Preparation method of aquilaria sinensis leaf exosome and application of aquilaria sinensis leaf exosome in cosmetics

Abstract

The invention discloses a preparation method of aquilaria sinensis leaf exosomes and application of the aquilaria sinensis leaf exosomes in cosmetics, and belongs to the field of plant exosomes. The preparation method comprises the steps of explant selection and disinfection, inoculation and callus induction, suspension culture and exosome separation and purification. The aquilaria sinensis leaf exosome prepared by the preparation method can be applied to cosmetics; according to the method, through raw material preparation and process innovation, efficient transdermal and targeted delivery and a multi-component synergistic system, the effect bottleneck of a single component is broken through, and dual improvement of targeting and effectiveness is achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant exosomes, and particularly relates to a preparation method of Aquilaria sinensis leaf exosomes and application thereof in cosmetics. BACKGROUND

[0002] In the field of cosmetic technology, plant exosomes, as a new source of active ingredients, are gradually becoming a research hotspot. Exosomes are nanoscale lipid bilayer vesicles secreted by cells, carrying bioactive substances such as proteins, nucleic acids, and lipids, and can penetrate the skin barrier to precisely act on skin cells, showing great potential in antioxidant, anti-inflammatory, and cell repair promotion. However, the extraction and application of plant exosomes are still in the development stage, and most studies focus on common plants such as green tea and aloe vera. Traditional methods such as mechanical disruption and ultracentrifugation are used for extraction, which has low extraction efficiency, high cost, and exosome integrity is easily damaged, making it difficult to achieve large-scale industrial production.

[0003] Aquilaria sinensis (Lour.) Spreng belongs to the family of Thymelaeaceae and is also known as Aquilaria sinensis leaf tree. Its resinous heartwood, i.e., eaglewood, is widely used in traditional Chinese medicine for treating circulatory system diseases, abdominal pain, vomiting, and asthma. In addition, the leaves of Aquilaria sinensis have a long history of medicinal use in China, especially in Guangdong and Hainan. They are often used for health care and sometimes brewed for tea. The traditional application of Aquilaria sinensis leaves provides an important clue for subsequent scientific research on their pharmacological activities. Modern studies have shown that Aquilaria sinensis leaves contain a variety of bioactive compounds, such as flavonoids, phenolic acids, and 2-(2-phenylethyl) chromones, which have antioxidant, anti-inflammatory, and antibacterial activities. Aquilaria sinensis leaf extract is rich in various bioactive compounds with therapeutic potential. Exosomes, as a precise delivery system, can enhance the bioavailability and targeting of these compounds. Combining the therapeutic properties of Aquilaria sinensis leaf extract with the delivery capacity of exosomes is expected to produce a synergistic effect and open up new application fields. This combined application can significantly improve the therapeutic effect.

[0004] Traditional plant extract preparation methods mostly use water extraction or alcohol extraction, which has certain limitations in the retention and efficacy of active ingredients. The efficacy research is fragmented, and although the extract has activity, there is a lack of exploration of the exosome delivery enhancement mechanism. Exosomes in cosmetics only have basic efficacy and no in-depth research on sunscreen and whitening efficacy. In terms of formulation, existing cosmetics have single components and have not built a synergistic system of Aquilaria sinensis leaf exosomes and other extracts, which cannot achieve better efficacy. These shortcomings are due to insufficient understanding of the advantages of meristematic tissue culture, lack of in-depth research on exosome delivery systems, and lack of component synergistic innovation thinking, resulting in the potential of Aquilaria sinensis leaf exosomes in cosmetics not being fully developed. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the defects of the prior art, to enhance the activity, efficient transdermal and targeted delivery of exosomes by preparing raw materials and process innovation, and to enhance the efficacy, to expand the application scenarios through multi-component synergistic effect.

[0006] To achieve the above invention purposes, the following technical solutions are adopted: A preparation method of bai-muxiang leaf exosomes, comprising the following steps:

[0007] Step (1): The collected stem tip / bud tip meristem is soaked in ethanol for 30-60 seconds, then disinfected with mercuric chloride solution for 5-10 minutes, and then washed with sterile water for 3-5 times to obtain disinfected meristem, so as to remove the microorganisms and impurities on the surface and ensure that the subsequent culture process is not contaminated;

[0008] Step (2): The disinfected meristem in step (1) is inoculated on the induction medium, and cultured for 20-30 days under the conditions of temperature 25±2℃, light intensity 1500-2000lx, and light time 12-16h / d, to induce the formation of clustered buds;

[0009] Step (3): The clustered buds in step (2) are inoculated into liquid medium, and suspended cultured for 10-15 days under the conditions of temperature 25±2℃, shaking speed 120-180g, and light intensity 1000-1500lx, to collect the culture filtrate. The synergistic regulation of speed and light intensity can significantly promote the secretion of exosomes;

[0010] Step (4): The filtrate in step (3) is centrifuged at 3000-5000g at 4℃ for 15-30 minutes, and then the supernatant is filtered through a filter membrane. Finally, the filtrate is ultracentrifuged at 100000-150000g at 4℃ for 70-90 minutes, and the precipitate is resuspended with PBS buffer to obtain bai-muxiang leaf exosomes, which are stored in a freezer. This process can make the purity of exosomes reach more than 92%, the particle size distribution is concentrated in 30-150nm (accounting for 85%), and the culture medium protein and other impurities are removed.

[0011] Further, the purified exosomes are diluted with PBS buffer to a concentration of 1×10 9 particles / mL (or 1mg / mL protein concentration), and stored in a-80℃ refrigerator after aliquot for standby.

[0012] Further, the mass fraction of ethanol in step (1) is 70%; the mass fraction of mercuric chloride solution is 0.1%; and the length of stem tip / bud tip meristem is 0.5-1.5cm. Ensure that the survival rate of explants is ≥85% and the contamination rate is ≤5%.

[0013] Further, the induction medium formula in step (2) is MS basic medium, 6-benzylaminopurine 0.5-2.0 mg / L, naphthalene acetic acid 0.1-0.5 mg / L, sucrose 20-30 g / L, agar 6-8 g / L, copper sulfate (CuSO4·5H2O) 0.01-0.05 mg / L, zinc sulfate (ZnSO4·7H2O) 0.1-0.5 mg / L, manganese sulfate (MnSO4·H2O) 0.2-0.8 mg / L, and the pH is adjusted to 5.8-6.0; the ratio is verified by experiments to make the induction rate of the cluster shoots reach 92%, which is 40% higher than the traditional callus induction method. The concentration ratio of 6-BA and NAA (1:0.2-1:0.5) directly affects the differentiation direction of the meristem.

[0014] Further, the inoculation density of the toxic meristem is 3-5 pieces per bottle (250 mL triangular bottle).

[0015] Further, the cluster shoots in step (3) are cut into small pieces of 0.2-0.5 cm, and the inoculation density is 1-1.5 g fresh weight / 50 mL medium.

[0016] Further, the formula of the liquid medium in step (3) is: MS basic medium, 6-benzylaminopurine 0.5-2.0 mg / L, naphthalene acetic acid 0.1-0.5 mg / L, sucrose 15-25 g / L, GA3 0.1-0.5 mg / L, copper sulfate 0.005-0.03 mg / L, zinc sulfate 0.05-0.3 mg / L, and manganese sulfate 0.1-0.5 mg / L. This system can make the exosome secretion amount reach 2.8 mg / L, which is 1.3 times higher than the conventional callus culture.

[0017] Add copper ions (Cu 2+ ): as a competitive inhibitor of PPO, by binding to the copper ion site of the enzyme active center, reducing the catalytic activity of PPO, reducing polyphenol oxidation (browning core reaction); add zinc ions (Zn 2+ ): involved in the synthesis of superoxide dismutase (SOD), enhancing the antioxidant capacity of cells, removing active oxygen (ROS) produced during browning, reducing oxidative damage; add manganese ions (Mn 2+ ): activate peroxidase (POD), promote the degradation of browning products, while stabilizing the intracellular membrane structure and maintaining the exosome secretion function.

[0018] Further, the pore size of the filter membrane in step (4) is 0.22 μm; the resuspension condition is ultracentrifugation at 100000-150000 g at 4°C for 70-90 minutes, repeated 2-3 times.

[0019] The white mulberry leaf exosome prepared by the preparation method of the white mulberry leaf exosome has applications in sunscreen whitening essence or anti-hair loss hair care shampoo.

[0020] Further, the sunscreen whitening essence is composed of, by mass fraction, white mulberry leaf exosome 1-3%, rhodiola extract 0.6-1%, sea buckthorn fruit extract 0.4-0.8%, glycerol 3-7%, sodium hyaluronate 0.1-0.5%, vitamin E 0.05-0.15%, phenoxyethanol 0.3-0.8%, and the balance is deionized water; the white mulberry leaf exosome has a protein concentration of 1.2 mg / mL, the rhodiola extract has a rhodioloside mass fraction of greater than or equal to 3%, and the sea buckthorn fruit extract has a vitamin C mass fraction of greater than or equal to 5%.

[0021] Further, the anti-hair loss hair care shampoo is composed of, by mass fraction, white mulberry leaf exosome 1-1.5%, radix falcaliae extract 0.5-0.9%, cypress leaf extract 0.4-0.8%, sodium lauryl sulfate 6-10%, sodium laureth sulfate 10-14%, cocamidopropyl betaine 4-6%, glycerol 2-4%, sodium chloride 0.5-1.5%, and sodium benzoate 0.3-0.7%, and the balance is deionized water.

[0022] Further, the white mulberry leaf exosome has a protein concentration of 1.2 mg / mL; the radix falcaliae extract has a stilbene glycoside mass fraction of greater than or equal to 2%; and the cypress leaf extract has a total flavone mass fraction of greater than or equal to 1.5%.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] 1. Improved exosome activity: By preparing raw materials and process innovation, stem tip / bud tip meristem is selected as raw material, and by optimizing the hormone ratio (6-BA, NAA, GA3) and culture parameters, the content of active ingredients such as flavonoids in the exosome is increased by 1.3 times compared with that of callus, and the culture period is shortened by 20 days.

[0025] 2. The present application precisely inhibits polyphenol oxidase activity, enhances cell antioxidant capacity, and stabilizes cell secretion function through the synergistic regulation of composite trace metal elements (copper, zinc, and manganese), reduces the browning rate from 45% to 8%-10%, increases the exosome yield by 40%, narrows the yield fluctuation range from ±20% to ±5%, and for the first time realizes large-scale preparation of white mulberry leaf exosomes with high stability and high yield, thereby providing a replicable technical solution for the industrialization of plant exosomes.

[0026] 3. Improve transdermal rate and targeted delivery: The nanoscale lipid bilayer structure of exosomes can efficiently enter cells through endocytosis, and its transdermal efficiency is 3-5 times higher than that of free extract, and its uptake rate in hair follicle cells is increased by 2.8 times, significantly enhancing the bioavailability of active ingredients.

[0027] 4. Multi-component synergistic effect: The combination with extracts of rhodiola, sea buckthorn fruit, etc. can improve the effect by 40% in the fields of sun protection and whitening; the combination with extracts of radix polygoni multiflori and biota leaf can extend the hair follicle growth period by 30%, and increase the hair density by 28%, thereby breaking through the bottleneck of single component efficacy.

[0028] 5. Significant advantages in stability and safety: The lipid membrane structure of exosomes can be stored at 4℃ for 6 months without significant decrease in activity, and can tolerate a pH range of 4-9. Skin irritation test shows that the sensitization rate is less than 0.1%, and the safety is better than that of traditional extracts. BRIEF DESCRIPTION OF DRAWINGS

[0029] The application will be further described below in combination with the drawings and examples, and the drawings are as follows:

[0030] Figure 1 is the average particle size distribution diagram of the bauhinia blakeana leaf exosome of example 1 of the application.

[0031] Figure 2 is the transmission electron microscope diagram of the bauhinia blakeana leaf exosome of example 1 of the application. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the application more clear, the application will be further described in detail below in combination with examples. It should be understood that the specific examples described herein are only used to explain the application, and are not used to limit the application.

[0033] Example 1

[0034] The example of the application provides a preparation method of bauhinia blakeana leaf exosome, which comprises the following steps:

[0035] (1) Taking and disinfecting: taking the stem tip meristem (length 1.2 cm) of bauhinia blakeana plants grown for 3 years, soaking in 70% ethanol for 45 seconds, and disinfecting with 0.1% mercuric chloride for 8 minutes, and washing with sterile water for 5 times.

[0036] (2) Cluster bud induction: inoculate 4 per bottle into 50 mL induction medium (250 mL flask), the induction medium formula is MS basic medium + 6-BA 1.0 mg / L + NAA 0.3 mg / L + sucrose 25 g / L + agar 7 g / L + copper sulfate (CuSO4·5H2O) 0.03 mg / L + zinc sulfate (ZnSO4·7H2O) 0.3 mg / L + manganese sulfate (MnSO4·H2O) 0.6 mg / L, pH 5.8, cultivate for 25 days at 25℃, 1800lx illumination (14h / d), and the induction rate reaches 94%.

[0037] (3) Suspension culture: cut the cluster buds into 0.5 cm small pieces, inoculate 1.2 g / 50 mL into a liquid medium prepared from MS basic medium + GA3 0.3 mg / L + sucrose 20 g / L + copper sulfate 0.01 mg / L + zinc sulfate 0.2 mg / L + manganese sulfate 0.3 mg / L, cultivate for 12 days at 25℃ on a 150g shaking table, and the illumination is 1200lx; collect the culture filtrate.

[0038] (4) Exosome extraction: centrifuge the culture filtrate at 4000g for 20 minutes, filter through a 0.22μm filter membrane, ultracentrifuge at 120000g for 80 minutes (4℃), resuspend in PBS and repeat the centrifugation twice, to obtain an exosome solution with a concentration of 1.2mg / mL, the particle size distribution is 30-150nm and accounts for 88%, and the flavonoid content is 2.3mg / g.

[0039] Example 2

[0040] The application embodiment provides a preparation method of white camphor leaf exosomes, which comprises the following steps:

[0041] (1) Material taking and disinfection: take stem tip meristems (length 1cm) of white camphor plants grown for 3 years, soak in 70% ethanol for 30 seconds, disinfect with 0.1% mercuric chloride for 5 minutes, and wash with sterile water for 3 times.

[0042] (2) Cluster bud induction: inoculate 4 per bottle into 50 mL induction medium (250 mL flask), the induction medium formula is MS basic medium + 6-BA 1.0 mg / L + NAA 0.3 mg / L + sucrose 25 g / L + agar 7 g / L + copper sulfate (CuSO4·5H2O) 0.01 mg / L + zinc sulfate (ZnSO4·7H2O) 0.1 mg / L + manganese sulfate (MnSO4·H2O) 0.2 mg / L, pH 5.8, cultivate for 20 days at 23℃, 1500lx illumination (12h / d), and the induction rate reaches 92%.

[0043] (3) Suspension culture: the tufted buds are cut into small pieces of 0.5 cm, inoculated into a liquid medium of MS basic medium + GA3 0.3 mg / L + sucrose 20 g / L + copper sulfate 0.005 mg / L + zinc sulfate 0.05 mg / L + manganese sulfate 0.1 mg / L at an inoculation amount of 1.2 g / 50 mL, cultured at 25 DEG C in a 120 g shaking bed for 15 days, the light is 1000 lx, and the culture filtrate is collected.

[0044] (4) Exosome extraction: the culture filtrate is centrifuged at 3000 g for 30 minutes, filtered through a 0.22 μm filter membrane, ultracentrifuged at 100000 g for 90 minutes (4 DEG C), resuspended in PBS and centrifuged twice, to obtain an exosome solution with a concentration of 1.2 mg / mL, a particle size distribution of 90% of 30-150 nm, and a flavonoid content of 2.5 mg / g.

[0045] Example 3

[0046] The preparation method of the exosome of the bauhinia championii leaf provided in the embodiments of the present application comprises the following steps:

[0047] (1) Sampling and disinfection: the stem tip meristem (length 1.5 cm) of a bauhinia championii plant grown for 3 years is soaked in 70% ethanol for 60 seconds, disinfected with 0.1% mercuric chloride for 10 minutes, and washed with sterile water for 6 times.

[0048] (2) Tufted bud induction: inoculate 4 per bottle into 50 mL of induction medium (250 mL triangular flask) at an inoculation amount of 4, the induction medium formula is MS basic medium + 6-BA 1.0 mg / L + NAA 0.3 mg / L + sucrose 25 g / L + agar 7 g / L + copper sulfate (CuSO4·5H2O) 0.05 mg / L + zinc sulfate (ZnSO4·7H2O) 0.5 mg / L + manganese sulfate (MnSO4·H2O) 0.8 mg / L, pH 5.8, and cultured at 27 DEG C under 2000 lx illumination (16 h / d) for 25 days, the induction rate reaches 93%.

[0049] (3) Suspension culture: the tufted buds are cut into small pieces of 0.5 cm, inoculated into a liquid medium of MS basic medium + GA3 0.3 mg / L + sucrose 20 g / L + copper sulfate 0.03 mg / L + zinc sulfate 0.3 mg / L + manganese sulfate 0.5 mg / L at an inoculation amount of 1.2 g / 50 mL, cultured at 25 DEG C in a 180 g shaking bed for 10 days, the light is 1500 lx, and the culture filtrate is collected.

[0050] (4)Exosome extraction: the culture filtrate was centrifuged at 5000g for 15 minutes, filtered through a 0.22 mu m filter membrane, ultracentrifuged at 150000g for 70 minutes (4 DEG C), resuspended in PBS and centrifuged twice again to obtain an exosome solution with a concentration of 1.2 mg / mL, the particle size distribution was 86% of 30-150 nm, and the flavonoid content was 2.4 mg / g.

[0051] Example 4

[0052] The application further provides a sunscreen whitening essence containing the Aquilaria sinensis leaf exosome, and the formula is as follows in terms of mass percentage: 1.5% of the Aquilaria sinensis leaf exosome (1.2 mg / mL), 0.8% of the Rhodiolae Radix extract (Rhodioloside is greater than or equal to 3%), 0.6% of the Hippophae Rhamnoides L. fruit extract (Vitamin C is greater than or equal to 5%), 5.0% of glycerol, 0.3% of sodium hyaluronate, 0.1% of vitamin E, 0.5% of phenoxyethanol, and deionized water accounting for the rest.

[0053] The preparation method of the sunscreen whitening essence containing the Aquilaria sinensis leaf exosome is as follows: the deionized water is heated to 75 DEG C, and the glycerol and the sodium hyaluronate are dissolved; the mixture is cooled to 45 DEG C, and the exosome, the Rhodiolae Radix extract, the Hippophae Rhamnoides L. fruit extract, and the vitamin E are added and stirred for 30 minutes; the phenoxyethanol is added, the pH is adjusted to 5.8, and the sunscreen whitening essence is obtained after filtration.

[0054] Example 5

[0055] The application further provides a hair loss prevention and hair care shampoo containing the Aquilaria sinensis leaf exosome, and the formula is as follows in terms of mass percentage: 1.2% of the Aquilaria sinensis leaf exosome (1.2 mg / mL), 0.7% of the Polygoni Multiflori Radix extract (Stilbene Glycoside is greater than or equal to 2%), 0.6% of the Platycladi Cacumen extract (total flavonoids are greater than or equal to 1.5%), 8.0% of sodium lauryl sulfate, 12.0% of sodium laureth sulfate, 5.0% of cocamidopropyl betaine, 3.0% of glycerol, 1.0% of sodium chloride, 0.5% of sodium benzoate, and deionized water accounting for the rest.

[0056] The preparation method of the hair loss prevention and hair care shampoo containing the Aquilaria sinensis leaf exosome is as follows: the deionized water is heated to 65 DEG C, and the surfactant is dissolved; the mixture is cooled to 40 DEG C, and the exosome, the Polygoni Multiflori Radix extract, the Platycladi Cacumen extract, and the glycerol are added and stirred for 20 minutes; the viscosity is adjusted by adding the sodium chloride, the pH is adjusted to 6.5, and the hair loss prevention and hair care shampoo containing the Aquilaria sinensis leaf exosome is obtained after filtration.

[0057] Comparative Example 1

[0058] The steps are the same as those in Example 1, except that the culture medium does not contain copper sulfate, zinc sulfate and manganese sulfate.

[0059] Comparative Example 2

[0060] The procedure is the same as example 1, the difference is that only copper sulfate is added in the culture medium, no zinc sulfate, manganese sulfate is added.

[0061] Comparative example 3

[0062] The procedure is the same as example 1, the difference is that copper sulfate 0.1 mg / L + zinc sulfate 1.0 mg / L + manganese sulfate 1.5 mg / L (far beyond the scope of the invention) is added in the induction medium.

[0063] Test example 1: comparison of the activity of exosomes from callus and meristem

[0064] Process difference:

[0065] Experimental group: example 1.

[0066] Control group: Take the leaves of Aquilaria sinensis to induce callus (2,4-D 2.0 mg / L + 6-BA 1.0 mg / L), and use 2,4-D 1.0 mg / L for suspension culture, and the rest of the steps are the same as example 1, and the culture period is 65 days.

[0067] Active ingredient detection and efficacy detection were carried out on the experimental group and the control group, and the results are shown in Table 1 and Table 2.

[0068] Table 1 is the active ingredient detection results of the experimental group and the control group

[0069] Index Experimental group Control group Difference rate Flavonoid content 2.5 mg / mg 1.1 mg / mg ↑127％ Luteolin derivative 1.56 mg / mg 0.48 mg / mg ↑225％ Exosome yield 1.5 mg / L 0.8 mg / L ↑87.5％

[0070] Table 2 is the efficacy results of the experimental group and the control group

[0071]

[0072] Test example 2: comparison of whitening efficacy of single exosome and complex system

[0073] Formulation design:

[0074] Experimental group: example 1

[0075] Control group 1: single exosome 1.5%;

[0076] Control group 2: Rhodiola + sea-buckthorn fruit extract complex (0.8% + 0.6%);

[0077] Control group 3: commercially available arbutin 2.0%.

[0078] In vitro experiment tests were carried out on the experimental group and control groups 1-3, and the results are shown in Table 3.

[0079] Table 3 is the in vitro experimental data results of the experimental group and control groups 1-3

[0080] Group Tyrosinase inhibition rate Melanin synthesis inhibition rate Cytotoxicity (IC 50 )]]> Experimental group 78％ 65％ > 1000 μg / mL Control group 1 52％ 41％ > 1000 μg / mL Control group 2 39％ 28％ > 1000 μg / mL Control group 3 62％ 50％ 500 μg / mL

[0081] As can be seen from Table 3, the tyrosinase inhibition rate of the complex system is increased by 50% compared with the single exosome, and by 61% compared with the simple component superposition, confirming that the synergistic effect is significant; although the inhibition rate of arbutin group is higher, the cytotoxicity is obvious, and the safety of the system in the experimental group is better.

[0082] Test Example 3: Comparison of anti-hair loss effects of traditional extract and exosome

[0083] Experimental design

[0084] Experimental group: Example 5

[0085] Control group 1: BaiMuxiang leaf water extract 5.0% (containing the same amount of flavonoids) + HeShouWu extract + Cephalotaxus fortunei extract;

[0086] Control group 2: commercially available minoxidil 5%;

[0087] Blank group: matrix control.

[0088] Animal experiments (SD rat alopecia model) were performed on the experimental group, control groups 1-2 and blank group, and the results are shown in Table 4.

[0089] Table 4: Animal experiment results of experimental group, control groups 1-2 and blank group

[0090]

[0091] As can be seen from Table 4, the hair follicle activation efficiency of the exosome group is increased by 81% compared with the traditional extract, which is attributed to the protection and targeting of the nano delivery system for active ingredients; although the minoxidil group has a certain effect, the inflammatory factor inhibition ability is weaker than the system of the present application, suggesting the unique advantage of exosome in improving the microenvironment of the scalp.

[0092] Test Example 4: Comparison test of active ingredients of BaiMuxiang leaf exosome and traditional extract

[0093] Materials and methods

[0094] Sample preparation: BaiMuxiang leaf exosome was prepared according to Example 1, and an equal amount of leaf was extracted by 70% ethanol reflux for 2h to prepare traditional extract.

[0095] Detection method: HPLC was used to determine the contents of flavonoids (luteolin-7-3', 4'-trimethyl ether) and phenolic acids (chlorogenic acid), and ELISA method was used to detect the level of anti-inflammatory factor IL-10, and the results are shown in Table 5.

[0096] Table 5: Test data of active ingredients of BaiMuxiang leaf exosome and traditional extract

[0097]

[0098] From Table 5, the exosome enrichment of active ingredients is significantly better than the traditional extraction process, and the level of anti-inflammatory factors is increased by 2.3 times, verifying its advantage in cell communication.

[0099] Test Example 5: Transdermal efficiency comparison test of white camphor leaf exosomes

[0100] Materials and Methods

[0101] Experimental model: Construct artificial synthetic membrane (MWCO 10kDa) and ex vivo pig skin transdermal model.

[0102] Sample treatment: Label the flavonoids in the exosomes and traditional extracts of Example 1, respectively, and determine the transdermal amount by fluorescence quantification. The results are shown in Table 6.

[0103] Table 6: Transdermal efficiency test data of white camphor leaf exosomes

[0104]

[0105] From Table 6, the nanoscale lipid membrane structure of exosomes significantly improves the transdermal efficiency, and the cumulative transdermal amount in 24h is 2.24 times that of traditional extracts, verifying the effectiveness of its delivery system.

[0106] Test Example 6: Synergistic antioxidant test of white camphor leaf exosome complex system

[0107] Materials and Methods

[0108] Experimental group: Complex system of Example 4.

[0109] Control group 1: White camphor leaf exosome group; Control group 2: Rhodiola extract group; Control group 3: Sea buckthorn fruit extract group; Simple mixing group 1: Exosomes mixed with Rhodiola; Simple mixing group 2: Exosomes mixed with sea buckthorn fruit.

[0110] Detection index: DPPH free radical scavenging rate, ABTS+ cation scavenging rate, SOD activity, and the results are shown in Table 7.

[0111] Table 7: Antioxidant detection results of experimental group and control groups 1-3

[0112]

[0113] From Table 7, the antioxidant activity of the complex system is significantly better than that of single components and simple mixing groups, and the DPPH clearance rate IC 50 is reduced by 32.4%, verifying the synergistic mechanism.

[0114] Test Example 7: Browning inhibition and exosome quality effect test

[0115] Table 8 Performance test results of Example 1, Comparative Examples 1-3

[0116]

[0117] As can be seen from Table 8, by synergistic regulation of composite trace metal elements (copper, zinc, manganese), the present application precisely inhibits polyphenol oxidase activity, enhances cell antioxidant capacity, stabilizes cell secretion function, reduces browning rate from 45% to 8%-10%, and increases exosome yield by 40%; the reduction of browning products directly reduces the damage to active ingredients. Compared with the traditional process without adding trace metal elements, the content of flavonoid ingredients in the exosomes prepared by the present application is increased by 20%-30% (up to 2.8 mg / g), and the content of luteolin derivatives is increased by 16%-17% (up to 1.82 mg / g).

[0118] The above description is merely preferred embodiments of the present application, but not to limit the present application, any modification, equivalent replacement or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for preparing exosomes from Aquilaria sinensis leaves, characterized in that, Includes the following steps: Step (1): Soak the collected stem tip / bud tip meristem in ethanol for 30-60 seconds, then disinfect with mercuric chloride solution for 5-10 minutes, and then rinse with sterile water 3-5 times to obtain sterilized meristem; Step (2): Inoculate the sterilized meristem from step (1) onto the induction medium and culture it for 20-30 days under the conditions of temperature 25±2℃, light intensity 1500-2000lx, and light duration 12-16h / d to induce the formation of clustered shoots; Step (3): Inoculate the clustered shoots from step (2) into liquid culture medium and suspend them for 10-15 days at a temperature of 25±2℃, a shaking speed of 120-180g, and a light intensity of 1000-1500lx. Collect the culture filtrate. Step (4): After centrifuging the filtrate from step (3) at 3000-5000g for 15-30 minutes at 4℃, pass the supernatant through the filter membrane, and finally centrifuge the filtrate at 100000-150000g for 70-90 minutes at 4℃. Collect the precipitate, resuspend it in PBS buffer, and obtain the exosomes from the leaves of Aquilaria sinensis. Freeze and store them.

2. The method for preparing exosomes from Aquilaria sinensis leaves according to claim 1, characterized in that, In step (1), the mass fraction of ethanol is 70%; the mass fraction of mercuric chloride solution is 0.1%; and the length of the shoot tip / bud tip meristem is 0.5-1.5 cm.

3. The method for preparing exosomes from Aquilaria sinensis leaves according to claim 1, characterized in that, The induction culture medium formula in step (2) is MS basic medium, 6-benzylaminopurine 0.5-2.0 mg / L, naphthaleneacetic acid 0.1-0.5 mg / L, sucrose 20-30 g / L, agar 6-8 g / L, copper sulfate 0.01-0.05 mg / L, zinc sulfate 0.1-0.5 mg / L, manganese sulfate 0.2-0.8 mg / L, and the pH is adjusted to 5.8-6.0; the meristematic tissue inoculation density is 3-5 pieces per bottle (250 mL Erlenmeyer flask).

4. The method for preparing exosomes from Aquilaria sinensis leaves according to claim 1, characterized in that, The process involves cutting the clustered buds in step (3) into small segments of 0.2-0.5cm, with an inoculation density of 1-1.5g fresh weight / 50mL culture medium.

5. The method for preparing exosomes from Aquilaria sinensis leaves according to claim 1, characterized in that, The formula for the liquid culture medium in step (3) is as follows: MS basic culture medium, 6-benzylaminopurine 0.5-2.0 mg / L, naphthaleneacetic acid 0.1-0.5 mg / L, sucrose 15-25 g / L, GA3 0.1-0.5 mg / L, copper sulfate 0.005-0.03 mg / L, zinc sulfate 0.05-0.3 mg / L, and manganese sulfate 0.1-0.5 mg / L.

6. The method for preparing exosomes from Aquilaria sinensis leaves according to claim 1, characterized in that, The filter membrane pore size in step (4) is 0.22 μm; the resuspension conditions are: centrifugation at 100,000-150,000 g for 70-90 minutes at 4°C, repeated 2-3 times.

7. The method for preparing exosomes from Aquilaria sinensis leaves according to claims 1-6, characterized in that, The exosomes prepared by the method described above can be used in sunscreen and whitening essences or anti-hair loss shampoos.

8. The method for preparing exosomes from Aquilaria sinensis leaves according to claim 7, characterized in that, The sunscreen and whitening essence, by mass fraction, is formulated with 1-3% Aucklandia lappa leaf exosomes, 0.6-1% Rhodiola rosea extract, 0.4-0.8% Hippophae rhamnoides fruit extract, 3-7% glycerin, 0.1-0.5% sodium hyaluronate, 0.05-0.15% vitamin E, 0.3-0.8% phenoxyethanol, and the balance being deionized water; the Aucklandia lappa leaf exosome protein concentration is 1.2 mg / mL, the Rhodiola rosea extract contains ≥3% rhodioloside by mass fraction, and the Hippophae rhamnoides fruit extract contains ≥5% vitamin C by mass fraction.

9. The method for preparing exosomes from Aquilaria sinensis leaves according to claim 7, characterized in that, The hair loss prevention and conditioning shampoo, by mass fraction, is formulated with 1-1.5% exosomes from Aucklandia lappa leaves, 0.5-0.9% extract of Polygonum multiflorum, 0.4-0.8% extract of Platycladus orientalis leaves, 6-10% sodium lauryl sulfate, 10-14% sodium laureth sulfate, 4-6% cocamidopropyl betaine, 2-4% glycerin, 0.5-1.5% sodium chloride, 0.3-0.7% sodium benzoate, and the balance being deionized water.

10. The method for preparing exosomes from Aquilaria sinensis leaves according to claim 9, characterized in that, The concentration of exosome protein in the Aucklandia lappa leaf is 1.2 mg / mL; the stilbene glycoside content in the Polygonum multiflorum extract is ≥2%; and the total flavonoid content in the Platycladus orientalis leaf extract is ≥1.5%.

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