Preparation method of artemisia annua extract and application thereof in cosmetics

Abstract

The application provides a preparation method of artemisia annua extract and application thereof in cosmetics, and belongs to the technical field of plant extraction. The whole artemisia annua is used as raw material, mixed with a eutectic solvent, sequentially subjected to high-pressure pulse electric field extraction and ultrasonic treatment, centrifuged to collect supernatant and diluted with water, and then purified through a ceramic membrane and a macroporous adsorption resin to obtain the artemisia annua extract. In the application, the eutectic solvent can effectively soften the plant cell wall and enhance the penetrating power of the solvent, and in combination with the irreversible electric pore effect of the high-pressure pulse electric field, the intracellular active ingredients are rapidly released, the extraction condition is mild, and the degradation of heat-sensitive components such as artemisinin is avoided. The ceramic membrane traps macromolecular allergenic proteins and plant waxes, and the X-5 type macroporous adsorption resin selectively adsorbs active ingredients, so that the purity of the extract is improved. The artemisia annua extract has the effects of mosquito repellent, bacteriostasis, soothing and anti-inflammation, has good safety, and is suitable for application in low-sensitivity products.

Description

Technical Field

[0001] This invention relates to the field of plant extraction technology, specifically to a method for preparing Artemisia annua extract and its application in cosmetics. Background Technology

[0002] Artemisia annua ( Artemisia annua Artemisia annua (L.) is an annual herb belonging to the genus Artemisia in the family Asteraceae. It is a traditional Chinese medicine, used internally for its heat-clearing, detoxifying, malaria-treating, and stomach-strengthening properties, and also has good efficacy when used externally. Artemisia annua contains abundant active ingredients, mainly including artemisinin and its derivatives, flavonoids, volatile oils, polysaccharides, and coumarins. In recent years, Artemisia annua extract has received widespread attention due to its significant antibacterial, anti-inflammatory, antioxidant, and immunomodulatory biological activities.

[0003] However, existing technologies for preparing Artemisia annua extract still have many shortcomings. In terms of extraction processes, traditional methods mainly include water extraction, steam distillation, organic solvent extraction, ultrasonic extraction, and supercritical fluid extraction. Among these, water extraction and alcohol extraction have relatively low extraction efficiency, with significant loss of active ingredients during the extraction process. While traditional organic solvent extraction is widely used, it often only selectively extracts certain types of components, making it difficult to achieve simultaneous and efficient extraction of multiple active ingredients from Artemisia annua. Although supercritical fluid extraction can obtain high-purity extracts, it requires large equipment investment and has high operating costs, hindering large-scale application. Furthermore, extracts obtained by the above methods often contain many impurities, including allergenic proteins, which can easily cause irritation when used directly in cosmetics.

[0004] Therefore, developing a preparation method that can efficiently enrich the effective components of Artemisia annua, effectively remove allergenic impurities, and whose extract itself has the triple effects of antiseptic, mosquito repellent, and soothing is of great significance for simplifying cosmetic formulations, reducing production costs, and improving product safety and user experience. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing Artemisia annua extract and its application in cosmetics. The Artemisia annua extract obtained by this invention has the characteristics of mosquito repellency, antibacterial properties, soothing effect, and low allergenicity.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing Artemisia annua extract, comprising: crushing whole Artemisia annua plants, mixing with a eutectic solvent, extracting under a high-voltage pulsed electric field, ultrasonicating, centrifuging, collecting the supernatant, mixing with water, and purifying sequentially through a ceramic membrane and a macroporous adsorption resin to obtain Artemisia annua extract; wherein the eutectic solvent is composed of choline chloride, benzoic acid and water.

[0007] Preferably, the method for preparing the eutectic solvent includes: heating and melting choline chloride and benzoic acid, then mixing them with water to obtain the eutectic solvent.

[0008] Preferably, the molar ratio of choline chloride to benzoic acid is (1.5-2.5):1, and the water is 12%-17% of the mass of the eutectic solvent.

[0009] Preferably, the mass ratio of the whole plant Artemisia annua to the eutectic solvent is 1:(10-15).

[0010] Preferably, the electric field strength of the high-voltage pulsed electric field is 5-8 kV / cm, the pulse width is 3-7 μs, and the number of pulses is 50-60.

[0011] Preferably, the ultrasonic treatment is performed at a frequency of 22-28 kHz, a temperature of 30-40 °C, and a time of 30-40 min.

[0012] Preferably, the volume ratio of the supernatant to water is 1:(3-5).

[0013] Preferably, the macroporous adsorption resin is an X-5 type macroporous adsorption resin.

[0014] The present invention also provides an extract of Artemisia annua prepared according to the above preparation method.

[0015] The present invention also provides an application of the above-described Artemisia annua extract in any one or more of the following: (1) Application in the preparation of mosquito repellent products; (2) Application in the preparation of antibacterial products; (3) Application in the preparation of soothing and anti-inflammatory products; (4) Application in the preparation of hypoallergenic cosmetics.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention employs a eutectic solvent as the extraction solvent, combined with a high-voltage pulsed electric field and ultrasonic treatment to achieve efficient dissolution of various active ingredients. The benzoic acid groups in the eutectic solvent soften the lignocellulose in plant cell walls, enhancing the solvent's penetration into cells; simultaneously, its excellent conductivity efficiently transmits pulsed electric field energy, strengthening the irreversible damage to the cell membrane caused by the electroporation effect. This allows intracellular active ingredients to be rapidly released into the solvent under the drive of the electric field and the influence of the concentration gradient. Combined with ultrasonic treatment, this further shortens the extraction time and avoids the degradation of heat-sensitive components such as artemisinin.

[0017] This invention combines ceramic membrane purification with macroporous adsorption resin purification methods, improving the safety, purity, and bioactivity of Artemisia annua extract. The ceramic membrane purification process traps large molecular sensitizing components that cause skin irritation and allergies; the X-5 type macroporous adsorption resin selectively adsorbs active ingredients, enhancing the extract's activity in mosquito repellency, antibacterial properties, and soothing anti-inflammatory effects.

[0018] This invention discloses Artemisia annua extract, which possesses triple activities of mosquito repellency, antibacterial activity, and anti-inflammatory and soothing effects. It exhibits excellent inhibitory effects on common skin pathogens such as Propionibacterium acnes, Malassezia, and Staphylococcus aureus, and can inhibit the release of inflammatory factors. It achieves a repellency rate of over 90% against Aedes albopictus. When applied to mosquito repellent products and repair cosmetics, it can reduce the need for additional preservatives, soothing agents, and mosquito repellent adjuvants, simplifying the formula components, reducing the risk of compounding conflicts between multiple raw materials, and improving the stability of the product formula. Detailed Implementation

[0019] This invention provides a method for preparing Artemisia annua extract, comprising: crushing whole Artemisia annua plants, mixing with a eutectic solvent, extracting under a high-voltage pulsed electric field, ultrasonicating, centrifuging, collecting the supernatant, mixing with water, and purifying sequentially through a ceramic membrane and a macroporous adsorption resin to obtain Artemisia annua extract; wherein the eutectic solvent is composed of choline chloride, benzoic acid and water.

[0020] This invention utilizes the whole plant of Artemisia annua, improving resource utilization and enabling complementary active ingredients from different tissues, resulting in extracts exhibiting broader-spectrum biological activity in mosquito repellency, antibacterial properties, and soothing anti-inflammatory effects. Furthermore, using the whole plant as raw material eliminates the need for stem and leaf separation, reducing labor costs.

[0021] In this invention, the method for preparing the eutectic solvent preferably includes: heating and melting choline chloride and benzoic acid, then mixing them with water to obtain the eutectic solvent; the molar ratio of choline chloride and benzoic acid is preferably (1.5-2.5):1, more preferably 2:1; the water is preferably 12%-17% of the mass of the eutectic solvent, more preferably 15%; the mass ratio of the whole plant Artemisia annua to the eutectic solvent is preferably 1:(10-15), more preferably 1:12.

[0022] The eutectic solvent of this invention can efficiently dissolve various active ingredients in Artemisia annua, such as artemisinin, flavonoid glycosides, and volatile oils. The benzoic acid groups soften the lignocellulose in plant cell walls, enhancing the penetration of the eutectic solvent into stem and leaf tissues and promoting the rapid release of intracellular active substances. The eutectic solvent of this invention has good conductivity, enabling efficient transfer of energy from high-voltage pulsed electric fields, enhancing the irreversible damage to cell membranes caused by electroporation, and significantly improving extraction efficiency. The eutectic solvent of this invention is non-volatile, has good stability, and remains stable during extraction, while the extraction conditions are mild, avoiding the degradation of heat-sensitive components such as artemisinin. The eutectic solvent of this invention is biodegradable, has low toxicity, and can be effectively removed by subsequent water elution, ensuring the safety of the Artemisia annua extract.

[0023] In this invention, the electric field strength of the high-voltage pulse electric field is preferably 5-8 kV / cm, more preferably 7 kV / cm, the pulse width is preferably 3-7 μs, more preferably 5 μs, and the pulse number is preferably 50-60, more preferably 55.

[0024] This invention places the whole plant of Artemisia annua in a eutectic solvent and uses high-voltage electric pulses to create irreversible electroporation on the cell membrane, increasing cell membrane permeability. This allows intracellular active ingredients to be rapidly released into the extraction solvent without a lengthy diffusion process. The high-voltage pulsed electric field of this invention results in a low extraction temperature, effectively avoiding thermal degradation of heat-sensitive components such as artemisinin; the extraction time is significantly shortened, improving extraction efficiency; it selectively disrupts plant cell membranes with minimal impact on the cell wall, reducing impurities from cell wall breakage and facilitating purification; after extraction in the eutectic solvent, the contents of Artemisia annua cells rapidly enter the solvent under the drive of the electric field and the effect of the concentration gradient, achieving efficient, gentle, and rapid extraction of active ingredients.

[0025] In this invention, the frequency of the ultrasonic treatment is preferably 22-28 kHz, more preferably 25 kHz, the temperature is preferably 30-40℃, more preferably 35℃, and the time is preferably 30-40 min, more preferably 35 min.

[0026] The ultrasonic treatment of this invention can promote the release of active ingredients tightly bound to cellulose and hemicellulose into the solvent, thereby increasing the total extraction rate. The ultrasonic treatment conditions are mild and the time is short, which can improve the extraction rate without causing significant degradation of the target active ingredients.

[0027] In this invention, the volume ratio of the supernatant to water is preferably 1:(3-5), and more preferably 1:4.

[0028] In this invention, the molecular weight cutoff of the ceramic membrane is preferably 500-2000 Da, more preferably 1000 Da.

[0029] This invention utilizes a ceramic membrane for purification, allowing the active ingredients in Artemisia annua to permeate while retaining allergens (large molecular proteins, plant waxes) that cause skin irritation and allergic reactions, thus achieving efficient separation of the active ingredients from the allergens. Ceramic membrane purification removes large molecular impurities such as pigments and colloids from the extract, significantly improving the purity and clarity of the extract. This creates excellent feeding conditions for subsequent macroporous adsorption resin purification, while also ensuring the safety and low allergenicity of the Artemisia annua extract from the source.

[0030] In this invention, the macroporous adsorption resin is preferably an X-5 type macroporous adsorption resin. The purification of the macroporous adsorption resin preferably includes: adsorbing the permeate purified by the ceramic membrane onto the X-5 type macroporous adsorption resin at 1-2 BV / h, eluting sequentially with 2-3 BV of deionized water and 3-5 BV of 65%-75% ethanol solution, collecting the ethanol eluent, concentrating under reduced pressure, and drying to obtain Artemisia annua extract. More preferably, the purification process includes: adsorbing the permeate purified by the ceramic membrane onto the X-5 type macroporous adsorption resin at 1.5 BV / h, eluting sequentially with 2.5 BV of deionized water and 4 BV of 70% ethanol solution, collecting the ethanol eluent, concentrating under reduced pressure, and drying to obtain Artemisia annua extract.

[0031] This invention utilizes macroporous adsorption resin purification to remove residual low-eutectic solvents, eliminate water-soluble impurities, and enrich target components. The invention selects X-5 type macroporous adsorption resin, which exhibits excellent adsorption selectivity and capacity for small molecule organic compounds (such as artemisinin, flavonoid aglycones, and volatile oil components). In the adsorption stage, the permeate treated with a ceramic membrane passes through the resin column, and the target active ingredient is adsorbed onto the resin surface and within the pores. In the water washing stage, deionized water is used to remove highly polar water-soluble impurities from the solution, such as choline chloride, benzoic acid, sugars, and inorganic salts. In the alcohol washing stage, ethanol disrupts the hydrophobic interaction between the target component and the resin, efficiently desorbing and recovering the adsorbed artemisinin, flavonoids, volatile oils, and other active ingredients, thus improving the purity of the active ingredients.

[0032] The present invention also provides an extract of Artemisia annua prepared according to the above preparation method.

[0033] The present invention also provides an application of the above-described Artemisia annua extract in any one or more of the following: (1) Application in the preparation of mosquito repellent products; (2) Application in the preparation of antibacterial products; (3) Application in the preparation of soothing and anti-inflammatory products; (4) Application in the preparation of hypoallergenic cosmetics.

[0034] This invention also provides a mosquito repellent product containing Artemisia annua extract, which is composed of the following raw materials in 100 parts by weight: 0.3-0.7 parts Artemisia annua extract, 1-1.5 parts caprylic / capric triglyceride, 1.2-1.8 parts polyglycerol-4-decanoate, 1.2-1.8 parts lactone-type sophorolipid, 0.08-0.12 parts low molecular weight sodium hyaluronate, 1-1.5 parts panthenol, 0.12-0.18 parts sodium polyglutamate, 0.4-0.6 parts hydrogenated polyisobutylene, 3-5 parts glycerin, 1.2-1.8 parts xylitol, 0.025-0.035 parts citric acid, 0.06-0.08 parts sodium citrate, and deionized water to make up to 100. More preferably, it is composed of the following raw materials: 0.5 parts Artemisia annua extract, 1.2 parts caprylic / capric triglyceride, 1.5 parts polyglycerol-4-decanoate, 1.5 parts lactone-type sophorolipid, 0.1 parts low molecular weight sodium hyaluronate, 1.2 parts panthenol, 0.15 parts sodium polyglutamate, 0.5 parts hydrogenated polyisobutylene, 4 parts glycerin, 1.5 parts xylitol, 0.03 parts citric acid, 0.07 parts sodium citrate, and deionized water to make up to 100. The preferred preparation method includes: mixing caprylic / capric glyceride and hydrogenated polyisobutylene, stirring at 38±2℃ and 300r / min for 20min, adding polyglycerol-4-decanoate and lactone-type sophorolipid and stirring continuously for 15min, adding Artemisia annua extract and stirring continuously for 20min to obtain an oil-soluble active phase; stirring glycerol, xylitol and water at 500r / min for 10min, then adding low molecular weight sodium hyaluronate, sodium polyglutamate and panthenol sequentially, and stirring continuously for 30min to obtain an aqueous phase; injecting the aqueous phase into the oil-soluble active phase at 38±2℃, stirring at 600r / min for 10min, homogenizing under a pressure of 300bar for 8min, cooling to 25±2℃, adding citric acid and sodium citrate, stirring at 300r / min for 5min, allowing to stand and mature for 12h, and filtering with a 0.22μm filter membrane to obtain the mosquito repellent product.

[0035] In this invention, the extract of Artemisia annua in the mosquito repellent product possesses a triple effect of mosquito repellency, broad-spectrum antibacterial activity, and anti-inflammatory and soothing properties, making it the core active ingredient for mosquito repellency and protection. Caprylic / capric triglyceride and hydrogenated polyisobutylene serve as oil-phase carriers, enhancing the solubility of the lipid-soluble active ingredients in Artemisia annua, assisting in film formation and sustained release, prolonging the duration of mosquito repellency and protection, and optimizing skin smoothness. The combination of lactone-type sophorolipid and polyglycerol-4-decanoate exhibits a synergistic effect; polyglycerol-4-decanoate efficiently matches the oil phase and reduces interfacial tension, while lactone-type sophorolipid enhances compatibility with the aqueous phase. The resulting microemulsion system exhibits no stratification or precipitation, solving the dispersion and precipitation problems of the lipid-soluble mosquito repellent active ingredients in Artemisia annua, improving the formulation's mosquito repellency duration and skin compatibility, and providing a refreshing and smooth skin feel, suitable for the application requirements of spray-type mosquito repellent liquids. Polyglycerol-4-decanoate... The emulsification system is composed of lactone-type sophorolipids. The combination of the two has a synergistic effect. Both are mild nonionic emulsifiers, non-irritating to the skin, and suitable for sensitive skin. Polyglycerol-4-decanoate can reduce the surface tension of the oil phase, improve the dispersibility of oil droplets, and has a moisturizing effect. Lactone-type sophorolipids are more hydrophilic and can enhance the stability of the oil-water interface. The combination of the two constructs a stable microemulsion system, which solves the problems of poor water solubility and easy precipitation of Artemisia annua active ingredients. At the same time, it significantly reduces the irritation risk of traditional emulsifiers, achieving the dual effect of system stability and skin gentleness. Low molecular weight sodium hyaluronate, sodium polyglutamate, panthenol, glycerin, and xylitol are moisturizing and repairing components, which work together to lock in moisture, repair the skin barrier, relieve redness and itching after mosquito bites, and form a breathable hydrating film to assist in the slow release of active ingredients.

[0036] The Artemisia annua of this invention is of variety Kehao No. 2, and the whole plant is fresh Artemisia annua in full bud stage. The crushing method is as follows: straighten the whole plant of Artemisia annua, cut it into small segments of 1.3±0.2cm, put it into a crusher for crushing, and pass it through a 10-mesh sieve to obtain crushed Artemisia annua.

[0037] Polyglycerol-4-decanoate, sourced from Hubei Mingya New Material Technology Co., Ltd., product code PEOFA TM MYPG1; Sodium polyglutamate, sourced from Shanxi Huatai Biotechnology Co., Ltd., catalog number ht-2025062032; Hydrogenated polyisobutylene, CAS number 59030-00-1, sourced from Guangdong Wengjiang Chemical Reagent Co., Ltd., catalog number PB90808.

[0038] Example 1 Preparation method of Artemisia annua extract After crushing the whole plant of Artemisia annua, 12 times its mass of eutectic solvent was added. Extraction was performed under a high-voltage pulsed electric field with an electric field strength of 7 kV / cm, a pulse width of 5 μs, and a pulse number of 55. The extract was then sonicated at a frequency of 25 kHz and a temperature of 35 °C for 35 min. After centrifugation at 4000 rpm for 10 min, the supernatant was collected and mixed with 4 times its volume of deionized water. The mixture was filtered through a ceramic membrane with a molecular weight cutoff of 1000 Da. The permeate was collected and adsorbed onto an X-5 macroporous adsorption resin column at a flow rate of 1.5 BV / h. The column was then eluted sequentially with 2.5 BV of deionized water and 4 BV of 70% ethanol solution. The ethanol eluent was collected, concentrated under reduced pressure, and freeze-dried under vacuum until the moisture content reached 2.83 wt%, yielding the Artemisia annua extract.

[0039] The method for preparing the eutectic solvent is as follows: choline chloride and benzoic acid are mixed at a molar ratio of 2:1, stirred at 65°C and 500 rpm for 30 min, and then mixed with deionized water to obtain a eutectic solvent with a water content of 15%.

[0040] Example 2 Preparation method of Artemisia annua extract After crushing the whole plant of Artemisia annua, 10 times its mass of eutectic solvent was added. Extraction was performed under a high-voltage pulsed electric field with an electric field strength of 5 kV / cm, a pulse width of 3 μs, and a pulse number of 60. The extract was then ultrasonically treated at a frequency of 22 kHz and a temperature of 30 °C for 40 min. After centrifugation at 3000 rpm for 13 min, the supernatant was collected and mixed with 3 times its volume of deionized water. The mixture was filtered through a ceramic membrane with a molecular weight cutoff of 500 Da. The permeate was collected and adsorbed onto an X-5 macroporous adsorption resin column at a flow rate of 1 BV / h. The column was then eluted sequentially with 2 BV of deionized water and 3 BV of 65% ethanol solution. The ethanol eluent was collected, concentrated under reduced pressure, and freeze-dried under vacuum until the moisture content reached 2.32 wt%, yielding the Artemisia annua extract.

[0041] The method for preparing the eutectic solvent is as follows: choline chloride and benzoic acid are mixed at a molar ratio of 1.5:1, stirred at 60℃ and 450rpm for 35min, and then mixed with deionized water to obtain a eutectic solvent with a water content of 12%.

[0042] Example 3 Preparation method of Artemisia annua extract After crushing the whole plant of Artemisia annua, 15 times its mass of eutectic solvent was added. Extraction was performed under a high-voltage pulsed electric field with an electric field strength of 8 kV / cm, a pulse width of 7 μs, and a pulse number of 50. The extract was then sonicated at a frequency of 28 kHz and a temperature of 40 °C for 30 min. After centrifugation at 5000 rpm for 8 min, the supernatant was collected and mixed with 5 times its volume of deionized water. The mixture was filtered through a ceramic membrane with a molecular weight cutoff of 2000 Da. The permeate was collected and adsorbed onto an X-5 macroporous adsorption resin column at a flow rate of 2 BV / h. The column was then eluted sequentially with 3 BV of deionized water and 5 BV of 75% ethanol solution. The ethanol eluent was collected, concentrated under reduced pressure, and freeze-dried under vacuum until the moisture content reached 3.06 wt%, yielding the Artemisia annua extract.

[0043] The method for preparing the eutectic solvent is as follows: choline chloride and benzoic acid are mixed at a molar ratio of 1.5:1, stirred at 70℃ and 650rpm for 28min, and then mixed with deionized water to obtain a eutectic solvent with a water content of 17%.

[0044] Comparative Example 1 Unlike Example 1, the whole plant of Artemisia annua was replaced with fresh Artemisia annua leaves, while other conditions remained the same.

[0045] Comparative Example 2 The difference from Example 1 is that the eutectic solvent was replaced with a 70% volume fraction ethanol solution, while other conditions remained the same.

[0046] Comparative Example 3 The difference from Example 1 is that high-voltage pulsed electric field extraction was replaced by heating extraction: the whole plant of Artemisia annua was crushed and 12 times its mass of eutectic solvent was added. The mixture was heated and extracted at 65°C and 500 rpm for 2 hours, while other conditions remained unchanged.

[0047] Comparative Example 4 Unlike Example 1, the ceramic membrane purification step was omitted, and the liquid obtained by mixing the supernatant with deionized water was directly purified by X-5 macroporous adsorption resin, while other conditions remained unchanged.

[0048] Comparative Example 5 The difference from Example 1 is that the X-5 type macroporous adsorption resin is replaced with the AB-8 type macroporous adsorption resin, while other conditions remain the same.

[0049] Comparative Example 6 Unlike Example 1, the purification step of X-5 macroporous adsorption resin was omitted. Instead, the ceramic membrane permeate was concentrated under reduced pressure and freeze-dried under vacuum to a moisture content of 2.73 wt% to obtain Artemisia annua extract, with other conditions remaining unchanged.

[0050] application I. Allergenicity testing The contents of 55 allergenic components, 100 hormones, 16 pesticide residues and clobetasolbutyrate in the Artemisia annua extracts prepared in Examples 1-3 were tested respectively.

[0051] The 55 allergenic components are: pentylcinnamyl alcohol, anisyl alcohol, benzyl alcohol, benzyl benzoate, benzyl cinnamate, benzyl salicylate, lily aldehyde, cinnamaldehyde, cinnamyl alcohol, citral, citronellol, farnesol, geraniol, hexylcinnamaldehyde, hydroxycitronellol, neolily aldehyde, isoeugenol, methyl 2-octynoyl acid, tree moss extract, oakmoss extract, rose ketone, propylphthalide, methyl salicylate, hexadecyl ring lactone, hexamethyl indopyran, linalyl acetate, menthol, ebony alcohol, salicylaldehyde, santalol, perillyl alcohol, trimethylphenylpropanol, butyl acetate. Phenolic esters, pinene, α / β-damascene, acetylacetone, amyl salicylate, anethole, acetylisoeugenol / isobutyl acetate, tetramethylacetyloctahydronaphthalene, dimethylbenzyl carboacetate (DMBCA), 6-methylcoumarin, turpentine oil, eugenol extract, lemon leaf essential oil, ylang-ylang flower oil / extract, cinnamon leaf oil, lemon peel oil, North American juniper oil, broad and narrow leaf hybrid lavender flower / leaf / stem extract, damask rose flower oil / extract, sandalwood oil, geraniol acetate, pentylcinnamaldehyde, α-isomethylionone.

[0052] The 100 hormone components are: triamcinolone, prednisolone, hydrocortisone, prednisone, cortisone, methylprednisolone, betamethasone, dexamethasone, flumethasone, beclomethasone, triamcinolone acetonide, flumethasone acetate, triamcinolone diacetate, prednisolone acetate, flumethasone, hydrocortisone acetate, dexamethasone acetate, flumethasone acetate, methylprednisolone acetate, betamethasone acetate, budesonide, hydrocortisone butyrate, dexamethasone acetate, flumethasone acetate, hydrocortisone valerate, triamcinolone acetonide, and more. Decanoate, Fluocinolone Acetate, Difluralasone Diacetate, Betamethasone Valerate, Prednisolone, Halcinonide, Aclomethasone Dipropionate, Ansinonide, Clobetasol Propionate, Fluticasone Propionate, Mometasone Furoate, Betamethasone Dipropionate, Beclomethasone Dipropionate, Clobetasol Butyrate, Fluocinolone, Dessonide, Cyclosone, Halometasone (Halometasone), Peramisone, Peramisone Acetate, Diflucolone Valerate, Isoflurane, Desoxymethasone, Halometasone Propionate, Flunisolone, Ecarboxylate, Flucolone, Remezoline Betamethasone benzoate, prednisolone valerate, betamethasone propionate, dexamethasone isonicotinate, dexamethasone valerate, flumethasone valerate, fluocinolone valerate, isoflurane prednisolone acetate, hydrocortisone cyclopentyl propionate, hydrocortisone succinate, methylprednisolone, methylprednisolone acetate, hexamethylenetetramine, difluralasone, fluocinolone, 16α-hydroxyprednisolone, 16α-hydroxyprednisolone acetate, 6α-fluoro-isofluoroprednisolone, methylprednisolone succinate, methylprednisolone acetate propionate, chlorclotropone valerate. Halobetasol, dexamethasone valerate, tecortisone valerate, cloprednisolone, difluprednisolone, tecortisone, diflumethasone acetate, hydrocortisone acetate, betamethasone butyrate propionate, methaqualone, desosonide 21-acetate, clobetasol, betamethasone 17-butyrate, betamethasone 21-propionate, beclomethasone 17-propionate, beclomethasone 21-propionate, fluticasone, mometasone, betamethasone diacetate, dexamethasone sodium phosphate, prednisolone sodium phosphate, hydrocortisone 21-octanoate, clotepone, betamethasone sodium phosphate.

[0053] The 16 pesticide residues are: trichlorfon, o,p'-DDD, o,p'-DDD, o,p'-DDD, p,p'-DDD, p,p'-DDD, α-endothelium, α-HCH, β-endothelium, β-HCH, γ-HCH, δ-HCH, aldrin, dieldrin, and chlordane.

[0054] The detection methods for allergenic components were based on the International Federation of Fragrance and Flavor Associations (IFRA) testing methods (published on September 12, 2007). The detection limits for all 55 allergenic components were 10 mg / kg.

[0055] The detection method for the first 41 hormone components is GB / T 24800.2-20094, and the detection method for the remaining hormone components is liquid chromatography-tandem mass spectrometry. The detection limit for all 100 hormone components is 0.1 mg / kg.

[0056] The detection methods for the 16 pesticide residues are GB 23200.8-2016. The detection limits for all 16 pesticide residues are 1 mg / kg.

[0057] The detection method for clobetasolbutyrate is liquid chromatography-tandem mass spectrometry, with a detection limit of 0.5 mg / kg.

[0058] Testing revealed that 55 allergenic components, 100 hormonal components, 16 pesticide residues, and clobetasolbutyrate were all undetectable (below the detection limit) in the Artemisia annua extracts of Examples 1-3. This indicates that the preparation method of this invention can remove most of the allergenic components. Ceramic membrane purification effectively removes large molecular weight allergenic proteins and plant waxes. X-5 macroporous adsorption resin purification not only efficiently enriches active ingredients such as artemisinin and flavonoid aglycones, but also thoroughly removes residual low-eutectic solvents (choline chloride / benzoic acid) and highly polar water-soluble impurities, thus avoiding the risk of contact allergies and chemical irritation from the source.

[0059] II. Antibacterial activity test The antibacterial effects of Artemisia annua extracts from Examples 1-3 and Comparative Examples 1-6 on Staphylococcus aureus (ATCC6538), Escherichia coli (8099), Candida albicans (ATCC10231), Pseudomonas aeruginosa (ATCC 15442), Aspergillus niger (ATCC15442), Propionibacterium acnes (ATCC11828), and Malassezia furfur (ATCC14521) were tested respectively.

[0060] The minimum inhibitory concentration (MIC) of Artemisia annua extract from Example 1 was tested using the agar dilution method (2002 edition of the "Disinfection Technical Specifications") in section 2.1.8.3. The MIC results against Staphylococcus aureus (ATCC 6538), Escherichia coli (8099), Candida albicans (ATCC 10231), Pseudomonas aeruginosa (ATCC 15442), Aspergillus niger (ATCC 6275), and Propionibacterium acnes (GDMCC 1.162) were determined by referring to section 2.1.8.3 of the "Disinfection Technical Specifications". The specific results are shown in Table 1.

[0061] Referring to WS / T 650-2019 "Evaluation Methods for Antibacterial and Bacteriostatic Effects" 5.1.1, the quantitative suspension antibacterial test was conducted to determine the antibacterial rate of Artemisia annua extracts from Examples 1-3 and Comparative Examples 1-6 (the Artemisia annua extracts were diluted to 0.5% by mass with 1% Span 80 aqueous solution for testing) against Staphylococcus aureus, Escherichia coli, Propionibacterium acnes, Candida albicans, and Malassezia furfur. Staphylococcus aureus, Escherichia coli, and Propionibacterium acnes were cultured for 48 h, while Candida albicans and Malassezia furfur were cultured for 72 h. The experimental results of the antibacterial rates of each Artemisia annua extract are shown in Table 2.

[0062] Table 1. Minimum inhibitory concentration of Artemisia annua extract Staphylococcus aureus 2500 E. coli 2500 Candida albicans 5000 Pseudomonas aeruginosa 5000 Propionibacterium acnes 5000 Aspergillus niger 7500 Table 2 Antibacterial results of Artemisia annua extract (%) Example 1 97.56 99.24 94.63 95.74 92.85 Example 2 96.84 98.48 93.37 94.66 92.13 Example 3 97.07 98.73 94.15 94.92 92.41 Comparative Example 1 84.90 87.51 79.21 81.47 78.69 Comparative Example 2 71.45 75.95 66.86 68.28 64.34 Comparative Example 3 75.71 79.17 70.40 72.53 69.92 Comparative Example 4 88.19 91.60 85.72 86.31 83.27 Comparative Example 5 82.68 84.36 77.09 79.19 75.50 Comparative Example 6 68.33 71.82 62.54 65.85 60.78 Table 1 shows that the Artemisia annua extract of Example 1 has a clear inhibitory effect on Staphylococcus aureus, Escherichia coli, Candida albicans, Pseudomonas aeruginosa, Aspergillus niger and Propionibacterium acnes, indicating that the Artemisia annua extract of the present invention has a broad-spectrum inhibitory ability against skin-related pathogens.

[0063] Table 2 shows that the inhibition rates of Examples 1-3 against the five tested bacteria were all above 92%, with the highest inhibition rate against *Escherichia coli*, exceeding that of all comparative examples. Comparative Example 1, using only leaf material, showed a lower inhibition rate compared to the examples, indicating that whole-plant extraction can achieve complementary active ingredients from different tissues. Comparative Examples 2-3, by replacing the extraction solvent and abandoning the high-voltage pulsed electric field process, showed a decrease in inhibition rate, indicating that the combination of eutectic solvent and high-voltage pulsed electric field can promote efficient dissolution of active ingredients. Comparative Examples 4-6, by abandoning ceramic membrane purification, replacing macroporous adsorption resin, and omitting the purification step, all showed a significant decrease in inhibition rate, indicating that the purification process can effectively enrich the target active ingredients, remove impurities, and improve the antibacterial activity of the *Artemisia annua* extract. The above data demonstrate that the preparation process of this invention can efficiently enrich the antibacterial active ingredients in *Artemisia annua*, and the extract possesses both broad-spectrum and highly effective antibacterial effects.

[0064] III. Soothing and Anti-inflammatory Test The soothing and anti-inflammatory effects of Artemisia annua extracts from Examples 1-3 and Comparative Examples 1-6 were tested respectively.

[0065] The testing method for soothing and anti-inflammatory effects refers to T / SHRH 034-2021, the group standard of Shanghai Daily Chemical Industry Association.

[0066] Cell source: RAW264.7 macrophages, purchased from the Chinese Academy of Sciences Cell Bank (TCM13).

[0067] Control group: Dissolve 100 mg of dexamethasone in 1000 μL DMSO, then dilute with DMEM basal medium to a dexamethasone solution of 100 μg / mL.

[0068] Sample group: Artemisia annua extract was diluted with DMEM basal medium to obtain sample solutions with a mass concentration of 0.00625%.

[0069] The system includes a blank control group, a negative control group, and a sample group.

[0070] Test: Press 1×10 5 Cells were seeded at a density of cells / well into 24-well plates and cultured overnight in an incubator (37℃, 5% CO2). When the cell confluence reached 50% ± 5%, cells were divided into groups and treated with DMEM basal medium (blank and negative control groups, sample groups were treated with sample solution). The drug volume per well was 999 μL, with 5 replicates per group. Cells were cultured for another 2 hours in an incubator (37℃, 5% CO2). Except for the blank control group, 1 μL of 1 μg / mL LPS solution was added to each well of the plates, and the blank control group was treated with an equal volume of DMEM basal medium. Cells were cultured for another 22 hours in an incubator (37℃, 5% CO2). 800 μL of cell culture supernatant was collected, and the levels of NO and TNF-α were detected using a NO kit (Beyotime, catalog number S0021S) and a TNF-α ELISA kit (Hangzhou Kelian Biotechnology Co., Ltd., catalog number EK201A), respectively. The inhibition rate was calculated, and the average value was taken. The specific results are shown in Table 3.

[0071] Inhibition rate (%) = [1 - (Inflammatory factor content in sample group - Inflammatory factor content in blank group) ÷ (Inflammatory factor content in negative control group - Inflammatory factor content in blank group)] × 100%.

[0072] Table 3. Inhibition rate (%) of inflammatory factors in each group Example 1 17.23 20.06 Example 2 16.55 18.76 Example 3 16.81 19.04 Comparative Example 1 13.16 14.42 Comparative Example 2 9.35 10.67 Comparative Example 3 11.24 12.5 Comparative Example 4 14.12 15.36 Comparative Example 5 12.87 13.05 Comparative Example 6 8.49 9.53 Table 3 shows that the NO inhibition rate and TNF-α inhibition rate of Examples 1-3 are higher than those of all comparative examples, indicating that the Artemisia annua extract prepared by the method of the present invention has excellent soothing and anti-inflammatory activity.

[0073] Comparative Example 1 used only leaves and lacked the anti-inflammatory components in the stem, resulting in a lower inhibition rate than the Example.

[0074] Eutectic solvents can soften lignocellulose, promote the release of intracellular active substances, and facilitate the dissolution of various anti-inflammatory components such as artemisinin, flavonoids, and volatile oils. Comparative Example 2, using 70% ethanol solution as a solvent, showed insufficient extraction efficiency for active components such as flavonoids and volatile oils, resulting in a lack of anti-inflammatory active ingredients and a reduced inhibition rate of inflammatory factors.

[0075] High-voltage pulsed electric field extraction was used to create irreversible electroporation pores in the cell membrane, enabling rapid release of intracellular components and preventing the degradation of heat-sensitive components such as artemisinin. Comparative Example 3 used 65℃ heating extraction, which led to the degradation of heat-sensitive active ingredients and a decrease in the inhibition rate of inflammatory factors.

[0076] Ceramic membranes can remove impurities such as large molecular proteins and waxes, reducing interference; X-5 type resin has high adsorption selectivity for active ingredients such as artemisinin, and can effectively enrich the target analytes. Comparative Example 4 discarded the ceramic membrane, as impurities interfered with the activity, reducing the purification degree and decreasing the inhibition rate; Comparative Example 5 used AB-8 macroporous adsorption resin instead, which reduced the inhibition rate of inflammatory factors; Comparative Example 6 discarded the resin purification step, as residual impurities such as choline chloride and benzoic acid may interfere with cell experiments, and the low purity of the active ingredients led to a decrease in the inhibition rate of inflammatory factors.

[0077] IV. Mosquito Repellency Test 1. Avoidance rate The repellency rates of Artemisia annua extracts from Examples 1-3 and Comparative Examples 1-6 were tested respectively.

[0078] The testing methods refer to GB / T 13917.9-2009 "Indoor Efficacy Tests and Evaluation of Sanitary Insecticides for Pesticide Registration - Part 9: Repellents" and GB / T 30126-2013 "Test and Evaluation of Mosquito Repellency Performance of Textiles".

[0079] Test insects: Aedes albopictus, female adult mosquitoes that have not fed on blood 3-5 days after emergence.

[0080] Test conditions: temperature (26±1)℃, relative humidity 65±10%.

[0081] Sample to be tested: Artemisia annua extract and caprylic / capric triglyceride (GTCC) were mixed at a mass ratio of 1:199 to obtain a 0.5% Artemisia annua extract dilution.

[0082] Experimental procedure: 300 Aedes albopictus mosquitoes were placed in a mosquito cage to conduct an attack test.

[0083] Four test subjects with adequate attack power were selected (half male and half female). A 50mm x 50mm area of ​​skin was drawn on the back of each of their hands. One hand was treated with 1.5μL / cm². 2 A 0.5% diluted Artemisia annua extract solution was evenly applied to a 40mm x 40mm area of ​​skin, with the remaining area tightly covered. The other hand served as a blank control. Control group: The hand with the sample applied was placed into a mosquito cage, and the number of mosquitoes landing on the exposed skin (B) was observed within 2 minutes. Test sample group: The hand with the sample applied was placed into a mosquito cage, and the number of mosquitoes landing on the exposed skin (A) was observed within 2 minutes. Each tester conducted the experiment once, and the mosquito repellency rate was calculated and averaged. The specific results are shown in Table 4.

[0084] Repellency rate (%) = (Number of mosquitoes in the control group - Number of mosquitoes in the test sample group) ÷ Number of mosquitoes in the control group × 100%.

[0085] A repellency rate > 70% is rated A, indicating a very strong repellency effect; a repellency rate of 50%-70% is rated B, indicating a good repellency effect; and a repellency rate of 30%-50% is rated C, indicating a repellency effect.

[0086] Table 4. Mosquito repellent effects of Artemisia annua extracts in each group Example 1 47 2 95.7 Example 2 48 3 93.8 Example 3 49 4 91.8 Comparative Example 1 50 13 74.0 Comparative Example 2 51 22 56.9 Comparative Example 3 49 19 61.2 Comparative Example 4 52 11 78.8 Comparative Example 5 45 16 64.4 Comparative Example 6 49 26 46.9 Table 4 shows that the repellency rates of Examples 1-3 were all higher than 91%, which was significantly higher than that of Comparative Examples 1-6, proving that the extract of the present invention has a very strong repellency effect on Aedes albopictus.

[0087] Benzoic acid in the eutectic solvent can penetrate the waxy layer of stems and leaves, promoting the dissolution of oily mosquito-repellent components. Simultaneously, the conductivity of the eutectic solvent allows for efficient transmission of the high-voltage pulsed electric field, causing cell membrane rupture and rapid release of the active ingredients. Comparative Example 2 used ethanol instead of the eutectic solvent; however, due to ethanol's weak penetration through the cell wall, the extraction rate of the active ingredients was low, resulting in a reduced repellency rate. While heating extraction in Comparative Example 3 could increase diffusion, prolonged heating led to the loss of volatile oils and the decomposition of artemisinin, resulting in lower efficiency than in the examples.

[0088] The examples used the whole plant of Artemisia annua. The composition of active ingredients differs among the roots, stems, and leaves, and whole-plant extraction can achieve complementary mosquito-repellent activity spectra. Comparative Example 1 used only the leaves, lacking the specific mosquito-repellent components found in the stems, resulting in a lower repellency rate, thus confirming the rationale for utilizing the whole plant.

[0089] In the examples, ceramic membranes and macroporous resins were used to purify the active ingredients, achieving enrichment of the mosquito-repellent active ingredients. Comparative Example 4 discarded the ceramic membrane; large molecular impurities competed with the active ingredients for resin adsorption sites, leading to loss of active ingredients and a decrease in repellency rate. Comparative Example 5 used AB-8 macroporous adsorption resin, which reduced hydrophobicity, decreasing the adsorption amount of active ingredients and lowering the repellency rate. Comparative Example 6 abandoned resin purification; the concentration of active ingredients was diluted, and residual low-eutectic solvent reduced the content of mosquito-repellent active ingredients, resulting in the lowest repellency rate. This demonstrates that resin purification is not only a removal of impurities but also a crucial step in the enrichment of active ingredients.

[0090] 2. Stability and duration of mosquito repellent use Artemisia annua extract was used in mosquito repellent products, and the formulation is shown in Table 5.

[0091] Table 5. Formulations of mosquito repellent products containing Artemisia annua extract (g) Artemisia annua extract 0.5 0.3 0.7 Caprylic / capric triglyceride 1.2 1.5 1 Polyglycerol-4-decanoate 1.5 1.2 1.8 Lactone-type sophorolipids 1.5 1.8 1.2 Low molecular weight sodium hyaluronate 0.1 0.08 0.12 Panthenol 1.2 1.5 1 Sodium polyglutamate 0.15 0.12 0.18 Hydrogenated polyisobutylene 0.5 0.6 0.4 glycerin 4 3 5 Xylitol 1.5 1.8 1.2 Citric acid 0.03 0.025 0.035 Sodium citrate 0.07 0.08 0.06 Deionized water Add to 100 Add to 100 Add to 100 Preparation method: Caprylic / capric glyceride and hydrogenated polyisobutylene were mixed and stirred at 38±2℃ and 300 r / min for 20 min. Polyglycerol-4-decanoate and lactone-type sophorolipid were added and stirred continuously for 15 min. Artemisia annua extract was added and stirred continuously for 20 min to obtain the oil-soluble active phase. Glycerin, xylitol, and water were stirred at 500 r / min for 10 min. Low molecular weight sodium hyaluronate, sodium polyglutamate, and panthenol were added sequentially and stirred for 30 min to obtain the aqueous phase. The aqueous phase was injected into the oil-soluble active phase at 38±2℃ and stirred at 600 r / min for 10 min. The mixture was then homogenized by circulation at 300 bar for 8 min, cooled to 25±2℃, and citric acid and sodium citrate were added. The mixture was stirred at 300 r / min for 5 min and allowed to stand for 12 h. The mixture was then filtered through a 0.22 μm filter membrane to obtain the mosquito repellent product.

[0092] Among them, the Artemisia annua extract in Application Example 1 is the Artemisia annua extract from Example 1; The Artemisia annua extract in Application Example 2 is the Artemisia annua extract from Example 2; The Artemisia annua extract in Application Example 3 is the Artemisia annua extract from Example 3.

[0093] Application Comparative Example 1: Replace "Artemisia annua extract of Example 1" with "Artemisia annua extract of Comparative Example 1" in Application Example 1, and keep everything else unchanged.

[0094] Application Comparative Example 2: Replace "Artemisia annua extract of Example 1" with "Artemisia annua extract of Comparative Example 2" in Application Example 1, and keep everything else the same.

[0095] Application Comparative Example 3: Replace "Artemisia annua extract of Example 1" with "Artemisia annua extract of Comparative Example 3" in Application Example 1, and keep everything else the same.

[0096] Application Comparative Example 4: Replace "Artemisia annua extract of Example 1" with "Artemisia annua extract of Comparative Example 4" in Application Example 1, and keep everything else the same.

[0097] Application Comparative Example 5: Replace "Artemisia annua extract of Example 1" with "Artemisia annua extract of Comparative Example 5" in Application Example 1, and keep everything else the same.

[0098] Application Comparative Example 6: Replace "Artemisia annua extract of Example 1" with "Artemisia annua extract of Comparative Example 6" in Application Example 1, and keep everything else unchanged.

[0099] Application Comparative Example 7: Replace “caprylic / capric triglyceride” with “mineral oil” and “hydrogenated polyisobutylene” with “hydrogenated polydecene” in Application Example 1, keeping the amounts the same and everything else the same.

[0100] Comparative Example 8: The lactone-type sophorolipid in Application Example 1 was discarded, and the amount of polyglycerol-4-decanoate was adjusted to 3.

[0101] Comparative Example 9: The polyglycerol-4-decanoate in Application Example 1 was discarded, and the amount of lactone-type sophorolipid was adjusted to 3.

[0102] The stability and effective protection time of mosquito repellent products made from Artemisia annua extract were tested.

[0103] (1) Stability The stability of the mosquito repellent products used in Application Example 1 and Comparative Examples 7-9 was tested.

[0104] High and low temperature thermal cycling stability test: Take 100mL of each group of samples, refrigerate at 4℃ for 12h, place in an oven at 45℃ for 12h, and cycle for 24h each time, and complete 5 cycles in a row. Each group of samples is placed in a high and low temperature test chamber at the same time, and the temperature is strictly controlled. After 5 cycles, take them out and let them stand at room temperature, and observe the appearance to see if there is oil-water separation, bottom sedimentation, surface floating oil, white flocculent precipitation, or turbidity and stratification.

[0105] Centrifugation acceleration stability test: Take 10 mL of each group of samples, put them into centrifuge tubes, centrifuge at 3000 r / min for 30 min, let stand for 5 min, and observe the appearance to see if oil-water separation, bottom sedimentation, surface floating oil, white flocculent precipitation, or turbidity and stratification occur.

[0106] The stability test results are shown in Table 6.

[0107] Table 6. Stability results of mosquito repellent products in each group Application Example 1 Clear and transparent, without layering, sediment, or flocculent matter. Uniform and transparent, with no floating oil or bottom sediment. Application Comparative Example 7 Oily surface layer, with trace amounts of waxy sediment at the bottom. The upper layer is clearly an oil layer, while the lower layer is a turbid aqueous phase. Application Comparative Example 8 Slightly cloudy, with no obvious stratification The overall color is milky white and cloudy, with no obvious oil-water separation. Application Comparison Example 9 A white flocculent precipitate appears and cannot be redissolved upon standing. A large amount of flocculent matter is suspended, and the system is heterogeneous. The results in Table 6 show that Application Example 1 remained clear, transparent, and uniform without stratification under both conditions, while the comparative examples showed varying degrees of instability. This indicates that the selection of the compound emulsion system and oil phase in mosquito repellent products is key to stability.

[0108] The choice of oil phase determines basic compatibility. In Application Example 1, a blend of caprylic / capric glyceride and hydrogenated polyisobutylene was used as the oil phase. Caprylic / capric glyceride possesses both polar and nonpolar characteristics, effectively dissolving the lipid-soluble mosquito-repellent active ingredients in Artemisia annua; hydrogenated polyisobutylene, a high-molecular-weight hydrocarbon, can aid film formation and adjust skin feel. The blend exhibits good interfacial compatibility with emulsifiers. In Comparative Example 7, the oil phase was replaced with mineral oil and hydrogenated polydecene. Mineral oil, with its pure hydrocarbon structure and extremely low polarity, has poor affinity for active ingredients containing ester and hydroxyl groups, as well as nonionic emulsifiers. This leads to the precipitation of floating oil due to solubility changes during hot and cold cycling, and significant oil-water separation during centrifugation due to density differences.

[0109] The formulation of the emulsion system is a key component in maintaining the microemulsion of mosquito repellent products. Application Example 1 uses a combination of polyglycerol-4-decanoate (strong lipophilicity, reducing oil-water interfacial tension) and lactone-type sophorolipid (strong hydrophilicity, enhancing compatibility with the aqueous phase). Both are nonionic, mild, and non-irritating. Through complementary hydrophilic-lipophilic balance values, they form a stable microemulsion interfacial film with low interfacial tension, resistant to temperature and centrifugal shear force. Comparative Example 8, omitting sophorolipid and using only polyglycerol-4-decanoate, results in insufficient hydrophilicity and a loose interfacial film, leading to slight turbidity after thermal cycling. After centrifugation, the entire system becomes milky white and turbid, indicating that a single emulsifier cannot cover the entire interface between the oil and water phases. Comparative Example 9, also omitting polyglycerol-4-decanoate and using only sophorolipid, suffers from strong hydrophilicity, resulting in a lack of lipid side chain support in the interfacial film, leading to white flocculent precipitation. After centrifugation, flocculent suspension occurs, indicating an inhomogeneous system. Both comparative studies show that a single emulsifier cannot achieve long-term kinetic stability of microemulsions, and only by combining them can a synergistic interfacial protection effect be obtained.

[0110] (2) Effective protection time Test insects: Aedes albopictus, female adult mosquitoes that have not fed on blood 3-5 days after emergence.

[0111] Test conditions: temperature (26±1)℃, relative humidity 65±10%.

[0112] Experimental equipment: mosquito cage, length 400mm, width 300mm, height 300mm.

[0113] Test samples: mosquito repellent products used in Application Example 1 and Comparative Examples 1-9.

[0114] Experimental Procedure: 300 Aedes albopictus mosquitoes were placed in a mosquito cage for an attack test. Four participants (half male and half female) with satisfactory attack capabilities were selected. A 50mm x 50mm area was drawn on the back of each participant's hand. One hand was treated with a concentration of 1.5 mg / cm². 2 Apply the sample evenly, exposing a 40mm x 40mm section of skin while completely covering the rest. The other hand remains untreated as a blank control. Two hours after sample application, place the hand into a mosquito cage with adequate attack power for 2 minutes, observing for any mosquitoes that land and feed. Repeat this process every hour thereafter; a sample is considered invalid if even one mosquito attempts to feed.

[0115] Record the effective protection time (h) of the samples and calculate the average value. The specific results are shown in Table 7. Note: For each control, the hand should first undergo a mosquito attack test. Test insects with qualified attack power can continue the test. Test insects with unqualified attack power need to be replaced with qualified test insects for the test.

[0116] Efficacy grades: Grade A, effective protection time ≥ 6.0h; Grade B, effective protection time ≥ 4.0h. The efficacy results are divided into two grades, A and B. Products that do not meet the Grade B standard are unqualified.

[0117] Table 7. Mosquito repellency duration (h) for each group of mosquito repellent products Application Example 1 7.2 Grade A Application Comparative Example 1 6.2 Grade A Application Comparative Example 2 5.6 Grade B Application Comparative Example 3 5.8 Grade B Application Comparative Example 4 6.4 Grade A Application Comparative Example 5 6.0 Grade A Application Comparative Example 6 4.7 Grade B Application Comparative Example 7 5.9 Grade B Application Comparative Example 8 5.6 Grade B Application Comparison Example 9 5.3 Grade B Table 7 shows that the effective protection time of Application Example 1 reaches Level A (≥6h); while the protection time of Application Comparative Examples 1-9 is significantly lower than that of Application Example 1.

[0118] The extracts of Comparative Examples 1-6 had insufficient content or purity of active ingredients due to changes in the extraction steps (changing raw materials, changing extraction solvents, replacing high-voltage pulse electric fields, replacing resins, etc.), resulting in a significant reduction in mosquito repellency time when added in the same amount in the formulation.

[0119] The formulation excipients prolong the protective time by providing sustained release and stabilization for the active ingredient. In Application Example 1, glyceryl caprylate and hydrogenated polyisobutylene form an oily film, reducing the evaporation rate of the active ingredient and achieving sustained release. The microemulsion system constructed from lactone-type sophorolipid and polyglycerol-4-decanoate ensures uniform dispersion of the active ingredient, avoiding rapid release due to excessively high local concentrations. In contrast, in Comparative Example 7, replacing the oil phase with mineral oil and hydrogenated polydecene resulted in decreased stability. Furthermore, due to the low solubility and poor film-forming properties of mineral oil for Artemisia annua active ingredient, the active ingredient could not adhere to the skin for a long time, leading to a reduction in the protective time. In Comparative Examples 8 and 9, the microemulsion structure had poor stability, and the active ingredients were prone to precipitation or aggregation, resulting in a decrease in the effective release concentration and a reduction in the protective time.

[0120] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing Artemisia annua extract, characterized in that, include: After crushing the whole plant of Artemisia annua, it was mixed with a eutectic solvent, extracted by a high-voltage pulsed electric field, ultrasonicated, centrifuged, and the supernatant was collected and mixed with water. The mixture was then purified by ceramic membrane and macroporous adsorption resin to obtain Artemisia annua extract. The eutectic solvent consists of choline chloride, benzoic acid, and water.

2. The preparation method according to claim 1, characterized in that, The method for preparing the eutectic solvent includes: heating and melting choline chloride and benzoic acid, then mixing them with water to obtain the eutectic solvent.

3. The preparation method according to claim 2, characterized in that, The molar ratio of choline chloride to benzoic acid is (1.5-2.5):1, and the water is 12%-17% of the mass of the eutectic solvent.

4. The preparation method according to claim 1, characterized in that, The mass ratio of the whole plant Artemisia annua to the eutectic solvent is 1:(10-15).

5. The preparation method according to claim 1, characterized in that, The electric field strength of the high-voltage pulsed electric field is 5-8 kV / cm, the pulse width is 3-7 μs, and the number of pulses is 50-60.

6. The preparation method according to claim 1, characterized in that, The ultrasonic treatment is performed at a frequency of 22-28 kHz, a temperature of 30-40 ℃, and a duration of 30-40 min.

7. The preparation method according to claim 1, characterized in that, The volume ratio of the supernatant to water is 1:(3-5).

8. The preparation method according to claim 1, characterized in that, The macroporous adsorption resin is X-5 type macroporous adsorption resin.

9. An extract of Artemisia annua prepared by the preparation method according to any one of claims 1-8.

10. An application of the Artemisia annua extract according to claim 9 in any one or more of the following: (1) Application in the preparation of mosquito repellent products; (2) Application in the preparation of antibacterial products; (3) Application in the preparation of soothing and anti-inflammatory products; (4) Application in the preparation of hypoallergenic cosmetics.