curve, the encapsulation efficiency of 1:5 is 60.5±1.79%; the encapsulation efficiency of 1:10 is 68.6±2.17%. Encapsulation efficiency (%) = astaxanthin content in nanoparticles / astaxanthin content added * 100%.

[0031] 3. Lactobacillus extracellular vesicles encapsulating astaxanthin DPPH free radical scavenging experiment: three groups were set, including astaxanthin group (PLGA and astaxanthin composition), vesicle group (Lactobacillus extracellular vesicle), and astaxanthin vesicle composition. DPPH powder was dissolved in anhydrous ethanol organic solvent to prepare a standard concentration of DPPH working solution. The three groups of experimental samples were weighed separately and added to the selected anhydrous ethanol organic solvent. The samples were stirred or ultrasonically treated until they were completely dissolved to prepare concentrations of 10μg / ml, 25μg / ml, and 50μg / ml. Ensure that the solution is stored in a dark environment to maintain its stability and activity. A certain amount of DPPH working solution was added to a suitable cuvette, followed by an equal volume of each sample solution. Mix immediately, measure the absorbance at a wavelength of 517nm, and record the initial absorbance value. By comparing the absorbance changes of the DPPH solution before and after the addition of the sample, the scavenging rate of the sample for DPPH free radicals was calculated. The free radical scavenging rate was used to evaluate the antioxidant capacity of the sample. Figure 3 A DPPH radical scavenging rate graph is shown, and the results show that the vesicle composition group (astaxanthin + vesicles), astaxanthin group, and vesicle group all have the highest clearance rates at a concentration of 50ug / ml, which are around 90%, both higher than the astaxanthin group (58%) and the vesicle group (60%).

[0032] 3. Cytotoxicity test (MTT test): HaCaT cells in the cell culture dish were collected by digestion with 0.25% trypsin and the cell concentration was adjusted to 1×10 5 / mL, 100uL / well was inoculated in a 96-well plate, 200uL / well of PBS buffer was added to the four wells of the 96-well plate, and the 96-well plate was placed in a cell culture incubator for overnight culture. The vesicle-encapsulated astaxanthin sample was diluted to different concentrations with complete culture medium, 100uL / well was added to the 96-well plate, and a normal control group and a blank control group were set up at the same time, and the cells were placed in a cell culture incubator for 24±2h. Take out the 96-well plate, add 20uL of 5mg / mL MTT solution to each well, and place it in a cell culture incubator for 4h. The liquid in the 96-well plate was aspirated, DMSO 150uL / well was added, and the OD value of each group at 490nm was determined using an enzyme marker. According to the OD value measurement results, the cell survival rate at each concentration was calculated, and the cell survival rate = (well to be tested-blank well) / (normal well-blank well), and the sample concentration with a cell survival rate ≥90% was selected as the sample concentration without obvious cytotoxicity for the next test.

[0033] 4. Oxidative stress test: HaCaT cells in the cell culture dish were collected by digestion with 0.25% trypsin and the cell concentration was adjusted to 2×10 5 / mL, 500uL / well inoculated in a 24-well plate, and the 24-well plate was placed in a cell culture incubator overnight. A normal control group (blank group), a model group, a vesicle composition group, and a positive control group (vitamin E) were set up, with 4 wells in each group. The sample to be tested and vitamin E were diluted with cell culture medium, respectively, and added to the corresponding well plate. Complete cell culture medium was added to the remaining wells, and each well was 250uL / well, so that the final concentration of the sample to be tested in the 24-well plate was a non-toxic concentration, and vitamin E was 5%. The 24-well plate was placed in a cell culture incubator for 0.5h. Take out the 24-well plate, add complete culture medium to the normal group, and add 250uM H2O2 solution to the remaining wells to induce cellular oxidative stress, with a volume of 250uL and an induction time of 0.5h.

[0034] Installed in fluorescent probe: Take out the 24-well plate from the cell culture incubator and wash the 24-well plate with PBS buffer for 3 minutes each time, for a total of 3 times. Dilute the DCFH-DA solution with PBS buffer to a final concentration of 10μmol / L, add 1mL / well to the 24-well plate, place it in a cell culture incubator and culture it in the dark for 0.5h, take it out again and wash the plate with PBS buffer for 3 times, 5 minutes each time.

[0035] Collect fluorescence images: Place the 24-well plate on the stage of a fluorescence microscope, use the FI TC channel, set the incident light wavelength to 525 nm, the excitation light wavelength to 488 nm, and set the same exposure time under a 20x objective lens to collect fluorescence images of each well.

[0036] The results of the cytotoxicity test are shown in Table 1;

[0037] Table 1

[0038]

[0039] Experimental results of induced oxidative stress model - fluorescence images such as Figure 4 The experimental results of the induced oxidative stress model - Density (mean) and IOD values ​​are shown in Table 2.

[0040] Table 2

[0041]

[0042] Note: Compared with the normal control group, # represents P < 0.05, ## Represents P < 0.01, compared with the model group, * represents P < 0.05, ** Represents P < 0.01.

[0043] Analysis of results: Compared with the normal control group, the fluorescence intensity of the cells in the model group was significantly increased (p < 0.05), which proved that the oxidative stress model was successfully established. Compared with the model group, the fluorescence intensity of the vesicle composition group and the vitamin E group was significantly decreased (both p < 0.01), which proved that the vesicle composition group and the vitamin E group had the effect of significantly reducing intracellular ROS. It can be considered that the vesicle composition can improve the antioxidant capacity of astaxanthin.

[0044] In summary, the present invention provides an antioxidant and application of astaxanthin encapsulated in extracellular vesicles of lactobacillus, extracts extracellular vesicles of lactobacillus by ultracentrifugation, and combines astaxanthin with vesicles by certain means to form a vesicle composition. Its antioxidant properties are analyzed by free radical scavenging experiments, and fluorescent images of cells are collected using a fluorescence microscope. It can be intuitively seen that the ROS content of vesicle-encapsulated astaxanthin is reduced, indicating that vesicle-encapsulated astaxanthin can improve the antioxidant capacity of astaxanthin.

[0045] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A vesicle composition, characterized in that The invention comprises lactobacillus vesicles and free radical scavenging compounds encapsulated in the lactobacillus vesicles.

2. The vesicle composition according to claim 1, characterized in that: The lactobacillus vesicles are prepared in the following manner: S11) inoculating lactobacillus into MRS culture medium and culturing at 37° C. to obtain lactobacillus fermentation liquid; S12) removing large particle impurities in the extract by low-speed centrifugation, and filtering by a filtering device to obtain a supernatant; S13) concentrating the collected supernatant by a concentrating device; S14) ultracentrifuging the filtrate after concentration to obtain a lactobacillus extracellular vesicle precipitate; S15) resuspending the obtained precipitate with a sterile PBS solution, and filtering by a filter membrane to obtain the final lactobacillus extracellular vesicles.

3. The vesicle composition according to claim 2, characterized in that The lactobacillus is plant lactobacillus.

4. The vesicle composition according to claim 2, characterized in that The rotation speed of the low-speed centrifugation in step S12) is 6000 rpm; steps S12)-S13) are performed under ice bath conditions; the ultracentrifugation condition of step S14) is ultracentrifugation at 100000 rpm for 2 hours; the filter membrane used for filtration in step S14) is 0.22 μm.

5. The vesicle composition according to claim 1, characterized in that The free radical scavenging compound is astaxanthin.

6. A method for preparing the vesicle composition according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: S21) dissolving PLGA and astaxanthin in dichloromethane, and dissolving by ultrasonication in a water bath to obtain an organic phase; S22) adding the organic phase solution into a polyvinyl alcohol solution, and crushing the organic phase solution by using an ultrasonic crusher to obtain an emulsion; S23) stirring the organic phase solution at room temperature until the organic solvent is completely evaporated; centrifuging the solution at low temperature and high speed to remove the supernatant, and resuspending the solution in PBS to obtain a PLGA and astaxanthin composition; S24) mixing the PLGA and astaxanthin composition with lactobacillus vesicles in a centrifuge tube, ultrasonicating the solution in a water bath, centrifuging the solution at low temperature and high speed to remove the supernatant, and resuspending the solution in PBS to obtain a vesicle composition.

7. The preparation method according to claim 6, characterized in that: The conditions of low-temperature high-speed centrifugation in steps S23 and S24 are 14000 rpm and 40 min.

8. The preparation method according to claim 6, characterized in that: The mass ratio of PLGA to astaxanthin is (10-30):1; preferably 20:1; preferably, the molecular weight of PLGA is 17 kDa; and the concentration of polyvinyl alcohol is 1wt%.

9. The preparation method according to claim 6, characterized in that: In step 24), the ratio of the PLGA and astaxanthin composition to the lactobacillus vesicles is 1:(5-10).

10. Use of the vesicle composition according to any one of claims 1 to 3.

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