Extraction and purification method of plant-derived exosome, plant-derived exosome and application of plant-derived exosome

Abstract

The invention discloses an extraction and purification method and application of plant-derived exosome-like nanoparticles, and relates to the technical field of plant exosome preparation. The extraction and purification method comprises the following steps: washing fresh ginseng, airing for later use, adding a proper amount of buffer solution, mixing, breaking walls, filtering, centrifuging filtrate to obtain supernate, carrying out ultracentrifugation on the supernate to obtain ginseng exosome nano-particle precipitates, resuspending the precipitates, respectively adding pure water sucrose solutions with different concentrations, and carrying out ultracentrifugation to obtain ginseng exosome nano-particles; and re-suspending the target strip, and carrying out ultracentrifugation to remove redundant cane sugar so as to obtain the purified ginseng exosome-like nano-particles. The ginseng exosome-like nano-particles disclosed by the invention have a remarkable effect when being applied to cosmetics or medicines with skin photoaging resistance, skin oxidation resistance and wrinkle resistance. In an anti-UVB (Ultraviolet B) induced cell injury experiment, the ginseng exosome-like nano-particles have an excellent anti-UVB induced cell injury effect.

Description

Technical Field

[0001] The present invention relates to the technical field of plant exosome preparation, and more specifically to a method for extracting and purifying plant-derived exosome-like nanoparticles and an application thereof in anti-photoaging. Background Art

[0002] Skin photoaging is a series of damages to skin tissue functions caused by long-term and repeated exposure to ultraviolet rays (UV), which manifests as erythema, roughness and thickening, wrinkle formation, pigmentation, etc. on the exposed parts of the skin. Among them, short-wave ultraviolet rays (UVB) are the most active in damaging the epidermis and dermis of the skin. Studies have reported that UVB irradiation induces an abnormal increase in intracellular reactive oxygen species (ROS), induces oxidative stress, DNA damage, and mediates the formation of inflammatory cytokines. By upregulating the expression of matrix metalloproteinases (MMPs), it leads to disorders in the synthesis and degradation of the dermal matrix, thereby causing overall photoaging of the skin. Therefore, the development of active substances that can treat photoaging is of great significance for the development of drugs and skin care products.

[0003] Plant-derived exosome-like nanoparticles have similar structures and functions to mammalian exosomes. Moreover, plant-derived ELNs contain relatively abundant miRNAs, so in addition to performing their own functions, they can also regulate cross-kingdom gene expression and play an important role in organisms. Studies have shown that plant-derived ELNs have anti-inflammatory, antiviral, anti-tumor effects, and participate in defense responses to pathogen invasion. Therefore, plant-derived ELNs may interact with mammalian systems as new bioactive ingredients.

[0004] Ginseng (Panax ginseng CA Mey) is a perennial herbaceous plant of the Araliaceae family. The root and rhizome of ginseng have been known as the "king of herbs" since ancient times. It has been used in Eastern countries for more than 1,600 years. It has clinical effects such as enhancing immunity, reducing oxidative stress, regulating the central nervous system, lowering blood sugar and anti-aging. It has always been a research hotspot in my country's medical industry. It is listed as a top grade in the "Shennong Bencao Jing". The "Compendium of Materia Medica" once recorded that ginseng can keep people young and youthful. Modern research shows that ginseng has anti-tumor, anti-inflammatory, antioxidant and anti-aging effects. Summary of the invention

[0005] In view of this, the present invention provides a method for extracting and purifying plant-derived exosome-like nanoparticles, plant-derived exosome-like nanoparticles and applications. The ginseng exosome-like nanoparticles provided by the present invention have excellent anti-skin photoaging, anti-wrinkle and anti-oxidation effects, and are applied to cosmetics or medicines with anti-skin photoaging, anti-skin oxidation and anti-wrinkle effects, and have significant effects.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for extracting and purifying plant-derived exosome-like nanoparticles, characterized in that it comprises the following steps:

[0008] S1, rinse fresh ginseng, dry it and set it aside, add appropriate amount of buffer solution, and put it into a homogenizer to make a homogenate;

[0009] S2, the homogenate was centrifuged at low speed several times to remove cell debris, fibers, and large particles to obtain the supernatant;

[0010] S3, centrifuge the supernatant obtained in S2, then resuspend the precipitate with Tris-HCl and vortex thoroughly;

[0011] S4, placing the suspension prepared in S3 in a centrifuge tube, adding sucrose solutions of different concentrations respectively, aspirating the target band after centrifugation, and then diluting with Tris-HCl to obtain a diluent;

[0012] S5, the diluted solution prepared in S4 is centrifuged to remove sucrose, thereby obtaining purified ginseng-derived exosome-like nanoparticles.

[0013] Preferably, the mass ratio of the fresh human ginseng to the buffer in step S1 is 1:(3-10); the buffer is PBS; after filtering the homogenate obtained in step S1, the operation of step S2 is performed.

[0014] Preferably, the multiple low-speed centrifugations in step S2 are: 300×g for 10 min, 2,000×g for 20 min, and 10,000×g for 30 min.

[0015] Preferably, the centrifugation in step S3 is ultracentrifugation at 100,000×g for 2 h; and the concentration of Tris-HCl is 20 mmol / L.

[0016] Preferably, the mass concentration of the sucrose solution in step S4 is 15%, 30%, 45%, and 60%, wherein the middle sample bands with concentrations of 30% and 45% are the target bands; and the concentration of Tris-HCl is 20 mmol / L.

[0017] Preferably, in step S5, the centrifugation is performed at 100,000×g for 2 h;

[0018] Another object of the present invention is to provide the use of the above-mentioned plant-derived exosome-like nanoparticles in anti-photoaging.

[0019] Preferably, plant-derived exosome-like nanoparticles are used to prepare a composition for treating photodamaged HacaT cells.

[0020] Preferably, plant-derived exosome-like nanoparticles are used for preparing medicines and / or cosmetics;

[0021] Preferably, in the cosmetics or medicines containing plant-derived exosome-like nanoparticles, the effective concentration of ginseng exosome-like nanoparticles is 10 μg / mL to 100 mg / mL.

[0022] The medicines and / or cosmetics include products for cell tissue repair, cell anti-photoaging, cell antioxidant or cell anti-inflammatory.

[0023] More preferably, the cosmetics include medical aesthetic supplies and / or skin care products and / or beauty products.

[0024] Preferably, the dosage form of the cosmetics includes any one of facial masks, creams, lotions, toners or essence.

[0025] Preferably, the dosage form of the medicines containing ginseng exosome-like nanoparticles includes external solution, ointment or patch.

[0026] It can be seen from the above technical solutions that, compared with the prior art, the present invention has the following beneficial effects:

[0027] In the present invention, the application of ginseng exosome-like nanoparticles in cosmetics or medicines with anti-skin photoaging, anti-skin oxidation and anti-wrinkle effects is remarkable. In the experiment of anti-UVB-induced cell damage, ginseng exosome-like nanoparticles have excellent anti-UVB-induced cell damage effects. The animal model experiment of the present invention shows that: the model group and the administration group take pictures of the back skin and take skin for pathological tissue analysis. By comparison, it can be seen that the skin damage and aging of the mice in the model group are serious, while the damage and aging of the administration group are significantly lighter. Mechanistically, it involves the Keap1 / Nrf2 antioxidant defense mechanism and the NF-κB inflammatory signaling pathway;

[0028] GELNs play an anti-photoaging role by regulating the Nrf2 / Keap1 antioxidant defense mechanism, increasing the activity of antioxidant enzymes, and reversing the ROS accumulation induced by UVB. At the same time, GELNs can inhibit the excessive generation of MMPs caused by oxidative stress and promote collagen production, alleviating UVB-induced skin photoaging. In addition, GELNs can also reduce the inflammatory response by regulating the NF-κB inflammatory signaling pathway, thereby improving photoaging. Description of the Drawings

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on the provided drawings.

[0030] Figure 1 It is a technical roadmap of a preferred method for preparing ginseng exosome-like nanoparticles disclosed by the present invention.

[0031] Figure 2 It is a transmission electron microscope image of the ginseng exosome-like nanoparticles prepared in Example 1.

[0032] Figure 3 It is a dynamic light scattering (DLS) particle size diagram of the ginseng exosome-like nanoparticles prepared in Example 1.

[0033] Figure 4 It is a dynamic light scattering (DLS) potential diagram of the ginseng exosome-like nanoparticles prepared in Example 1.

[0034] Figure 5 It is the particle size distribution of the ginseng exosome-like nanoparticles prepared in Example 1.

[0035] Figure 6 It is the test result of the scratch repair ability of HaCaT cells by different concentrations of the ginseng exosome-like nanoparticles prepared in Example 1.

[0036] Figure 7 It is the effect of different doses of UVB radiation on the viability of HaCaT cells.

[0037] Figure 8 It is the test result of the proliferation of HaCaT cells after UVB treatment by different concentrations of the ginseng exosome-like nanoparticles prepared in Example 1.

[0038] Figure 9 It is the test result of the effect of different concentrations of the ginseng exosome-like nanoparticles prepared in Example 1 on the total antioxidant capacity of HaCaT cells after UVB treatment.

[0039] Figure 10 It is the test result of the effect of different concentrations of the ginseng exosome-like nanoparticles prepared in Example 1 on the MDA content of HaCaT cells after UVB treatment.

[0040] Figure 11 It is the test result of the effect of different concentrations of the ginseng exosome-like nanoparticles prepared in Example 1 on the GSH-Px content of HaCaT cells after UVB treatment.

[0041] Figure 12Test results of the effects of different concentrations of ginseng exosome-like nanoparticles prepared in Example 1 on the SOD content in HaCaT cells after UVB treatment.

[0042] Figure 13 Test results of the effects of different concentrations of ginseng exosome-like nanoparticles prepared in Example 1 on the ROS content in HaCaT cells after UVB treatment.

[0043] Figure 14 Test results of the effects of different concentrations of ginseng exosome-like nanoparticles prepared in Example 1 on the epidermal morphology of photoaged skin.

[0044] Figure 15 Test results of the effects of different concentrations of ginseng exosome-like nanoparticles prepared in Example 1 on the thickness of photoaged skin.

[0045] Figure 16 Test results of the effects of different concentrations of ginseng exosome-like nanoparticles prepared in Example 1 on the tissue structure of photoaged skin.

[0046] Figure 17 Test results of the effects of different concentrations of ginseng exosome-like nanoparticles prepared in Example 1 on the expression of proteins related to the Keap1 / Nrf2 pathway in HaCaT cells after UVB treatment.

[0047] Figure 18 Test results of the effects of different concentrations of ginseng exosome-like nanoparticles prepared in Example 1 on the expression of NF-κB protein in HaCaT cells after UVB treatment.

[0048] Figure 19 Test results of the effects of different concentrations of ginseng exosome-like nanoparticles prepared in Example 1 on the expression of MMPs proteins in HaCaT cells after UVB treatment. Detailed implementation manners

[0049] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. 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.

[0050] Experimental Example 1: Extraction and purification of ginseng exosome-like nanoparticles

[0051] According to the extraction and purification technical route as Figure 1 described, ginseng exosome-like nanoparticles were successfully extracted and purified. The main steps are as follows:

[0052] (1)Rinse the fresh ginseng, dry it, add an appropriate amount of PBS, and make a homogenate in a homogenizer.

[0053] (2)After filtration, use differential centrifugation to centrifuge at 300×g, 2,000×g, and 10,000×g for 10 min, 20 min, and 30 min respectively to remove cell debris, fibers, and large particles.

[0054] (3)Take the supernatant and add it to an ultracentrifuge tube, centrifuge at 100,000×g for 2 h (SW32Ti rotor), resuspend the precipitate with 20 mmol / L Tris-HCl, and vortex thoroughly.

[0055] (4)Continue purification using the sucrose density gradient method. Place the sample in a centrifuge tube, add sucrose solutions with different mass concentrations (15%, 30%, 45%, 60%) of pure water respectively. After centrifuging at 100,000×g for 2 h, gently aspirate the sample band between 30% and 45%. Dilute it with 20 mmoL / L Tris-HCl and then centrifuge at 100,000×g for 2 h to remove sucrose, and the purified ginseng exosome-like nanoparticles are obtained and stored at -80 °C for further analysis.

[0056] Morphological identification of ginseng exosome-like nanoparticles was carried out by transmission electron microscopy. Transmission electron microscopy (TEM) detected that the ginseng exosome-like nanoparticles had a cup-shaped structure and no aggregated distribution. The results are shown in Figure 2 .. The particle size and zeta potential of ginseng exosome-like nanoparticles were measured by dynamic light scattering method (DLS). The average particle size was 110.0 nm and the zeta potential was -17.8 mV. The results are shown in Figure 3 、 Figure 4 , and the particle size distribution range of ginseng exosome-like nanoparticles was measured by a nano-flow cytometer. The proportion of nanoparticles with a diameter range of 30 - 150 nm in ginseng exosome-like nanoparticles was 99.7%. The results are shown in Figure 5 .

[0057] Experimental Example 2: Test on the skin cell repair performance of ginseng exosome-like nanoparticles

[0058] The cell migration and repair ability after treatment with ginseng exosome-like nanoparticles for a period of time was judged by detecting the healing rate of artificial scratches on HaCaT cells (human immortalized epidermal cells). The results are shown in Figure 6 , both the blank control group and the ginseng exosome-like nanoparticle group showed a healing trend within 24 h. Compared with the blank control group, the healing rate of the ginseng exosome-like nanoparticle group showed an upward trend and reached the maximum healing rate of 55.44 ± 1.72% at 50 μg / mL. The scratch experiment proved that ginseng exosome-like nanoparticles can promote cell growth and migration, showing cell repair ability.

[0059] Experimental Example 3: Testing the Proliferation Performance of Ginseng Exosome-like Nanoparticles on Skin Cells

[0060] To successfully establish a photoaging model of HaCaT cells, in this experiment, HaCaT cells were irradiated with UVB at doses of 625, 1250, 2500, and 5000 mJ / cm 2 . After culturing for 24 h, the cell viability was detected by the CCK8 method. The results are shown in Figure 7 . Compared with the control group, as the UVB radiation dose increased, the cell survival rates decreased by 17.49 ± 3.60%, 46.68 ± 4.82%, 59.67 ± 1.02%, and 68.41 ± 2.89% respectively, all showing significant differences (P < 0.001). This indicates that UVB radiation can cause cytotoxicity to HaCaT cells, thereby inhibiting their proliferation. Therefore, 1250 mJ / cm 2 was selected as the radiation dose for subsequent experiments.

[0061] Figure 8 The following are the test results of the proliferation of HaCaT cells treated with UVB by different concentrations of ginseng exosome-like nanoparticles. Compared with the UVB group, as the dose increased, the cell survival rates in the ginseng exosome-like nanoparticle groups increased by 7.11 ± 1.87%, 15.33 ± 1.45%, 28.71 ± 3.58%, and 6.07 ± 1.68% respectively. The results show that 12.5, 25, and 50 μg / mL of ginseng exosome-like nanoparticles not only have no obvious toxic damage to HaCaT cells, but also can promote cell proliferation and reverse the cell damage induced by UVB.

[0062] Experimental Example 4: Determination of the Total Antioxidant Capacity of Ginseng Exosome-like Nanoparticles by the ABTS Method

[0063] Prepare an appropriate amount of ABTS working solution. Mix the ABTS solution and the oxidant at a volume ratio of 1:1 to form the ABTS working mother liquor. After storing it in the dark at room temperature for 12 - 16 h, dilute the ABTS working mother liquor 30 - 55 times with PBS to obtain the ABTS working solution. Take an appropriate amount of 10 mM Trolox solution and dilute it with PBS to 0.05, 0.15, 0.3, 0.6, 0.9, 1.2, and 1.5 mM. Take a 96-well plate and set blank wells, standard wells, and measurement wells. Add 280 μL of ABTS working solution and 7 μL of distilled water to the blank wells, add 280 μL of ABTS working solution and 7 μL of different concentrations of Trolox standard solutions to the standard wells, and add 280 μL of ABTS working solution and 7 μL of the supernatant of each group of cells to the measurement wells. Set 2 parallel duplicate wells for each concentration in the standard group. React at room temperature for 2 - 6 min, and measure the absorbance at 405 nm with an enzyme-linked immunosorbent assay (ELISA) reader.

[0064] Using the Trolox standard as the abscissa and the A405 value as the ordinate, a standard curve of the total antioxidant capacity was plotted. The antioxidant capacity of the sample relative to the Trolox standard was calculated based on the absorbance value of the sample measurement well. The results are shown in Figure 9 , and showed a dose-dependence (P < 0.01). Ginseng exosome-like nanoparticles could significantly reduce the total antioxidant capacity, thus alleviating UVB-induced photoaging.

[0065] Experimental Example 5: Detection of MDA content

[0066] Weighed an appropriate amount of TBA, prepared an appropriate amount of TBA stock solution with a concentration of 0.37% using the TBA preparation solution, heated it to 70 °C and vortexed vigorously to promote its complete dissolution. Prepared an appropriate amount of MDA detection working solution according to the volume ratio of TBA dilution solution: TBA stock solution: antioxidant of 150:50:3, heated it to 70 °C and vortexed vigorously to promote its complete dissolution. Diluted an appropriate amount of standard product with distilled water to 1, 2, 5, 10, 20, 50, 100 μM for making the standard curve. Took several PCR tubes with heat lids and set up blank control tubes, standard tubes, and sample tubes. The blank control tubes were added with 0.1 mL of PBS and 0.2 mL of MDA detection working solution, the standard tubes were added with 0.1 mL of different concentrations of standard product and 0.2 mL of MDA detection working solution, and the sample tubes were added with 0.1 mL of the cell supernatant of each group and 0.2 mL of MDA detection working solution. Each of the blank control tubes and standard tubes was set with 2 parallel replicates. After mixing, heated at 100 °C for 15 min, then cooled to room temperature in a water bath, centrifuged at 1000 g at room temperature for 10 min, took 200 μL of the supernatant and added it to a 96-well plate, and measured the absorbance at 532 nm with an enzyme-labeled instrument. Calculated the MDA concentration of the sample according to the standard curve, and expressed the MDA content of the sample in terms of protein content (µmol / mg prot);

[0067] The results are shown in Figure 10 , compared with the Control group, the MDA content in the UVB group increased significantly (P < 0.001), and the MDA content in the 12.5, 25, 50 μg / mL ginseng exosome-like nanoparticle groups decreased significantly (P < 0.001), and showed a dose-dependence (P < 0.01). The results indicated that ginseng exosome-like nanoparticles could inhibit the production of lipid peroxides, thus alleviating UVB-induced photoaging.

[0068] Experimental Example 6: Detection of GSH-Px enzyme activity

[0069] Prepare a 0.08 μmol / mL reduced glutathione standard solution using distilled water. Take several 1.5 mL EP tubes, set up assay tubes and control tubes. In the assay tubes, add 20 μL of the cell supernatant from each group and 20 μL of reagent one working solution (NADPH solution). In the control tubes, only add 20 μL of reagent one working solution. After preheating at 37°C for 5 min, add 10 μL of reagent two working solution (GSH solution) to both. React at 37°C for 5 min. In the assay tubes, only add 200 μL of reagent three (glutathione peroxidase detection buffer). In the control tubes, add 200 μL of reagent three and 20 μL of the cell supernatant from each group. Mix well, centrifuge at 4000 rpm at room temperature for 5 min, and take 100 μL of the supernatant and transfer it to a 96-well plate.

[0070] Set up assay wells, control wells, standard wells, and blank wells. In the standard wells, add 100 μL of the standard solution. In the blank wells, add 100 μL of distilled water. Then, add 100 μL of reagent four and 25 μL of reagent five (peroxide reagent solution) to each well. After mixing the well plate thoroughly, let it stand at room temperature for 15 min, measure the absorbance at 412 nm. Set up 2 parallel replicates for the blank wells and the standard wells. Calculate ΔAassay = A 对照孔 - A 测定孔 ,ΔAstandard = A 标准孔 - A 空白孔 。Define one enzyme activity unit as catalyzing the oxidation of 1 nmol GSH per minute per mg of protein in the reaction system. Cpr: protein concentration in the supernatant, mg / mL.

[0071] The results are shown in Figure 11 ,compared with the Control group, the GPX activity in the UVB group decreased significantly (P < 0.001). Compared with the UVB group, the GPX activities in the 12.5, 25, and 50 μg / mL ginseng exosome-like nanoparticle groups increased significantly (P < 0.001) and showed a dose-dependent manner (P < 0.01). The results indicate that ginseng exosome-like nanoparticles can increase the antioxidant enzyme activity in cells, thereby alleviating UVB-induced photoaging.

[0072] GPX (U / mg prot) = 200 × ΔAassay ÷ ΔAstandard ÷ Cpr.

[0073] Experimental Example 7: Detection of SOD Enzyme Activity

[0074] Prepare an appropriate amount of NBT / enzyme working solution according to the volume ratio of SOD detection buffer: NBT: enzyme solution = 158:1:1, and prepare an appropriate amount of reaction start working solution according to the volume ratio of reaction start solution (40X): SOD detection buffer = 39:1. Store both at 4°C for later use.

[0075] In a 96-well plate, set up sample wells, blank control wells 1, 2, and 3. Add 20 μL of the cell supernatant of each group, 160 μL of the NBT / enzyme working solution, and 20 μL of the reaction initiation solution to the sample wells. Add 20 μL of the SOD detection buffer, 160 μL of the NBT / enzyme working solution, and 20 μL of the reaction initiation solution to blank control well 1. Add 40 μL of the SOD detection buffer and 160 μL of the NBT / enzyme working solution to blank control well 2. Add 20 μL of the cell supernatant of each group, 20 μL of the SOD detection buffer, and 160 μL of the NBT / enzyme working solution to blank control well 3. Incubate at 37 °C for 30 min and measure the absorbance at 560 nm.

[0076] When the inhibition percentage in the reaction system is defined as 50%, the SOD enzyme activity is one enzyme activity unit (unit). The results are shown in Figure 12 , compared with the Control group, the SOD activity in the UVB group decreased significantly (P < 0.001). Compared with the UVB group, the SOD activities in the 12.5, 25, and 50 μg / mL ginseng exosome-like nanoparticle groups increased significantly (P < 0.001) and showed a dose-dependent relationship (P < 0.01). The results indicate that ginseng exosome-like nanoparticles can increase the antioxidant enzyme activity in cells, thereby alleviating UVB-induced photoaging.

[0077] Inhibition percentage = [(Ablank control 1 - Ablank control 2) - (Asample - Ablank control 3)] / (Ablank control 1 - Ablank control 2) × 100%.

[0078] Experimental Example 8: ROS Detection

[0079] After successfully establishing the cell photoaging model, for the samples loaded with probes after collecting the cells, remove the cell culture medium, wash the adherent cells once with PBS, and add 200 μL of trypsin to digest the cells. Incubate at room temperature until the adherent cells can be detached by gently pipetting, then add 1 mL of complete medium to terminate the digestion. Gently pipette the cells down, transfer them to a centrifuge tube, centrifuge at 1000 g for 5 minutes, discard the supernatant, collect the cells and resuspend them in the diluted DCFH-DA at a cell concentration of 1×10 6 / mL, incubate in a 37 °C cell culture incubator for 20 minutes, and invert and mix every 5 minutes to ensure sufficient contact between the probe and the cells. Wash the cells three times with serum-free cell medium to fully remove the DCFH-DA that has not entered the cells. Finally, add 500 μL of PBS to resuspend the cell pellet, and detect the ROS levels in the cells of each group using a flow cytometer. Process the data using FlowJo software.

[0080] The results are shown in Figure 13, it was found by analyzing the fluorescence intensity that the ROS level in the UVB group was 2.18 times that of the Control group, and the ROS levels in the 12.5, 25, and 50 μg / mL ginseng exosome-like nanoparticle groups were 1.88, 1.27, and 1.02 times that of the Control group, respectively. The ROS level in the 50 μg / mL GELN group had recovered to a level similar to that of the Control group, indicating that ginseng exosome-like nanoparticles can dose-dependently reduce the excessive intracellular ROS level induced by UVB, thereby preventing oxidative damage caused by ROS accumulation.

[0081] Experimental Example 9: Study on the effect of UVB-induced skin photoaging in rats

[0082] Thirty female SD rats were randomly divided into 5 groups, with 6 rats in each group. The specific grouping is as follows: Control group (shaved, untreated, not treated), UV group (shaved, irradiated, not treated), 0.25, 0.5, 1 mg / mL ginseng exosome-like nanoparticle groups (shaved, irradiated, treated). Before the start of modeling, the rats were shaved, and the area was 4 cm × 4 cm. A photoaging model was established by irradiating the bare back skin of the rats with ultraviolet light (UVA + UVB). Combining the test situation and the irradiation protocol in the literature, except for the Control group, the shaved rats were placed in a self-made ultraviolet light box, the fixed irradiation distance was 20 cm, and ultraviolet light (UVA + UVB) was irradiated. In the first week, it was 20 min / day, in the second week it was 40 min / day, in the third week it was 60 min / day, and then it was maintained at 60 min / day until the back skin of the rats showed dryness, peeling, and even local ulceration, which indicated the successful establishment of the photoaging model. After the modeling was completed, for the affected skin on the back of the rats, the ginseng exosome-like nanoparticle groups were treated with ginseng exosome-like nanoparticles at different concentrations and the same dose (1 mL / day), and the Control group and the UVB group were smeared with the same dose of PBS. After continuous administration for 7 days, after the treatment was completed, the rats were anesthetized and blood was collected from the abdominal aorta, and the bare target skin was taken.

[0083] During the administration period, the skin changes of the test rats in each group were photographed and recorded. The results are as Figure 14 , the back skin status of the rats in each group after 7 days of administration. The back skin of the rats in the Control group was in good condition, the surface skin was relatively smooth, there was no rough peeling phenomenon, and occasional fine wrinkles could be seen, but they would disappear with the movement of the rats. The rats in the UV group showed photoaging skin characteristics such as rough skin, obvious redness and swelling, persistent wrinkles, and abnormal skin thickness. 0.25, 0.5, and 1 mg / mL ginseng exosome-like nanoparticles could all improve these symptoms and reverse the photo-damage. The skin status of the high-dose ginseng exosome-like nanoparticle group was significantly improved, and there was no significant difference from the skin status of the Control group.

[0084] The back skin thickness of each group of experimental rats was measured. The changes in skin thickness after treatment of each group of rats are as Figure 15 shown. Compared with the Control group, the skin thickness of the UV group increased significantly. Compared with the UV group, the skin thickness decreased after treatment with ginseng exosome-like nanoparticles, and the effect was most significant in the 1 mg / mL ginseng exosome-like nanoparticle group. The results showed that acceptable therapeutic effects were obtained in all ginseng exosome-like nanoparticle groups, and ginseng exosome-like nanoparticles had a significant effect on the treatment of photoaging.

[0085] H&E and Masson staining were used to detect the changes in the epidermal skin structure and dermal collagen level of skin tissues, and the results are shown in Figure 16 . By observing the skin morphology with H&E staining, compared with the Control group, ultraviolet irradiation led to epidermal thickening, accompanied by obvious infiltration of inflammatory cells. Treatment with ginseng exosome-like nanoparticles significantly restored the epidermal thickness, and the effect was enhanced in a dose-dependent manner. The arrangement of dermal collagen fibers was observed by Masson staining. Compared with the Control group, UV irradiation led to sparse and disordered partial dermal collagen fibers and a decrease in the collagen space density. After treatment with ginseng exosome-like nanoparticles, the arrangement of partial dermal collagen fibers was neater and the collagen density increased. In summary, ginseng exosome-like nanoparticles can effectively alleviate ultraviolet-induced skin photoaging.

[0086] Experimental Example 10: Effect of ginseng exosome-like nanoparticles on the expression of proteins related to the Keap1 / Nrf2 pathway in UVB-induced HaCaT cells

[0087] The Nrf2 signaling pathway is a crucial antioxidant defense mechanism. Nrf2 is regulated by the Keap1 protein and induces the expression of a series of antioxidant genes. The Keap1 / Nrf2 system is the main node for cells to resist exogenous and endogenous oxidative stress. The results are shown in Figure 17 . Compared with the Control group, the expression level of Nrf2 protein in the UVB group decreased significantly, and the expression level of Keap-1 protein increased significantly. Compared with the UVB group, ginseng exosome-like nanoparticles could significantly inhibit the expression of Keap1 protein and promote the expression of Nrf2 (P < 0.01), and showed a dose-dependent manner. The results indicated that GELNs exerted antioxidant ability by inducing the expression of antioxidant genes through the Nrf2 signaling pathway, thereby playing a role in delaying skin aging.

[0088] Experimental Example 11: Effect of ginseng exosome-like nanoparticles on the expression of NF-κB protein in UVB-induced HaCaT cells

[0089] NF-κB is a dimer composed of p65 and p50 that participates in the regulation of inflammatory and immune responses. It can be activated by cytokines, free radicals, and ultraviolet radiation. The activation of NF-κB can increase the level of TNF-α and the expression of MMPs, induce the degradation of the extracellular matrix (ECM), and accelerate skin aging. The results are shown in Figure 18 , compared with the Control group, the expression level of NF-κB p65 protein in the UVB group was significantly increased, and the expression level of NF-κB p65 protein in the GELNs group was significantly decreased in a dose-dependent manner. The results indicate that GELNs may improve UVB-induced photoaging by inhibiting the NF-κB inflammatory signaling pathway in UVB-induced HaCaT cells.

[0090] Experimental Example 12: Effect of ginseng exosome-like nanoparticles on the expression of MMPs-related proteins in UVB-induced HaCaT cells

[0091] Matrix metalloproteinases (MMPs) are a type of collagen hydrolase that can degrade almost all protein components in the extracellular matrix (ECM). Irradiation with ultraviolet light and oxidative stress can directly or indirectly cause an upregulation of the expression of MMPs in keratinocytes, leading to the degradation of type I collagen, which is the core link of photoaging. MMP-1 can specifically degrade type I collagen initially, and its degradation products can also inhibit the expression of collagen. MMP-9 can further decompose the degraded collagen fragments and damage the basal layer of the epidermis. We detected the protein expression of MMP-1 and MMP-9, and the results are shown in Figure 19 , after UVB irradiation, the protein expression levels of MMP-1 and MMP-9 in HaCaT cells were significantly increased, and the collagen fiber structure was damaged. After treatment with GELNs, the increased expression could be significantly inhibited, and it showed a dose-dependent manner. The results indicate that GELNs can inhibit the excessive production of MMPs caused by oxidative stress and promote the regeneration of collagen, thereby alleviating UVB-induced photoaging.

[0092] In this specification, each embodiment is described in a progressive manner. The key point of each embodiment is to illustrate the differences from other embodiments. For the same or similar parts among the embodiments, reference can be made to each other. For the devices disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple, and reference can be made to the description in the method part for the relevant parts.

[0093] The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for extracting and purifying plant-derived exosome-like nanoparticles, characterized in that: The following steps are involved: S1, rinse fresh ginseng, dry it and set it aside, add buffer solution, and put it into a homogenizer to make a homogenate; S2, the homogenate was centrifuged at low speed several times to remove cell debris, fibers, and large particles to obtain the supernatant; S3, centrifuge the supernatant obtained in S2, then resuspend the precipitate with Tris-HCl and vortex thoroughly; S4, placing the suspension prepared in S3 in a centrifuge tube, adding sucrose solutions of different concentrations respectively, aspirating the target band after centrifugation, and then diluting with Tris-HCl to obtain a diluent; S5, the diluted solution prepared in S4 is centrifuged to remove sucrose, thereby obtaining purified ginseng-derived exosome-like nanoparticles.

2. The method for extracting and purifying plant-derived exosome-like nanoparticles according to claim 1, characterized in that: The mass ratio of the fresh human ginseng to the buffer in step S1 is 1:(3-10); the buffer is PBS; after filtering the homogenate obtained in step S1, the operation of step S2 is performed.

3. The method for extracting and purifying plant-derived exosome-like nanoparticles according to claim 1, characterized in that: The multiple low-speed centrifugations in step S2 are: 300×g for 10 min, 2,000×g for 20 min, and 10,000×g for 30 min.

4. The method for extracting and purifying plant-derived exosome-like nanoparticles according to claim 1, characterized in that: The centrifugation in step S3 is ultracentrifugation at 100,000×g for 2 h; the concentration of Tris-HCl is 20 mmol / L.

5. The method for extracting and purifying plant-derived exosome-like nanoparticles according to claim 1, characterized in that: In step S4, the mass concentrations of the sucrose solution are 15%, 30%, 45%, and 60%, wherein the middle sample bands with concentrations of 30% and 45% are the target bands; and the concentration of the Tris-HCl is 20 mmol / L.

6. The method for extracting and purifying plant-derived exosome-like nanoparticles according to claim 1, characterized in that: In step S5, the centrifugation is performed at 100,000×g for 2 h.

7. A plant-derived exosome-like nanoparticle, characterized in that: The product is prepared by the extraction and purification method according to any one of claims 1 to 6.

8. Use of the plant-derived exosome-like nanoparticles as claimed in claim 7 in anti-photoaging.

9. The use of plant-derived exosome-like nanoparticles in anti-photoaging according to claim 8, characterized in that: Plant-derived exosome-like nanoparticles were used to prepare a composition for treating photodamaged HacaT cells.

10. The use of plant-derived exosome-like nanoparticles in anti-photoaging according to claim 8, characterized in that: Using plant-derived exosome-like nanoparticles for the preparation of medicines and / or cosmetics; The medicines and / or cosmetics include cell tissue repair, cell anti-photoaging, cell anti-oxidation or cell anti-inflammatory products.

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