Fermented artemisia apiacea oil with post-medical repair effect and application of fermented artemisia apiacea oil

Abstract

The invention provides a post-medical repair application of fermented artemisia apiacea oil. The fermented artemisia apiacea oil is prepared by the following steps: (a) carrying out supercritical CO2 extraction on artemisia apiacea medicinal materials; (b) fermenting, namely fermenting by adopting bifidus yeast; (c) separating to obtain fermented artemisia apiacea oil; wherein the bifidobacterium is selected from bifidobacterium adolescentis, bifidobacterium animalis subspecies, bifidobacterium animalis subspecies lactis, bifidobacterium bifidum, bifidobacterium breve, bifidobacterium longum subspecies longum or bifidobacterium longum subspecies infantis. The medical art is caused by dot matrix laser. The invention also relates to an application of the fermented artemisia apiacea oil in preparation of an external skin preparation with a post-medical repair effect.

Description

TECHNICAL FIELD

[0001] The present application relates to a yeast-fermented artemisia oil and its use in a repair cosmetic composition, wherein the yeast-fermented artemisia oil is obtained by fermentation in artemisia oil using a two-line yeast as a strain. BACKGROUND

[0002] Artemisia annua L. as a traditional Chinese medicine, first appeared in "Fifty-two Disease Treatments", and later was recorded in many herbal classics, widely distributed in Chongqing, Anhui, Hubei, Shandong and other places in China. Artemisia annua is used as a traditional Chinese medicine to treat fever and cold, which is recorded in "Shennong's Classic of Materia Medica", "Elbow Post- Emergency Prescription" and "Compendium of Materia Medica". Artemisia annua has the effects of clearing heat and resolving toxicity, cooling blood and invigorating the stomach, and contains flavonoids, polysaccharides, polyphenols, volatile oils and other bioactive components.

[0003] China has rich resources of Chinese herbal medicines, and there have been many research reports on cosmetic raw materials with significant skin care effects obtained by microbial fermentation of Chinese herbal medicines. However, the research on the fermentation of Artemisia annua is limited.

[0004] As the core technology of skin reconstruction, fractional laser shows significant efficacy in repairing acne scars, fading wrinkles and remodeling the epidermis. However, the controllable thermal damage to the skin barrier function during the treatment process has become a clinical concern. Fractional laser emits micrometer-sized beams to form micro-columnar thermal damage zones (MTZs) and construct a high-density grid-like micropore on the skin surface. These micropores penetrate the stratum corneum and reach the superficial dermis, and their thermal effect not only directly vaporizes local keratinocytes, but also destroys the lamellar arrangement of intercellular lipids (ceramides, cholesterol, free fatty acids) to disrupt the barrier integrity. Ceramides, as the main structural component of the lipid bilayer, their rupture leads to the loss of continuity of "grout" in the "brick wall structure" of the stratum corneum, and the physical protection function of the barrier is significantly reduced, which is manifested as a sharp rise in TEWL.

[0005] Interleukin 1 (IL-1), interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) are key proinflammatory cytokines. All three are involved in the first phase of the wound healing process, the inflammatory phase. IL-1β is an important member of the IL-1 family and has been the focus of much attention due to its important role in inflammation-related diseases. IL-1β has strong proinflammatory activity and can induce a variety of proinflammatory mediators, such as cytokines and chemokines. IL-1β has multiple functions. It has multiple effects on various cells and ultimately leads to a wide range of inflammatory events. TNF-α levels can rapidly increase when damaged by proinflammatory factors. IL-6, as the main mediator of the acute phase response, is continuously present in the formation of new tissues and remodeling processes.

[0006] The present application first explores the repair effect of fermented artemisia oil after, for example, fractional laser surgery and its application in cosmetic compositions. SUMMARY

[0007] In one aspect, the present application provides a post-medical cosmetic treatment repair application of fermented artemisia oil, which is prepared by the following method:

[0008] (a) Artemisia annua is extracted by supercritical CO2 extraction;

[0009] (b) fermentation, using Schizosaccharomyces fermenting;

[0010] (c) obtaining fermented artemisia oil after separation;

[0011] Among them, the Schizosaccharomyces is a bifidobacterium selected from: Bifidobacterium adolescentis, Bifidobacterium animalis subsp. animalis, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum subsp. longum or Bifidobacterium longum subsp. infantis.

[0012] In a preferred embodiment, the content of artemisia acid in the fermented artemisia oil is ≥0.03% by weight, the acid value of the fermented artemisia oil is ≤2.5 mg / g, the peroxide value of the fermented artemisia oil is ≤0.650 g / 100 g, and the density of the fermented artemisia oil is 0.9-0.95 g / cm 3 .

[0013] In a preferred embodiment, the post-medical cosmetic treatment is caused by fractional laser.

[0014] In a preferred embodiment, the repair includes relieving the changes in skin TEWL and / or SCH after medical cosmetic treatment.

[0015] In a preferred embodiment, the repair includes reducing skin inflammation. Preferably, the repair includes reducing the expression levels of genes selected from IL-1β, IL-6, or TNF-α.

[0016] On the other hand, the present invention relates to the application of fermented artemisia annua oil in the preparation of topical skin agents with post-medical aesthetic repair effects, characterized in that the fermented artemisia annua oil is prepared by the following method:

[0017] (a) Artemisia annua was extracted using supercritical CO2 extraction;

[0018] (b) Fermentation, using Bifida ferment lysate;

[0019] (c) Fermented artemisia oil was obtained after separation;

[0020] The bifida ferment lysate is a bifidobacterium, selected from: Bifidobacterium adolescentis, Bifidobacterium animalis subsp. animalis, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum subsp. longis, or Bifidobacterium longum subsp. infantis.

[0021] In a preferred embodiment, the content of fermented artemisia oil in the topical skin preparation is 0.001-100% by weight, preferably 0.01-5% by weight.

[0022] In a preferred embodiment, the topical skin agent is selected from: face cream, lotion, gel, toner, serum, face mask, eye cream, aerosol cleansing foam, spray, shower gel, or facial cleanser. Brief description of the attached figures

[0023] Figure 1 The images show the results of photographs taken at 0h, 12h, 24h, 36h, 48h, 3 days, 4 days, 5 days, and 7 days after AFL in the normal control group (without AFL) and mice. After fractional laser treatment, redness of the skin and pinholes from the fractional laser microneedles were visible. Re-epithelialization of the basal cell layer began after 24 hours, and the skin remained red during the inflammatory phase. New epidermal coverage was completed in 3 days, and the scab fell off with epidermal recovery on day 5. The wound was completely closed with no significant scarring on day 7.

[0024] Figure 2 The results show the effects of applying fermented artemisia oil twice daily (100 μL each time, 12 hours apart) after fractional laser treatment, with photographs taken on days 0, 12, 24, 36, 48, 3, 4, 5, and 7. Compared to the AFL group, the AFL + fermented artemisia oil group showed reduced skin redness at 24 hours, and from 36 hours to day 5, scabs formed and fell off, with less severe scab formation, indicating that fermented artemisia oil promotes epidermal recovery.

[0025] Figure 3The results of assessing TEWL changes in mice using the GPSkin device are shown. As shown in the figure, compared with the CON group, TEWL continued to increase after AFL, indicating that the epidermal barrier function was disrupted. Applying fermented artemisia oil after AFL significantly inhibited the increase of TEWL at 12, 24, and 36 hours. This regulatory effect did not show significant differences in TEWL measurement from 48 hours to day 7 due to the influence of crusting and repair processes. Therefore, fermented artemisia oil has a significant effect on improving epidermal barrier function.

[0026] Figure 4 The results of assessing stratum corneum water content (SCH) in mice using the GPSkin device are shown. As shown in the figure, compared with conjugation (CON), ALF resulted in skin edema, manifested as a sustained increase in SCH. However, applying fermented artemisia oil could improve the edema caused by ALF and promote skin repair.

[0027] Figure 5 This study demonstrated the effect of fermented artemisia annua oil on the gene expression levels of IL-1β, IL-6, and TNF-α. Pro-inflammatory cytokines interleukin-1β (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-alpha) play important roles in the early and late stages of inflammation, trauma, and wound healing. The inflammatory period is 1-3 days after fractional laser treatment, with the first day showing the most severe inflammation. Due to epidermal crusting and re-epithelialization in subsequent days, it was impossible to obtain complete and sufficient epidermal samples for testing; therefore, the experiment was conducted at the 24-hour time point. As shown in the figure, 24 hours after AFL, the expression levels of pro-inflammatory cytokines interleukin-1β (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-alpha) were significantly increased compared to the CON group, indicating an increase in inflammation levels. Conversely, compared to the ALF group, the expression levels of inflammatory factors were significantly decreased in the AFL + fermented artemisia annua oil group, indicating that fermented artemisia annua oil has anti-inflammatory effects.

[0028] Figure 6 Confocal micrographs of the normal control group (without AFL), the AFL group, and the AFL + fermented artemisia oil group are shown. As shown in the figure, the sections were stained with Oil Red O 7 days after fractional laser ablation. The normal skin stratum corneum of the CON group showed continuously distributed lipid-positive areas, while the lipid signal was completely absent in the AFL-treated areas. This indicates that laser ablation can disrupt the spatial continuity of the epidermal lipid layer. The AFL + fermented artemisia oil group significantly improved the lipid signal loss caused by fractional laser ablation and restored the spatial continuity of the epidermal lipid layer. Detailed Implementation

[0029] This invention is the first to discover that fermented artemisia oil obtained by fermenting artemisia oil with Diplosperidium brevicornu as a strain has a skin repair effect after fractional laser treatment, and is particularly suitable for the preparation of cosmetic compositions with skin repair effects.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar or equivalent to those described herein may be used to practice or test the invention, preferred methods and materials are described herein. For the purposes of this invention, the following terms are defined.

[0031] As used herein, the term "about" means a quantity, level, value, dimension, size, or amount that differs from that of a reference by as much as 30%, 20%, or 10%. Percentages used herein, unless otherwise stated, are by weight.

[0032] Throughout this specification and claims, unless otherwise required, the words “comprising” and its variations “containing” and “including” shall be understood to mean including the said whole or step, or a group of whole or steps, but not excluding any other whole or step, or other group of whole or steps.

[0033] Fermented artemisia oil

[0034] This invention uses fermented artemisia annua oil purchased from Hangzhou Shiguang Xinya Biotechnology Co., Ltd., commercially known as Bifida ferment lysate fermented artemisia annua oil. The artemisia annua oil of this invention is obtained by fermenting artemisia annua oil using Bifida ferment lysate as the inoculum. In this application, four different batches of fermented artemisia annua oil will be used for experiments: batch 1, batch 2, batch 3, and batch 4.

[0035] The fermented artemisia oil in this application is obtained by separating artemisia annua medicinal material through supercritical CO2 extraction and fermentation with Bifidobacterium bifida. The Bifidobacterium bifida strain is Bifidobacterium bifidum, including one of the following: Bifidobacterium adolescentis, Bifidobacterium animalis subsp. Animalis, Bifidobacterium animalis subsp. Lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum subsp. Longum, and Bifidobacterium longum subsp. infantis.

[0036] In a specific embodiment, the artemisinin acid content in the fermented artemisia oil of the present invention is ≥0.03% by weight, the acid value of the fermented artemisia oil is ≤2.5 mg / g, the peroxide value of the fermented artemisia oil is ≤0.650 g / 100g, and the density of the fermented artemisia oil is 0.9-0.95 g / cm³. 3 .

[0037] In embodiments of the present invention, the fatty acid composition of the fermented artemisia annua oil is greater than 90% by weight. In embodiments of the present invention, the relative content of linoleic acid in the fatty acid composition of the fermented artemisia annua oil is ≥35% by weight, preferably ≥45% by weight.

[0038] In an embodiment of the present invention, the content of artemisinin in the fermented artemisia oil is ≥0.03% by weight, preferably ≥0.05% by weight.

[0039] GC-MS analysis of fermented artemisia annua oil revealed a total of 54 compounds, including lipids, aldehydes, terpenes, sterols, vitamins, and others. Mass content analysis showed that fatty acid glycerides were the dominant compounds (93.4%-99.4%), with other trace components including squalene (0.01%-0.02%), vitamin E (0.02%-0.04%), and γ-sitosterol (0.08%-0.14%). Further relative mass content analysis revealed three aldehydes, including 2-heptenal, trans-2-decenal, and a high content of trans-2,4-decadienal (7.38%); six terpenoids, represented by β-caryophyllene (8.97%), β-serlingene, and caryophyllin; and three sterols, with γ-sitosterol (6.52%), campesterol (10.11%), and stigmasterol (5.36%) forming the sterol system. Vitamin E (3.99%) was the only vitamin component detected. Notably, the triterpenoid cycloartenol was the most abundant component at 40.27%, while α-amyrin and 24-methylenecycloartenol further enriched the diversity of terpenoids. Other components included organic acids such as hydroxyvaleric acid and sesquiterpene lactones such as artemisinin b (1.53%), collectively forming a complex component system.

[0040] In addition, fermented artemisia annua oil contains dozens of fatty acids. The saturated fatty acids are mainly palmitic acid (C16:0, 5.87%), stearic acid (C18:0, 3.49%), and behenic acid (C22:0, 0.749%). Oleic acid (C18:1n9c, 26.1%) is the dominant monounsaturated fatty acid. Among the polyunsaturated fatty acids, linoleic acid (C18:2n6c, 53%) is the most abundant component, followed by α-linolenic acid (C18:3n3, 0.11%), while γ-linolenic acid (C18:3n6) is present in low amounts (≤0.0026%). Furthermore, trace amounts of long-chain fatty acids such as arachidic acid (C20:0, 0.259%) and lignotaric acid (C24:0, 0.259%) were also detected in the fatty acid glycerides.

[0041] Skin external agent

[0042] The fermented artemisia oil of this invention can be used as an efficacy additive in topical skin agents to improve skin condition.

[0043] In some embodiments, the content of fermented artemisia oil in the topical skin preparation is 0.001-20% by weight, preferably 0.01-5% by weight.

[0044] In some embodiments, the concentration of fermented artemisia annua oil used is at least 0.01% by weight. In some embodiments, the concentration of fermented artemisia annua oil used is 0.01-1% by weight, preferably 0.01-0.5% by weight. In a preferred embodiment, the concentration of fermented artemisia annua oil used is 0.02-0.25% by weight. In some embodiments, the concentration of fermented artemisia annua oil used is 0.02-0.1% by weight. In a specific embodiment, the concentration of fermented artemisia annua oil used is 0.03% by weight. In some embodiments, the concentration of fermented artemisia annua oil used is 0.1-0.25% by weight.

[0045] In some embodiments, the topical skin agent is selected from: face creams, lotions, gels, toners, serums, face masks, eye creams, aerosols (cleansing foams), shower gels, and facial cleansers. Different dosages are added depending on the type of formulation.

[0046] The term "topical skin agent" is a general term encompassing all ingredients typically used on the external surface of the skin, such as cosmetic compositions. These cosmetic compositions can include basic cosmetics, facial makeup cosmetics, body cosmetics, hair care cosmetics, etc., with no specific restrictions on their dosage forms; they can be rationally selected according to different purposes. Depending on the dosage form and purpose, these cosmetic compositions may also contain different cosmetically permissible media or matrix excipients.

[0047] Topical skin preparations contain dermatologically acceptable carriers or mediators (e.g., lotions, creams, ointments, cleansers, etc.). Those skilled in the art can select carriers capable of dissolving or dispersing these components at the concentrations described above, based on common knowledge in the art. When using a carrier, it should not cause inactivation of fermented artemisinin oil and should not produce any adverse effects on the skin during application.

[0048] Those skilled in the art can select suitable carriers based on common knowledge and their ability to dissolve or disperse in the active component at the concentration most suitable for processing, such as water, alcohols, oils, etc.

[0049] The topical skin agents of the present invention can be in the form of topical application products that can be applied externally to the skin and can be prepared using common techniques known in the art. The carrier can have various practical forms, such as creams, dressings, gels, lotions, ointments, or liquids, including compositions that are applied and washed off, and materials incorporated into them using methods known in the art, such as dry or wet applicators, hydrogel matrices, or adhesive (or non-adhesive) patches. Preferably, the carrier is a gel or a moisture-enhancing lotion, or an applicator in dry or wet form.

[0050] Typical carriers include emulsions containing water and / or alcohols and emollients, where the emollients are, for example, oils and waxes of hydrocarbons, silicone oils, hyaluronic acid, fats or oils from plants, animals, or marine organisms, glyceryl ester derivatives, fatty acids, or fatty acid esters or alcohols or alcohol ethers, lanolin and its derivatives, polyols or esters, wax esters, sterols, phospholipids, etc., and generally include emulsifiers (nonionic, cationic, or anionic), although some emollients themselves have emulsifying properties. Additionally, these same components can be formulated into creams, gels, or solid bars by utilizing different proportions of their components and / or by incorporating thickeners such as gums or other forms of hydrophilic colloids.

[0051] The topical skin preparation of the present invention may include additional components commonly found in skin care compositions, such as emollients, skin conditioners, emulsifiers, preservatives, antioxidants, fragrances, chelating agents, etc., as long as they are physically and chemically compatible with other components in the topical skin preparation and do not affect the efficacy of the fermented artemisia oil of the present invention.

[0052] In some embodiments of the topical skin formulation of the present invention, one or more preservatives may be used. Suitable preservatives include p-hydroxyacetophenone, C1-C4 alkyl p-hydroxybenzoate, and phenoxyethanol. Based on the total weight of the composition, the amount of preservative used is from about 0.5 to about 2% by weight, preferably from about 0.5 to 1% by weight.

[0053] In one example of the topical skin preparation of the present invention, one or more antioxidants may be used. Suitable antioxidants include butylated hydroxytoluene (BHT), ascorbyl palmitate (BHA), butylated hydroxyanisole, phenyl-α-naphthylamine, hydroquinone, propyl gallate, nordihydroguaiac acid, vitamin E or a derivative of vitamin E, vitamin C and its derivatives, calcium pantothenate, green tea extract, and mixed polyphenols, as well as mixtures of the substances described above. The antioxidant used is approximately 0.02 to 0.5% by weight of the total weight of the composition, more preferably in the range of approximately 0.002 to 0.1% by weight.

[0054] In one example of the topical skin preparation of the present invention, one or more emollients may be used, which, by virtue of their ability to remain on the skin surface or in the stratum corneum, act as lubricants to reduce exfoliation and improve the appearance of the skin. Typical emollients include fatty esters, fatty alcohols, mineral oils, polyether silicone copolymers, and the like. Examples of suitable emollients, not limited to, include polypropylene glycol (“PPG”)-15 octadecyl ether, PPG-10 hexadecyl ether, Steareth-10, Oleth-8, PPG-4 dodecyl ether, vitamin E acetate, lanolin, cetyl alcohol, cetearyl alcohol ethylhexanoate, cetearyl alcohol, glyceryl stearate, octyl hydroxystearate, dimethyl polysiloxane, and combinations thereof. Cetyl alcohol, cetearyl alcohol ethylhexanoate, cetearyl alcohol, glyceryl stearate, and combinations thereof are preferred. When used, the emollient is applied in an amount ranging from about 0.1% to about 30% by weight, preferably from about 1% to about 30% by weight, based on the total weight of the composition.

[0055] In one example of the topical skin preparation of the present invention, one or more moisturizers may be used. Moisturizers, also known as humectants, help enhance the effectiveness of emollients, reduce exfoliation, stimulate the removal of scaly skin, and improve skin feel. Polyols may be used as moisturizers, including, but not limited to, glycerin, polyalkylene glycols, alkylene polyols and their derivatives, including butylene glycol, propylene glycol, dipropylene glycol, glycerol, polyethylene glycol and their derivatives, sorbitol, hydroxypropyl sorbitol, hexanediol, 1,3-dibutylene glycol, 1,2,6-hexanetriol, ethoxylated glycerin, propoxylated glycerin, and combinations thereof. When used, the amount of moisturizer is from about 0.1% to about 20% by weight, preferably from about 1% to about 15% by weight, based on the total weight of the composition.

[0056] In one example of the topical skin formulation of the present invention, one or more emulsifiers may be used. The emulsifier may be used within a range of effective stable amounts. Preferably, the emulsifier is used at an amount of about 1.0 to about 10.0% by weight, more preferably about 3.0 to about 6.0% by weight, based on the total weight of the composition. Any emulsifier compatible with the components in the composition may be used. Suitable emulsifiers include stearic acid, cetyl alcohol, glyceryl stearate, lecithin, octadecyl alcohol, Steareth-2, Steareth-20, acrylate / C10-30 alkanol acrylate crosspolymers, and combinations thereof.

[0057] In one example of the topical skin formulation of the present invention, one or more pH adjusters may be used. Beneficial pH adjusters in the topical skin formulation of the present invention include tromethamine. When used, the amount of pH adjuster is approximately 0.1 to approximately 2% by weight, preferably approximately 0.1 to approximately 1% by weight, based on the total weight of the composition.

[0058] In one specific embodiment of the invention, the topical skin agent comprises an acrylate / C10-30 alkanol acrylate crosspolymer, glycerin, p-hydroxyacetophenone, glyceryl stearate and lecithin, cetearyl alcohol, cetearyl ethylhexanoate, tromethamine or a combination thereof.

[0059] In some embodiments of the present invention, the amount of fermented artemisia oil in the topical skin preparation is 0.001%-20% (w / w), preferably 0.01%-20% (w / w), more preferably 0.01%-10% (w / w), and most preferably 0.1%-5% (w / w).

[0060] In one specific embodiment of the present invention, the amount of fermented artemisia oil in the topical skin preparation is 0.1-5% by weight. In a preferred embodiment, the amount of fermented artemisia oil in the topical skin preparation is 0.13% by weight.

[0061] Example

[0062] The present invention will be further illustrated below with reference to specific embodiments. It is important to note that these embodiments are for illustrative purposes only and should not be construed as limiting the scope of protection of the invention. Those skilled in the art can make non-essential improvements and adjustments based on the above description of the invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, all percentages and parts are by weight.

[0063] Example 1: Component Detection of Fermented Artemisia annua Oil

[0064] (The method refers to the normalization method, the third method in GB 5009.168-2016)

[0065] Weigh 0.1 g to 10 g of fermented artemisia annua oil into a 250 mL flat-bottomed flask, add approximately 100 mg of pyrogallol, a few boiling chips, and 2 mL of 95% ethanol, and mix well. Add 10 mL of hydrochloric acid solution and mix well. Place the flask in a water bath at 70℃ to 80℃ for 40 min to hydrolyze. Shake the flask every 10 min to incorporate particles adhering to the flask wall into the solution. After hydrolysis, remove the flask and cool to room temperature. Add 8 mL of 2% sodium hydroxide methanol solution to the hydrolysate, connect a reflux condenser, and reflux in a water bath at 80℃ ± 1℃ until the oil droplets disappear. Add 7 mL of 15% boron trifluoride methanol solution from the top of the reflux condenser, and continue reflux in a water bath at 80℃ ± 1℃ for 2 min. Rinse the reflux condenser with a small amount of water. Stop heating, remove the flask from the water bath, and quickly cool to room temperature. Add 10 mL to 30 mL of n-heptane accurately, shake for 2 min, then add saturated sodium chloride aqueous solution and allow to stand for separation. Pipette approximately 5 mL of the upper n-heptane extract into a 25 mL test tube, add approximately 3 g to 5 g of anhydrous sodium sulfate, shake for 1 min, let stand for 5 min, and then pipette the upper solution into a sample vial for analysis.

[0066] Chromatographic determination: Single fatty acid methyl ester standard solutions and mixed fatty acid methyl ester standard solutions were injected separately into the gas chromatograph for qualitative analysis of the chromatographic peaks. Capillary column: Poly(dicyandipropylsiloxane) strongly polar stationary phase, column length 100 m, inner diameter 0.25 mm, film thickness 0.2 μm. Injector temperature: 270 ℃. Detector temperature: 280 ℃. Temperature program: Initial temperature 100 ℃, hold for 13 min; 100 ℃ ~ 180 ℃, ramp rate 10 ℃ / min, hold for 6 min; 180 ℃ ~ 200 ℃, ramp rate 1 ℃ / min, hold for 20 min; 200 ℃ ~ 230 ℃, ramp rate 4 ℃ / min, hold for 10.5 min. Carrier gas: Nitrogen. Split ratio: 100:1. Injection volume: 1.0 μL.

[0067] The percentage of a specific fatty acid in the total fatty acids in a sample, Yi, is calculated using the following formula. The content of a given component i is calculated by measuring the percentage of the corresponding peak area relative to the sum of the peak areas of all components:

[0068]

[0069] In the formula:

[0070] Y i —The percentage of a specific fatty acid in the total fatty acids in the sample, expressed as %.

[0071] A Si ———Peak areas of each fatty acid methyl ester in the sample test solution;

[0072] F FAMEi-FAi ——— The coefficient for the conversion of fatty acid methyl ester i into fatty acids;

[0073] ∑A Si ———The sum of the peak areas of each fatty acid methyl ester in the sample test solution.

[0074] The result should be rounded to 3 significant figures.

[0075] Test results show that batch 2 of fermented artemisia annua oil mainly contains oily components, with linoleic acid (cis-9,12-octadecadienoic acid) accounting for approximately 50%, which is considered a medium-to-high level among plant oils. Linoleic acid, an Omega-6 polyunsaturated fatty acid, is an essential component that the human body cannot synthesize on its own. Studies have found that linoleic acid has various effects in skincare, including improving the skin barrier, soothing inflammation, and anti-oxidation. The high linoleic acid content in fermented artemisia annua oil provides a material basis for its skincare effects. As the main component of fermented artemisia annua oil, when used as a product testing parameter, a relative content of linoleic acid ≥35% in the fatty acid composition of fermented artemisia annua oil meets the requirements. The results are shown in Tables 1-2.

[0076] Table 1

[0077]

[0078] Table 2: Fatty acid composition and relative content in fermented Artemisia annua oil

[0079]

[0080] HPLC method for determining the content of artemisinin in fermented artemisia annua oil

[0081] Experimental apparatus:

[0082] 1. Waters ARC high-performance liquid chromatograph with diode array detector (Waters);

[0083] 2. XS205 analytical balance (METTLER TOLEDO);

[0084] 3. KQ-800DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.)

[0085] Chromatographic conditions:

[0086] Column: Agilent ZORBAX SB-C18, 250 mm × 4.6 mm × 5 μm;

[0087] Mobile phase: Acetonitrile (90%) - 0.1% phosphoric acid aqueous solution (10%);

[0088] Flow rate: 1.0 mL / min;

[0089] Column temperature: 30℃;

[0090] Wavelength: 220 nm;

[0091] Injection volume: 10 μL.

[0092] Testing steps:

[0093] Preparation of artemisinin standard stock solution (400 mg / L): Weigh 20 mg of artemisinin standard (Nature Standard, purity ≥98.0%) (accurate to 0.01 mg), dissolve in methanol and dilute to 50 mL.

[0094] Preparation and determination of artemisinin standard working solution: Accurately transfer 1 mL of artemisinin standard stock solution and dilute to 10 mL with methanol to obtain a standard working solution with a concentration of 40 mg / L. Perform the determination according to the chromatographic conditions described above.

[0095] Sample processing and determination:

[0096] Weigh 0.25 g (accurate to 0.1 mg) of the sample into a 10 mL stoppered colorimetric tube, add 8 mL of methanol, and extract by sonication for 20 min. Make up to volume with methanol, shake well, and filter the liquid fraction through a 0.45 μm filter membrane. Then, determine the chromatographic content according to the above conditions. Calculate the artemisinic acid content in the sample using the single-point external standard method.

[0097] The results are shown in Table 3.

[0098] Table 3

[0099]

[0100] As shown in the table above, all batches of fermented artemisia annua oil contained artemisinic acid, a characteristic component of artemisia annua, with a content greater than 0.03% by weight, which can be used as a characteristic indicator of fermented artemisia annua oil. Artemisinic acid is an important sesquiterpene compound extracted from artemisia annua (Artemisia annua), possessing various biological activities, including antimalarial activity, antipyretic effects, antibacterial and anti-inflammatory activities, and anti-glycation effects. The presence of a certain amount of artemisinic acid in fermented artemisia annua oil may be one of the reasons why fermented artemisia annua oil exhibits new therapeutic effects.

[0101] Acid value, peroxide value, and density testing

[0102] Detection method:

[0103] The acid value test method is based on GB / T 2716, the peroxide value test method is based on GB 5009.227-2023, and the density test method is based on GB / T 611.

[0104] The test results are shown in Table 4.

[0105] Table 4

[0106]

[0107] Example 2: Post-medical aesthetic treatment application

[0108] Experimental materials:

[0109] Fermented Artemisia annua oil: purchased from Hangzhou Shiguang Xinya Biotechnology Co., Ltd.;

[0110] Mice with the SC57BL / 6J genetic background, Shanghai Jiao Tong University.

[0111] Experimental apparatus:

[0112] The ablative fractional laser (AFL) was performed using the Shanghai Laser LJL35-CS carbon dioxide laser therapy machine.

[0113] Test sample:

[0114]

[0115] Experimental methods:

[0116] This experiment used 50-day-old C57BL / 6J mice in the rudimentary hair stage. After shaving the back hair of the mice, depilatory cream was applied. The next day, an ablative fractional laser (AFL) was used on the back of the mice to simulate the effect of clinical laser treatment. The single-point energy was 10 mJ, the interval was 0.7 mm, and the area of ​​a single fractional laser was 15 mm × 15 mm. The mice were randomly divided into three groups as follows: (I) Normal control group (no fractional laser, CON); (II) AFL control group (fractional laser, AFL group); (III) AFL and 100 μl fermented artemisia oil application group (AFL + fermented artemisia oil). After fractional laser treatment on the back of the mice, fermented artemisia oil was applied twice daily, 100 μL each time, for 7 consecutive days. Mice were photographed and their TEWL and SCH levels were assessed using a GPSkin device at 0h, 12h, 24h, 36h, 48h, 3d, 4d, 5d, and 7d after AFL.

[0117] frozen sections of skin tissue

[0118] 1. Pre-cooling the machine: Set the object temperature (OT) of the cryostat to -22°C and the chamber temperature (CT) to -20°C in advance.

[0119] 2. Tissue fixation: Isolated mouse back skin tissue was fixed in 4% PFA for 24 hours and washed with PBS for 10 minutes three times.

[0120] 3. 30% sucrose solution: Place the tissue in a 30% sucrose solution (prepared with PBS) at 4°C and shake at 4°C for 24 hours.

[0121] 4. OCT: Place the tissue in an OCT chamber at 4°C overnight.

[0122] 5. Embedding: Immerse the tissue in a plastic embedding cassette containing OCT, remove air bubbles, and then place the plastic embedding cassette horizontally at -20°C. After the OCT solidifies, store it at -80°C as soon as possible.

[0123] 6. Sectioning: Remove the OCT block from the embedding cassette and section it. The thickness of the section should be 15-20 μm after Oil Red O staining.

[0124] Oil Red O Staining

[0125] Reagent preparation

[0126] 1. Prepare Oil Red O stock solution by mixing 0.5 g of Oil Red O powder with 100 mL of 99% isopropanol. Store at 4°C protected from light.

[0127] 2. Preparation of Oil Red O working solution: Dilute the Oil Red O staining stock solution with ddH2O at a ratio of 3:2, i.e., 60 ml Oil Red O staining solution + 40 ml ddH2O. Filter through a 0.2 μm sterile filter / filter paper (or 8000 rpm / 2 min, let stand for 10 min), and allow to stand at room temperature for >10 min. The working solution should be wine-red and free of precipitate. Prepare fresh before use and store at 4°C protected from light.

[0128] 3. Preparation of 60% isopropanol: 30 ml isopropanol + 20 ml ddH2O, store at 4℃.

[0129] Oil Red O staining

[0130] Frozen tissue sections were carefully washed three times in PBS for five minutes each time.

[0131] In a fume hood, fix the sections in 4% PFA for 30 minutes, discard the solution, and wash twice with PBS for 5 minutes each time.

[0132] 3. Place the slices in 60% isopropanol for 5 minutes to facilitate staining with Oil Red O working solution.

[0133] 4. Immerse the slides in Oil Red O working solution for 15-30 minutes for staining.

[0134] 5. Wash in 60% isopropanol for 1 minute to remove background staining.

[0135] 6. Rinse with running water for 5 minutes to remove any residual isopropyl alcohol.

[0136] 7. Hematoxylin staining: Filter hematoxylin through a 0.22 μm filter membrane. Immerse the tissue in the hematoxylin staining solution for 5 minutes, taking care to avoid light. Rinse with running water for 5 minutes to remove any excess stain.

[0137] 8. Differentiation: Immerse in hydrochloric acid and ethanol for 10 seconds (until the slices turn red and the nuclei are clear). Blueing: Rinse with running water for 15 minutes.

[0138] 9. Mounting and Observation: Blot dry, add 20 μL of glycerol-gelatin mounting medium (melted in a water bath beforehand) to each tissue, cover with a coverslip, taking care to avoid air bubbles. Observe under a bright field microscope (Nikon Eclipse 80i) with a 20x objective lens.

[0139] RNA extraction and reverse transcription

[0140] This study used the Trizol method to extract total RNA. Throughout the experiment, consumables such as RNA-free pipette tips, centrifuge tubes, and EP tubes were used. Before the operation, the laboratory table and instruments were thoroughly wiped with a cleaning agent containing RNase / DNase inactivating agent (Labshark, 130114007) to strictly prevent RNase contamination.

[0141] 1. Skin sample / cell processing:

[0142] A. Epidermal Sample Preparation: After removing the subcutaneous fat layer, the epidermis was laid flat with the surface facing upwards on a 0.25% trypsin solution (digested at 37°C for 1 h) to separate the epidermis from the dermis. After mechanical separation with ophthalmic scissors, the tissue was lysed at room temperature for 20 min (with intermittent shaking) at a ratio of 50-100 mg tissue / 500 μL TRIZOL. The volume of the tissue sample was strictly controlled to be less than 10% of the TRIZOL volume.

[0143] B. Whole Skin Tissue: After obtaining whole skin tissue, use ophthalmic scissors to cut the tissue into small pieces as much as possible, then use a tissue homogenizer to further break it up. Add 500 μL of TRIZOL to the whole skin tissue and incubate at room temperature for 20 min to fully lyse, shaking the tissue multiple times during this period. Ensure that the sample volume does not exceed 10% of the TRIZOL volume.

[0144] C. Cells: After aspirating the cell culture medium, gently wash three times with PBS for 5 minutes each time. Then add 300 μL of TRIZOL to each well and lyse at room temperature for 20 minutes. After the cells have completely detached from the culture dish, transfer them to 1.5 mL centrifuge tubes for subsequent RNA extraction.

[0145] 2. Chloroform phase separation: Add chloroform to TRIZOL liquid at a 1:1 volume ratio, shake vigorously for 15 seconds to form an emulsion, and let stand at room temperature for 10 min. Centrifuge at 12000 g for 15 min at 4℃ to separate the phases.

[0146] 3. RNA precipitation: Pipette 250 μL of supernatant into a new EP tube, add isopropanol at a 1:1 volume ratio, gently invert to mix, and incubate at room temperature for 10 min after thorough mixing with isopropanol. Centrifuge at 12000 g for 15 min at 4℃ to obtain the precipitate.

[0147] 4. Ethanol washing: Discard the supernatant, add 1 mL of 75% ethanol (RNase-free ethanol:DEPC water = 3:1) to resuspend the precipitate, gently pipette, centrifuge at 7500 g for 5 min at 4°C, repeat the washing twice, and a white precipitate can be seen after centrifugation.

[0148] 5. RNA dissolution: Remove residual ethanol with a pipette, and air dry for 20 min to evaporate the remaining ethanol until the white RNA precipitate becomes clear; add 20-50 μL of DEPC water preheated to 55°C, and then place in a 4°C refrigerator for 30 min to dissolve.

[0149] 6. Quality Control: After mixing, use a Nanodrop instrument to test purity. After zeroing with DEPC water, test RNA concentration. Note that the RNA solution should be thoroughly mixed before testing. The A260 / 280 ratio should be close to 2.0, and the A260 / 230 ratio should be greater than 2.0. Qualified samples should be used immediately for cDNA synthesis or frozen at -80℃.

[0150] 7. Reverse transcription was performed using the SuperScript First-Strand Synthesis Kit (Invitrogen).

[0151] a. Prepare the reactants according to the following proportions, and gently mix the liquid with a shaker.

[0152]

[0153] b. After the first step of the reaction, add 4 μl of 5×HiScript III qRT SuperMix to each tube of reaction mixture. After low-speed centrifugation, set the PCR instrument program as follows: maintain 37℃ for 15 min to complete reverse transcription in the first stage, followed by inactivation of reverse transcriptase at 85℃ for 5 sec. Aliquot the cDNA product into sterile EP tubes, label them, and store them at -20℃ for a short period.

[0154] Real-time quantitative PCR (RT-PCR)

[0155] 1. The primers for real-time PCR are as follows:

[0156] Real-time PCR primer sequences

[0157]

[0158] cDNA template normalization: Adjust the cDNA concentration according to the expression abundance of the target gene to ensure that the 18S internal reference Ct value is controlled within the 8-13 cycle range.

[0159] Amplification system construction: The reaction system was prepared according to the manufacturer's instructions using SYBR Green premixed buffer (FastStart). Three replicates were used in a 96-well qPCR plate. After amplification, the relative expression level of the target gene was calculated using the ΔΔCt method.

[0160] RT-PCR reaction system

[0161]

[0162] 4. Apply the sealing film and use a scraper to remove air bubbles.

[0163] 5. After centrifuging the 5.96-well plates at 1000 rpm for 1 min, the reaction was carried out on a Varioskan LUX machine.

[0164] RT-PCR reaction conditions

[0165]

[0166] Experimental results:

[0167] Table 5 shows the experimental group information.

[0168] Table 5

[0169]

[0170] Figure 1 The results of taking pictures at 0h, 12h, 24h, 36h, 48h, 3d, 4d, 5d and 7d after fractional laser imaging are shown.

[0171] Figure 2 The image shows the results of applying fermented artemisia oil after fractional laser treatment, 100 μL twice daily, 12 hours apart, for 7 consecutive days. Images were taken at 0h, 12h, 24h, 36h, 48h, 3d, 4d, 5d, and 7d after applying 100 μL of fermented artemisia oil.

[0172] like Figures 1-2 As shown, after fractional laser treatment, the skin was red and showed puncture marks from the fractional laser microneedles. After 24 hours, the basal cell layer initiated re-epithelialization, and the skin remained red during the inflammatory phase. New epidermal growth was completed within 3 days, and the scab fell off on day 5 along with epidermal recovery. The wound completely closed on day 7 with no significant scarring. Compared to the AFL group, the AFL + fermented artemisia oil group showed reduced skin redness at 24 hours, and from 36 hours to day 5, scabs formed and fell off, with less pronounced scab formation, indicating that fermented artemisia oil promoted epidermal recovery.

[0173] Figure 3 The results show the changes in TEWL in mice at 12h, 24h, 36h, 48h, 5d, and 7d, as assessed using the GPSkin device.

[0174] Figure 4 The results show the changes in SCH (stratum corneum water content) in mice at 12h, 24h, and 36h, as assessed using the GPSkin device.

[0175] Combination Figures 3-4 The results showed that AFL led to an increase in TEWL and SCH, while applying fermented artemisia oil could alleviate the phenotypic changes.

[0176] Figure 5 This study demonstrated the effects of fermented artemisia annua oil on the gene expression levels of IL-1β, IL-6, and TNF-α. The results showed that AFL (artemisia annua fermentation oil) led to an increase in the expression levels of IL-1β, IL-6, and TNF-α in the epidermis. Fermented artemisia annua oil significantly reduced the gene expression levels of IL-1β, IL-6, and TNF-α, alleviating skin inflammation.

[0177] Figure 6 The image shows a photograph of the sebum layer after AFL application followed by fermented artemisia oil. The results indicate that applying fermented artemisia oil after AFL application can restore the skin's sebum layer and increase lipid levels.

[0178] The fermented artemisia oil of this invention can be used as a functional additive in topical skin agents, preferably cosmetic compositions, including but not limited to the preparation of products in dosage forms such as creams, lotions, gels, toners, serums, masks, eye creams, aerosols (cleansing foams), shower gels, and facial cleansers. The weight percentage of fermented artemisia oil in the topical skin agent is 0.0001%-100% (w / w). A preferred weight percentage is 0.001%-20% (w / w). A more preferred weight percentage is 0.001%-5% (w / w). The most preferred weight percentage is 0.01%-5% (w / w).

[0179] The following are specific examples of the application of fermented artemisia annua oil in topical skin preparations, along with the formulations and preparation methods of these dosage forms. Specific applications are as follows:

[0180] Application Example 1: Preparation of Face Cream

[0181]

[0182] Application Example 2: Emulsion Preparation

[0183]

[0184] Application Example 3: Preparation of Eye Cream

[0185]

[0186] Application Example 4: Preparation of Facial Masks

[0187]

[0188] Application Example 5: Preparation of Serum

[0189]

[0190] Application Example 6: Preparation of Essential Oils

[0191]

Claims

1. The application of fermented artemisia oil in post-medical aesthetic procedures, characterized by: The fermented artemisia oil was prepared by the following method: (a) Artemisia annua was extracted using supercritical CO2 extraction; (b) Fermentation, using Bifida ferment lysate; (c) Fermented artemisia oil was obtained after separation; The bifida ferment lysate is a bifidobacterium, selected from: Bifidobacterium adolescentis, Bifidobacterium animalis subsp. animalis, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum subsp. longis, or Bifidobacterium longum subsp. infantis.

2. The application as described in claim 1, characterized in that, The fermented artemisia oil contains artemisinin acid at a content ≥0.03% by weight, has an acid value ≤2.5 mg / g, a peroxide value ≤0.650 g / 100g, and a density of 0.9-0.95 g / cm³. 3 .

3. The application as described in claim 1, characterized in that, The cosmetic procedure described was performed using fractional laser.

4. The application as described in claim 1, characterized in that, The repair includes alleviating changes in skin TEWL and / or SCH after cosmetic procedures.

5. The application as described in claim 1, characterized in that, The repair includes reducing skin inflammation.

6. The application as described in claim 5, characterized in that, The repair involves reducing the expression levels of genes selected from IL-1β, IL-6, or TNF-α.

7. Application of fermented artemisia annua oil in the preparation of topical skin agents with post-medical aesthetic repair effects, wherein the fermented artemisia annua oil is prepared by the following method: (a) Artemisia annua was extracted using supercritical CO2 extraction; (b) Fermentation, using Bifida ferment lysate; (c) Fermented artemisia oil was obtained after separation; in, The Bifida Ferment Lysate is a Bifidobacterium, selected from: Bifidobacterium adolescentis, Bifidobacterium animalis subsp. animalis, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum subsp. longis, or Bifidobacterium longum subsp. infantis.

8. The application as described in claim 7, characterized in that, The content of fermented artemisia oil in topical skin preparations is 0.001-100% by weight.

9. The application as described in claim 7, characterized in that, Artemisia annua oil is present in pharmaceuticals or topical skin preparations at a concentration of 0.01-5% by weight.

10. The application as described in claim 7, characterized in that, The topical skin agent is selected from: face cream, lotion, gel, toner, serum, face mask, eye cream, aerosol cleansing foam, spray, shower gel, or facial cleanser.

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