Ashitaba exovesicles, their preparation methods, and their application in the preparation of skin wound repair products

Abstract

This invention discloses an exovesicle from *Ashitsubaki* (Tomorrow's Leaf), its preparation method, and its application in the preparation of skin wound repair products, belonging to the field of plant exovesicle technology. The method uses fresh *Ashitsubaki* leaf stems and leaves as raw materials. After crushing, the material is filtered through a filter bag. The filtrate is then enzymatically hydrolyzed, centrifuged, and finally filtered through a tangential flow filtration system and further filtered to remove impurities and bacteria, yielding uniformly sized *Ashitsubaki* exovesicles. The preparation method of this invention is simple, and the obtained *Ashitsubaki* exovesicles exhibit excellent cell migration and skin healing promotion abilities, showing broad application prospects in the preparation of skin wound repair products.

Description

Technical Field

[0001] This invention belongs to the field of plant exovesicle technology, specifically relating to an Ashitaba exovesicle, its preparation method, and its application in the preparation of skin wound repair products. Background Technology

[0002] As the largest barrier organ in the human body, the skin plays a crucial role in resisting microbial invasion, maintaining internal homeostasis, and regulating body temperature. Wound formation is essentially a pathological process in which the skin and subcutaneous tissues are damaged by external factors such as physical, chemical, and biological agents, leading to impaired barrier function.

[0003] Skin wound repair is a physiological healing process in response to tissue damage. It is a complex physiological process involving three coordinated stages: inflammation, new tissue formation, and remodeling, ultimately leading to wound closure and restoration of tissue function. Several hours to three days after injury, hemostasis and inflammation begin simultaneously, blood clots seal the wound, and immune cells clear foreign bodies. From 3 to 14 days, the proliferative phase begins, with fibroblasts secreting collagen to form granulation tissue, angiogenesis, and epithelial cell migration completing epithelialization. Two weeks to several months later, the remodeling phase occurs, with collagen rearrangement, scar softening, and gradual recovery of skin function. This process is regulated by cells, cytokines, and the microenvironment; abnormalities can easily lead to delayed healing or scar hyperplasia. Traditional wound treatment drugs (such as iodine, alcohol, and growth factor preparations) generally suffer from single-function limitations and poor targeting; antibacterial agents easily damage surrounding normal tissue cells and disrupt local microenvironment homeostasis; growth factor drugs have short half-lives and are easily inactivated by antibacterial drugs. Therefore, the development of a safe, efficient, and low-cost novel wound repair agent is urgently needed.

[0004] Extracellular vesicles (EVs) are small, membrane-bound vesicles released from cells into the extracellular matrix, ranging in diameter from 50 to 500 nm. They can carry components such as proteins, lipids, and nucleic acids of parental origin. Furthermore, as naturally occurring nanoparticles, EVs possess immune tolerance, circulatory stability, and the ability to cross biological barriers to reach distant organs. Therefore, EVs can serve as transport carriers for cellular bioactive molecules. Plant-derived EVs offer advantages such as a wide availability of raw materials, high biocompatibility, rich biological functions, and significant application value.

[0005] Ashitaba (Angelica keiskei) is a perennial herb belonging to the genus Angelica L. of the family Umbelliferae. It contains a variety of beneficial chemical components, such as chalcones, flavonoids, coumarins, vitamins, minerals, amino acids, dietary fiber, and volatile oils. In traditional East Asian medicine, it is believed to have the effects of detoxification, swelling reduction, blood circulation promotion, and pain relief. Summary of the Invention

[0006] The purpose of this invention is to provide an Ashitaba (Tomorrow Leaf) exovesicle, its preparation method, and its application in the preparation of skin wound repair products. This invention uses the leaf stems and leaves of Ashitaba as raw materials, which are crushed, enzymatically hydrolyzed, and filtered to prepare uniformly sized Ashitaba exovesicles, which exhibit excellent effects in promoting skin wound healing.

[0007] The technical solution for achieving the objective of this invention is as follows:

[0008] The method for preparing extravesicles of Ashitaba includes the following steps:

[0009] (1) Chop the stems and leaves of fresh Ashitaba and put them into a blender, add PBS, and blend until homogenized. Then pour the slurry into a 200-mesh filter bag for filtration. Add pectinase to the collected filtrate to reduce the viscosity of the sample.

[0010] (2) Centrifuge the enzymatically hydrolyzed filtrate at 10000~13000×g and 4℃ to remove impurities, and then filter the supernatant with a 0.45 μm filter head to further remove fragments and large vesicles;

[0011] (3) Most of the impurities and proteins in the supernatant after filtration were removed by a tangential flow filtration system and concentrated to obtain vesicle collection solution. The working conditions of the tangential flow filtration system were: a 300 kDa hollow fiber column was used, the transmembrane pressure was maintained at 3±1 psi, the flow rate was 35±5 mL / min, and the concentrated supernatant was replaced with 10 times the volume of PBS.

[0012] (4) Centrifuge the vesicle collection solution at 12000~13000 ×g and 4℃, collect the supernatant, and finally filter it with a 0.22μm filter head to further remove impurities and bacteria, and obtain the outer vesicles of Ashitaba.

[0013] Furthermore, in step (1), the crushing time is 30~40s.

[0014] Further, in step (1), the amount of pectinase added is 1 mg pectinase / 10 L slurry.

[0015] Furthermore, in step (2), the centrifugation time is 15~20 min.

[0016] Furthermore, in step (4), the centrifugation time is 10~15 min.

[0017] This invention provides Ashitaba exovesicles prepared by the above-described method.

[0018] The present invention also provides the application of the above-mentioned Ashitaba exovesicles in the preparation of skin wound repair products.

[0019] Furthermore, skin wound repair products include, but are not limited to, skin care products, pharmaceutical compositions, medical devices, biomaterials, and tissue-engineered products.

[0020] The present invention provides a composition or article comprising the above-mentioned Ashitaba exovesicles.

[0021] Furthermore, the compositions include, but are not limited to, skin care products, pharmaceutical compositions, etc.; the products include, but are not limited to, medical devices, biomaterials, tissue-engineered products, etc.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] (1) The present invention uses fresh Ashitaba leaves as raw materials. After crushing, enzymatic hydrolysis, and centrifugal filtration, the outer vesicles are prepared by a tangential flow filtration system. By adjusting the working conditions of the tangential flow filtration system, Ashitaba leaf outer vesicles with uniform particle size are obtained.

[0024] (2) The extravesicles of Ashitaba prepared by this invention have excellent cell migration promotion ability and show excellent skin wound repair effect in a full-thickness skin defect model on the back of mice. They have broad application prospects in wound repair and regeneration, chronic wound treatment, anti-oxidation, anti-aging, and repair skin care products. Attached Figure Description

[0025] Figure 1 The images show the morphology and particle size analysis results of the *Ashita no Yoru* exovesicles prepared in Example 1. Figure 1 Image (a) is a transmission electron microscope (TEM) image. Figure 1 (b) in the figure is the NTA particle size-concentration distribution diagram;

[0026] Figure 2 The results of cell experiments on Ashitaba exovesicles and Ashitaba extracts are shown. Figure 2 Image (a) shows the results of the Hacat cell scratch assay. Figure 2 In the figure, (b) represents the relative migration rate, and "**" indicates that the statistical significance level of the difference between groups is p≤0.01;

[0027] Figure 3 These are the results of animal experiments on the extravesicles of *Ashita no Yotsuba*, among which... Figure 3 (a) shows the effect of promoting wound healing in a mouse model of full-thickness skin defect on the back. Figure 3 (b) shows the results of quantitative analysis of the wound area on the 12th postoperative day. Figure 3 (c) in the figure is a graph showing the relative change in wound area. In the figure, "****" indicates that the statistical significance level of the difference between groups is p<0.0001.

[0028] Figure 4This section describes the wound healing process in a mouse model of full-thickness skin defect on the back after treatment with Ashitaba exovesicles. Figure 4 Image (a) shows the results of H&E and Masson staining. Figure 4 (b) in the figure is a wound length analysis diagram. Figure 4 (c) in the figure is a collagen density analysis graph. In the figure, "*" indicates that the statistical significance level of the difference between groups is p≤0.05, and "***" indicates that the statistical significance level of the difference between groups is p≤0.001.

[0029] Figure 5 The effect of *Ashita no Yogatsuma* exovesicle treatment on the expression of genes related to wound healing, among which... Figure 5 (a) in the figure shows the results of the effect on the expression of angiogenesis-related genes. Figure 5 (b) in the figure shows the results of the effect on the expression of inflammation-related genes. In the figure, "*" indicates that the statistical significance level of the difference between groups is p≤0.05, and "***" indicates that the statistical significance level of the difference between groups is p≤0.01. Detailed Implementation

[0030] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and should not be construed as limiting the scope of protection of the present invention. Where specific techniques or conditions are not specified in the following embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or in accordance with the product manual.

[0031] Example 1

[0032] After washing the fresh leaves of *Shijidai* (a type of leaf), chop them with a knife, centrifuge the mixture to obtain the supernatant, and use a tangential flow system to prepare *Shijidai* exovesicles, as follows:

[0033] (1) Wash the leaves and stems of fresh Ashitaba with water, chop them with a knife and put them into a blender. Add an equal amount of PBS and blend into a homogenate. Then filter it through a 200-mesh filter bag. Add pectinase to the collected filtrate at a ratio of 1 mg pectinase / 10 L of slurry to reduce the viscosity of the sample.

[0034] (2) Dispense the enzymatically hydrolyzed filtrate into centrifuge tubes and centrifuge at 12000 × g and 4℃ for 15 min to remove leaf cell residues and other impurities. Then filter the resulting supernatant through a 0.45 μm filter to remove leaf fragments and large vesicles.

[0035] (3) Use a tangential flow filtration system to further remove most of the impurities in the supernatant and concentrate it to obtain vesicle collection solution. The specific operation is as follows: use a 300 kDa hollow fiber column, maintain a transmembrane pressure of 3 psi, control the flow rate at 35 mL / min, and replace the concentrated supernatant with 10 times the volume of PBS.

[0036] (4) Centrifuge the vesicle collection solution at 12000 × g and 4℃ for 10 min and collect the supernatant. Finally, filter with a 0.22 μm filter to further remove impurities and bacteria, and obtain Ashitaba exovesicles. After aliquoting, store in a -80 ℃ freezer.

[0037] The extravesicles of Ashitaba prepared in Example 1 are as follows Figure 1 As shown, its size is 112.5 nm and its particle size is uniform.

[0038] Comparative Example 1

[0039] This comparative example is roughly the same as Example 1, except that (3) a tangential flow filtration system is used to further remove most of the impurities in the supernatant and concentrate it to obtain vesicle collection solution. The specific operation is as follows: a 300 kDa hollow fiber column is used to maintain a transmembrane pressure of 6 psi and the flow rate is controlled at 70 mL / min. The concentrated supernatant is replaced with 10 times the volume of PBS.

[0040] Comparative Example 2

[0041] Preparation of Ashitaba extract:

[0042] (1) Wash the leaves and stems of fresh Ashitaba with water, chop them with a knife and put them into a blender. Add an equal amount of PBS and blend into a homogenate. Then filter it through a 200-mesh filter bag. Add pectinase to the collected filtrate at a ratio of 1 mg pectinase / 10 L of slurry to reduce the viscosity of the sample.

[0043] (2) The filtrate after enzymatic hydrolysis was dispensed into centrifuge tubes and centrifuged at 12000 ×g and 4℃ for 15 min to remove leaf cell residues and other impurities. The resulting supernatant was then filtered with a 0.45 μm filter to remove leaf fragments, thus obtaining Ashitaba extract.

[0044] Example 2

[0045] To test the cell migration-promoting ability of Ashitaba exovesicles obtained by different extraction processes and Ashitaba extracts, a cell scratch assay was conducted. The specific implementation is as follows:

[0046] HaCaT cell line (human immortalized keratinocytes) was used for cell experiments. First, parallel reference lines (at least two intersecting lines per well) were drawn on the back of the bottom of a 24-well plate for subsequent image positioning and scratch width calibration. Then, 3 × 10⁶ cells were seeded into each well. 5HaCaT cells were cultured for 24 h until cell confluence reached over 90%. Using a sterile 10 μL pipette tip, a uniform scratch was made perpendicular to the reference line at the bottom of the well. After gently washing away the cell debris, the intersection of the reference line was selected as the fixed observation area under an inverted microscope, and an image of the initial scratch state was taken and its initial width recorded. Each group of cells was then treated with a final concentration of 10 μg / mL of the *Ashitsubaki* exovesicles prepared in Example 1 and Comparative Example 1, and the *Ashitsubaki* extract dilution prepared in Comparative Example 2. After another 24 h of routine culture, the cells were positioned using the pre-marked reference line at the bottom of the well. The cell migration status of the scratched area was photographed under an inverted microscope in the same field of view as the initial image. ImageJ software was used to quantitatively analyze the scratch images of the initial and 24 h intervention periods within the same field of view.

[0047] The results are as follows Figure 2 As shown, 10 μg / mL Ashitaba vesicles prepared under a transmembrane pressure of 3 psi and a flow rate controlled at 35 mL / min (Example 1) significantly promoted cell migration compared to 10 μg / mL Ashitaba vesicles prepared under a transmembrane pressure of 6 psi and a flow rate controlled at 70 mL / min (Comparative Example 1) and 10 μg / mL Ashitaba extract (Comparative Example 2). These results indicate that Ashitaba vesicles prepared under a transmembrane pressure of 3 psi and a flow rate controlled at 35 mL / min can significantly accelerate HaCaT cell scratch healing.

[0048] Example 3

[0049] To test the ability of different concentrations of *Shitsurea costata* exovesicles to promote skin wound repair, a mouse test experiment was conducted. The specific implementation is as follows:

[0050] 1. Establishment of a full-thickness skin defect model on the back of a mouse:

[0051] BALB / C mice were used to create a full-thickness skin defect model on the back. After one week of acclimatization, the mice were separated into cages of four. After isoflurane inhalation anesthesia, the mice were fixed in a prone position, and the backs were shaved using an electric small animal shaver. A suitable amount of depilatory cream was applied evenly to the shaved area, and after about 1 minute, it was reapplied with a cotton swab. The cream was then gently wiped off with a damp gauze to minimize residue and wetting of other fur. Excess water was wiped away with a dry gauze. An 8mm skin punch was used to press directly onto the back, and a small mark was made using a marker. The removed portion of the full-thickness skin was lifted with ophthalmic forceps, and ophthalmic scissors were used to cut the full-thickness skin according to the mark. The mice were then placed in a cage without bedding, and bedding was added after the wound had dried.

[0052] 2. Grouping and Experimentation:

[0053] The experimental group was divided into three groups: PBS group (control group), low-dose Ashitaba exovesicle group (5 mg / mL, PEVs-L), and high-dose Ashitaba exovesicle group (25 mg / mL, PEVs-H). On the day of modeling, PBS or different concentrations of Ashitaba exovesicles were applied to the back wound of the mice and the wound condition was recorded by taking pictures. Then, the back skin wound of the mice was applied for 12 days and the condition was recorded by taking pictures. After 12 days, the mice were sacrificed by cervical dislocation and the back tissue of the mice was taken.

[0054] 3. Experimental Results:

[0055] (1) Wound healing status: Results are as follows Figure 3 As shown, the initial wound condition was consistent across all groups on postoperative day 0 (D0). Over time, the wound healing process in the control group was slow, with noticeable defects still visible on day 12. The wound contraction rate in the PEVs-L group was slightly faster than that in the control group. The wound healing rate in the PEVs-H group was the fastest, with significant contraction observed on day 4 postoperatively, and the wound essentially closed by day 12. Dynamic thermal imaging of wound healing further visually demonstrated that the wound healing rate in the low- and high-dose Epimedium extracellular vesicle groups was significantly faster than that in the PBS control group. Quantitative analysis of wound area on postoperative day 12 showed that the residual wound area in the control group remained at approximately 18%, decreased to approximately 8% in the PEVs-L group, and was only approximately 4% in the PEVs-H group, showing a highly significant difference compared to the control group.

[0056] (2) H&E and Masson staining:

[0057] On the 12th day after surgery, the mice were euthanized by cervical dislocation, and full-thickness skin tissue from the center of the wound was taken. The tissue was rinsed with physiological saline to remove blood and was immediately fixed in 4% paraformaldehyde fixative. After fixation, the tissue was dehydrated with graded ethanol, cleared with xylene, embedded in paraffin, and then 4 μm thick serial sections were prepared using a microtome. The sections were mounted on glass slides and baked for later use.

[0058] For H&E staining, paraffin sections were dewaxed twice in xylene (10 min each time), then rehydrated stepwise with 100%, 95%, 85%, and 75% ethanol, and finally rinsed with distilled water. The sections were then stained with hematoxylin for 5 min, rinsed with tap water, differentiated with 1% hydrochloric acid ethanol for 30 s, and then soaked in tap water for 5 min to regain their blue color. The sections were then stained with eosin for 2 min, dehydrated with 75%, 85%, 95%, and 100% ethanol, and cleared with xylene. Neutral resin was added for mounting, and the tissue morphology was observed under an optical microscope. The cell nuclei were stained blue-purple by hematoxylin, and the cytoplasm and extracellular matrix were stained red by eosin.

[0059] The dewaxing and rehydration process for Masson staining is the same as for HE staining. First, the cells are stained with Weigert iron hematoxylin solution for 5 min to stain the nuclei. After rinsing with tap water, they are stained with Ponceau S and acid fuchsin solution for 10 min to stain the cytoplasm and collagen fibers. Then, they are differentiated with phosphomolybdic acid solution for 5 min, rinsed with distilled water, and stained with aniline blue solution for 5 min. After treatment with 1% glacial acetic acid for 1 min, the cells are dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin. Under a microscope, collagen fibers appear blue, cell nuclei appear blue-purple, and cytoplasm appears red, clearly distinguishing collagen distribution and tissue morphology.

[0060] The results are as follows Figure 4 As shown, H&E staining confirmed that the epidermis regeneration in the Ashitaba vesicle treatment group was complete, forming a continuous stratified squamous epithelial structure. Dermal neovascularization density increased, and inflammatory cell infiltration significantly decreased. Quantitative analysis showed that, compared to the control group, the wound length in the Ashitaba vesicle treatment group decreased in a concentration-dependent manner; the wound length in the PEVs-H group was significantly shorter than that in the control group. Masson staining confirmed that the collagen fibers in the Ashitaba vesicle treatment group were more densely and orderly arranged, with a significantly increased proportion of blue mature collagen. The collagen density in the PEVs-H group was significantly higher than that in the control group, indicating that it can effectively promote epidermal regeneration, inhibit inflammatory response, and accelerate dermal collagen deposition and maturation, thereby significantly improving wound healing quality, and this effect is concentration-dependent. These results demonstrate that the Ashitaba vesicles prepared in Example 1 can significantly promote the repair of full-thickness skin defects.

[0061] (3) Real-time quantitative PCR (qPCR) detection of gene expression related to wound healing in mice:

[0062] Total RNA was extracted from wound tissue on postoperative day 12. After reverse transcription, the relative mRNA expression levels of angiogenesis-related factors (VEGF, bFGF) and inflammation-related factors (IL-4, IL-10, IL-13, IL-6) were detected by qPCR. Normalization analysis was performed with the control group as the baseline.

[0063] The results are as follows Figure 5As shown, compared with the Control group, the *Epiphyllum oxypetalum* vesicle treatment group significantly regulated the expression of angiogenesis and inflammation-related genes in wound tissue: Regarding angiogenesis-related genes, VEGF expression was slightly downregulated in the PEVs-L group, while VEGF expression was significantly upregulated in the PEVs-H group; BFGF expression was significantly increased in both the PEVs-L and PEVs-H groups, with the highest expression level in the PEVs-H group, showing a concentration-dependent upregulation. Regarding inflammation-related genes, the expression of anti-inflammatory factors IL-4 and IL-13 was significantly increased in both the PEVs-L and PEVs-H groups compared with the control group; IL-10 expression was extremely significantly increased in the PEVs-H group, and also showed an upregulation trend in the PEVs-L group; while the expression of pro-inflammatory factor IL-6 showed a downregulation trend in both the PEVs-L and PEVs-H groups. The above results suggest that Ashitaba exovesicles can synergistically accelerate the healing of full-thickness skin defects by promoting angiogenesis and regulating the inflammatory microenvironment of the wound, and that high concentrations of Ashitaba exovesicles have a better effect.

Claims

1. A method for preparing extravesicles of Ashitaba, characterized in that, Includes the following steps: (1) Chop the stems and leaves of fresh Ashitaba and put them into a blender, add PBS, and blend until homogenized. Then pour the slurry into a 200-mesh filter bag for filtration. Add pectinase to the collected filtrate to reduce the viscosity of the sample. (2) Centrifuge the enzymatically hydrolyzed filtrate at 10000~13000×g and 4℃ to remove impurities, and then filter the supernatant with a 0.45 μm filter head to further remove fragments and large vesicles; (3) Most of the impurities and proteins in the supernatant after filtration were removed by a tangential flow filtration system and concentrated to obtain vesicle collection solution. The working conditions of the tangential flow filtration system were: a 300 kDa hollow fiber column was used, the transmembrane pressure was maintained at 3±1 psi, the flow rate was 35±5 mL / min, and the concentrated supernatant was replaced with 10 times the volume of PBS. (4) Centrifuge the vesicle collection solution at 12000~13000 ×g and 4℃, collect the supernatant, and finally filter it with a 0.22 μm filter head to further remove impurities and bacteria, and obtain the outer vesicles of Ashitaba.

2. The preparation method according to claim 1, characterized in that, In step (1), the crushing time is 30~40s, and the amount of pectinase added is 1mg pectinase / 10L slurry.

3. The preparation method according to claim 1, characterized in that, In step (2), the centrifugation time is 15-20 min; in step (4), the centrifugation time is 10-15 min.

4. The Ashitaba exovesicles prepared by any one of claims 1 to 3.

5. The use of the Ashitaba exovesicles according to claim 4 in the preparation of skin wound repair products.

6. The application according to claim 5, characterized in that, Skin wound repair products include skin care products, pharmaceutical compositions, medical devices, biomaterials, or tissue engineering products.

7. A composition, characterized in that, It includes the extravesicular vesicles of the leaf as described in claim 4.

8. The composition according to claim 7, characterized in that, The composition is a skin care product or a pharmaceutical composition.

9. An article, characterized in that, It includes the extravesicular vesicles of the leaf as described in claim 4.

10. The article of claim 9, characterized in that, The products are medical devices, biomaterials, or tissue engineering products.

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