An extraction method and application of artemisia extract from artemisia leaves

Abstract

This invention discloses a method for extracting Artemisia annua extract from Artemisia annua leaves and its application. The method includes the following steps: taking Artemisia annua leaves, packaging and drying them; placing the dried Artemisia annua leaves into a wide-mouth bottle, ensuring the leaves are fully soaked, and then placing the fully soaked leaves into an ultrasonic instrument for ultrasonic-assisted extraction; after ultrasonic-assisted extraction, filtering to obtain filter residue, and then repeating ultrasonic-assisted extraction to obtain all the filtrate; rotary evaporating all the filtrate to obtain a concentrated solution, which is then dissolved again in an appropriate amount of pure water, subjected to chromatographic separation, and gradient elution; collecting the specified eluent fraction, and then rotary evaporating it again to obtain Artemisia annua extract. This invention provides a method for extracting Artemisia annua extract from Artemisia annua leaves and its application, which can extract other active ingredients from Artemisia annua, improve the bioavailability of other active ingredients in Artemisia annua, and expand its application in pharmaceuticals and cosmetics.

Description

Technical Field

[0001] This invention relates to the field of plant active ingredient extraction, and in particular to an extraction method and application of artemisia annua extract from artemisia annua leaves. Background Technology

[0002] Atopic dermatitis (AD) is a common chronic inflammatory skin disease, clinically characterized by dry skin, intense itching, and eczematous rashes. Its pathogenesis is complex, involving multiple aspects such as genetics, immunity, epidermal barrier dysfunction, environmental factors, and neuroimmune interactions. In recent years, the prevalence of atopic dermatitis in my country has been on the rise, especially among children, with a lifetime prevalence reaching up to 20%, severely impacting patients' quality of life.

[0003] Currently, treatments for atopic dermatitis mainly include topical corticosteroids, immunosuppressants (such as tacrolimus), antihistamines, and biologics. However, traditional treatments often have limited efficacy, high relapse rates, and long-term use may cause side effects such as skin atrophy and immunosuppression. Furthermore, some novel biologics are expensive and difficult to widely implement. Therefore, developing a safe, effective, and economical new treatment strategy has significant clinical and social value.

[0004] Artemisia annua is an annual herb belonging to the genus Artemisia of the Asteraceae family. It is also a traditional Chinese medicine, recorded in the Chinese Pharmacopoeia. It is bitter and cold in nature, possessing various effects such as clearing deficiency heat, relieving summer heat, relieving malaria, reducing jaundice, and cooling the blood. Its main active ingredient, artemisinin, is widely known for its remarkable effects in the treatment of malaria. Recent studies have found that Artemisia annua and its extracts also possess various biological activities such as anti-inflammatory, immunomodulatory, and antiviral activity (Wang Hongbo, Xiao Wan, Hua Huiming, et al. Research progress on the chemical components of Artemisia annua [J]. Modern Drugs and Clinical, 2011, 26(6): 430-433), showing potential therapeutic value in inflammatory skin diseases and skin barrier repair. However, the skin barrier repair effect of Artemisia annua extract has not been reported. Extraction methods for active substances in Artemisia annua include water extraction and traditional organic solvent extraction (ethanol extraction), mainly including extraction... Artemisinin and its derivatives, artemisia polysaccharides, and artemisia essential oil are the main active ingredients in Artemisia annua extract. Artemisinin compounds are primarily fat-soluble and present in low concentrations in Artemisia annua. Artemisinin compounds are generally sensitive to heat and almost insoluble in water, requiring extraction with alcohol or organic solvents. Improper extraction methods can easily damage their structure or result in the failure to extract artemisinin compounds. Currently, research on the activity of artemisinin and its derivatives, besides their antimalarial properties, mainly focuses on their antioxidant and anti-inflammatory effects.

[0005] Therefore, developing an extraction method that preserves the active ingredients in Artemisia annua and improves the bioavailability of Artemisia annua extract is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of the above-mentioned deficiencies of the prior art, the present invention provides an extraction method and application for extracting Artemisia annua extract from Artemisia annua leaves, which can extract other active ingredients in Artemisia annua, improve the bioavailability of other active ingredients in Artemisia annua, and extract a natural extract that improves the source of the immune microenvironment and promotes the repair of the skin barrier.

[0007] To achieve the above objectives, the present invention provides a method for extracting artemisia extract from artemisia leaves, comprising the following steps:

[0008] Artemisia annua leaves are collected, packaged, and then dried.

[0009] The dried Artemisia annua leaves were placed in a wide-mouthed bottle and allowed to soak fully. Then, the soaked Artemisia annua leaves were placed in an ultrasonic instrument for ultrasonic-assisted extraction.

[0010] After ultrasonic-assisted extraction, the residue was filtered to obtain filter residue, and then ultrasonic-assisted extraction was repeated to obtain all the filtrate.

[0011] All the filtrate was rotary evaporated to obtain a concentrated solution, which was then dissolved in an appropriate amount of pure water. After chromatographic separation, gradient elution was performed, and the specified eluent fraction was collected and rotary evaporated again to obtain artemisia annua extract.

[0012] Further, Artemisia annua leaves are taken, packaged, and then dried. Specifically, Artemisia annua leaves are taken, placed in kraft paper envelopes, and dried in an oven at 50°C for 48 hours.

[0013] Further, the dried Artemisia annua leaves were placed in a wide-mouthed bottle and allowed to fully soak. Then, the fully soaked Artemisia annua leaves were placed in an ultrasonic instrument for ultrasonic-assisted extraction, as follows:

[0014] After drying for 48 hours, weigh 1000g of dried Artemisia annua leaves and put them into a wide-mouthed bottle.

[0015] Add 7L of 70% ethanol to a wide-mouthed bottle and stir to allow the ethanol to fully soak the Artemisia annua leaves;

[0016] After soaking for 2 hours, the wide-mouth bottle was placed in an ultrasonic instrument for ultrasonic-assisted extraction.

[0017] Furthermore, the temperature for ultrasound-assisted extraction was 50℃, the power was 60W, and the ultrasound time was 1 hour.

[0018] Further, after the ultrasound-assisted extraction is completed, the residue is filtered to obtain a filter cake, and then the ultrasound-assisted extraction is repeated to obtain all the filtrate; the details are as follows:

[0019] After ultrasound, filter with gauze and wring out the filter residue;

[0020] Add the filter residue back into 4L of 70% ethanol and repeat the ultrasonic-assisted extraction process. Filter and wring out the filter residue, collecting as much filtrate as possible.

[0021] Further, the entire filtrate was rotary evaporated to obtain a concentrated solution, which was then dissolved again in an appropriate amount of pure water. After chromatographic separation, gradient elution was performed, and the specified eluent fraction was collected and rotary evaporated again to obtain artemisinin extract, as detailed below:

[0022] All the above filtrates were placed in batches into a rotary evaporator for rotary evaporation. The resulting concentrate was dissolved again in an appropriate amount of pure water. Then, HPD-100 macroporous adsorption resin was used to perform chromatographic separation of the solution. The solution was eluted sequentially with pure water and ethanol with volume fractions of 30%, 50%, 75%, and 90%. The 75% eluent was collected and then concentrated by rotary evaporation.

[0023] Collect the Artemisia annua extract obtained by rotary evaporation.

[0024] Furthermore, during rotary evaporation, the temperature was 60°C and the rotation speed was 60 rpm.

[0025] Furthermore, the process also includes placing the collected artemisia extract into a stainless steel dish, freeze-drying it in a pre-cooled freeze dryer for 48 hours, collecting it into 50mL centrifuge tubes, sealing them, and storing them at -20℃.

[0026] In a preferred embodiment of the present invention, an application of artemisia extract extracted from artemisia leaves in the preparation of a drug for treating atopic dermatitis is provided.

[0027] Furthermore, it promotes the proliferation of macrophages and epidermal cells.

[0028] Technical effect

[0029] This invention discloses a method for extracting Artemisia annua extract from Artemisia annua leaves and its applications. The extracted Artemisia annua extract significantly promotes the cell viability of macrophages and epidermal cells; shows a trend towards increasing the phagocytic capacity of macrophages; significantly inhibits the release of nitric oxide from macrophages; significantly inhibits the migration of epidermal cells; significantly inhibits the mRNA expression of macrophage inflammatory factors; and has complex effects on the mRNA expression in an epidermal cell atopic dermatitis model. The method for extracting Artemisia annua extract from Artemisia annua leaves provided by this invention successfully prepares a rich and highly active Artemisia annua extract through steps such as drying at 50℃, ultrasonic-assisted extraction with 70% ethanol, chromatographic separation with macroporous adsorption resin, and freeze-drying. Metabolomics analysis identified 710 substances in the extract, with sesquiterpenes as the core (relative content 30.05%), including 45% artemisinin. It is also rich in phenolic acids, coumarins, phenylpropanoids, and fatty acids, forming a natural product system with both antioxidant and anti-inflammatory activities. Pharmacological experiments showed that the extract could promote the proliferation of macrophages (RAW264.7) and keratinocytes (HaCaT) in a concentration-dependent manner, and enhance the phagocytic capacity of macrophages; it significantly inhibited lipopolysaccharide-induced macrophage nitric oxide release and the mRNA expression of inflammatory factors such as IL-6, TNF-α, and IL-1β; in an atopic dermatitis cell model, it could regulate the expression of skin barrier-related genes, promote the recovery of tight junction protein (OCLN) and filaggrin (FLG), and inhibit excessive migration of keratinocytes. The extraction method of this invention is simple, environmentally friendly, and suitable for industrial production. The resulting artemisinin extract has broad application prospects in the preparation of drugs for treating atopic dermatitis or skin care products.

[0030] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0031] Figure 1 This is an experimental result showing the effect of Artemisia annua extract on the viability of RAW264.7 macrophage cells according to a preferred embodiment of the present invention;

[0032] Figure 2 This is an experimental result showing the effect of Artemisia annua extract on the viability of human immortalized keratinocytes (HaCaT cells) according to a preferred embodiment of the present invention.

[0033] Figure 3 This is an experimental result showing the effect of artemisia annua extract on the phagocytic capacity of macrophages, according to a preferred embodiment of the present invention.

[0034] Figure 4This is an experimental result showing the effect of artemisia annua extract on the migration ability of human immortalized keratinocytes (HaCaT cells) according to a preferred embodiment of the present invention.

[0035] Figure 5 The following are experimental results from a preferred embodiment of the present invention regarding the effect of Artemisia annua extract on nitric oxide release in RAW264.7 macrophage cells:

[0036] Figure 6 This is an experimental result showing the effect of Artemisia annua extract on the mRNA expression levels of related inflammatory factors in RAW264.7 macrophage cells, according to a preferred embodiment of the present invention.

[0037] Figure 7 This is an experimental result of the effect of Artemisia annua extract on the mRNA expression level of skin barrier protein associated with human immortalized keratinocytes (HaCaT cells) according to a preferred embodiment of the present invention.

[0038] In the attached figures, statistically significant differences between the control group and other groups, and between the control group and other groups, were obtained using t-test statistical analysis. In the figures, "*" indicates a p-value less than 0.05 compared to the control group, "**" indicates a p-value less than 0.01 compared to the control group, and "***" indicates a p-value less than 0.001 compared to the control group. Detailed Implementation

[0039] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0040] In the following description, specific details, such as particular internal procedures and techniques, are set forth for illustrative purposes and not for limitation, in order to provide a thorough understanding of the embodiments of the invention. However, those skilled in the art will understand that the invention may be practiced in other embodiments without these specific details.

[0041] This invention provides a method for extracting artemisia extract from artemisia leaves, comprising the following specific steps:

[0042] Step 1: Take Artemisia annua leaves, put them in a kraft paper envelope, and dry them in a 50℃ oven for 48 hours.

[0043] Step 2: After 48 hours, remove the leaves from the oven and weigh 1000g of dried Artemisia annua leaves and put them into a wide-mouthed bottle.

[0044] Step 3: Add 7L of 70% ethanol to a wide-mouthed bottle and stir to ensure the ethanol fully soaks the Artemisia annua leaves. After soaking for 2 hours, place the bottle in an ultrasonic extractor for ultrasonic-assisted extraction at 50℃ and 60W for 1 hour.

[0045] Step 4: After sonication, filter the solution through gauze and wring out the residue. Add 4L of 70% ethanol to the residue and repeat the sonic-assisted extraction process. Filter again, wring out the residue, and collect as much filtrate as possible.

[0046] Step 5: Divide all the above filtrates into batches and perform rotary evaporation in a rotary evaporator (60℃, 60rpm). Dissolve the resulting concentrate again in an appropriate amount of pure water. Then, perform chromatographic separation using HPD-100 macroporous adsorption resin, eluting sequentially with pure water, 30%, 50%, 75%, and 90% ethanol (v / v). Collect the 75% eluent fraction and concentrate it again by rotary evaporation. Recover the ethanol, determine its concentration using an alcohol meter, and collect and store it. Collect the Artemisia annua extract obtained by rotary evaporation.

[0047] The embodiments of the present invention also include the following steps

[0048] Step 6: The collected artemisia extract is placed in a stainless steel tray and freeze-dried in a pre-cooled freeze dryer for 48 hours. The extract is then collected into 50mL centrifuge tubes, sealed, and stored at -20℃.

[0049] Step 7: Artemisia annua extract containing 5% artemisinin was obtained by redissolving the extract in anhydrous ethanol and ultrapure water at a ratio of 1:1 and then detecting the solution by ultra-high performance liquid chromatography. This extract was used for subsequent experiments.

[0050] In the following examples, mouse macrophages (RAW264.7) and human immortalized keratinocytes (HaCaT) were purchased from the Cell Bank of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and stored in liquid nitrogen. Resuscitated RAW264.7 cells were added to fresh DMEM medium containing 10% fetal bovine serum (FBS), and resuscitated HaCaT cells were added to fresh DMEM medium containing 10% FBS. Both were seeded into T25 cell culture flasks and cultured routinely at 37°C in a 5% CO2 incubator. Seeding density was maintained according to the experimental requirements, and the medium was replaced with fresh medium every other day.

[0051] Experimental reagents: Freeze-dried Artemisia annua extract was dissolved in anhydrous ethanol: ultrapure water = 1:1 to prepare a stock solution, which was stored at room temperature and diluted with fresh DMEM medium containing 10% fetal bovine serum (FBS) before use in experiments.

[0052] The specific implementation method is as follows:

[0053] Example 1: Metabolomics

[0054] (1) After dissolving the lyophilized sample in methanol, take 100 μL and evaporate to dryness. Add 100 μL of extraction solution (methanol:acetonitrile = 1:1), which contains an isotope-labeled internal standard. Mix well and sonicate for 10 min. Let stand at -40 ℃ for 1 h.

[0055] (2) Centrifuge the sample at 4 ℃ and 12000 rpm for 15 min, and take the supernatant into the sample bottle for testing.

[0056] (3) Take HE during the logarithmic growth phase to target artemisinin, precisely prepare artemisinin standard methanol solution, create artemisinin concentration gradient, and determine the content by HPLC.

[0057] (4) For nonpolar metabolites, a Vanquish ultra-high performance liquid chromatograph was used to separate the target compounds by chromatographic separation using a Phenomenex Kinetex C18 liquid chromatography column. Phase A of the liquid chromatography was aqueous phase containing 0.01% acetic acid, and phase B was isopropanol:acetonitrile (1:1).

[0058] The results showed that, based on the peak area of ​​artemisinin in the sample calculated from the standard curve, the artemisinin content in the Artemisia annua extract was approximately 5%.

[0059] Raw data were converted to mzXmL format using ProteoWizard software, and metabolite identification was performed using a collaboratively developed R package. The databases used were BiotreeDB (V3.0, standard library) and BT-Plant (V1.1, plant-specific library). Non-targeted metabolomics analysis of the Artemisia annua ethanol extract revealed a rich and diverse chemical composition, identifying 710 substances matched with standards. Sesquiterpenes were the dominant metabolites, accounting for 30.05% of the total detected metabolites. This is consistent with the high artemisinin content found in the extract, highlighting Artemisia annua as an important source of sesquiterpenoid active components. Furthermore, phenolic acids, coumarins, and phenylpropanoids accounted for 8.80%, 9.30%, and 8.12% of the extract, respectively. These compounds typically exhibit significant antioxidant and anti-inflammatory activities, enhancing the overall bioactivity profile of the extract. Fatty acids and their derivatives accounted for 9.54%, reflecting the presence of certain lipid-soluble components in the extract. Other components, such as flavonoids, small peptides, and monoterpenes, although present in lower proportions, still collectively constitute the complex metabolite basis of Artemisia annua extract, providing a chemical basis for its multifaceted pharmacological effects. Overall, this extract is a natural product system with sesquiterpenes as its core and containing multiple bioactive components, holding significant value in drug development and phytochemical research. The top 50 substances detected in the original data are as follows:

[0060] name mass-to-charge ratio Retention time Molecular formula Material classification relative content <![CDATA[(1R,2R,6R,9R)-2,11,11-trimethyl-3-oxatricyclo[4.3.2.0¹, 5 undecane-9-carboxylic acid]]> 249.1499 245.5 C15H22O3 Sesquiterpenes 3.68% Isoscopolamine 193.0495 173 C10H8O4 Coumarins 3.25% Ethyl 3,5-dihydroxybenzoate 181.0508 206.9 C9H10O4 Phenolic acids 2.62% 2-(4-hydroxy-4,8-dimethyl-6-oxo-7-isopropylbicyclo[3.2.1]octane-1-yl)acetic acid 267.1605 222.5 C15H24O4 Sesquiterpenes 2.27% 3-O-feruloquinic acid 367.1039 161 C17H20O9 Phenylacetylene 2.15% Usnea acid 345.097 235.4 C18H16O7 Aromatic polyketones 1.95% chlorogenic acid 353.0881 137.4 C16H18O9 Phenylacetylene 1.79% (2S,4aR,8aS)-2-hydroxy-4a-methyl-8-methylenedehydronaphthyl-2-ylacrylic acid 249.1499 263 C15H22O3 Sesquiterpenes 1.75% sebacic acid 201.1134 216.7 C10H18O4 Fatty acids and their derivatives 1.61% <![CDATA[(1R,5R,8S)-2,3-dihydroxy-2,10,10-trimethyltricyclo[6.3.0.0¹, 5 undec-6-en-6-carboxylic acid]]> 265.1448 217 C15H22O4 Sesquiterpenes 1.59% 5-Hydroxyvalproic acid 159.1028 215.8 C8H16O3 Fatty acids and their derivatives 1.42% 8-Hydroxy-6-methyl-2-(4-methylpent-3-enyl)oct-2,6-dienoic acid 251.1655 238.7 C15H24O3 Monoterpenes 1.02% 3,4-Dehydro-6-hydroxyhoneycomb 191.0351 172.3 C10H8O4 Coumarins 0.85% <![CDATA[5-[6-(hydroxymethyl)-7-oxatricyclo[4.3.0.0³, 9 nonan-9-yl]-2-methyl-4-oxopentanoic acid]]> 281.1398 216.5 C15H22O5 Sesquiterpenes 0.83% choline 104.107 24.9 C5H14NO pseudoalkaloids 0.80% 1-Deoxypebranolide 415.2117 247.6 C24H30O6 Sesquiterpenes 0.73% Tianming styrolactone 249.1485 225.8 C15H20O3 Sesquiterpenes 0.66% <![CDATA[(4E)-8-Hydroxy-4-(2-hydroxy-1-methylethylidene)-10-oxatricyclo[7.2.1.0¹, 5 dodecane-8-carboxylic acid]]> 281.1398 200.3 C15H22O5 Sesquiterpenes 0.58% Dehydroauric acid lactone 231.1379 221.7 C15H18O2 Sesquiterpenes 0.54% Phenylalanine 166.0863 35.8 C9H11NO2 Small peptides 0.53% Pure mushroom extract 343.0827 235.6 C18H16O7 Polycyclic aromatic polyketones 0.51% Quinic acid 191.0561 59.6 C7H12O6 Phenolic acids 0.51% Tryptophan 205.0971 54.4 C11H12N2O2 Small peptides 0.49% betaine 118.0863 27.1 C5H11NO2 Small peptides 0.43% 6-Hydroxy-3,8a-dimethyl-5-methylene-4a,6,7,8,9,9a-hexahydro-4H-benzo[f]benzofuran-2-one 249.1485 203.5 C15H20O3 Sesquiterpenes 0.40% Alpha-linolenic acid 277.2177 302.7 C18H30O2 Fatty acids and their derivatives 0.39% Butyl lactate 145.0871 194.6 C7H14O3 Fatty acid esters 0.37% (1aS,3aS,6aS,6bR)-2-formyl-5,5,6b-trimethyl-3a,4,6,6a-tetrahydro-1H-cyclopropano[e]indene-1a-carboxylic acid 247.1342 256.3 C15H20O3 Sesquiterpenes 0.37% azelaic acid 187.0977 195.8 C9H16O4 Fatty acids and their derivatives 0.35% 2,6-Dihydroxy-3-methyl-4-(2-hydroxy-1-methylpropyl)benzoic acid 239.0927 164.2 C12H16O5 Phenolic acids 0.34% 10-Hydroxydecanoic acid 187.1341 260.7 C10H20O3 Fatty acids and their derivatives 0.34% 5-Hydroxy-2-methyl-7-O-β-D-glucopyranoside-4-chromogenone 355.1023 142.6 C16H18O9 Chromones 0.32% Pomodoro 471.3487 283.2 C30H48O4 Triterpenoids 0.32% stearic acid 283.2646 341.8 C18H36O2 Fatty acids and their derivatives 0.28% (3S,3aR,5R,7aS)-5-(2-carboxypropyl-2-en-1-yl)-3a-acetyl-3-hydroxy-7a-methylhexahydro-1H-indene 265.1447 241.6 C15H22O4 Sesquiterpenes 0.28% fulroacetone 183.0652 206.6 C9H10O4 Phloroglucinol 0.27% Kaempferol 3,7,4'-trimethyl ether 329.1021 257.8 C18H16O6 Flavonoids 0.25% DL-Leucine 132.1019 27.9 C6H13NO2 Fatty acids and their derivatives 0.22% Umbelliferone 161.0244 181.9 C9H6O3 Coumarins 0.22% salicylic acid 137.0245 173.1 C7H6O3 Phenolic acids 0.22% 5,7-Dihydroxy-2-(4-hydroxyphenyl)-6-O-β-D-glucopyranoside-8-O-β-D-xyloside-4-chromogenone 565.1552 165.3 C26H28O14 Flavonoids 0.19% Ash tree spirit 223.0601 171.7 C11H10O5 Coumarins 0.19% 9α,11-Dihydroxy-7-en-6-one 251.1655 259.6 C15H24O3 Sesquiterpenes 0.19% 5-(β-D-glucosoxy)-2-hydroxybenzoic acid 315.0724 32 C13H16O9 Phenolic acids 0.18% Xia Futa Gan 563.1416 166.1 C26H28O14 Flavonoids 0.18% callus acid 227.129 226.3 C12H20O4 Fatty acids and their derivatives 0.18% Poisonous horse flavonoids 359.0775 225.5 C18H16O8 Flavonoids 0.17% Poria cocos acid A 249.1498 223.5 C15H22O3 Sesquiterpenes 0.17% trans-2-octenic acid 141.0922 216.7 C8H14O2 Fatty acids and their derivatives 0.15% adenine 136.0618 31.8 C5H5N5 pseudoalkaloids 0.15%

[0061] Example 2: Cell viability experiment

[0062] (1) Take RAW264.7 cells and HaCaT cells in the logarithmic growth phase and collect them in 2mL centrifuge tubes, centrifuge, and discard the supernatant.

[0063] (2) Cells were counted using a cell counter, and the centrifuged cells were resuspended. The RAW264.6 cell count was adjusted to 1×10⁶ cells. 6 HaCaT cells were adjusted to 5 × 10⁶ cells / mL. 5 Cells were seeded at a density of 1 / mL into 96-well plates and cultured at 37°C in a 5% CO2 incubator until the cell confluence reached approximately 80%, at which point cell drug delivery was performed.

[0064] (3) The original culture medium was dried, and the cells were divided into a control group and a drug treatment group. The control group was replaced with fresh DMEM medium containing 10% fetal bovine serum (FBS), and the drug treatment group was treated with Artemisia annua extract diluted in a gradient at the maximum concentration of 100 μg / mL. The cells were cultured for another 24 hours.

[0065] (4) After 24 hours, add 10 μl of the CCK-8 kit to each well and incubate at 37°C in a 5% CO2 incubator for 1 hour. Then, measure the absorbance at 450 nm using a microplate reader. The data are presented as the mean ± standard deviation of three independent experiments. The relationship between cell growth inhibition rate and Artemisia annua extract concentration was derived from the absorbance values.

[0066] like Figure 1 As shown, compared with the control group, at a lower concentration gradient, the viability of RAW264.7 cells gradually increased with decreasing Artemisia annua extract concentration, exhibiting a certain trend. This indicates that as the drug concentration decreases, its inhibitory effect on cell viability diminishes and it exhibits a certain promoting effect, demonstrating a dose-response relationship. The promoting effect reached its peak at a concentration of 3.125 μg / mL. Subsequent experiments selected 25 μg / mL, which had no effect on cell viability, as the maximum drug treatment concentration.

[0067] like Figure 2 As shown, compared with the control group, at a lower concentration gradient, the viability of HaCaT cells gradually increased with decreasing Artemisia annua extract concentration, exhibiting a certain trend. This indicates that as the drug concentration decreases, its inhibitory effect on cell viability decreases and it exhibits a certain promoting effect, demonstrating a dose-response relationship. Subsequent experiments selected 12.5 μg / mL as the maximum drug concentration, which had no effect on cell viability, for treatment.

[0068] Example 3: Cell phagocytosis experiment

[0069] (1) Collect RAW264.7 cells in the logarithmic growth phase into a 2mL centrifuge tube, centrifuge, and discard the supernatant.

[0070] (2) Cells were counted using a cell counter, and the centrifuged cells were resuspended. The RAW264.6 cell count was adjusted to 1×10⁶ cells. 6 Cells were seeded at a density of 1 / mL into 96-well plates and cultured at 37°C in a 5% CO2 incubator until the cell confluence reached approximately 80%, at which point cell drug delivery was performed.

[0071] (3) After incubation for 24 hours, the changes in the phagocytic capacity of RAW264.7 cells treated with different concentrations of Artemisia annua extract were detected by neutral red staining.

[0072] (4) Blot dry the old culture medium, wash with PBS to remove residual culture medium, add 100 μL of 1 mg / mL neutral red solution to each well, and incubate at 37℃ and 5% CO2 for 30 min.

[0073] (5) Aspirate the neutral red staining solution, wash with PBS, add 100 μl of cell lysis buffer to each well, and incubate at 37°C on a shaker for 30 min.

[0074] (6) The absorbance was measured at OD540nm using an ELISA reader. The data are presented as the mean ± standard deviation of three independent experiments. The data were analyzed to determine the relationship between the phagocytic capacity of macrophages and the concentration of Artemisia annua extract treatment based on the absorbance.

[0075] like Figure 3 As shown, the phagocytic capacity of RAW264.7 cells increased slowly with decreasing concentration of Artemisia annua extract, but the promoting effect was not significant. Preliminary data indicate that Artemisia annua extract treatment tends to improve the phagocytic capacity of RAW264.7 cells, although this did not reach statistical significance under the current experimental conditions. This trend suggests that it may have mild immunomodulatory potential, which warrants further verification in subsequent studies with larger sample sizes and optimized conditions.

[0076] Example 4: Cell Migration Experiment

[0077] (1) First, use a marker pen to draw horizontal lines evenly on the back of the 6-well plate to mark the area to be photographed. Collect HaCaT cells in the logarithmic growth phase into a 2mL centrifuge tube, centrifuge, and discard the supernatant.

[0078] (2) Cells were counted using a cell counter, and the centrifuged cells were resuspended. The HaCaT cell count was adjusted to 2.5 × 10⁻⁶. 5 Cells were seeded at a density of 1 / mL into 6-well plates and cultured at 37°C in a 5% CO2 incubator until the cell confluence reached approximately 90%.

[0079] (3) Remove the culture medium, use a 200 μL yellow pipette tip to streak the well plate vertically, wash away excess cell debris and floating cells with PBS, ensure the streak area is clean, take a picture under a microscope, and record 0h of drug administration.

[0080] (4) Add serum-free (FBS) DMEM containing 12.5 μg / mL Artemisia annua extract, incubate for 24 h, wash off the culture medium with PBS, and take pictures under a microscope according to the markings.

[0081] (5) Cell migration rate = (initial scratch area – scratch area after 24 h) / initial scratch area x 100%.

[0082] like Figure 4 As shown in the figure, A represents the control group, B represents the group treated with 12.5 μg / mL Artemisia annua extract, C represents the group treated with 6.25 μg / mL Artemisia annua extract, and D is a bar chart showing the significance of the experimental and control groups. In this experimental system, the migration ability of HaCaT cells was detected using the scratch assay. After 24 hours of culture, the cell migration rate of the untreated control group was 23.39%, while the cell migration rate of the experimental group treated with 6.25 μg / mL Artemisia annua extract was 19.7%, and the cell migration rate of the experimental group treated with 12.5 μg / mL Artemisia annua extract was 16.2%. The data indicate that Artemisia annua extract treatment reduced the in vitro migration rate of HaCaT cells to some extent. This inhibitory effect suggests that Artemisia annua extract may be involved in regulating the rate and process of skin repair.

[0083] Example 5: Nitric Oxide Detection Experiment

[0084] (1) RAW264.7 with 1×10 5 The cells were seeded at a density of 1 / mL in 96-well plates and cultured at 37°C in a 5% CO2 incubator until the cell confluence reached 80%.

[0085] (2) Artemisia annua extract was administered to cells at the maximum concentration of 12.5 μg / mL. After incubation for 24 h, the NO content in the supernatant was determined using a nitric oxide assay kit (Beyotime, China).

[0086] (3) In a new 96-well plate, collect 50 μl of supernatant from each well, and add equal volumes of room temperature GriessReagent I and II sequentially. Measure the absorbance at 540 nm. The NO content was evaluated by preparing a standard curve using a standard diluted with complete culture medium.

[0087] like Figure 5As shown, the inhibitory effect of artemisinin extract on nitric oxide release from RAW 264.7 cells increased with increasing concentration, indicating that artemisinin extract is an effective macrophage NO release inhibitor with a concentration-dependent effect, and its mechanism points to intervention in classical inflammatory pathways. This is not only key evidence for its role as an anti-inflammatory substance, but also lays a solid foundation for subsequent identification of active ingredients, in-depth analysis of mechanisms, and drug development for specific inflammatory diseases.

[0088] Example 6: qRT-PCR experiment

[0089] (1) RAW264.7 cells and HaCaT cells were grown at a rate of 3 × 10⁻⁶. 5 The cells were seeded at a density of 1 / mL in 6-well plates and cultured at 37°C in a 5% CO2 incubator until the cell confluence reached 70-80%.

[0090] (2) Aspirate the culture medium. RAW264.7 cells were used to establish a macrophage inflammation model in serum-free medium containing 1 μg / mL LPS (lipopolysaccharide), and were treated with serially diluted Artemisia annua extract at the maximum concentration of 25 μg / mL. HaCaT cells were used to establish an atopic dermatitis cell model in serum-free medium containing 100 ng / mL IL-4 / IL-13, and were treated with serially diluted Artemisia annua extract at the maximum concentration of 12.5 μg / mL.

[0091] (3) After 48 hours in the incubator, total RNA was extracted from the cells using the RNA Easy Fast Animal Tissue / Cell Total RNA Extraction Kit.

[0092] (4) RNA concentration and purity were measured using NanoDrop2000. The total RNA concentration was adjusted to 500 ng / 20 μL and RNA was reverse transcribed using 5×PrimeScript RT Master Mix (Takara, China) and RNA-free ddH2O.

[0093] (5) Reverse transcription was performed using a LightCycler® 96 real-time quantitative PCR instrument (Roche Pharmaceuticals Shanghai Co., Ltd., China) at 42℃ for 15 s, 37℃ for 20 min, and 85℃ for 5 s. cDNA was used for quantitative qPCR. The cDNA was diluted 200-fold, and 8.8 μL was combined with 10 μL of 2×FastFire qPCR PreMix and 0.6 μL (10 pmol / mL) of upstream and downstream specific primers. qPCR was performed under the following conditions: pre-denaturation at 95℃ for 2 min; 40 cycles: 95℃ for 10 s; 60℃ for 20 s; 72℃ for 30 s.

[0094] like Figure 6As shown, when the RAW264.7 inflammation model was treated with 25 μg / mL Artemisia annua extract, the expression levels of mRNAs of inflammatory factors IL-6, TNF-α, IL-1β, and iNOS were significantly reduced compared to the model group. This indicates that Artemisia annua extract can effectively inhibit the expression of inflammatory factors, confirming it as an effective inflammatory transcriptional inhibitor at the gene expression level. It likely targets core pathways such as NF-κB, synergistically inhibiting the production of key inflammatory mediators, thereby exerting anti-inflammatory and immunomodulatory effects at the root cause.

[0095] like Figure 7 As shown, artemisia annua extract can preferentially regulate barrier and remodeling-related genes in an AD cell model in a concentration-dependent manner. Its core characteristics are: at lower concentrations, it exhibits a tendency to restore tight junction proteins (especially OCLN) and a mild ameliorative effect on terminal differentiation proteins (FLG), but its repair effect on CLDN1 is limited, and it may have complex effects on collagen metabolism.

[0096] In summary, this invention provides a simple, efficient, environmentally friendly method for extracting Artemisia annua extract from Artemisia annua leaves suitable for industrial production. Using Artemisia annua leaves as raw material, the method involves drying at 50°C, ultrasonic-assisted extraction with 70% ethanol, rotary evaporation to recover ethanol, and freeze-drying, successfully preparing a component-rich Artemisia annua extract. The extraction process effectively preserves the active ingredients in Artemisia annua, avoids structural damage, and the raw materials are readily available and the cost is controllable.

[0097] From the perspective of compositional characteristics, metabolomics analysis of this artemisia extract identified 710 substances, with sesquiterpenes as the core (relative content 30.05%), of which artemisinin content reached 45%. It also contains a variety of active ingredients such as phenolic acids, coumarins, phenylpropanoids, fatty acids and their derivatives, forming a natural product system with comprehensive biological activities such as anti-oxidation and anti-inflammation, providing a solid chemical basis for its multifaceted pharmacological effects.

[0098] Pharmacological experiments have confirmed that the artemisia annua extract has significant biological activity: First, it can promote the activity of macrophages (RAW264.7) and immortalized human keratinocytes (HaCaT) in a concentration-dependent manner, enhance the phagocytic capacity of macrophages, and exert a mild immunomodulatory effect; Second, it can effectively inhibit the release of nitric oxide (NO) from macrophages and the mRNA expression of inflammatory factors such as IL-6, TNF-α, and IL-1β, targeting and intervening in inflammatory pathways to improve the immune microenvironment from the source; Third, it can inhibit HaCaT cell migration and, in an atopic dermatitis cell model, regulate the expression of skin barrier-related genes in a concentration-dependent manner, promote the recovery of expression of tight junction proteins (such as OCLN) and terminal differentiation proteins (FLG), and possess skin barrier repair function.

[0099] In terms of application scenarios, addressing the pain points of limited efficacy, significant side effects, or high cost of existing treatments for atopic dermatitis, the artemisia extract of this invention, as a natural extract, is safe, effective, economical, and readily available. It not only broadens the development and utilization of artemisia leaves and enhances the medicinal value of artemisia, but also provides a novel natural candidate solution for the treatment of inflammatory skin diseases. It has important clinical significance and social value in drug development, skin care, and other fields, and has broad application prospects.

[0100] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. An extraction method for extracting artemisinin extract from leaves of Artemisia annua, characterized by, The method comprises the following steps: Take the leaves of Artemisia annua, and perform drying treatment after packaging; Put the dried leaves of Artemisia annua into a wide-mouth bottle, and make the leaves fully infiltrate, then put the fully infiltrated leaves into an ultrasonic instrument for ultrasonic-assisted extraction; After the ultrasonic-assisted extraction is completed, filter to obtain filter residue, then repeat the ultrasonic-assisted extraction to obtain all filtrates; Perform rotary evaporation on all the filtrates, then dissolve the concentrated liquid in a proper amount of pure water again, perform chromatographic separation, use gradient elution, collect specified elution parts, and perform rotary evaporation again to obtain Artemisia annua extract.

2. The extraction method according to claim 1, wherein the extraction method is characterized by, Take the leaves of Artemisia annua, and perform drying treatment after packaging, specifically including taking the leaves of Artemisia annua, putting them into a cowhide paper envelope, and putting them into a 50℃ oven for drying for 48 hours.

3. The extraction method according to claim 1, wherein the extraction method is characterized by, Put the dried leaves of Artemisia annua into a wide-mouth bottle, and make the leaves fully infiltrate, then put the fully infiltrated leaves into an ultrasonic instrument for ultrasonic-assisted extraction, specifically as follows: Put 1000g of the dried leaves of Artemisia annua into a wide-mouth bottle after drying for 48 hours; Add 7L of 70% ethanol into the wide-mouth bottle, and stir to make the ethanol fully infiltrate the leaves of Artemisia annua; After soaking for 2 hours, put the wide-mouth bottle into an ultrasonic instrument for ultrasonic-assisted extraction.

4. The extraction method according to claim 3, wherein the extraction method is characterized by, The temperature of the ultrasonic-assisted extraction is 50℃, the power is 60W, and the ultrasonic time is 1 hour.

5. The extraction method according to claim 3, wherein the extraction method is characterized by, After the ultrasonic-assisted extraction is completed, filter to obtain filter residue, then repeat the ultrasonic-assisted extraction to obtain all filtrates; specifically as follows: After the ultrasonic ends, filter with gauze and wring out the filter residue; Repeat the above ultrasonic-assisted extraction by adding 4L of 70% ethanol into the filter residue, filtering and wringing out the filter residue, and collecting all the filtrates as much as possible.

6. The method for extracting Artemisia annua extract from Artemisia annua leaves as described in claim 1, characterized in that, Perform rotary evaporation on all the filtrates, then dissolve the concentrated liquid in a proper amount of pure water again, perform chromatographic separation, use gradient elution, collect specified elution parts, and perform rotary evaporation again to obtain Artemisia annua extract, specifically as follows: Put all the filtrates into a rotary evaporator in batches for rotary evaporation, dissolve the concentrated liquid obtained again in a proper amount of pure water, then use HPD-100 type macroporous adsorption resin to perform chromatographic separation on the solution, use pure water, 30%, 50%, 75%, and 90% ethanol gradient elution in sequence, collect the 75% elution part, and perform rotary evaporation again on it; Collect the Artemisia annua extract obtained by rotary evaporation.

7. The extraction method according to claim 6, wherein the extraction method is characterized by, The temperature is 60℃ and the rotation speed is 60rpm during the rotary evaporation.

8. The method for extracting Artemisia annua extract from Artemisia annua leaves as described in claim 1, characterized in that, Further include putting the collected Artemisia annua extract into a stainless steel tray, putting it into a pre-cooled freeze dryer for freeze drying for 48 hours, collecting it into a 50mL centrifuge tube, sealing, and storing at -20℃.

9. The method of claim 1-8, wherein the extraction method is characterized by, The content of sesquiterpenes in the obtained Artemisia annua extract is more than 30%.

10. The use of Artemisia annua extract extracted from the leaves of Artemisia annua in the preparation of a medicament for treating atopic dermatitis and in the preparation of a cosmetic product with soothing and anti-inflammatory functions.

11. Use according to claim 10, wherein the compound is ###0002### Inhibit macrophage inflammation and promote epidermal cell proliferation, thereby restoring the skin barrier.

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