Application of eclipta-derived extracellular vesicles

Abstract

The invention discloses an application of eclipta source extracellular vesicles. The extracellular vesicles are extracted from the eclipta by adopting an overspeed combined ultrafiltration centrifugation method, and the extracted vesicles are complete in morphology and good in dispersity, and have the characteristics of high yield, high separation speed, low cost and easiness in large-scale production. The extracted vesicles can be successfully taken by animal cells. The yerbadetajo herb alcohol extract has the advantages that the yerbadetajo herb alcohol extract has antioxidant and anti-aging effects after being taken in, ultraviolet-induced matrix metalloproteinase 1 (MMP1) expression can be obviously reduced, collagen I (COL-I) generation can be promoted, ultraviolet-induced skin light aging can be improved, and the effect is better than that of the yerbadetajo herb alcohol extract, so that the yerbadetajo herb alcohol extract can be used for preparing the skin The eclipta-derived extracellular vesicles provided by the invention provide a new strategy for skin aging and anti-aging treatment.

Description

Technical Field

[0001] This invention belongs to the field of plant-derived vesicle application technology, specifically relating to the application of extracellular vesicles derived from Eclipta prostrata. Background Technology

[0002] The skin is composed of the epidermis, dermis, and subcutaneous tissue. Skin aging can be broadly divided into two types based on its causes. Natural aging is accompanied by the continuous decline of skin structure and physiological function with age. External aging is caused by the cumulative effects of external stresses such as sunlight. Photoaging occurs when the skin is exposed to ultraviolet radiation for a long time, leading to collagen degradation, increased melanin, and causing the skin to become dry, rough, and loose, resulting in wrinkles, age spots, leptomeningeal lesions, and in severe cases, even skin cancer. Existing products for preventing and treating photoaging mainly include ingredients such as antioxidants and hyaluronic acid. However, these methods suffer from problems such as poor stability, poor absorption, limited effectiveness, toxicity, and intolerance. Therefore, a better treatment method is still lacking.

[0003] Extracellular vesicles (EVs) are tiny vesicles secreted by various cells. EVs mainly include exosomes, microvesicles, and apoptotic bodies. Exosomes are the smallest, typically with a diameter between 30-150 nm, while microvesicles have a wide size range, with diameters from 100-1000 nm. EVs carry various bioactive molecules, such as nucleic acids, proteins, and metabolites, and are crucial mediators of intercellular signaling, regulating the physiological activities of recipient cells. However, using mammalian cell-derived EVs as therapeutic drugs presents a key problem: clinical applications require large quantities of EVs, and large-scale production of EVs involves high production costs and safety risks. Synthetic nanoparticles also have drawbacks such as immunogenicity, cytotoxicity, and complex manufacturing processes.

[0004] Active ingredients derived from natural medicinal plants have attracted attention due to their low toxicity and multi-target effects; however, the skin barrier limits their penetration efficiency. Plant exosome-like nanovesicles (PELNVs)—small vesicles released by plant cells containing bioactive molecules such as lipids, proteins, enzymes, and nucleic acids, as well as plant-specific chemical components—have structures similar to mammalian exosomes and exhibit good biocompatibility. Compared to mammalian exosomes, PELNVs are widely available, have low immunogenicity, high biosafety, and tissue-targeting properties. Another major advantage of PELNVs is that they are themselves excellent transdermal systems, capable of crossing the skin barrier to penetrate deep into the stratum corneum, achieving better diffusion, absorption, and action. Recent studies have shown that plant vesicles exhibit a variety of biological activities.For example, vesicles derived from shiitake mushrooms, broccoli, etc., have shown good anti-tumor, immunomodulatory, intestinal disease regulation, and hepatoprotective effects. For instance: 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. ActaPharm 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 DendriticCell AMP-Activated Protein Kinase. Mol Ther. 2017 Jul ;25(7):1641-1654. Liu B, Lu Y, Chen X, Muthuraj PG, Li

[0005] Eclipta prostrata L. is a traditional Chinese medicine for "darkening hair and beard," and modern research shows that it is rich in flavonoids, triterpenoid saponins, and polyphenols, which have significant protective effects against liver damage, detoxification, antioxidant properties, and can lower blood lipids, as well as antibacterial, hair growth-promoting, and wound-healing effects. However, no research has been reported on the anti-aging effects, especially anti-photoaging, of extracellular vesicles derived from Eclipta prostrata. Summary of the Invention

[0006] The purpose of this invention is to provide an application of extracellular vesicles derived from the plant *Eclipta prostrata*, specifically for the preparation of anti-photoaging drugs. These plant-derived extracellular vesicles provide a new drug source for the field of anti-photoaging medical aesthetics.

[0007] The present invention provides a method for preparing extracellular vesicles derived from Eclipta prostrata, comprising 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 extracellular vesicles derived from Eclipta prostrata.

[0008] Furthermore, The buffers used in each step include at least one of the following: PBS, Tris buffer, Tris-HCl buffer, or TBST buffer; all centrifugation-related operations are performed at 4-8°C.

[0009] Furthermore, 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.

[0010] When adding Eclipta prostrata raw materials to the buffer solution, the ratio should be 1-3g: 1-2mL.

[0011] The present invention also provides extracellular vesicles derived from *Eclipta prostrata* prepared by the method, with a diameter between 50-200 nm. The *Eclipta prostrata* extracellular vesicles are spherical vesicles with a lipid bilayer; the protein concentration is 40-400 μg / mL.

[0012] Anti-photoaging drug formulations include at least one of the following topical preparations: hydrogel, cream, and transdermal patch.

[0013] Preferably, when applied to exert its anti-photoaging effect, the protein concentration of the extracellular vesicles of *Eclipta prostrata* is 20-800 μg / mL. After 24 hours of treatment, it can be successfully taken up by animal cells. More preferably, the protein concentration of the extracellular vesicles of *Eclipta prostrata* is 40-400 μg / mL. These *Eclipta prostrata*-derived extracellular vesicles can be used in therapeutic products for skin aging and anti-aging. This invention provides a new direction for the in-depth utilization of *Eclipta prostrata* in the treatment and prevention of skin aging and anti-aging, which not only promotes the further development of *Eclipta prostrata* but also lays the foundation for future applications in related fields.

[0014] This invention utilizes a UV-induced human skin keratinocyte and fibroblast damage model to investigate the antioxidant, anti-inflammatory, and collagen-promoting anti-photoaging effects of extracellular vesicles from *Eclipta prostrata* on UV-induced human skin cells in in vitro cell and animal experiments. The *Eclipta prostrata* extracellular vesicles provided in this invention, upon uptake of SA-β-Gal (β-galactosidase), significantly improve the UV-induced decrease in the viability of immortalized human skin keratinocytes (HaCaT), effectively reduce UV-induced levels of reactive oxygen species (ROS) and malondialdehyde (MDA) in HaCaT cells, increase superoxide dismutase (SOD) activity, significantly reduce UV-induced matrix metalloproteinase 1 (MMP1) expression, promote collagen I (COL-I) production, and decrease SA-β-Gal levels in immortalized human skin fibroblasts (HSF).

[0015] The beneficial effects of this invention are: 1. The separation and purification method used in this invention is simple to operate, and the obtained Eclipta prostrata extracellular vesicles have high purity and yield. Compared with most vesicles using ultra-high speed centrifugation, density gradient centrifugation and other methods, it also has the advantages of low cost, high yield, easy industrial transformation, and high safety risk.

[0016] 2. This invention uses ultra-fast combined with ultrafiltration centrifugation to extract extracellular vesicles from Eclipta prostrata. These morphologically exhibit membrane-structured nanoparticles with diameters between 50-200 nm and an average concentration of 4.35 × 10⁻⁶. 11 1 / mL; extracellular vesicles derived from Eclipta prostrata can be successfully taken up by animal cells after 24 hours of treatment.

[0017] 3. The extracellular vesicles derived from *Eclipta prostrata* prepared in this invention, upon uptake, can significantly improve the UV-induced decrease in HaCaT cell viability, effectively reduce UV-induced ROS and MDA levels in HaCaT cells, and increase SOD activity; significantly counteract UV-induced MMP1 upregulation, significantly increase COL-I content, and reduce SA-β-Gal production in HSF cells. Therefore, the extracellular vesicles derived from *Eclipta prostrata* can enhance the skin's antioxidant capacity, maintain skin structure and function, effectively reduce UV radiation-induced cell aging and apoptosis, and have a significant anti-UV-induced photoaging effect. The extracellular vesicles derived from *Eclipta prostrata* described in this invention provide a new strategy for skin aging and anti-aging treatment. Attached Figure Description

[0018] Figure 1 Comparative results of the methods for preparing extracellular vesicles from Eclipta prostrata in Example 1; among which Figure 1 A: Representative particle size distribution obtained by NTA detection of ultrafiltration centrifugation; Figure 1 B: Representative particle size distribution obtained by NTA detection of ultracentrifugation method; Figure 1 C: Representative particle size distribution prepared by NTA detection of sucrose density gradient centrifugation; Figure 1 D: Average particle size of the three methods; Figure 1 E: Average yield of the three methods; Figure 1 F: Comparison of purity among the three methods.

[0019] Figure 2 Characterization results of the extracellular vesicles derived from *Eclipta prostrata* prepared in Example 1; wherein... Figure 2 A: Morphological image of extracellular vesicles extracted and prepared from Eclipta prostrata using transmission electron microscopy (TEM); Figure 2 B: Zeta potential map of extracellular vesicles extracted and prepared from Eclipta prostrata by dynamic light scattering (DLS).

[0020] Figure 3 The results of the in vitro anti-photoaging effect of extracellular vesicles from *Eclipta prostrata* in Example 2 are shown; wherein... Figure 3 A: HaCaT cells internalize DIO-labeled extracellular vesicles derived from Eclipta prostrata; Figure 3 B: Effect of extracellular vesicles from Eclipta prostrata on UV-induced HaCaT proliferation activity; Figure 3 C: Effect of extracellular vesicles from Eclipta prostrata on UV-induced SOD activity in HaCaT cells; Figure 3 D: Effects and quantitative results of extracellular vesicles from Eclipta prostrata on UV-induced ROS levels in HaCaT cells; Figure 3 E: Effect of extracellular vesicles from Eclipta prostrata on the SA-β-Gal activity level of UV-induced HSF cells; Figure 3F: Quantitative results of the effect of extracellular vesicles from Eclipta prostrata on UV-induced MDA concentration in HaCaT cells; Figure 3 G: Results and quantification of MMP1 protein expression in HaCaT cells induced by ultraviolet light from extracellular vesicles of Eclipta prostrata using Western blotting (WB); Figure 3 H:ELISA assay results of extracellular vesicles from Eclipta prostrata cells on ultraviolet-induced COL-I expression in HaCaT cells.

[0021] Figure 4 : The in vivo anti-photoaging effect of extracellular vesicles from Eclipta prostrata; among which Figure 4 A: Masson staining image of mouse skin; Figure 4 B: Immunohistochemical images of COL-1 and MMP1 in mouse skin. Detailed Implementation

[0022] The present invention will be illustrated below through specific embodiments, without limiting the scope of the invention.

[0023] The HaCaT and HSF cells used in this invention were purchased from Wuhan Saiweier Biotechnology Co., Ltd. The female BALB / c mice (6–8 weeks old) used in this invention were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.

[0024] Example 1: Isolation and characterization of Eclipta prostrata-derived vesicles

[0025] This embodiment describes the preparation and morphological observation of extracellular vesicles derived from Eclipta prostrata.

[0026] 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.

[0027] 1.2 Preparation of Eclipta prostrata alcohol extract: To compare the effects, Eclipta prostrata extract was prepared according to conventional extraction methods. The whole herb of Eclipta prostrata was dried and pulverized into powder. The powder was extracted three times by reflux with 95% methanol. The extracts were combined, filtered, and recovered under reduced pressure to obtain a crude extract. After vacuum drying, the Eclipta prostrata alcohol extract was obtained in powder form. This was used in the extract group in Examples 2 and 3.

[0028] Example 2: This example illustrates the in vitro anti-photoaging effect of extracellular vesicles from Eclipta prostrata.

[0029] Confocal microscopy was used to observe whether extracellular vesicles from Eclipta prostrata cells could be taken up by HaCaT cells. Figure 3 As shown in Figure A, confocal microscopy confirmed that the vesicles could be taken up by cells. HaCaT cells induced by ultraviolet light were treated with different concentrations of vesicles (0, 50, 100, 200, 400, 800 μg / mL) for 24 h, and the results were analyzed using a CCK-8 assay. Figure 3 B, in which the control group was not subjected to ultraviolet irradiation for modeling, the model group was ultraviolet induced without vesicles, and the rest were treated with different concentrations of vesicles (0, 50, 100, 200, 400, 800 μg / mL) in the ultraviolet-induced HaCaT group. It was found that extracellular vesicles from Eclipta prostrata promoted the proliferation of ultraviolet-induced HaCaT cells in a concentration-dependent manner.

[0030] Ultraviolet radiation induces oxidative stress, leading to high levels of reactive oxygen species (ROS), resulting in inflammation and lipid peroxidation, ultimately causing skin aging. Eclipta prostrata vesicles (400 μg / mL) and extracts (10 mg / mL) were used to treat UV-induced HaCaT and HSF cells. SA-β-Gal levels were measured using the DCFH-DA probe method under a fluorescence microscope and a SA-β-Gal assay kit. The results are as follows: Figure 3 Compared with the control group and the extract group, the CF and vesicle groups significantly reduced ROS in HaCaT cells and SA-β-Gal levels in HSF cells. Combined with SOD and MDA results, the extracellular vesicles of *Eclipta prostrata* showed stronger antioxidant effects than the alcohol extract. Overexpression of MMP1 degrades type I collagen in the skin, disrupting the integrity of collagen and elastin fiber structures. WB and ELISA results ( Figure 3 GH analysis confirmed that extracellular vesicles derived from Eclipta prostrata significantly counteracted UV-induced MMP1 upregulation and promoted COL-I production, with a stronger effect than the alcohol extract. These results indicate that extracellular vesicles derived from Eclipta prostrata can effectively alleviate UV-induced cell damage and promote collagen production.

[0031] Example 3: This example illustrates the in vivo anti-photoaging effect of extracellular vesicles from Eclipta prostrata.

[0032] 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.

[0033] 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 in the preparation of drugs to combat skin photoaging; The method for preparing extracellular vesicles derived from Eclipta prostrata includes the following steps: Eclipta prostrata raw material is added to a buffer solution and then crushed, sieved to obtain a pulp, and then filtered; then high-speed centrifugation is performed at least three times, and the supernatant is collected after each centrifugation for the next centrifugation, and the supernatant is collected after the last centrifugation, followed by ultrafiltration centrifugation, and the resulting upper suspension is resuspended in a buffer solution; the resuspended liquid is membrane filtered to obtain the extracellular vesicles derived from Eclipta prostrata.

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, 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.

5. 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.

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

Images