Application of garlic extracellular vesicles in the preparation of drugs for treating acne

Abstract

The present invention discloses the use of garlic extracellular vesicles in the preparation of a drug for treating acne. Fresh Yunnan purple-skinned garlic cloves are peeled, the rhizomes removed, and then washed and juiced. The squeezed garlic juice is subjected to ultracentrifugation. The resulting solution is subjected to sucrose gradient purification. The resulting solution is mixed and packaged to obtain garlic extracellular vesicles. The extracted garlic extracellular vesicles are applied to acne as an experimental acne treatment drug. Retinoic acid cream is used as a positive control group, and normal saline is used as a model group. The therapeutic effect of garlic extracellular vesicles on acne healing in rats is observed. The results show that garlic extracellular vesicles can effectively increase the reduction rate of acne thickness, significantly increase the reduction rate of acne area, significantly increase the smoothness and flatness of skin healing, and significantly shorten the acne healing period.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and particularly relates to application of garlic extracellular vesicles in preparing a medicine for treating acne. Background Art

[0002] Acne vulgaris is a chronic inflammatory skin disease affecting the sebaceous follicles, most commonly occurring during adolescence. It typically presents as black and white comedones, papules, pustules, nodules, and scars. The pathogenesis of acne is currently believed to include androgen-induced excessive sebaceous gland lipid secretion, abnormal keratinization of the sebaceous ducts, proliferation of sebaceous microorganisms such as Propionibacterium acnes, and a complex inflammatory response. Current clinical treatments, such as tretinoin cream, have issues such as a long healing period, the formation of epidermal crystals during the healing process, and difficulty in removing acne scars after healing.

[0003] Extracellular vesicles (EVs) are nanoscale, vesicle-like substances secreted by various cell types. They carry a variety of bioactive substances, including proteins, lipids, functional miRNAs, and lncRNAs, and play a crucial role in intercellular communication, information transfer, and immune regulation. EVs may attach to or fuse with the membranes of target cells, thereby delivering surface proteins and cytoplasmic components to the cell. EVs may also be internalized by recipient cells through mechanisms such as endocytosis, becoming extracellular messengers. Research has shown that EVs participate in cell-to-cell communication by transferring their bioactive substances to distant cells and are thought to regulate gene and protein expression in recipient cells. There are also studies that have engineered EVs into drug-carrying vehicles for targeted disease treatment. Furthermore, increasing evidence indicates that EV-like substances derived from edible plants can be absorbed by mammals. Plant-derived edible nanoparticles have been shown to exhibit excellent cross-species communication capabilities and have attracted significant interest as natural treatments for a variety of diseases.

[0004] Garlic, the bulb of the Allium species of the Liliaceae family, is a widely consumed food and medicine, rich in nutrients and essential amino acids. Purple garlic, a selenium-rich agricultural product, boasts antibacterial, anticancer, and antimutagenic properties, boosts bioactivity, and regulates the immune system, earning it the nickname "natural antibiotic." Its rhizome, the primary edible and medicinal part, contains numerous beneficial substances such as calcium, iron, and zinc, making it highly effective in preventing cancer and boosting immunity. It also contains several essential amino acids. Allicin is not present in fresh garlic. To extract it, the garlic tissue must be crushed. The alliin in the cytoplasm reacts with endogenous allinase in the vacuole to form allicin. Modern medical experiments have shown that allicin has numerous pharmacological benefits, including antimicrobial, antitumor, and antiparasitic properties, as well as blood pressure and lipid lowering and immune enhancement. Allicin has high medicinal value, effectively preventing and treating various diseases. Its development potential is high, earning it the nickname "plant gold," and it holds broad research and application prospects. However, there are currently no reports on the use of plant extracellular vesicles, especially garlic extracellular vesicles (GL-EVs), to treat and repair acne. Summary of the Invention

[0005] The present invention aims to provide application of garlic extracellular vesicles in preparing a drug for treating acne.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The present invention provides application of garlic extracellular vesicles in preparing a medicine for treating acne.

[0008] Garlic extracellular vesicles (EVs) were applied to acne-affected rats as an experimental treatment. Retinoic acid cream served as a positive control group, and saline was used as a model group (negative control group). The results showed that EVs effectively increased the reduction rate of acne thickness and significantly increased the reduction rate of acne area, significantly increased the smoothness of skin healing, and significantly shortened the acne healing period.

[0009] The method for extracting garlic extracellular vesicles comprises the following steps:

[0010] S1. Take fresh Yunnan purple-skinned single-headed garlic, peel it, remove the rhizome, wash it, and squeeze the juice;

[0011] S2, the squeezed garlic juice was centrifuged continuously at 4 ° C, 1000g, 10 min; 3000g, 20 min; 10000g, 40 min; after centrifugation, the supernatant was taken, the precipitate was discarded, and the supernatant was further subjected to ultracentrifugation in an ultracentrifuge at 4 ° C, 150000g, centrifuged for 90 min, and the supernatant was discarded after centrifugation to obtain a precipitate suspension;

[0012] S3. Add sucrose solution to a centrifuge tube in a gradient of 8%, 30%, 45%, and 60%. Then, add the precipitate suspension obtained in step S2 to the top layer of the centrifuge tube, and then ultracentrifuge at 150,000 g for 120 min.

[0013] S4. Mix the obtained solution and divide it into portions.

[0014] Preferably, the specific steps of mixing and packaging the solution in step S4 are: collecting the layers separately and placing them into cleaned centrifuge tubes, adding ice PBS solution and mixing evenly, centrifuging in an ultraspeed refrigerated centrifuge at 150,000g for 90 minutes, resuspending the precipitate with 1-3 mL of PBS solution, filtering with a filter membrane, and taking the filtrate for packaging.

[0015] Preferably, the filter membrane is 0.22 μm.

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

[0017] (1) The garlic extracellular vesicles extracted by the present invention have small particle size and uniform size distribution, simple extraction steps, readily available raw materials, and low cost;

[0018] (2) The garlic extracellular vesicles extracted by the present invention can effectively increase the reduction rate of acne thickness, significantly increase the reduction rate of acne area, significantly increase the smoothness of skin healing, and significantly shorten the acne healing period; therefore, garlic extracellular vesicles have the potential to be used as a drug for clinical treatment of acne. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a flow chart of garlic extracellular vesicle extraction;

[0020] Figure 2 This is a transmission electron micrograph of garlic extracellular vesicles;

[0021] Figure 3 is the particle size distribution of garlic extracellular vesicles;

[0022] Figure 4 is the smoothness of rat acne skin;

[0023] Figure 5 is the overall changes in rat acne skin at different times;

[0024] Figure 6 Figure 1 shows the changes in thickness of rat acne skin at different times: A is a general comparison of acne thickness reduction rates; B is a comparison of acne thickness reduction rates between the tretinoin cream group and the drug-treated group;

[0025] Figure 7The following are the changes in the area of ​​rat acne skin at different times: A is the overall comparison of the acne area reduction rate; B is the comparison of the acne area reduction rate between the tretinoin cream group and the drug-treated group. DETAILED DESCRIPTION

[0026] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0027] Example 1: A method for extracting extracellular vesicles of garlic, such as Figure 1 As shown, the following steps are included:

[0028] S1. Take an appropriate amount of fresh Yunnan purple-skinned single-headed garlic, peel it, remove the rhizome, wash it three times with tap water and then three times with deionized water, each time for about 2-3 minutes, wait until the water is dry, and squeeze the juice;

[0029] S2, the squeezed garlic juice was centrifuged continuously at 4 ° C, 1000g, 10 min; 3000g, 20 min; 10000g, 40 min; after centrifugation, the supernatant was taken, the precipitate was discarded, and the supernatant was further subjected to ultracentrifugation in an ultracentrifuge at 4 ° C, 150000g, centrifuged for 90 min, and the supernatant was discarded after centrifugation to obtain a precipitate suspension;

[0030] S3. Add sucrose solution in a gradient to a centrifuge tube, add the precipitate suspension obtained in step S2 to the top layer, and then ultracentrifuge at 150,000 g for 120 min;

[0031] S4. After centrifugation, collect the layers and place them into clean centrifuge tubes. Add ice-cold PBS solution and mix evenly. Centrifuge at 150,000 g for 90 minutes in an ultracentrifuge. Resuspend the precipitate with 1-3 mL of PBS solution and filter with a 0.22 μm filter membrane. Take the filtrate and package it.

[0032] Figure 2 This is a transmission electron micrograph of extracellular vesicles extracted from garlic. Figure 2 It can be seen that the extracted extracellular vesicles have a typical round appearance.

[0033] Figure 3 This is the particle size distribution of extracellular vesicles extracted from garlic. Figure 3 It can be seen that the average particle size of the extracted extracellular vesicles is 139.6 nm, which further confirms the size distribution of nanovesicles.

[0034] Example 2: Effects of garlic extracellular vesicles in treating acne and skin tissue repair in rats

[0035] 2.1 Materials

[0036] 2.1.1 Experimental animals

[0037] Twelve healthy adult Xuzhou rats weighing 200 ± 20 g were obtained from the Experimental Animal Center of Xuzhou Medical University. The animals were housed in the rat room of the School of Medical Technology of Xuzhou Medical University at a constant temperature of 22 ± 4°C and acclimated for one week before the experiment.

[0038] 2.1.2 Experimental instruments

[0039] (1) ABS animal gas anesthesia machine: Shanghai Yuyan

[0040] (2) Induction cooker: Midea model C21-simple103

[0041] (3) Micropipette: Gilson

[0042] (4) Ultra-speed refrigerated centrifuge: Model Optima XPN-100*100

[0043] (5) Cryostat: Model Leica CM1950

[0044] (6) Multifunctional microplate (enzyme reader): Model Synergy2

[0045] (7) Research-grade upright fluorescence microscope: Model Olympus BX43

[0046] (8) Inverted fluorescence microscope: Model Olympus IX73

[0047] (9) Micro-oscillator: Model QL-901

[0048] (10) Ultra-low temperature refrigerator: Thermo

[0049] (11) JA2003N electronic balance: Shanghai Precision Scientific Instruments

[0050] (12) pH meter: Shanghai Leici

[0051] (13) Decolorization shaker: Shanghai Xibashi

[0052] 2.1.3 Experimental drugs

[0053] (1) Garlic juice

[0054] (2) Retinoic acid cream

[0055] (3) Garlic extracellular vesicles (prepared in Example 1)

[0056] 2.1.4 Experimental reagents

[0057] (1) Propionibacterium acnes: purchased from Beina Biotechnology

[0058] (2) 1:5000 potassium permanganate solution: self-prepared

[0059] (3) 0.9% normal saline: self-prepared

[0060] (5) PBS: self-prepared

[0061] (6) 95% ethanol solution

[0062] (7) 15% and 30% sucrose solutions: self-prepared

[0063] (8) 1% hydrochloric acid-ethanol solution: self-prepared

[0064] (11) Blocking solution

[0065] (12) BCA protein detection kit: Beyotime

[0066] 2.1.5 Preparation of main reagents

[0067] (1)PBS

[0068] Take 9.6g of PBS powder and dilute to 1000ml with distilled water. Store in a refrigerator at 4℃ until use.

[0069] (2) 1% hydrochloric acid-ethanol solution

[0070] Take 99 ml of 70% alcohol solution and 1 ml of concentrated hydrochloric acid to prepare 100 ml of 1% hydrochloric acid-ethanol solution, and place it in a 4°C refrigerator for use.

[0071] (3) Garlic juice

[0072] Peel and core the fresh garlic, wash it, and squeeze it with a juicer to get the garlic juice.

[0073] 2.2 Methods

[0074] 2.2.1 Establishment of rat ear acne model

[0075] (1) Establishment of acne model

[0076] Mice were housed in separate cages and divided into four groups: model + saline (negative control group), model + tretinoin cream (positive control group), model + garlic juice control group, and model + garlic extracellular vesicles administration group. Rats were anesthetized using an animal anesthesia machine and placed on an operating table. 50 μL of Propionibacterium acnes solution (6 × 107 cfu / mL) was injected intradermally into the auricle once daily for 14 days. After the rat acne tissue model was established, the following indicators were used to verify its efficacy:

[0077] Apparent indicators: Level I indicators (core indicators):

[0078] ① Local tissue thickening and hardening

[0079] ② There are black keratin plugs at the mouth of the hair follicles, forming blackheads

[0080] ③ Hair follicles enlarge, become raised papules, and develop pustules

[0081] ④Accompanied by erythema and edema

[0082] ⑤ Pigmentation appears, etc.

[0083] 2.2.2 Applying medicine

[0084] The corresponding drugs were applied on the wound surface (model group: 0.9% normal saline; tretinoin cream group: tretinoin cream; drug administration group: garlic extracellular vesicles; garlic juice control group: garlic juice). After application, the rats were put back into the cage and applied with drugs twice a day for 10 consecutive days. The recovery and healing of the wound surface of each group of rats (acne thickness, acne area, epidermal smoothness, and healing period) were observed.

[0085] 2.3 Experimental Results

[0086] 2.3.1 Observation of the appearance of the wound healing process of rats in each group: see attached Figure 4 、 5

[0087] (1) On the first day after drug application, the epidermis of all groups was swollen, white in the middle and red on the outside, with no significant difference.

[0088] (2) On the third day after medication, the epidermis of each group recovered to varying degrees, with the medication group recovering the fastest, followed by the retinoic acid cream group and the garlic juice control group, and the model group recovering the slowest.

[0089] (3) On the 7th day after medication, the drug group recovered fastest, followed by the tretinoin cream group and the garlic juice control group, and the model group recovered slowest. It was also found that the tretinoin cream group and the garlic juice control group showed varying degrees of acne hyperplasia, with the garlic juice control group showing the most severe acne.

[0090] (4) On the 9th day after the application of the medication, the difference in recovery rate between the medication group and the other groups reached its maximum. The acne in the medication group was completely healed: the skin was flat and smooth, and the ear cartilage tissue was visible; while the tretinoin cream group and the garlic juice control group showed varying degrees of epidermal crystal formation.

[0091] 2.3.2 Reduction rate of acne thickness in rats of each group at different time points: see Figure 6

[0092] Calculation formula: Thickness reduction rate = (ab) / a

[0093] Note: a is the thickness of acne on day n in group X, b is the thickness of acne on day n+1 in group X

[0094] As shown in the figure, with twice-daily dosing, the thickness reduction rate in the dosing group on day 3 was approximately 17.8%, while the reduction rates in the model group, garlic juice control group, and tretinoin cream group were approximately 7.6%, 6.5%, and 9.6%, respectively, showing minimal changes in thickness. The thickness reduction rate in the dosing group on days 5-7 was approximately 29.0%, while the reduction rates in the model group, garlic juice control group, and tretinoin cream group were approximately 10.3%, 7.6%, and 15.5%, respectively. This indicates that early healing in the dosing group was significantly better than in the other groups. On day 9, the thickness reduction rate in the dosing group was approximately 30.4%, while the reduction rates in the model group, garlic juice control group, and tretinoin cream group were approximately 14.3%, 6.0%, and 15.5%, respectively. The dosing group had fully recovered, and the other groups also recovered to varying degrees. In terms of overall thickness reduction, the dosing group had the fastest healing, and the best early recovery was observed.

[0095] 2.3.3 Changes in acne area in rats of each group at different time points

[0096] Calculation formula: Area reduction rate = (xy) / x

[0097] Note: x is the acne area of ​​group A on day n, and y is the acne area of ​​group A on day n+1.

[0098] As can be seen from the figure: in the case of two doses of medication per day, the thickness reduction rate of the medication group on the 3rd day was 15.4%, and the thickness reduction rates of the model group, garlic juice control group, and tretinoin cream group were approximately 8.2%, 10.1%, and 12.3%, respectively; the acne thickness reduction rate of the medication group on the 5th to 7th day was approximately 49.5%, and the acne area reduction rates of the model group, garlic juice control group, and tretinoin cream group were approximately 10.9%, 14.4%, and 24.3%, respectively. It can be seen that the healing of the medication group was significantly better than that of the other groups; the acne thickness reduction rate of the medication group on the 9th day was approximately 44.1%, and the acne area reduction rates of the model group, garlic juice control group, and tretinoin cream group were approximately 10.7%, 23.9%, and 24.1%, respectively. The medication group has fully recovered, and the other groups have also recovered to varying degrees. In terms of the overall thickness reduction, the acne healing in the medication group was the fastest, and the early stage was the best. (See Figure 7 ).

[0099] Conclusion: The experiment confirmed that garlic extracellular vesicles have a significant repairing effect on acne without scarring, which is mainly reflected in:

[0100] (1) Garlic extracellular vesicles can effectively increase the reduction rate of acne thickness and area: the acne thickness and area of ​​the early administration group were most significantly reduced, and completely recovered in the later stage, with visible cartilage tissue.

[0101] (2) Garlic extracellular vesicles can significantly increase the smoothness of skin healing: the skin epidermis of the drug-treated group remained smooth and flat throughout the healing period, without acne marks, while the skin epidermis of the garlic juice control group and the tretinoin cream group easily formed crystals during the healing process, and the skin surface was rough.

[0102] (3) Garlic extracellular vesicles can significantly shorten the healing period of acne: in the later stage of the experiment, the drug-treated group had fully recovered, while the other groups had incomplete recovery.

[0103] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. Application of garlic extracellular vesicles in the preparation of drugs for treating acne.

2. The use according to claim 1, characterized in that The method for extracting garlic extracellular vesicles comprises the following steps: S1. Take fresh Yunnan purple-skinned single-headed garlic, peel it, remove the rhizome, wash it, and squeeze the juice; S2, the squeezed garlic juice was centrifuged continuously at 4 ° C, 1000g, 10 min; 3000g, 20 min; 10000g, 40 min; after centrifugation, the supernatant was taken, the precipitate was discarded, and the supernatant was further subjected to ultracentrifugation in an ultracentrifuge at 4 ° C, 150000g, centrifuged for 90 min, and the supernatant was discarded after centrifugation to obtain a precipitate suspension; S3. Add sucrose solution to a centrifuge tube in a gradient of 8%, 30%, 45%, and 60%. Then, add the precipitate suspension obtained in step S2 to the top layer of the centrifuge tube, and then ultracentrifuge at 150,000 g for 120 min. S4. Mix the obtained solution and divide it into portions.

3. The use according to claim 2, characterized in that The specific steps of mixing and packaging the solution in step S4 are: collecting the layers separately and placing them into cleaned centrifuge tubes, adding ice-cold PBS solution and mixing them evenly, centrifuging in an ultraspeed refrigerated centrifuge at 150,000g for 90 minutes, resuspending the precipitate with 1-3 mL of PBS solution, filtering with a filter membrane, and taking the filtrate and packaging it.

4. The use according to claim 3, characterized in that The filter membrane is a 0.22 μm filter membrane.

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