Berberis thunbergii plant extracellular vesicles with effects of preventing and improving white hair as well as preparation method and application thereof

Abstract

The invention discloses a preparation method of a berberis plant extracellular vesicle, which comprises the following steps: adding a berberis plant into a PBS (Phosphate Buffer Solution) for homogenizing, and then putting into a centrifugal tube; centrifuging 300-600g, taking the supernatant, precipitating, adding the PBS solution again, homogenizing, centrifuging again, taking the supernatant, and filtering; centrifuging 2000-4000 g of the filtered solution, and taking the supernatant; centrifuging 8000-12000g, taking the supernatant, and putting the supernatant into another clean centrifugal tube; and filtering to obtain the product. The research finds that the natural berberis plant extracellular vesicles can obviously prevent and improve the white hair.

Description

Technical Field

[0001] The present invention relates to a plant extracellular vesicle, in particular to a Berberis plant extracellular vesicle having the efficacy of preventing and improving gray hair, and a preparation method and application thereof. Background Art

[0002] Berberine, an active alkaloid isolated from the Chinese medicinal herbs Coptis chinensis and Berberis, has inhibitory effects on a variety of pathogenic bacteria and can modulate pathophysiological processes such as inflammation, oxidative stress, and metabolism. Berberine has been shown to play a role in skin diseases such as melanoma, squamous cell carcinoma, atopic dermatitis, allergic contact dermatitis, psoriasis, acne, and vitiligo.

[0003] However, berberine itself has poor solubility, instability, and low bioavailability, limiting its effectiveness in topical skin applications. Currently, there have been reports of nanoformulations using liposomes, microemulsions, nanoemulsions, chitosan, albumin, microspheres, and polymers to enhance berberine's oral absorption and achieve sustained release, but these efforts have had minimal impact on enhancing the efficacy of berberine topical applications.

[0004] More notably, the irritation potential of berberine has yet to receive sufficient attention. Studies have shown that when applied topically, either single times or cumulatively, at concentrations exceeding 50%, it can disrupt the skin's barrier function, leading to increased transepidermal water loss (TEWL) and inducing transient irritation reactions such as erythema and itching. While existing nanocarriers (such as liposomes) can improve solubility, their rapid release properties prevent them from effectively controlling the instantaneous concentration of berberine on the skin surface, and the risk of irritation persists.

[0005] Berberine, a pigment found in natural barberry extracts, produces a bright yellow color and can be used as a natural dye for textile dyeing. However, this pigment also limits its topical use. Long-term skin use can easily lead to pigmentation. Factors such as epidermal barrier disruption, basement membrane damage, imbalanced skin microbiota, ultraviolet radiation, and the accumulation of oxygen free radicals can exacerbate this pigmentation, resulting in a tan to dark brown discoloration.

[0006] Graying hair is often considered a natural sign of aging, but recent studies have found that stress can also accelerate graying by activating the sympathetic nervous system and releasing norepinephrine to melanocyte stem cells within hair follicles. The β2-adrenergic receptor (β2AR), a mediator of norepinephrine (NE) signals, plays a key role in this process. Given the current lack of specific blockers for this receptor and the complexity of traditional drug development, the search for natural β2AR blockers to disrupt norepinephrine signaling in hair follicle melanocyte stem cells has become an effective approach for preventing and treating stress-induced graying hair. However, existing topical preparations still have significant shortcomings in addressing the two major bottlenecks of berberine: irritation and pigmentation, severely restricting its clinical application. Summary of the Invention

[0007] The primary technical problem to be solved by the present invention is to provide a method for preparing Berberis plant extracellular vesicles that have the efficacy of preventing and improving gray hair.

[0008] Another technical problem to be solved by the present invention is to provide a Berberis plant extracellular vesicle prepared by the above method.

[0009] Another technical problem to be solved by the present invention is to provide a new use of the Berberis plant extracellular vesicles in preparing a product having the efficacy of preventing and improving gray hair.

[0010] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0011] A method for preparing Berberis truncatula extracellular vesicles having the efficacy of preventing and improving gray hair comprises the following steps:

[0012] (1) Berberis plants were homogenized in PBS solution and then placed in a centrifuge tube;

[0013] (2) Centrifuge at 300-600g, take the supernatant, add PBS solution again after precipitation and homogenize, centrifuge again to take the supernatant, and filter; centrifuge the filtered solution at 2000-4000g, take the supernatant; centrifuge at 8000-12000g, take the supernatant and place it in another clean centrifuge tube; filter to obtain.

[0014] Preferably, the Berberis plant is the leaf and / or stem bark of the Berberis plant.

[0015] Preferably, the homogenization in step (1) is high-pressure homogenization with a pressure range of 50-100 MPa.

[0016] Preferably, the centrifugation temperature during the berberine extracellular vesicle extraction process is 4-25°C.

[0017] Preferably, the PBS solution used in steps (1) and (2) contains NaCl, and the NaCl concentration is 10-50 mM.

[0018] Preferably, the preparation method further comprises step (3): mixing the extracellular vesicle solution obtained in step (2) with a lyophilization protectant, and freeze-drying to obtain extracellular vesicle lyophilized powder; the lyophilization protectant is selected from a combination of one or more of trehalose, sucrose, mannitol, and polyvinyl pyrrolidone.

[0019] A Berberis plant extracellular vesicle capable of preventing and improving gray hair is prepared by the above method.

[0020] Use of the Berberis plant extracellular vesicles in preparing cosmetics or medicines for preventing and improving gray hair.

[0021] Preferably, the cosmetic is a scalp wash-off and / or leave-on product; and the medicine is a tablet, capsule, pill, external liquid preparation, cream, powder or patch.

[0022] Preferably, the scalp wash-off and / or leave-on product is shampoo, conditioner, scalp or hair care essence.

[0023] The extracellular vesicles of the external Berberis plant extract are used for external use in preparing cosmetics and medicines with low irritation and low skin staining.

[0024] Compared with the prior art, the present invention has the following technical effects:

[0025] (1) The present invention has found through research that natural Berberis plant extracellular vesicles can significantly prevent and improve gray hair in a mouse model of stress-induced gray hair.

[0026] (2) The present invention uses extracellular vesicles of Berberaceae plants to encapsulate berberine in situ. Berberine extracellular vesicles serve as natural nanocarriers to encapsulate berberine. By optimizing the extracellular vesicle extraction process, the number of particles and lipid content in the extracellular vesicles are increased, thereby improving the encapsulation rate and transdermal absorption efficiency of berberine, thereby improving its bioavailability and efficacy, and enhancing its thermal stability in the application system.

[0027] (3) By using extracellular vesicles from the Berberis holly plant to encapsulate berberine in situ, the skin pigmentation and irritation caused by berberine when applied topically can be effectively reduced. Therefore, the application of natural berberine in skin care products has been greatly expanded.

[0028] (4) Berberine is encapsulated in extracellular vesicles to achieve a sustained release effect, significantly reducing the concentration of berberine that directly contacts the skin, thereby reducing skin sensitivity, redness and other irritation reactions caused by high concentration or rapid penetration. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Extracellular vesicle morphology. DETAILED DESCRIPTION

[0030] The present invention is further described below with reference to specific examples. These examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. Experimental methods in the following examples, where specific conditions are not specified, are generally performed under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods provided herein. The preferred embodiments and materials described herein are for exemplary purposes only.

[0031] Example 1: External use Berberis plant extract extracellular capsules with the efficacy of preventing and improving gray hair provided by the present invention Preparation method of foam

[0032] (1) Take 20 g of Berberis oleifera stem bark, wash it with ultrapure water, cut it into small pieces, then add PBS solution (containing 50 mM NaCl concentration) and perform high-pressure homogenization (pressure: 60 MPa), and place it in a 50 ml centrifuge tube.

[0033] (2) Centrifuge at 500g for 10 minutes, take the supernatant and place it in another clean centrifuge tube, add PBS solution (containing 50mM NaCl concentration) to the precipitate again and homogenize, centrifuge again and take the supernatant. Place a 70μm filter on a new centrifuge tube, mix the supernatant and pour it in, and filter. Centrifuge the filtered solution at 2000g for 20 minutes, take the supernatant and place it in another clean centrifuge tube. Centrifuge at 10000g for 20 minutes, take the supernatant and place it in another clean centrifuge tube. After filtering with a 0.8μm filter, filter with a 0.22μm filter in a clean centrifuge tube to obtain 100mL of extract, which is purified using a 100kDa ultrafiltration tube. The above centrifugation temperature is controlled at 4°C.

[0034] (3) The extracellular vesicle solution obtained in the above step was mixed with 5% trehalose at a volume ratio of 1:1, and freeze-dried to obtain extracellular vesicle freeze-dried powder Q1.

[0035] Example 2: External use of Berberis plant extract extracellular capsules with the efficacy of preventing and improving gray hair provided by the present invention Preparation method of foam

[0036] The present invention can also be prepared by the following method:

[0037] (1) Take 20 g of the bark of Berberis oleifera, wash it with ultrapure water, cut it into small pieces, then add PBS solution (containing 10 mM NaCl concentration) and perform high-pressure homogenization (pressure: 50 MPa), and place it in a 50 ml centrifuge tube.

[0038] (2) Centrifuge at 300g for 10 minutes, take the supernatant and place it in another clean centrifuge tube, add PBS solution (containing 10mM NaCl concentration) to the precipitate again and homogenize, centrifuge again and take the supernatant. Place a 70μm filter on a new centrifuge tube, mix the supernatant and pour it in, and filter. Centrifuge the filtered solution at 3000g for 20 minutes, take the supernatant and place it in another clean centrifuge tube. Centrifuge at 8000g for 20 minutes, take the supernatant and place it in another clean centrifuge tube. After filtering with a 0.8μm filter, filter with a 0.22μm filter in a clean centrifuge tube to obtain 100mL of extract, which is purified using a 100kDa ultrafiltration tube. The above centrifugation temperature is controlled at 25℃.

[0039] (3) The extracellular vesicle solution obtained in the above step was mixed with 5% mannitol at a volume ratio of 1:1, and freeze-dried to obtain extracellular vesicle freeze-dried powder Q2.

[0040] Example 3: External use of Berberis plant extract extracellular capsules with the efficacy of preventing and improving gray hair provided by the present invention Preparation method of foam

[0041] The present invention can also be prepared by the following method:

[0042] (1) Take 20 g of Berberis oleifera stem bark, wash it with ultrapure water, cut it into small pieces, then add PBS solution (containing 10 mM NaCl concentration) and perform high-pressure homogenization (pressure: 100 MPa), and place it in a 50 ml centrifuge tube.

[0043] (2) Centrifuge at 600g for 10 minutes, take the supernatant and place it in another clean centrifuge tube, add PBS solution (containing 10mM NaCl concentration) to the precipitate again and homogenize, centrifuge again and take the supernatant. Place a 70μm filter on a new centrifuge tube, mix the supernatant and pour it in, and filter. Centrifuge the filtered solution at 4000g for 20 minutes, take the supernatant and place it in another clean centrifuge tube. Centrifuge at 12000g for 20 minutes, take the supernatant and place it in another clean centrifuge tube. After filtering with a 0.8μm filter, filter with a 0.22μm filter in a clean centrifuge tube to obtain 100mL of extract, which is purified using a 100kDa ultrafiltration tube. The above centrifugation temperature is controlled at 4°C.

[0044] (3) The extracellular vesicle solution obtained in the above step was mixed with 5% sucrose at a volume ratio of 1:1, and freeze-dried to obtain extracellular vesicle freeze-dried powder Q3.

[0045] Example 4: External use of Berberis plant extract extracellular capsules with the efficacy of preventing and improving gray hair provided by the present invention Preparation method of foam

[0046] The present invention can also be prepared by the following method:

[0047] (1) Take 20 g of the bark of Berberis oleifera, wash it with ultrapure water, cut it into small pieces, then add PBS solution (containing 50 mM NaCl concentration) and perform high-pressure homogenization (pressure: 100 MPa), and place it in a 50 ml centrifuge tube.

[0048] (2) Centrifuge at 600g for 10 minutes, take the supernatant and place it in another clean centrifuge tube, add PBS solution (containing 50mM NaCl concentration) to the precipitate again and homogenize, centrifuge again and take the supernatant. Place a 70μm filter on a new centrifuge tube, mix the supernatant and pour it in, and filter. Centrifuge the filtered solution at 4000g for 20 minutes, take the supernatant and place it in another clean centrifuge tube. Centrifuge at 12000g for 20 minutes, take the supernatant and place it in another clean centrifuge tube. After filtering with a 0.8μm filter, filter with a 0.22μm filter in a clean centrifuge tube to obtain 100mL of extract, which is purified using a 100kDa ultrafiltration tube. The above centrifugation temperature is controlled at 25℃.

[0049] (3) The extracellular vesicle solution obtained in the above step was mixed with 2% PVP at a volume ratio of 1:1, and freeze-dried to obtain extracellular vesicle freeze-dried powder Q4.

[0050] Example 5 Preparation of Extracts from Different Parts of Berberis oleifera and Extracellular Vesicle Solutions and Berberine Content Testing

[0051] Wash the Berberis fruit, leaves, and stems briefly, peel them, and cut the stems and leaves into small pieces. Weigh 20g of each part and homogenize them with PBS (50mM NaCl). Place the mixture in a 50ml centrifuge tube. Centrifuge at 500g for 10 minutes, remove the supernatant, and place it in another clean centrifuge tube. Homogenize the pellet again with PBS, centrifuge again, and remove the supernatant. Place a 70μm filter on a new centrifuge tube, mix the supernatant, and filter. Centrifuge the filtered solution at 2000g for 20 minutes, remove the supernatant, and place it in another clean centrifuge tube. Centrifuge at 10,000g for 20 minutes, and remove the supernatant in another clean centrifuge tube. After filtering through a 0.8 μm filter, filter through a 0.22 μm filter into a clean centrifuge tube to obtain 100 mL of extract from each part. Take 50 mL of the extract and purify it using a 100 kDa ultrafiltration tube to obtain extracellular vesicle solutions from various parts of Berberis ilex. Then mix them with an equal volume of 5% trehalose solution and freeze-dry.

[0052] The berberine content in extracts from different parts of Berberis hollyii and in their extracellular vesicles was tested. The lyophilized powder obtained from each part was reconstituted in 50 mL of deionized water to obtain an extracellular vesicle solution for testing. The specific berberine detection method was as follows: liquid chromatography, chromatographic column: C18 column, 250 mm × 4.6 mm, 5 μm; detection wavelength: 345 nm; mobile phase A: acetonitrile; mobile phase B: 0.05 mol / L potassium dihydrogen phosphate buffer (pH adjusted to 3.0 with phosphoric acid); isocratic elution: 25% A, 75% B.

[0053] The berberine contents in the extracts of different parts of Berberis oleifera and in its extracellular vesicles are shown in Table 1.

[0054] Self-encapsulation rate = berberine content in extracellular vesicle solution / total berberine content in extract × 100%

[0055] Table 1

[0056]

[0057] From the above results, it can be seen that the berberine content is lowest in the fruit of Berberis oleifera, followed by the stem. The berberine content in the leaves and stem bark is relatively high. The stem bark will be selected for extraction in subsequent experiments.

[0058] Example 6 Berberis extracellular vesicle extraction process and optimization

[0059] Research Objective: The membrane of extracellular vesicles (EVs) is composed of a lipid bilayer. Increasing the lipid content can improve EV stability during storage and transportation. Lipids can enhance berberine encapsulation efficiency and transdermal absorption efficiency, thereby increasing its bioavailability. Therefore, the lipid content of EVs is particularly important in improving product efficacy and commercial performance.

[0060] The effects of centrifugation temperature (4°C, 25°C), homogenization pretreatment method and ionic strength in the extract on the extracellular vesicle extraction yield and lipid were investigated respectively.

[0061] The number of extracellular vesicle particles was detected by nanoflow cytometry.

[0062] The phospholipid content in lipids is measured using the Phospholipid (PLIP) (oxidase method) assay kit. Phospholipids are hydrolyzed by phospholipase D to produce choline and phosphatidic acid. Choline is oxidized by choline oxidase to produce hydrogen peroxide, which reacts with 4-aminoantipyrine and DAOS to produce a blue dye. This dye has a maximum absorption peak at 600 nm, and the absorption intensity is proportional to the phospholipid content in the sample. The absorption intensity can be used to determine the phospholipid content.

[0063] Under the premise of a centrifugation temperature of 4°C, the effects of different plant tissue pretreatment methods were investigated, and the test results are as follows.

[0064] Table 2 Effects of different homogenization methods on particle number and lipid content

[0065]

[0066] The results show that high-pressure homogenization (50-100 MPa) produces the highest number of extracellular vesicle particles and the highest lipid content. This is presumably due to the strong shear, impact, and cavitation effects generated under high pressure, which fragment the tissue and generate less heat, resulting in less damage to the extracellular vesicles. A pressure range of 50-100 MPa is recommended, as this effectively fragments plant tissue without causing excessive damage to the vesicle structure. From an energy-saving and environmental perspective, a pressure of 60 MPa is recommended. Subsequently, high-pressure homogenization was used to fragment the plants, and on this basis, the effects of different temperatures during the centrifugation process were explored.

[0067] Table 3 Effects of different temperatures on particle number and lipid content

[0068]

[0069] As can be seen from the above, the number of extracellular vesicle particles and the lipid content can be obtained well at temperatures between 4°C and 25°C. The number of extracellular vesicle particles and the lipid content are the highest at 4°C. It is speculated that the main reasons for the good effect of low temperature are: (1) low temperature reduces the fluidity of the vesicle membrane, reduces the collision and fusion between vesicles, and avoids the formation of large aggregates that are discarded by centrifugation; (2) the extracellular vesicle membrane structure is mainly lipids. Some membrane lipids may approach the phase transition temperature at 25°C, resulting in vesicle membrane instability. 4°C can maintain the orderly arrangement of lipids and promote the precipitation of complete vesicles.

[0070] Plant extracellular vesicles (EVs) typically carry a negative charge, and changes in ionic strength can affect the charge distribution on the vesicle surface. High ionic strength can shield the vesicle surface charge, making vesicles more susceptible to aggregation. Appropriate ionic strength, however, can make vesicles more stable.

[0071] Different concentrations of NaCl were added to the PBS extraction solution to observe the effect on the number of vesicle particles and lipid content. The centrifugation temperature was 4°C and high-pressure homogenization was performed (pressure: 60 MPa).

[0072] Table 4 Effects of different NaCl concentrations on particle number and lipid content

[0073]

[0074] The above results indicate that the addition of 10-50 mM NaCl significantly increases the number of extracellular vesicles (EVs) and lipid content. However, as the sodium chloride concentration continues to increase, the number of EVs and lipid content decrease. This is presumably due to the increased ionic strength causing vesicle aggregation, which is then removed during subsequent centrifugation.

[0075] Example 7 Effect of Lyoprotectants on Extracellular Vesicle Integrity During Lyophilization

[0076] The freeze-drying process exerts considerable stress on biological samples. Therefore, if not properly protected, more ice crystals will form during the freezing process as the ionic strength increases, causing extracellular vesicles to be damaged during freeze-drying and rehydration.

[0077] Lyoprotectants can form a protective layer that reduces ice crystal formation during freezing, protecting the structural integrity of extracellular vesicles and thus maintaining their biological activity. This experiment investigated the effects of different lyoprotectants: trehalose, sucrose, mannitol, and polyvinylpyrrolidone (PVP) on the integrity of extracellular vesicles during the freeze-drying process.

[0078] The extracellular vesicle solution was mixed with 5% trehalose, 5% sucrose, 5% mannitol, and 2% PVP in a 1:1 volume ratio. A blank control was prepared by mixing the extracellular vesicle solution with deionized water in equal proportions. The mixtures were then freeze-dried and stored at -20°C for 30 days. The mixtures were then reconstituted with an equal volume of deionized water and subjected to exosome size measurement. The vesicle morphology was also observed under a transmission electron microscope.

[0079] Before freeze-drying, the average particle size was 72 nm and the particle size range was 44-120 nm.

[0080] Particle size testing results (see Table 5) show that the particle size of the blank group decreased significantly compared to before lyophilization, while the particle size of the trehalose and mannitol groups remained unchanged. The particle size of the sucrose and PVP groups decreased slightly compared to before lyophilization, but was significantly higher than that of the blank group. Furthermore, electron microscopy revealed significantly more membrane fragments and aggregates in the blank group than in the other groups containing lyoprotectants. These results suggest that trehalose and mannitol are effective in preventing extracellular vesicle membrane fragmentation during the freeze-drying process, followed by PVP and sucrose.

[0081] Table 5

[0082]

[0083] Example 8 Preparation and Detection of Berberis Root Extracellular Vesicles Lyophilized Powder

[0084] (1) Preparation of lyophilized powder of extracellular vesicle particles, as described in Example 1.

[0085] (2) Extracellular vesicle identification test

[0086] The particle size distribution and particle number were determined using a nanoflow cytometry analyzer (nanoFCM) (N30E, Xiamen Fuliu Biotechnology Co., Ltd.); the morphology was observed using a transmission electron microscope (JEM-2100Plus, JEOL); and the Zeta potential was measured using a nanoparticle size zeta potential analyzer (SZ-100VZ, HORIBA).

[0087] Test results:

[0088] The particle size of the Berberis ilex extracellular vesicles Q1 in Example 1 was detected by nanoflow cytometry. The particle size of the purified extracellular vesicles was concentrated in the range of 40-120 nm, with an average particle size of 73 nm; the particle count was 4.56E+10 particles / ml, and the Zata potential was 12 mV. Figure 1 As shown, TEM microscopic results show that the purified extracellular vesicles have a uniform morphology, with an overall saucer-like or hemispherical structure of varying sizes, consistent with the microscopic identification characteristics of plant extracellular vesicles. The results of products prepared in other examples of the present invention are consistent with the above and are not repeated here.

[0089] Example 9 Sustained Release Test of Berberis Extracellular Vesicles

[0090] (1) Take the extracellular vesicle freeze-dried powder Q2 prepared in Example 2, add an appropriate amount of physiological saline to obtain an extracellular vesicle solution containing 0.175 mg / mL berberine, which is sample S-1;

[0091] (2) 17.5 mg of berberine standard (B21379, Shanghai Yuanye Biotechnology Co., Ltd.) was dissolved in 100 mL of normal saline to obtain a berberine concentration of 0.175 mg / mL, which is sample S-2;

[0092] (3) The permeability of samples S-1 and S-2 was tested through diffusion cell experiments.

[0093] Specific experimental methods: Prepared porcine skin was fixed between the dosing chamber and the receptor chamber of a diffusion cell. The receptor solution was normal saline. 2 mL each of sample S-1 and sample S-2 was placed in the dosing chamber and sealed with parafilm. This was repeated three times. The water bath temperature was 37.4 ± 0.5°C.

[0094] The test started at time 0, and samples were taken at set times 2, 4, 6, 8, 12, and 24. The berberine content in the sample solution was detected by liquid chromatography tandem mass spectrometry (LC-MS / MS), and the cumulative amount of berberine per unit area released through the skin was calculated.

[0095] The calculation formula for the cumulative transmittance per unit area is:

[0096]

[0097] Q: Cumulative permeation volume; V0: Volume of receptor fluid in the receiving chamber; Cn: Concentration measured at the nth sampling point; V: Volume of each sampling; A: Transdermal area.

[0098] The following table shows the cumulative permeation of berberine per unit area (μg / cm2)

[0099] Table 6 Cumulative permeation of berberine per unit area

[0100] time Sample S-1 Sample S-2 2h 0.001 0.005 4h 0.013 0.065 6h 0.167 0.235 8h 0.387 0.686 12h 0.512 0.781 24h 1.561 2.782

[0101] The above results indicate that the release rate of berberine from the extracellular vesicle nanoparticles of sample S-1 is slower than that of sample S-2. This demonstrates that berberine encapsulated in extracellular vesicles exhibits sustained release, controlling the release rate and prolonging the release duration, thereby reducing the risk of transient irritation to the skin barrier. This characteristic, along with the lack of skin staining in Example 10, demonstrates that exosome encapsulation technology can simultaneously address the irritation and pigmentation issues associated with traditional topical berberine applications. The results of products prepared in other examples of the present invention are consistent with those described above and are not detailed here.

[0102] Example 10 Testing the Skin Coloring of Berberine in Berberis Extracellular Vesicles after Self-Encapsulation

[0103] 1. Sample:

[0104] (1) Take the extracellular vesicle freeze-dried powder Q2 prepared in Example 2, add an appropriate amount of physiological saline, and obtain an extracellular vesicle solution containing 0.175 mg / mL berberine, which is sample S-1.

[0105] (2) 17.5 mg of berberine standard (B21449, Shanghai Yuanye Biotechnology Co., Ltd.) was dissolved in 100 mL of normal saline to obtain a berberine concentration of 0.175 mg / mL, which was sample S-2.

[0106] 2. Test method:

[0107] A total of 30 volunteers with fair skin and no scars participated in the test, and no volunteer withdrew during the test period.

[0108] The subjects cleaned the inner forearms of both hands uniformly by wiping them with dry tissue paper, and sat quietly in a laboratory with a temperature of 21±1°C and a humidity of 50±10% RH for 20 minutes, with their forearms exposed and remaining relaxed.

[0109] Laboratory technicians tested the changes in skin colorimetric b-values ​​on the subjects' arms before and after 30 minutes of use and washing. (The b-value represents the yellow-blue color of an object: a positive value indicates yellow, and the larger the value, the yellower the skin color.) The testing instrument was a spectrophotometer (CM-700d / 600d spectrophotometer, KONICA MINOLTA, Japan). Each area was 3 cm × 3 cm. Three areas were marked on the same arm at the same time, with at least 1 cm between them. The sample area and blank area were marked. The sample areas S-1 and S-2 were used at a dosage of 2 μL / cm 2 ; Blank area, product not in use.

[0110] Table 7 Changes in chromaticity value b value before and after use of the product

[0111] Group Average b* value before product use Average b* value after product use Blank group 16.6 16.7 Sample S-1 group 16.7 16.8 Sample S-2 group was used 16.6 18.9

[0112] The results showed that 30 minutes after the sample was used, the berberine S-1 that had been self-encapsulated by extracellular vesicles did not show any skin coloring (the b* value did not increase), while the S-2 sample group showed an increase in the b* value, that is, the skin as a whole showed a slight yellowing. It is speculated that this may be due to the slow release of berberine by self-encapsulation of extracellular vesicles, which reduces the permeability per unit time, thereby reducing the coloring of the skin. In addition, the slow release feature can also avoid the irritation reaction (such as erythema and itching) caused by the rapid aggregation of berberine on the skin surface, further verifying the advantages of exosome encapsulation technology in improving safety and tolerability. The results of the products prepared in other embodiments of the present invention are consistent with the above and are not repeated here.

[0113] Example 11 Testing the stability of berberine encapsulated in Berberis rubrum extracellular vesicles

[0114] The berberine S-1 and berberine solution S-2 encapsulated by extracellular vesicles were placed at room temperature, 4°C, and 40°C for observation for 3 months. The results showed that the S-2 sample placed at 4°C showed precipitation of the sample, and after heating, the precipitate redissolved. It is speculated that the temperature is too low, which affects the solubility of berberine. At the same temperature, berberine in S-1 did not precipitate. It is speculated that this may be due to the self-encapsulation of extracellular vesicles, which increases its stability. The berberine content was tested by liquid chromatography, and there was no significant change in the content at 4°C and room temperature. At 40°C, the berberine in S-2 decreased significantly, and the berberine in S-1 did not change significantly. This shows that the self-encapsulation of extracellular vesicles increases the stability of berberine.

[0115] Table 8 Changes in berberine content at different temperatures

[0116]

[0117] Example 12 Prevention of stress-induced gray hair with Berberis oleracea extracellular vesicle particles based on a mouse experimental model effect

[0118] Resiniferatoxin (RTX) acts as a stressor, activating the sympathetic nerves of mice, leading to the release of norepinephrine (NE) and binding to the β2-adrenergic receptor (β2AR) of melanocyte stem cells (McSCs). This triggers the downstream Gs protein-AC-cAMP-PKA pathway, leading to calcium homeostasis imbalance and mitochondrial apoptosis (e.g., caspase activation), while also inhibiting the expression of melanin genes (MITF, DCT). Sustained β2AR signaling leads to apoptosis and functional loss of McSCs, disrupting the pigment supply to hair follicles and forming a stress-induced gray hair model.

[0119] 3-4 week old C57BL / 6 female mice (partial hair growth phase) were selected and placed in a specific pathogen-free animal center with free access to water and food. The mice were depilated and then topically applied with 200 μL of 1 mg / mL propranolol, Berberis hollyleaf plant extracellular vesicle solution (calculated as berberine 1 mg / mL) (prepared by Example 3 sample Q3), and berberine standard solution 1 mg / mL. The negative control group was the untreated group. Once a day in the morning and evening, for 30 days. RTX (AdipoGen Life Sciences, AG-CN2-0534) was prepared in PBS containing 2% DMSO and 0.15% Tween 80 and injected into the flanks of the depilated mice at a dose of 20 μg / kg for 7 consecutive days. After 30 days, the mice were photographed and the white hair area was counted.

[0120] Table 9 Hair area and white hair ratio of mice

[0121]

[0122] The results showed that mice injected with RTX and not given any medication had widespread white hair on their backs, indicating that the white hair model was successfully established. In the experimental group treated with extracellular vesicles from Berberis ilex and a standard berberine solution for 30 days, the amount of white hair observed was significantly lower than that in the untreated group. To more intuitively observe the results, all photos of the mice were used to measure and calculate the proportion of white hair on the entire back area of ​​the mice using ImageJ software. The results showed that the area of ​​white hair in the control group treated with RTX was 34.54%, and that in the positive control group was 12.09%. After treatment with the extracellular vesicle solution and berberine, the area of ​​white hair was significantly reduced to 12.57% and 13.34%, respectively, as shown in Table 9.

[0123] From the above results, it can be seen that extracellular vesicles and berberine can effectively prevent the formation of gray hair through external application on the skin.

[0124] Example 13: Improvement of stress-induced gray hair with extracellular vesicles of berberis oleifera based on a mouse experimental model effect

[0125] 3-4 week old C57BL / 6 female mice (partial hair growth phase) were selected and placed in a specific pathogen-free animal center with free access to water and food. RTX (AdipoGen Life Sciences, AG-CN2-0534) was prepared in PBS containing 2% DMSO and 0.15% Tween80 and injected into the flank of the depilated mice at a dose of 20 μg / kg for 7 consecutive days. The inhibition group of mice was treated with 200 μL of topical application twice a day, 1 hour before and 11 hours after RTX injection, respectively. The positive control group was 1 mg / mL of propranolol, Berberis hollyleaf plant extracellular vesicle solution (calculated as berberine 1 mg / mL) (prepared by Example 3 Sample Q3), and berberine standard solution 1 mg / mL. The negative control group was the non-administered group. After 30 days, the mice were photographed and the white hair area was counted.

[0126] According to the experimental results, mice injected with RTX alone had widespread white hair on their backs, indicating that the white hair model was successfully established. After treatment with propranolol, Berberis oleracea plant extracellular vesicles, and berberine, the observed white hair was significantly reduced. In order to observe the results more intuitively, all photos of the mice were taken using ImageJ software to measure and calculate the proportion of white hair in the entire back area of ​​the mice. The results showed that the proportion of white hair area in the control group treated with RTX was 31.90%, and that in the positive control group was 12.44%. After treatment with Berberis oleracea plant extracellular vesicle particles, the proportion of white hair area was significantly reduced to 12.68%, and the proportion of white hair area treated with berberine was reduced to 14.37%, as shown in the table below.

[0127] The above results show that the Berberis ectocellular vesicles and berberine prepared in this example can effectively reduce gray hair by external application to the skin. The results of the products prepared in other examples of the present invention are consistent with the above and are not repeated here.

[0128] Table 10 Hair area and white hair ratio of mice

[0129]

Claims

1. A method for preparing Berberis plant extracellular vesicles with the efficacy of preventing and improving gray hair, characterized in that The steps include: (1) Berberis plants were homogenized in PBS solution and then placed in a centrifuge tube; (2) Centrifuge at 300-600g, take the supernatant, add PBS solution again after precipitation and homogenize, centrifuge again to take the supernatant, and filter; centrifuge the filtered solution at 2000-4000g, take the supernatant; centrifuge at 8000-12000g, take the supernatant and place it in another clean centrifuge tube; filter to obtain.

2. The method for preparing extracellular vesicles from a Berberis plant extract according to claim 1, wherein: The barberry plant is the leaf and / or stem bark of the barberry plant.

3. The method for preparing extracellular vesicles from a Berberis plant extract according to claim 1, wherein: The homogenization in step (1) is high-pressure homogenization with a pressure range of 50-100 MPa.

4. The method for preparing extracellular vesicles from a Berberis plant extract according to claim 1, wherein: The centrifugation temperature during the berberine extracellular vesicle extraction process is 4-25°C.

5. The method for preparing extracellular vesicles from a Berberis plant extract according to any one of claims 1 to 4, wherein: The PBS solution used in steps (1) and (2) contains NaCl, and the NaCl concentration is 10-50 mM.

6. The method for preparing extracellular vesicles from Berberis chinensis plant extract according to claim 5, characterized in that Also includes step (3): The extracellular vesicle solution obtained in step (2) is mixed with a freeze-drying protective agent and freeze-dried to obtain a freeze-dried extracellular vesicle powder; the freeze-drying protective agent is selected from a combination of one or more of trehalose, sucrose, mannitol, and polyvinyl pyrrolidone.

7. A Berberis plant extracellular vesicle having the efficacy of preventing and improving gray hair, characterized in that Obtained by the preparation method according to any one of claims 1 to 6.

8. Use of the Berberis rubrum extracellular vesicles according to claim 7 in the preparation of cosmetics or medicines for preventing and improving gray hair.

9. The use according to claim 8, characterized in that: The cosmetics are scalp wash-off and / or leave-on products; the medicines are tablets, capsules, pills, external liquid preparations, creams, powders or patches.

10. The use according to claim 9, characterized in that: The scalp wash-off type and / or leave-on type product is shampoo, conditioner, scalp or hair care essence.

Images