Application of eustachys japonicus extracellular vesicles

Abstract

The application discloses application of extracellular vesicles from Eclipta prostrata L. The extracellular vesicles are extracted from Eclipta prostrata L. by using ultracentrifugation and ultrafiltration, and the extracted vesicles are complete in appearance and good in dispersibility, and have the characteristics of high yield, fast separation, low cost and easy scaling. The extracted vesicles can be successfully taken by animal cells. After being taken, the vesicles play the roles of antioxidation and anti-aging, can significantly reduce ultraviolet-induced matrix metalloproteinase 1 (MMP1) expression, promote collagen I (COL-I) generation, improve ultraviolet-induced skin photoaging, and the effect is stronger than that of Eclipta prostrata L. alcohol extract, so that the extracellular vesicles from Eclipta prostrata L. provide a new strategy for skin aging and anti-aging treatment.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant-derived vesicle application, and particularly relates to application of cell extracellular vesicles derived from Eclipta prostrata L. BACKGROUND

[0002] Skin is composed of epidermis, dermis and subcutaneous tissue. Skin aging can be roughly divided into two types according to its causes. Natural aging is accompanied by continuous decline in skin structure and physiological function with aging. Extrinsic aging is caused by the accumulation of external stress such as sunlight. Photoaging is the long-term exposure of skin to ultraviolet light, which leads to the degradation of collagen and the increase of melanin, making the skin dry, rough, loose, wrinkled and pigmented, and even leading to skin cancer. Existing products for preventing and treating photoaging mainly include antioxidants, hyaluronic acid and other ingredients, but these methods have problems such as poor stability, poor absorption, limited effect, toxicity, intolerance and the like. Therefore, there is still a lack of better treatment methods.

[0003] Extracellular vesicles (EVs) are microvesicles secreted by various cells. EVs mainly include exosomes, microvesicles and apoptotic bodies, among which exosomes are the smallest, with a diameter of usually 30-150 nm, and the size range of microvesicles is extensive, with a diameter of 100-1000 nm. EVs carry various bioactive molecules such as nucleic acids, proteins and metabolites, and are extremely important mediators of intercellular signal transmission, regulating the physiological activities of recipient cells. However, there is a key problem in using EVs derived from mammalian cells as therapeutic drugs. A large number of EVs are needed for clinical application, and large-scale production of EVs has high production cost and safety risk. Synthetic nanoparticles also have disadvantages such as immunogenicity, cytotoxicity and complex manufacturing process requirements.

[0004] Natural medicinal plants are attracting more and more attention due to their low toxicity and multi-targets. However, the skin barrier limits the penetration efficiency of active ingredients from natural medicinal plants. Plant exosome-like nanovesicles (PELNVs) are small vesicles containing bioactive molecules such as lipids, proteins, enzymes, nucleic acids, and plant-specific chemical components, which are released by plant cells. PELNVs have a similar structure to mammalian exosomes and good biocompatibility. Compared with mammalian exosomes, PELNVs have a wide range of sources, low immunogenicity, high biological safety, and tissue targeting. Another advantage of PELNVs is that they are excellent transdermal systems that can cross the skin barrier and penetrate the stratum corneum, achieving better diffusion, absorption, and action. Recent studies have shown that plant vesicles exhibit a variety of biological activities.For example, the vesicles derived from shiitake mushroom, broccoli, etc. show good anti-tumor, immune regulation, regulation of intestinal diseases and liver protection, etc. effects, such as: Chen Q, Li Q, Liang Y, Zu M, Chen N, Canup BSB, Luo L, Wang C, Zeng L, Xiao B. Natural exosome-like nanovesicles from edible tea flowers suppress metastatic breast cancer via ROS generation and microbiota modulation. Acta Pharm Sin B. 2022 Feb;12(2):907-923. Deng Z, Rong Y, Teng Y, Mu J, Zhuang X, Tseng M, Samykutty A, Zhang L, Yan J, Miller D, Suttles J, Zhang HG. Broccoli-Derived Nanoparticle Inhibits Mouse Colitis by Activating Dendritic Cell AMP-Activated Protein Kinase. Mol Ther. 2017 Jul;25(7): 1641-1654. Liu B, Lu Y, Chen X, Muthuraj PG, Li X, Pattabiraman M, Zempleni J, Kachman SD, Natarajan SK, Yu J. Protective Role of Shiitake Mushroom-Derived Exosome-Like Nanoparticles in D-Galactosamine and Lipopolysaccharide-Induced Acute Liver Injury in Mice. Nutrients. 2020 Feb;12(2):477.

[0005] Eclipta prostrata L. is a traditional medicine for black beard and hair. Modern studies have shown that it is rich in flavonoids, triterpenoid saponins and polyphenols, which have significant protective effects on liver damage, detoxification, antioxidant and lipid-lowering, antibacterial, hair growth promotion, and wound healing. However, there is no report on the anti-aging, especially the anti-photoaging of extracellular vesicles derived from Eclipta prostrata L. SUMMARY

[0006] The purpose of the present application is to provide an application of plant-derived extracellular vesicles from Eclipta prostrata L., specifically for preparing anti-skin photoaging drugs. The plant-derived extracellular vesicles provide a new drug source for the field of anti-photoaging medical cosmetology.

[0007] The preparation method of the extracellular vesicles derived from Eclipta prostrata L. of the present application comprises the following steps:

[0008] The Eclipta prostrata L. raw material is added to the buffer solution and broken, and the original slurry is obtained after sieving and filtering. Then at least three high-speed centrifugations are performed, and the supernatant is collected after each centrifugation for the next centrifugation. The supernatant is collected after the last centrifugation, and then ultrafiltration centrifugation is performed. The upper suspension obtained is resuspended with a buffer solution. The resuspension is subjected to membrane filtration to obtain the extracellular vesicles derived from Eclipta prostrata L.

[0009] Further,

[0010] The buffer solution in each step comprises at least one of PBS, Tris buffer, Tris-HCl buffer and TBST buffer. All centrifugation-related operations are performed in an environment of 4-8 DEG C.

[0011] Further,

[0012] The Eclipta prostrata L. raw material is Eclipta prostrata L. whole grass. A juicer is used for breaking, and the rotation speed of the juicer is 8000-20000 rpm, and the breaking time is 20-150 seconds. The screen used for sieving the extracted juice is 100-200 mesh, and the filter membrane pore size for filtering after sieving is 1-5 pm. The three centrifugal forces are 600-800xg, 5000-7000xg and 10000-20000xg, respectively, and the centrifugation time for each is 30-180 minutes. The ultrafiltration centrifugal force is 2500-7000xg, the ultrafiltration filter membrane is 100 kDa, and the centrifugation time is 30-120 minutes. The membrane filtration of the resuspension is first passed through a filter membrane with a pore size of 0.45 pm, and then through a filter membrane with a pore size of 0.22 pm.

[0013] The Eclipta prostrata L. raw material is added to the buffer solution in a ratio of 1-3 g:1-2 mL.

[0014] The application also provides the ECVs from Eclipta prostrata L. with a diameter of 50-200nm.

[0015] The anti-skin photoaging pharmaceutical dosage form comprises an external preparation of at least one of the following: a hydrogel, a cream and a transdermal patch.

[0016] Preferably, the protein concentration of the ECVs from Eclipta prostrata L. is 20-800ug / mL when the ECVs are applied to play the anti-photoaging effect, and the ECVs can be successfully taken by animal cells after 24h of treatment.

[0017] The application uses an ultraviolet-induced human skin keratinocyte and fibroblast damage model to investigate the anti-oxidation, anti-inflammation and collagen generation promoting anti-photoaging effects of the ECVs from Eclipta prostrata L. on ultraviolet-induced human skin cells in vitro cell and animal experiments.

[0018] The application has the following advantages:

[0019] 1. The separation and purification method adopted in the application is simple in operation, and the obtained ECVs from Eclipta prostrata L. are high in purity and yield.

[0020] 2. The ECVs from Eclipta prostrata L. extracted by the ultra-speed combined ultrafiltration centrifugation method are nanometer particles with a membrane structure in morphology, with a diameter of 50-200nm and an average concentration of 4.35x10 11 The ECVs from Eclipta prostrata L. can be successfully taken by animal cells after 24h of treatment.

[0021] 3, The ECVs prepared by the method can significantly improve the decrease in HaCaT cell activity induced by ultraviolet, effectively reduce the ROS and MDA levels of HaCaT cells induced by ultraviolet, increase the activity of SOD, significantly offset the up-regulation of MMP1 induced by UV, obviously increase the content of COL-I, and reduce the SA-β-Gal production of HSF cells. Therefore, the ECVs can enhance the antioxidant capacity of the skin, maintain the structure and function of the skin, effectively reduce the cell senescence and apoptosis induced by ultraviolet radiation, and have obvious anti-skin photoaging effect induced by ultraviolet. The ECVs prepared by the method provide a new strategy for skin aging and anti-aging treatment. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 : Comparative results of the preparation method of ECVs from Eclipta prostrata in Example 1; wherein Figure 1 A: Representative particle size distribution graph of the ECVs prepared by the ultrafiltration centrifugation method detected by NTA; Figure 1 B: Representative particle size distribution graph of the ECVs prepared by the ultracentrifugation method detected by NTA; Figure 1 C: Representative particle size distribution graph of the ECVs prepared by the sucrose density gradient centrifugation method detected by NTA; Figure 1 D: Average particle size of the three methods; Figure 1 E: Average yield of the three methods; Figure 1 F: Purity comparison of the three methods.

[0023] Figure 2 : Characterization results of the ECVs from Eclipta prostrata prepared in Example 1; wherein Figure 2 A: Morphology graph of the ECVs from Eclipta prostrata prepared by extraction observed by transmission electron microscopy (TEM); Figure 2 B: Zeta potential graph of the ECVs from Eclipta prostrata prepared by extraction detected by dynamic light scattering (DLS).

[0024] Figure 3 : In vitro anti-photoaging effect results of the ECVs from Eclipta prostrata in Example 2; wherein Figure 3 A: Endocytosis of DIO-labeled ECVs from Eclipta prostrata in HaCaT cells; Figure 3 B: Effect of the ECVs from Eclipta prostrata on the proliferation activity of HaCaT cells induced by ultraviolet; Figure 3 C: Effect of the ECVs from Eclipta prostrata on the SOD activity of HaCaT cells induced by ultraviolet; Figure 3 D: Effect of the ECVs from Eclipta prostrata on the ROS level of HaCaT cells induced by ultraviolet and quantitative results; Figure 3 E: Effect of the ECVs from Eclipta prostrata on the SA-β-Gal activity level of HSF cells induced by ultraviolet; Figure 3F: Quantitative results of the effect of ECVs from E. prostrata on the MDA concentration of HaCaT cells induced by UV; Figure 3 G: Results and quantitative results of the effect of ECVs from E. prostrata on the MMP1 protein expression of HaCaT cells induced by UV by Western blot (WB); Figure 3 H: Results of the effect of ECVs from E. prostrata on the COL-I expression of HaCaT cells induced by UV by ELISA.

[0025] Figure 4 In vivo anti-photoaging effect of ECVs from E. prostrata; wherein Figure 4 A: MASSON staining diagram of mouse skin; Figure 4 B: Immunohistochemical diagram of COL-1 and MMP1 of mouse skin. DETAILED DESCRIPTION

[0026] The present application will be described below through specific examples, without forming a limitation on the present application.

[0027] HaCaT and HSF cells of the present application were purchased from Wuhan Saiver Biological Technology Co., Ltd;

[0028] Female BALB / c mice (6-8 weeks old) of the present application were purchased from Hunan Slike Jingda Experimental Animal Co., Ltd.

[0029] Example 1: Isolation and characterization of ECVs from E. prostrata

[0030] In this example, ECVs from E. prostrata were prepared and morphologically observed.

[0031] 1.1 Vesicle separation and purification of Eclipta prostrata: The whole plant of Eclipta prostrata was washed with distilled water to remove dust and soil. After being chopped, the juice was extracted using a juicer at a speed of 10,000 rpm for 120 seconds. The obtained juice was filtered through a 100-mesh sieve with a pore size of 4 μm. Then, it was centrifuged at 600×g for 30 minutes, 5000×g for 30 minutes, and 10000×g for 60 minutes to remove large fragments. After the final centrifugation, the supernatant was collected. Different separation methods were compared. For ultrafiltration centrifugation, a 100kDa (Millipore, USA) ultrafiltration centrifuge filter was used to concentrate the juice. The ultrafiltration centrifugation force was 6000×g, and the centrifugation time was 60 minutes. The separated upper suspension vesicles were resuspended in PBS and sterilized by filtration through 0.45μm and 0.22μm filters (Millipore, USA), and stored at -80℃. For high-speed centrifugation, an Optima TL ultracentrifuge from Beckman Coulter was used. Centrifugation was performed at 4℃, 120000×g for 2 hours. The supernatant was discarded, and the vesicles in the precipitate were resuspended. For density gradient centrifugation, sucrose densities of 60%, 45%, 20%, and 8% were layered sequentially. Centrifugation was performed at 4℃, 150000×g for 2 hours. The 45% to 25% layer was collected and centrifuged again at 4℃, 120000×g for 2 hours. Particle size distribution and concentration were determined using NTA, and protein concentration was determined using the BCA method. Figure 1 The AC results show that the NTA measurements indicate that samples obtained by ultracentrifugation and density gradient centrifugation exhibit multiple peaks (heterogeneous), while samples obtained by ultrafiltration centrifugation show a single peak with a narrow distribution (homogeneous). Figure 1 The DF results showed that the average particle size was 148.23 nm for ultrafiltration centrifugation, 161.3 nm for ultracentrifugation, and 140.07 nm for density gradient centrifugation. The yield (total number of particles) obtained by ultrafiltration centrifugation was lower than that by ultracentrifugation, but the difference between ultrafiltration centrifugation and density gradient centrifugation was not statistically significant. Purity, expressed as "particles / mg protein," was 2.52 × 10⁻⁶ for ultrafiltration centrifugation. 10 The concentrations per mg were obtained by ultracentrifugation (1.35 × 10⁻⁶ mg / mg). 10 (units / mg) and density centrifugation (1.43×10⁻⁶) 10 The concentrations of vesicles (per mg) were 1.79 times and 1.67 times higher than those obtained by ultrafiltration centrifugation, respectively, demonstrating the significant advantages of ultrafiltration centrifugation. The morphology and electrical potential of the vesicles obtained by ultrafiltration centrifugation were characterized by TEM and DLS, respectively. Figure 2 Results A showed that no aggregation was observed by TEM, and the nanovesicles were round or elliptical, similar to the lipid bilayer membrane structure of exosomes. Figure 2 The average zeta potential in sample B is -33.5 mV, indicating that the sample carries a negative charge and is stable.

[0032] 1.2 Preparation of J. curcas alcohol extract: To compare the effects, J. curcas extract was prepared according to the conventional extraction method. The whole plant of J. curcas was dried and crushed into powder. The powder was extracted with 95% methanol by reflux extraction for 3 times. The extract was combined, filtered, and recovered under reduced pressure to obtain the crude extract. The extract was vacuum dried to obtain the J. curcas alcohol extract in powder form. The extract was used in Examples 2 and 3.

[0033] Example 2: This example is used to illustrate the in vitro anti-photoaging effect of J. curcas-derived extracellular vesicles.

[0034] The confocal microscope was used to observe whether the J. curcas-derived extracellular vesicles could be taken up by HaCaT cells, as shown in FIG. 2A. The confocal microscope confirmed that the vesicles could be taken up by cells. Figure 3 The HaCaT cells induced by ultraviolet were treated with different concentrations of vesicles (0, 50, 100, 200, 400, and 800 pg / mL) for 24 h, and CCK-8 experiment was performed for detection, as shown in FIG. 2B. Among them, the control group was not subjected to ultraviolet irradiation modeling, the model group was ultraviolet induction without adding vesicles, and the rest were HaCaT groups treated with different concentrations of vesicles (0, 50, 100, 200, 400, and 800 pg / mL) after ultraviolet induction. It was found that the J. curcas-derived extracellular vesicles promoted the proliferation of ultraviolet-induced HaCaT cells in a concentration-dependent manner. Figure 3

[0035] Ultraviolet radiation causes oxidative stress, which produces high levels of ROS, leading to inflammation and lipid oxidation, and ultimately triggering skin aging. The J. curcas vesicles (400 pg / mL) and extract (10 mg / mL) were used to treat ultraviolet-induced HaCaT and HSF cells. The DCFH-DA probe method was used and observed under a fluorescence microscope, and the SA-β-Gal assay kit was used to determine SA-β-Gal, as shown in FIG. 3C-F. Compared with the control group and the extract group, the vesicle group could significantly reduce the ROS in HaCaT cells and the SA-β-Gal level in HSF cells. Combined with the results of SOD and MDA, it was shown that the J. curcas-derived extracellular vesicles had a stronger antioxidant effect than the alcohol extract. The overexpression of MMP1 could degrade skin type I collagen and damage the integrity of the collagen and elastic fiber structure. The WB and ELISA results (FIG. 3G-H) confirmed that the J. curcas-derived extracellular vesicles significantly offset the upregulation of ultraviolet-induced MMP1 and promoted the generation of COL-I, and the effect was stronger than that of the alcohol extract. The above results showed that the J. curcas-derived extracellular vesicles could effectively alleviate ultraviolet-induced cell damage and promote the generation of collagen. Figure 3 Figure 3

[0036] Example 3: This example is used to illustrate the in vivo anti-photoaging effect of J. curcas-derived extracellular vesicles. ​​​

[0037] Photoaging leads to a deterioration in skin appearance, including symptoms such as wrinkles, sagging, redness, and pigmentation. UVB radiation exacerbates these conditions by inducing collagen and elastin degradation, oxidative stress, and inflammation. A mouse model of photoaging was established using ultraviolet radiation.

[0038] After removing dorsal hair, female BALB / c mice (6–8 weeks old) were randomly divided into 6 groups (n=6 per group) for 4 weeks, with ethical approval granted by the Second Xiangya Hospital of Central South University (approval number: AP-C230806). The control group remained normal, while the model, extract, and vesicle groups received UVB irradiation once daily for 4 weeks. The vesicle and extract groups received treatment after each irradiation session. The model group was treated with saline as a placebo. UV irradiation intensity started at 1 MED (600 mJ / cm²) for the first two weeks, increasing to 2 MED in the third and fourth weeks. Skin changes were recorded weekly by photograph. After 4 weeks, the dorsal skin was excised, fixed in 10% formaldehyde, and embedded in paraffin for Masson staining and immunohistochemistry. Masson staining was used to observe the effects of vesicles on dorsal skin structure changes and collagen deposition. Figure 4 Results A showed that dense collagen in the dermis of mice in the model group was significantly reduced, and the vesicle group showed a stronger effect in increasing collagen content compared with the control group and the extract group. Furthermore, immunohistochemical results ( Figure 4 B) shows that COL-I expression was decreased and MMP1 expression was increased in the model group. In contrast, MMP1 expression was decreased and COL-I expression was significantly increased in both the vesicle group and the extract group, with the vesicle group showing a stronger effect than the extract group. These findings indicate that ultraviolet radiation significantly degrades skin collagen and accelerates the structural changes associated with cellular aging. Treatment with Eclipta prostrata-derived extracellular vesicles effectively mitigates these changes and maintains skin structure and function. The results from animal and in vitro experiments are consistent, and these data collectively demonstrate that Eclipta prostrata-derived extracellular vesicles can inhibit ultraviolet-induced photoaging by reducing the loss of skin collagen fibers and enhancing the skin's antioxidant capacity.

Claims

1. The application of extracellular vesicles derived from Eclipta prostrata, characterized in that, Used for preparing anti-skin photoaging drugs; the method for preparing extracellular vesicles derived from Eclipta prostrata includes the following steps: The raw material of Eclipta prostrata was added to a buffer solution and crushed. After sieving, the pulp was filtered. Then, it was centrifuged at least three times. After each centrifugation, the supernatant was collected for the next centrifugation. After the last centrifugation, the supernatant was collected and then ultrafiltration centrifugation was performed. The resulting upper suspension was resuspended in a buffer solution. The resuspended liquid was then filtered through a membrane to obtain the extracellular vesicles of Eclipta prostrata. The raw material for Eclipta prostrata is the whole herb of Eclipta prostrata. It is crushed using a juicer at a speed of 8000-20000 rpm for 20-150 seconds. The extracted juice is sieved through a 100-200 mesh screen, and the resulting filter membrane has a pore size of 1-5 μm. Three centrifugation processes are performed at forces of 600-800×g, 5000-7000×g, and 10000-20000×g, with each centrifugation lasting 30-180 minutes. Ultrafiltration is performed using a centrifugation force of 2500-7000×g, with a 100 kDa ultrafiltration membrane, and a centrifugation time of 30-120 minutes. Resuspension membrane filtration involves first passing the solution through a 0.45 μm pore size membrane, followed by a 0.22 μm pore size membrane.

2. The application of the extracellular vesicles derived from Eclipta prostrata according to claim 1, characterized in that, Anti-photoaging drug formulations include at least one of the following topical preparations: hydrogel, cream, and transdermal patch.

3. The application of the extracellular vesicles derived from Eclipta prostrata according to claim 1, characterized in that, The buffers used in each step include at least one of the following: PBS, Tris buffer, Tris-HCl buffer, or TBST buffer; all centrifugation operations are performed at 4-8°C.

4. The application of the extracellular vesicles derived from Eclipta prostrata according to claim 1, characterized in that, When adding Eclipta prostrata raw materials to the buffer solution, the ratio should be 1-3g: 1-2mL.

5. The application of the extracellular vesicles derived from Eclipta prostrata according to claim 1, characterized in that, It consists of spherical vesicles formed by a lipid bilayer; the protein concentration is 40-400 μg / mL.

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