Preparation method of fructus forsythiae exosome and application of fructus forsythiae exosome in cosmetics

Abstract

The invention discloses a preparation method of fructus forsythiae exosomes and application of the fructus forsythiae 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 fructus forsythiae exosome suspension prepared by the preparation method can be applied to cosmetics; according to the method, immature fruit tissue culture, extraction technology innovation and compound system optimization are adopted, a technical path with novelty and creativity is provided for innovative development of fructus forsythiae in the cosmetic industry, and particularly, the fructus forsythiae has wide application prospects in functional skin care products, professional acne removal series and oil-control and anti-dandruff washing and care products.

Description

TECHNICAL FIELD

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

[0002] In the field of cosmetics, plant exosomes as a new source of active ingredients have become 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 and precisely act on skin cells, showing great potential in antioxidant, anti-inflammatory and cell repair. However, the extraction and application of plant exosomes are still in the development stage. Most studies focus on common plants such as green tea and aloe, and the extraction methods mostly use traditional means such as mechanical disruption and ultrahigh-speed centrifugation. These methods have low extraction efficiency, high cost and are prone to damage the integrity of exosomes, making it difficult to achieve large-scale industrial production.

[0003] Forsythia (Forsythia suspensa) is a traditional Chinese medicinal material, which is recorded in the Pharmacopoeia of the People's Republic of China and has the effects of clearing heat and resolving toxicity, reducing swelling and resolving nodules. Its active ingredients such as forsythoside, forsythoside A and volatile oil have been confirmed in modern pharmacology to have anti-inflammatory, antibacterial and antioxidant effects. In the field of cosmetics, the current application of Forsythia mainly uses traditional methods such as water extraction and alcohol extraction to obtain crude extracts, which are then added to cosmetic matrices. However, this method has obvious defects: on the one hand, traditional extraction only uses the fruit part of Forsythia, and the branches and leaves are not fully utilized, resulting in waste of resources and limited fruit yield, making it difficult to meet the demand of large-scale production; on the other hand, traditional extraction methods cannot specifically obtain exosomes and other specific functional ingredients, resulting in impurities in the extract, low purity, unstable efficacy in cosmetics, inaccurate action, and difficulty in fully exerting the skin care potential of Forsythia.

[0004] Forsythia is only used in the form of crude extract in cosmetics, and the raw material is limited to fruit, and the branches and leaves are not fully utilized. So far, there is no report in the prior art on obtaining Forsythia exosomes through plant tissue culture and applying them to cosmetics to achieve specific effects such as acne removal, repair, oil control and dandruff removal. In view of this, the present application is proposed. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the defects of the prior art. By adopting immature fruit tissue culture, extraction technology innovation and complex system optimization, the present application provides a novel and creative technical path for the innovative development of Forsythia in the cosmetics industry, which has a broad application prospect in functional skincare products, professional acne-removing series and oil-controlling and dandruff-removing shampoo and conditioner products.

[0006] To achieve the above-mentioned purposes of the application, the following technical solutions are adopted: A preparation method of forsythia suspensa exosome comprises the following steps:

[0007] Step (1): Select immature forsythia fruit with a diameter of 0.9-1.1 cm, sterilize the fruit with ethanol for 20-40 seconds, then treat the fruit with a mercuric chloride solution for 6-10 minutes, and then rinse the fruit with sterile water for 4-6 times to obtain sterilized fruit;

[0008] Step (2): Cut the sterilized fruit in step (1) into small pieces with a size of 5 mm x 5 mm x 3 mm, inoculate and spread the small pieces on a modified MS culture medium, and place the inoculated culture medium in a condition of a temperature of 25±1℃, a light intensity of 1500-2000 lux, and a light cycle of 12-16 hours / day to induce callus, and form callus after 20-28 days;

[0009] Step (3): Cut the callus in step (2) into 1-2 mm particles, inoculate the particles into a liquid culture medium according to an inoculation amount of 1:5 (g / mL), and then culture the particles in a condition of a temperature of 22±1℃ and a rotation speed of 130-140 rpm for 28-32 days, and replace 1 / 2 of the culture medium every 7 days;

[0010] Step (4): Collect the supernatant of the culture in step (3), centrifuge the supernatant at a rotation speed of 2800-3500 rpm for 10-20 minutes, filter the supernatant through a filter membrane, and then perform ultrahigh-speed centrifugation on the filtered supernatant at a centrifugal force of 80000-120000 g for 60-80 minutes under a condition of 3-5℃, resuspend the precipitate with a PBS buffer to obtain forsythia suspensa exosome suspension.

[0011] Further, the mass fraction of ethanol in step (1) is 75%; and the mass fraction of the mercuric chloride solution is 0.1%.

[0012] Further, the inoculation density in step (2) is 8-10 pieces per bottle (250 mL triangular flask).

[0013] Further, the formula of the modified MS culture medium in step 2 is as follows: 2.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, 6.5 g / L agar, 0.02-0.08 mg / L cobalt chloride (CoCl2·6H2O), 0.5-2.0 mg / L sodium silicate (Na=SiO3·9H2O), and 0.1-0.5 mg / L titanium sulfate (Ti(SO4)2), and the pH value is adjusted to 5.8.

[0014] The addition of cobalt ions (Co 2+) as a non-competitive inhibitor of PPO, by binding to the enzyme's cofactor site, reduces its catalytic activity, inhibits phenolic material oxidation (browning core reaction); adding silicon ions (SiO32-): deposited on the cell wall to form a protective layer, enhance cell membrane stability, reduce intracellular phenolic material exosmosis, reduce the concentration of browning substrates from the source; adding titanium ions (Ti 4+ ) : activate intracellular superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity, accelerate the removal of active oxygen (ROS) produced during the browning process, reduce oxidative damage.

[0015] Further, the induction rate of the step (2) callus reaches more than 85%; the fresh weight of callus induced per gram of explant is not less than 3.2g.

[0016] Further, the liquid medium formula of the step (3) is prepared from 2.0mg / L 6-BA, 0.5mg / L NAA, 30g / L sucrose, 1.5mg / L 2,4-D, 0.3mg / L KT, 20g / L glucose, 0.01-0.05mg / L cobalt chloride, 0.3-1.5mg / L sodium silicate and 0.05-0.3mg / L titanium sulfate.

[0017] Further, the protein concentration of the step (4) forsythia exosome suspension is ≥0.8mg / mL; the pore size of the filter membrane in the step (4) is 0.22μm.

[0018] The forsythia exosome suspension prepared by the preparation method of the forsythia exosome has the application in the acne-removing repair face cream or oil-controlling dandruff-removing shampoo.

[0019] Further, the acne-removing repair face cream is composed of forsythia exosome suspension 10-14%, peach extract 0.3-0.8%, sophoramine 0.1-0.3%, squalane 8-12%, shea butter 6-10%, glycerol 4-8%, xanthan gum 0.3-0.8% and water 55-70% by mass fraction.

[0020] Further, the oil-controlling dandruff-removing shampoo is composed of forsythia exosome suspension 3-8%, olive extract 1-5%, white willow bark extract 1-3%, sodium laureth sulfate 10-20%, guar hydroxypropyltrimonium chloride 0.5-1.5%, sodium chloride 0.3-0.8%, phenoxyethanol 0.3-0.8% and water 70-80% by mass fraction.

[0021] Compared with the prior art, the beneficial effects of the present application are:

[0022] 1. Raw material utilization innovation: Breakthrough the limitation of traditional use of mature fruits, select unripe fruits as starting material, combine with plant tissue culture technology to realize year-round production. Expand to branches and leaves through induction of callus, solve the problem of fruit harvesting season restriction and raw material waste, and build a sustainable raw material supply system.

[0023] 2. The present application reduces the browning rate of forsythia exosome culture process from 42% to 6%-8% through the synergistic regulation of cobalt-silicon-titanium complex trace metal elements, increases the yield by 40%, and narrows the batch purity fluctuation from ±15% to ±3%, providing an industrializable technical solution for the large-scale and standardized production of forsythia exosomes, and solving the core pain points of traditional process "high loss and low stability".

[0024] 3. The present application uses complex trace metal elements to inhibit oxidation and protect cell structure, so that the content of forsythia ester glycoside A in exosomes is increased from 1.0 mg / g to 1.85 mg / g (increased by 85%), and the retention rate of antioxidant ingredients such as flavonoids is increased by more than 30%.

[0025] 4. Preparation technology breakthrough: Using improved culture medium formula and three-stage separation and purification technology, compared with traditional mechanical crushing and single centrifugation method, the exosome yield and purity are significantly improved. Optimize the culture temperature, rotation speed and other parameters to ensure the integrity of the exosome structure, and break through the technical bottleneck of low efficiency and high cost in industrial production.

[0026] 5. Efficacy mechanism upgrade: Utilizing the nanoscale delivery characteristics of exosomes, active ingredients are precisely carried to penetrate the skin barrier and exert anti-inflammatory and repair effects in the dermis. Different from the blind effect of traditional crude extract, a targeted regulation mechanism is formed to solve the industry pain points of unstable efficacy and inaccurate action of existing products.

[0027] 6. Cosmetic application system innovation: Compound with natural active ingredients to build a multi-target synergistic system, simultaneously realize oil control, repair, microecological regulation and other effects. Through hair follicle targeted delivery technology, the ingredient utilization rate is improved, and the risk of using chemical additives is reduced, forming a solution with safety and effectiveness in the field of acne and dandruff removal. BRIEF DESCRIPTION OF DRAWINGS

[0028] The present application will be further described below in combination with the drawings and examples. In the drawings:

[0029] Figure 1 is the forsythia callus culture of example 1 of the present application for two weeks.

[0030] Figure 2 is the forsythia callus culture of example 1 of the present application for four weeks. DETAILED DESCRIPTION

[0031] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only intended to explain the present application and not to limit the present application.

[0032] Example 1

[0033] The present application provides a preparation method of forsythia exosome, comprising the following steps:

[0034] (1) Selection and disinfection of explants: immature forsythia fruits with a diameter of 0.9 cm are selected, which are in the S3 stage of the development stage and present a yellow-green color. The fruits are first surface-disinfected with 75% ethanol for 30 seconds, then treated with 0.1% mercuric chloride solution for 8 minutes, and then rinsed with sterile water for 5 times to completely remove the residual disinfectant.

[0035] (2) Inoculation and callus induction: the disinfected fruits are cut into small pieces of 5 mm x 5 mm x 3 mm, inoculated at a density of 8 pieces per bottle (250 mL triangular flask), and laid on modified MS medium. The medium is added with 2.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, 6.5 g / L agar, 0.02 mg / L cobalt chloride (CoCl2·6H2O), 0.5 mg / L sodium silicate (Na2SiO3·9H2O), and 0.1 mg / L titanium sulfate (Ti(SO4)2), and the pH value is adjusted to 5.8. The inoculated triangular flask is placed in a culture environment with a temperature of 25°C, a light intensity of 1600 lux, and a light cycle of 14 h / d. After 28 days of culture, callus is successfully induced, the induction rate reaches 88%, and the fresh weight of callus induced per gram of explant is 3.5 g.

[0036] (3) Suspension culture: the induced callus is cut into 1-2 mm particles, and inoculated into liquid medium at a inoculation amount of 1:5 (g / mL). The liquid medium is prepared by removing agar from the modified MS medium and adding 1.5 mg / L 2,4-D, 0.3 mg / L KT, 20 g / L glucose, 0.01 mg / L cobalt chloride, 0.3 mg / L sodium silicate, and 0.05 mg / L titanium sulfate. The culture is carried out in a shaker at a temperature of 22°C and a rotation speed of 130 rpm for 30 days, and the culture solution is replaced by 1 / 2 every 7 days.

[0037] (4) Exosome isolation and purification: The supernatant of the suspension culture was collected, first centrifuged at 3000 rpm for 15 min to remove cell debris, then filtered through a 0.22 μm filter, and finally ultracentrifuged at 100,000 g for 70 min at 4°C. The precipitate was resuspended in PBS buffer to obtain a Forsythia suspensa exosome suspension with a protein concentration of 0.9 mg / mL and an exosome purity of 96% (NTA detection of impurity particles <4%).

[0038] Example 2

[0039] The present application provides a preparation method of Forsythia suspensa exosomes, comprising the following steps:

[0040] (1) Selection and disinfection of explants: immature Forsythia suspensa fruits with a diameter of 1.1 cm were selected, which were in the S3 stage of development and showed yellow-green color. The fruits were first surface-disinfected with 75% ethanol for 20 seconds, then treated with 0.1% mercuric chloride solution for 6 minutes, and then rinsed with sterile water for 4 times to completely remove the residual disinfectant.

[0041] (2) Inoculation and callus induction: 9 pieces of explants were inoculated per bottle (250 mL flask) and plated on modified MS medium. The medium was added with 2.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, 6.5 g / L agar, 0.05 mg / L cobalt chloride (CoCl2·6H2O), 0.15 mg / L sodium silicate (Na2SiO3·9H2O), and 0.3 mg / L titanium sulfate (Ti(SO4)2), and the pH value was adjusted to 5.8. The inoculated flasks were placed in a culture environment with a temperature of 25°C, a light intensity of 1600 lux, and a light cycle of 12 h / d. After 20 days of culture, callus was successfully induced, with an induction rate of 87% and a fresh weight of 3.4 g of callus induced per gram of explants.

[0042] (3) Suspension culture: The induced callus was cut into 1-2 mm particles, and inoculated into liquid medium at a ratio of 1:5 (g / mL). The liquid medium was prepared by removing agar from the modified MS medium and adding 1.5 mg / L 2,4-D, 0.3 mg / L KT, 20 g / L glucose, 0.03 mg / L cobalt chloride, 1 mg / L sodium silicate, and 0.15 mg / L titanium sulfate. The culture was carried out in a shaker at a temperature of 23°C and a rotation speed of 140 rpm for 30 days.

[0043] (4) Exosome isolation and purification: The supernatant of the suspension culture was collected, first centrifuged at 2800 rpm for 20 minutes to remove cell debris, then filtered through a 0.22 μm filter, and finally subjected to ultracentrifugation at 80,000 g at 4°C for 80 minutes. The precipitate was resuspended in PBS buffer to obtain a Forsythia suspensa exosome suspension with a protein concentration of 1.0 mg / mL and an exosome purity of 98%.

[0044] Example 3

[0045] The embodiment of the present application provides a preparation method of Forsythia suspensa exosomes, comprising the following steps:

[0046] (1) Selection and disinfection of explants: immature Forsythia suspensa fruits with a diameter of 1.0 cm were selected, which were in the S3 stage of the development stage and showed yellow-green color. The fruits were first surface-disinfected with 75% ethanol for 40 seconds, then treated with 0.1% mercuric chloride solution for 8 minutes, and then rinsed with sterile water for 6 times to completely remove the residual disinfectant.

[0047] (2) Inoculation and callus induction: 10 pieces of explants were inoculated per bottle (250 mL flask) and plated on modified MS medium. The medium was added with 2.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, 6.5 g / L agar, 0.08 mg / L cobalt chloride (CoCl2·6H2O), 2.0 mg / L sodium silicate (Na2SiO3·9H2O), and 0.5 mg / L titanium sulfate (Ti(SO4)2), and the pH value was adjusted to 5.8. The inoculated flasks were placed in a culture environment with a temperature of 25°C, a light intensity of 1600 lux, and a light cycle of 16 h / d. After 25 days of culture, callus was successfully induced, the induction rate reached 86%, and the fresh weight of callus induced per gram of explant was 3.3 g.

[0048] (3) Suspension culture: The induced callus was cut into 1-2 mm particles, and inoculated into liquid medium at a ratio of 1:5 (g / mL). The liquid medium was prepared by removing agar from the modified MS medium and adding 1.5 mg / L 2,4-D, 0.3 mg / L KT, 20 g / L glucose, 0.05 mg / L cobalt chloride, 1.5 mg / L sodium silicate, and 0.3 mg / L titanium sulfate. The culture was carried out in a shaker at a temperature of 23°C and a rotation speed of 140 rpm for 30 days.

[0049] (4) Exosome separation and purification: collect the supernatant of the suspension culture, first centrifuge at 3500 rpm for 10 minutes to remove cell debris, then filter through a 0.22 μm filter, and finally perform ultracentrifugation at 120,000 g at 4°C for 60 minutes. Resuspend the precipitate with PBS buffer to obtain Forsythia suspensa exosome suspension, and the protein concentration is 0.85 mg / mL, and the exosome purity reaches 97%.

[0050] Example 4

[0051] The application also provides a acne-removing repair cream, which has the following formula: 12% of the Forsythia suspensa exosome solution of Example 2, 0.5% of tocopheol, 0.2% of matrine, 10% of squalane, 8% of shea butter, 6% of glycerol, 0.5% of xanthan gum, and 62.8% of water.

[0052] The preparation method of the acne-removing repair cream is as follows: heat squalane and shea butter to 60°C to melt, and dissolve glycerol and xanthan gum in water and heat to 60°C. Slowly add the water phase to the oil phase, emulsify under stirring, and cool to 40°C. Then add the Forsythia suspensa exosome solution of Example 2, tocopheol and matrine, stir uniformly, and fill after cooling.

[0053] Example 5

[0054] The application also provides an oil-controlling and dandruff-removing shampoo, which has the following formula: 5% of the Forsythia suspensa exosome solution of Example 2, 3% of olive extract, 2% of white willow bark extract, 15% of sodium laureth sulfate, 1% of guar hydroxypropyltrimonium chloride, 0.5% of sodium chloride, 0.5% of phenoxyethanol, and 73% of water.

[0055] The preparation method of the oil-controlling and dandruff-removing shampoo is as follows: add deionized water to a stirring kettle, start stirring, and set the stirring speed to 30 r / min. Then add sodium laureth sulfate, guar hydroxypropyltrimonium chloride and sodium chloride in sequence, stir uniformly, and heat to 60°C to promote dissolution. After the above components are completely dissolved, add olive extract and white willow bark extract, and continue stirring for 15 minutes. Finally, add the Forsythia suspensa exosome suspension of Example 2 and phenoxyethanol, stir uniformly, and cool to room temperature to obtain the shampoo containing Forsythia suspensa exosomes.

[0056] Comparative Example 1

[0057] Traditional mature fruit extraction method:

[0058] (1) Raw material treatment: select mature Forsythia fruit (diameter 1.5-2.0 cm, brownish brown), cut into pieces after 75% ethanol disinfection for 30 seconds.

[0059] (2) Extraction process: 70% ethanol was added according to the ratio of material to liquid 1:10 (g / mL), and 80°C reflux extraction was performed twice, each for 2 hours, and the extraction liquid was combined.

[0060] (3) Purification step: The extraction liquid was concentrated under reduced pressure until there was no alcohol smell, centrifuged (3000 rpm x 15 min) and filtered through a 0.45 μm filter membrane to obtain the forsythia suspensa extract.

[0061] Comparative Example 2

[0062] Using the conventional MS culture medium culture method:

[0063] Culture medium formula: using the conventional MS culture medium (without 6-BA and NAA concentration optimization), i.e. MS basic ingredients + 30 g / L sucrose + 6.5 g / L agar, pH 5.8.

[0064] Culture parameters: callus induction stage temperature 28°C, light intensity 1000 lux, suspension culture rotation speed 100 rpm.

[0065] Separation process: single high-speed centrifugation (100,000 g x 70 min), without purification.

[0066] The rest of the experimental conditions are the same as in Example 2.

[0067] Comparative Example 3

[0068] Preparation of single-component acne cream (I)

[0069] The formula is: 0.5% peperomia tetraphylla and 0.2% matrine, and the rest of the auxiliary materials (emulsifiers, emollients, etc.) are the same as in Example 2, without the addition of forsythia suspensa exosome.

[0070] Comparative Example 4

[0071] The steps are the same as in Example 2, with the difference being that the culture medium does not add cobalt chloride, sodium silicate, and titanium sulfate.

[0072] Comparative Example 5

[0073] The steps are the same as in Example 2, with the difference being that only cobalt chloride is added to the culture medium.

[0074] Comparative Example 6

[0075] The steps are the same as in Example 2, with the difference being that in the induction medium, cobalt chloride 0.2 mg / L + sodium silicate 5.0 mg / L + titanium sulfate 1.0 mg / L (far beyond the scope of the present application).

[0076] Test Example 1

[0077] The effects of Example 2 and Comparative Examples 1-2 were detected, and the results are shown in the table below.

[0078] Table 1 Effect data of Example 2 and Comparative Example 1

[0079]

[0080] Table 2 Effect data of Example 2 and Comparative Example 2

[0081]

[0082] Test Example 2

[0083] In vitro experiment of acne-removing repair effect

[0084] (1) Experimental design

[0085] Cell model: human keratinocyte (HaCaT) and P. acnes co-culture model

[0086] Group setting: blank group: culture medium

[0087] Model group: P. acnes (1 x 10 6 CFU / mL)

[0088] Example 4: sample group: 12% forsythia suspensa exosome + 0.5% peatopan + 0.2% sophoramine

[0089] Comparative Example 3: control group: 0.5% peatopan + 0.2% sophoramine (without exosome)

[0090] (2) Performance detection index

[0091] Inflammatory factor level: ELISA detection of IL-8 and TNF-α content after 24h culture

[0092] Cell viability: CCK-8 method to detect HaCaT cell proliferation rate

[0093] Antibacterial activity: colony counting method to determine P. acnes survival rate

[0094] (3) Performance detection index results are shown in Table 3.

[0095] Table 3 Performance detection results of Example 4 and Comparative Example 3

[0096]

[0097] From Table 3, it can be seen that the sample group of Example 4 significantly reduces the inflammatory factor level (IL-8↓32.5%, TNF-α↓23.6%) compared with the control group of Comparative Example 3, improves the cell viability by 14%, and enhances the antibacterial effect by 417%, proving the synergistic acne-removing repair effect of exosome and peatopan and sophoramine.

[0098] Test Example 3

[0099] In vitro experiment of oil control and dandruff removal efficacy

[0100] (1) Experimental design

[0101] Scalp model: in vitro human scalp tissue (containing sebaceous glands and hair follicles)

[0102] Group setting:

[0103] Blank group: PBS buffer

[0104] Model group: Malassezia (1 x 10 5 CFU / mL) treatment

[0105] Sample group: shampoo containing 5% forsythia exosome + 3% olive extract + 2% white willow bark extract (Example 5)

[0106] Control group: commercially available oil control and dandruff removal shampoo (containing 1% ZPT)

[0107] (2) Performance detection index

[0108] Sebum secretion: measured by oil detector after 48h of culture

[0109] Malassezia number: qPCR detects ITS1 gene copy number

[0110] Stratum corneum water content: measure skin water content by capacitance method

[0111] (4) Performance detection index results are shown in Table 4

[0112] Table 4 Performance detection results of blank group, model group, sample group and control group

[0113]

[0114] From Table 4, the sample group of Example 5 has a 6.9% reduction in sebum secretion, an 8.6% reduction in fungus number, and a 13.0% increase in water content compared to the control group, and no skin dryness phenomenon occurs in the control group, proving the synergistic advantage of the natural ingredient combination in oil control and dandruff removal.

[0115] Test Example 4

[0116] Transdermal absorption and safety test

[0117] (1) Experimental design

[0118] Transdermal model: Franz diffusion cell (pig ear skin model)

[0119] Safety evaluation:

[0120] Patch test: 30 volunteers (18-45 years old)

[0121] Cytotoxicity: L929 cell MTT method

[0122] Example 2 is the sample group: 12% Forsythia exosome serum

[0123] Comparative Example 1 is the control group: 12% Forsythia extract serum

[0124] (2) Test index

[0125] Transdermal rate: HPLC determination of Forsythoside A permeation (24h)

[0126] Skin irritation index: observe skin reaction after 48h patch

[0127] Cell survival rate: detection after 24h treatment with different concentrations of samples

[0128] (3) Experimental results

[0129] Transdermal performance: exosome group permeation is 0.57±0.06 μg / cm 2 ·h, which is 375% higher than the traditional ethanol extract group (0.12±0.02 μg / cm 2 ·h), and shows sustained release characteristics (12h cumulative permeation accounts for 82%).

[0130] Safety data:

[0131] Patch test: 0 cases of erythema / edema in the sample group, 3 cases of mild irritation in the control group.

[0132] Cytotoxicity: cell survival rate >95% when sample concentration ≤100 μg / mL, survival rate of traditional extract group decreased to 82% at 50 μg / mL

[0133] (4) Conclusion

[0134] Forsythia exosomes significantly improve transdermal efficiency through nanoscale lipid membrane structure, and have better safety than traditional extracts and chemical active ingredients, providing an ideal carrier for precise delivery of cosmetics.

[0135] Test Example 5: Inhibition of browning and comparison of exosome quality

[0136] Table 5 Test results of Example 2 and Comparative Examples 4-6

[0137]

[0138] As shown in Table 5, by synergistically regulating the cobalt-silicon-titanium complex trace metal elements, the browning rate of forsythia exosome in the culture process is reduced from 42% to 6%-8% for the first time, the yield is increased by 40%, and the purity fluctuation of batches is narrowed from ±15% to ±3%; by inhibiting oxidation and protecting cell structure, the content of forsythia ester glycoside A in the exosome is increased from 1.0 mg / g to 1.85 mg / g (increased by 85%), and the retention rate of antioxidant ingredients such as flavonoids is increased by more than 30%.

[0139] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement or improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing Forsythia exosome, characterized in that, It comprises the following steps: Step (1): Select immature forsythia fruit with a diameter of 0.9-1.1 cm, disinfect it with ethanol for 20-40 seconds, then treat it with a mercuric chloride solution for 6-10 minutes, and then rinse it with sterile water for 4-6 times to obtain disinfected fruit; Step (2): Cut the disinfected fruit of step (1) into small pieces with a size of 5mm*5mm*3mm, inoculate and spread them on a modified MS medium, and then place the inoculated medium in a temperature of 25±1℃, a light intensity of 1500-2000 lux, and a light cycle of 12-16 hours per day to induce callus, and callus is formed after 20-28 days; Step (3): Cut the callus of step (2) into 1-2 mm particles, inoculate them into a liquid medium at an inoculation amount of 1:5 (g / mL), and then culture them under a temperature of 22±1℃ and a rotation speed of 130-140 rpm, and replace 1 / 2 of the culture solution every 7 days, and culture for 28-32 days; Step (4): Collect the supernatant of the culture of step (3), centrifuge it at a rotation speed of 2800-3500 rpm for 10-20 minutes, filter it through a filter membrane, centrifuge it at a centrifugal force of 80000-120000 g for 60-80 minutes under a condition of 3-5℃, resuspend the precipitate with a PBS buffer to obtain a forsythia exosome suspension.

2. The preparation method of forsythia exosome according to claim 1, characterized in that, The mass fraction of ethanol in step (1) is 75%; the mass fraction of the mercuric chloride solution is 0.1%. 3.The method of claim 1, wherein the forsythia exosome is prepared by the steps of, The inoculation density in step (2) is 8-10 pieces per bottle (250 mL triangular flask).

4. The method of claim 1, wherein the Forsythia exosome is prepared by the method comprising the steps of, The formula of the modified MS medium in step 2 is: 2.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, 6.5 g / L agar, 0.02-0.08 mg / L cobalt chloride, 0.5-2.0 mg / L sodium silicate, 0.1-0.5 mg / L titanium sulfate, and the pH value is adjusted to 5.

8. 5.The method of preparing for forsythia exosome according to claim 1, characterized in that, The induction rate of the callus in step (2) is more than 85%; the fresh weight of the callus induced from each gram of explant is not less than 3.2 g. 6.The method of preparing for forsythia exosome according to claim 1, characterized in that, The formula of the liquid medium in step (3) is: 2.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, 1.5 mg / L 2,4-D, 0.3 mg / L KT, 20 g / L glucose, 0.01-0.05 mg / L cobalt chloride, 0.3-1.5 mg / L sodium silicate, and 0.05-0.3 mg / L titanium sulfate. 7.The method of claim 1, wherein the forsythia exosome is prepared by the steps of, The protein concentration of the forsythia exosome suspension in step (4) is ≥0.8 mg / mL; the pore size of the filter membrane in step (4) is 0.22 μm.

8. The method for preparing Forsythia exosomes according to claims 1-7, characterized in that, The forsythia exosome suspension prepared by the preparation method of the forsythia exosome can be applied in an acne-removing repair face cream or an oil-controlling and dandruff-removing shampoo. 9.The method of claim 8, wherein the forsythia exosome is prepared by the steps of, The acne-removing repair face cream, according to the mass fraction, is composed of 10-14% forsythia exosome suspension, 0.3-0.8% peach kernel oil, 0.1-0.3% sophoramine, 8-12% squalane, 6-10% shea butter, 4-8% glycerol, 0.3-0.8% xanthan gum, and 55-70% water. 10.The method of claim 8, wherein the forsythia exosome is prepared by the steps of, The oil control and dandruff removal shampoo comprises, by mass fraction, 3-8% of forsythia suspensa exosome suspension, 1-5% of olea europaea extract, 1-3% of salix babylonica bark extract, 10-20% of sodium laureth sulfate, 0.5-1.5% of guar hydroxypropyltrimonium chloride, 0.3-0.8% of sodium chloride, 0.3-0.8% of phenoxyethanol and 70-80% of water.

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