Application of Chinese mugwort leaf alcohol extract in preparation of medicine for resisting oxidative stress injury

Abstract

The invention discloses application of a Chinese mugwort leaf alcohol extract in preparation of a medicine for resisting oxidative stress injury, and belongs to the technical field of biological medicines. It is found for the first time that the Chinese mugwort leaf alcohol extract significantly prolongs the average life of wild type caenorhabditis elegans under the oxidative stress condition and significantly improves the level and / or content of superoxide dismutase, catalase and reduced glutathione in wild type caenorhabditis elegans. Meanwhile, an antioxidant key transcription factor SKN-1 in transgenic caenorhabditis elegans LD1 is promoted to enter a nucleus, the expression of genes skn-1, gst-4, gcs-1, sod-2 and ctl-1 related to oxidative stress resistance is remarkably up-regulated, the expression of resistant proteins GCS-1 and GST-4 is further remarkably improved, and finally, an SKN-1 antioxidant signal channel is activated, so that the in-vivo antioxidant effect is enhanced, and therefore, the antioxidant activity of the caenorhabditis elegans is improved. The Chinese mugwort leaf alcohol extract has a good application prospect in preparation of medicines for resisting oxidative stress injury.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of the ethanol extract of Artemisia argyi Levl. var. argyi in the preparation of a drug for antioxidative stress injury. Background Art

[0002] Artemisia argyi Levl. var. argyi ( Artemisia argyi Levl. et Vant. cv. Qiai) is a cultivated variety of Artemisia argyi, a genus of Compositae, produced in Qichun, Hubei. Because of its significantly better quality than Artemisia argyi from other producing areas, it has now become a national geographical indication product in China. Artemisia argyi Levl. var. argyi is a traditional Chinese medicinal and edible plant in China, with a long history of medicinal and edible use. Flavonoids and polyphenols are important active ingredients in Artemisia argyi Levl. var. argyi, with various biological activities such as anti-inflammatory, antioxidant, anti-tumor, and antibacterial. Song et al. (SONGX W, XIANG W, HE J W, et al. Phytochemical components and biological activities of Artemisia argyi [J]. Journal of Functional Foods, 2019, 52: 648-662.) reported that Artemisia argyi Levl. var. argyi contains at least more than 30 flavonoids with different structures. In addition, it has been reported that three active ingredients, chlorogenic acid, naringin, and kaempferol, are contained in Artemisia argyi Levl. var. argyi samples of different batches, which can be used for the quality evaluation and quality control of Artemisia argyi Levl. var. argyi.

[0003] Caenorhabditis elegans, abbreviated as C. elegans, is widely used in the research of aging, oxidative stress tolerance, lipid metabolism, toxicology evaluation, neurodegenerative diseases, and behavior. Compared with traditional animal models, C. elegans has the advantages of small size (the body length of adult worms is about 1 mm), short life cycle (wild-type C. elegans is about 21 days), transparent body, easy cultivation, strong reproductive ability, detailed genomic sequence annotation, and 60-80% of its genes being highly conserved with humans. Research has shown that the excessive accumulation of free radicals in the body will destroy the redox homeostasis in the body, which is one of the factors promoting aging and inducing chronic diseases. Some natural plant extracts have increasingly become a research hotspot due to their good antioxidant activity, rich sources, and low side effects. Recent research reports have shown that the ethanol extract of citrus peel and the extract of papaya leaves have good free radical scavenging ability and have become important sources of natural antioxidants.

[0004] So far, there has been no report on whether the active ingredients of Artemisia argyi Levl. var. argyi have in vivo antioxidant effects and related mechanisms of action. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of the ethanol extract of Artemisia argyi Levl. var. argyi in the preparation of a drug for antioxidative stress injury, so as to solve the problem of the lack of the ethanol extract of Artemisia argyi Levl. var. argyi with a high in vivo antioxidative stress injury effect in the prior art.

[0006] In a first aspect, the present invention provides the use of the ethanol extract of Artemisia argyi Levl. et Vant. var. qiai in the preparation of a medicament for antioxidative stress injury. The preparation of the ethanol extract of Artemisia argyi Levl. et Vant. var. qiai comprises the following steps: providing the crude powder of Artemisia argyi Levl. et Vant. var. qiai; mixing the crude powder of Artemisia argyi Levl. et Vant. var. qiai with an organic solvent and then performing microwave treatment, filtering to obtain a filtrate; removing the organic solvent from the filtrate and then performing freeze-drying to obtain the ethanol extract of Artemisia argyi Levl. et Vant. var. qiai.

[0007] In the present invention, the inventors for the first time found through research that the ethanol extract of Artemisia argyi Levl. et Vant. var. qiai significantly prolongs the average lifespan of wild-type Caenorhabditis elegans under oxidative stress conditions, significantly increases the levels and / or contents of superoxide dismutase, catalase, and reduced glutathione in wild-type Caenorhabditis elegans, promotes the nuclear entry of the antioxidant key transcription factor SKN-1 in transgenic Caenorhabditis elegans LD1, and significantly upregulates genes skn-1 、 gst-4 、 gcs-1 、 sod-2 、 ctl-1 related to oxidative stress resistance, and further significantly increases the expression of the resistance proteins GCS-1 and GST-4, ultimately activating the SKN-1 antioxidant signaling pathway, thereby enhancing the antioxidant effect in vivo. Therefore, the ethanol extract of Artemisia argyi Levl. et Vant. var. qiai has good application prospects in the preparation of a medicament for antioxidative stress injury.

[0008] The preparation method of the ethanol extract of Artemisia argyi Levl. et Vant. var. qiai provided by the present invention is simple, and the raw materials used are cheap and easily available, which is suitable for large-scale industrial production applications.

[0009] In some embodiments, the preparation of the crude powder of Artemisia argyi Levl. et Vant. var. qiai comprises: drying, pulverizing, and sieving Artemisia argyi Levl. et Vant. var. qiai to obtain the crude powder of Artemisia argyi Levl. et Vant. var. qiai.

[0010] In some preferred embodiments, the mesh number of the sieve for sieving is 40 - 80 meshes.

[0011] In some more preferred embodiments, the mesh number of the sieve for sieving is 6o meshes.

[0012] In some embodiments, the effective concentration of the ethanol extract of Artemisia argyi Levl. et Vant. var. qiai is 50 - 400 μg / mL.

[0013] In some preferred embodiments, the effective concentration of the ethanol extract of Artemisia argyi Levl. et Vant. var. qiai is 200 μg / mL.

[0014] In some embodiments, during the process of mixing the crude powder of Artemisia argyi Levl. et Vant. var. qiai with an organic solvent and then performing microwave treatment, the material-liquid ratio of the crude powder of Artemisia argyi Levl. et Vant. var. qiai to the organic solvent is (0.5 - 1.5 g):(70 - 90 mL).

[0015] In some preferred embodiments, during the microwave treatment after mixing crude Artemisia argyi Levl. & Vant. powder with an organic solvent, the material-liquid ratio of the crude Artemisia argyi Levl. & Vant. powder to the organic solvent is 1 g: 80 mL.

[0016] In some embodiments, during the microwave treatment after mixing crude Artemisia argyi Levl. & Vant. powder with an organic solvent, the organic solvent includes ethanol, and the microwave treatment specifically includes: under the condition of a microwave power of 300 - 500 W, treating for 3 - 7 min.

[0017] In some preferred embodiments, during the microwave treatment after mixing crude Artemisia argyi Levl. & Vant. powder with an organic solvent, the organic solvent includes ethanol, and the microwave treatment specifically includes: under the condition of a microwave power of 400 W, treating for 5 min.

[0018] In some more preferred embodiments, the volume concentration of ethanol is 50%.

[0019] It can be understood that the organic solvent can be selected from conventional organic solvents in the prior art according to actual usage needs, as long as it can extract the active ingredients from the crude Artemisia argyi Levl. & Vant. powder. In the present invention, the organic solvent preferably includes ethanol, and the volume concentration of ethanol is preferably 50%.

[0020] In some embodiments, the ethanol extract of Artemisia argyi Levl. & Vant. includes flavonoids and polyphenols; among them, the flavonoid content is 100 - 104 mg / g, and the polyphenol content is 46 - 50 mg / g.

[0021] In some embodiments, the ethanol extract of Artemisia argyi Levl. & Vant. prolongs the average lifespan of wild-type Caenorhabditis elegans under oxidative stress conditions, and the prolongation of the average lifespan of wild-type Caenorhabditis elegans under oxidative stress conditions depends on skn-1 , and by increasing the levels and / or contents of superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) in wild-type Caenorhabditis elegans, thereby achieving the antioxidant effect in vivo.

[0022] In some embodiments, the ethanol extract of Artemisia argyi Levl. & Vant. promotes the nuclear entry of the antioxidant key transcription factor SKN-1 in transgenic Caenorhabditis elegans LD1, and up-regulates the genes related to oxidative stress resistance skn-1 , gst-4 , gcs-1 , sod-2 , ctl-1 expression, and at the same time increases the expression of the resistance proteins GCS-1 and GST-4, ultimately activating the SKN-1 antioxidant signaling pathway, thereby enhancing the antioxidant effect in vivo.

[0023] In a second aspect, the present invention provides a pharmaceutical composition for preventing and / or treating oxidative stress injury, which includes the above-mentioned ethanol extract of Artemisia argyi Levl. & Vant.

[0024] In some embodiments, in the pharmaceutical composition, the effective concentration of the ethanol extract of Artemisia argyi Levl. et Vant. var. qiai (Levl.) Hsiao is 50 - 400 μg / mL.

[0025] In some preferred embodiments, the effective concentration of the ethanol extract of Artemisia argyi Levl. et Vant. var. qiai (Levl.) Hsiao is 200 μg / mL.

[0026] In some embodiments, the above-mentioned pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0027] In the present invention, the term "pharmaceutically acceptable carrier" refers to excipients widely used in the field of drug production. Excipients are mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide methods to enable the active ingredient to dissolve at a desired rate after the subject receives the administration, or to promote the effective absorption of the active ingredient after the subject receives the composition administration. Pharmaceutical excipients can be inert fillers or provide certain functions, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. Pharmaceutical excipients can include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesives, glidants, wetting agents, gelling agents, absorption retardants, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweetening agents.

[0028] The pharmaceutical composition provided by the present invention can be prepared by any method known to those skilled in the art according to the disclosed content. For example, including but not limited to conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or freeze-drying processes.

[0029] In some embodiments, the dosage form of the pharmaceutical composition includes at least one of solid preparations, semi-solid preparations and liquid preparations.

[0030] The pharmaceutical composition provided by the present invention can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra - arterial, intramuscular) administration. The pharmaceutical composition of the present invention can also be in a controlled - release or sustained - release dosage form (such as liposomes or microspheres). Examples of solid oral preparations include, but are not limited to, powders, capsules, caplets, soft capsules and tablets. Examples of liquid preparations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs and solutions. Examples of topical preparations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or serum preparations. Examples of preparations for parenteral administration include, but are not limited to, injection solutions, dry powder preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, injection suspensions and injection emulsions. Examples of other suitable preparations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalants; liquid dosage forms suitable for parenteral administration; suppositories and lozenges.

[0031] The beneficial effects of the present invention are as follows: Different from the prior art, the present invention for the first time discovers that the ethanol extract of Artemisia argyi Levl. et Vant. var. qiai (Levl.) Hsiao can significantly prolong the average lifespan of wild - type Caenorhabditis elegans under oxidative stress conditions, and significantly increase the levels and / or contents of superoxide dismutase, catalase, and reduced glutathione in wild - type Caenorhabditis elegans. At the same time, it promotes the nuclear entry of the key antioxidant transcription factor SKN - 1 in transgenic Caenorhabditis elegans LD1, and significantly up - regulates the genes skn-1 、 gst-4 、 gcs-1 、 sod-2 、 ctl-1 expression, and further significantly increases the expression of the resistance proteins GCS - 1 and GST - 4, ultimately activating the SKN - 1 antioxidant signaling pathway, thereby enhancing the antioxidant effect in vivo. Therefore, the ethanol extract of Artemisia argyi Levl. et Vant. var. qiai (Levl.) Hsiao has good application prospects in the preparation of drugs for antioxidative stress injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 shows the results of the effect of the ethanol extract of Artemisia argyi Levl. et Vant. var. qiai (Levl.) Hsiao on the lifespan of wild - type Caenorhabditis elegans under oxidative stress conditions in Example 2 of the present invention; Figure 2 shows the results of the effect of the ethanol extract of Artemisia argyi Levl. et Vant. var. qiai (Levl.) Hsiao on the activities of SOD enzyme, CAT enzyme, and the level of reduced glutathione GSH in N2 nematodes in Example 3 of the present invention. Among them, " " represents p <0.05, " " represents p <0.01, " " represents p <0.001, all compared with the control group; Figure 3 This is the result of the effect of the ethanol extract of Artemisia argyi Levl. et Vant. from Qichun on the nuclear localization of SKN-1 protein in Caenorhabditis elegans in Example 4 of the present invention. Among them, " " indicates p <0.001, compared with the control group, ns indicates no significant difference; Figure 4 This is the result of the effect of the ethanol extract of Artemisia argyi Levl. et Vant. from Qichun on the lifespan of EU1 nematodes under oxidative stress conditions in Example 5 of the present invention; Figure 5 This is the result of the effect of the ethanol extract of Artemisia argyi Levl. et Vant. from Qichun on the expression of antioxidant-related genes in wild-type nematodes in Example 6 of the present invention. Among them, " " indicates p <0.05, " " indicates p <0.01, all compared with the control group; Figure 6 This is the result of the effect of the ethanol extract of Artemisia argyi Levl. et Vant. from Qichun on the expression of GCS-1::GFP in the LD1171 nematode strain in Example 7 of the present invention. Among them, " " indicates p <0.001, compared with the control group; Figure 7 This is the result of the effect of the ethanol extract of Artemisia argyi Levl. et Vant. from Qichun on the expression of GST-4::GFP in the CL2166 nematode strain in Example 8 of the present invention. Among them, " " indicates p <0.001, compared with the control group. Detailed implementation manners

[0033] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0034] For the experimental methods without specific conditions in the embodiments, they are usually carried out according to the conventional conditions and the conditions described in the manual, or according to the conditions recommended by the manufacturer. For the general equipment, materials, reagents, etc., if there is no special description, they can all be obtained from commercial channels.

[0035] In the present invention, three-year-old Artemisia argyi Levl. et Vant. from Qichun was purchased from Qichun Qaikang Herb Technology Co., Ltd.; wild-type Caenorhabditis elegans strain (N2), transgenic Caenorhabditis elegans EU1 skn-1 (zu67)Ⅳ , transgenic Caenorhabditis elegans CL2166 (pAF15)gst- 4p::GFP::NLS , transgenic Caenorhabditis elegans LD1 [ldls7(skn-1b / c::GFP + rol-6(su1006)) 1. Transgenic Caenorhabditis elegans LD1171 [ldIs3 (gcs-1p::GFP + rol-6(su1006)) was purchased from the Caenorhabditis Genetics Center (CGC), University of Minnesota; sodium nitrite, aluminum nitrate, Folin-Ciocalteu phenol, and sodium carbonate were purchased from the National Pharmaceutical Group; gallic acid standard and rutin standard were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; dimethyl sulfoxide (DMSO) and 5-fluoro-2'-deoxyuridine (FudR) were purchased from Sigma, USA; SOD kit, GSH kit, CAT kit, and BCA kit were purchased from Nanjing Jiancheng Bioengineering Institute; RNA extraction kit was purchased from Beijing Aidlab Biotechnologies Co., Ltd.; cDNA reverse transcription kit and qPCR kit SYBR were purchased from Novoprotein Scientific Inc.

[0036] Example 1 Preparation of ethanol extract of Artemisia argyi Levl. var. argyi H. Lév. This example provides a method for preparing the ethanol extract of Artemisia argyi Levl. var. argyi H. Lév., specifically including the following steps: The three-year-old Artemisia argyi Levl. var. argyi H. Lév. was dried, crushed, and sieved through a 60-mesh sieve to obtain the crude powder of Artemisia argyi Levl. var. argyi H. Lév.; then a certain amount of the crude powder of Artemisia argyi Levl. var. argyi H. Lév. was weighed and added to a 50% ethanol solution according to a solid-liquid ratio of 1 g:80 mL for microwave extraction. Among them, the microwave power was 400 W, and the microwave extraction time was 5 min. After extraction, it was filtered to obtain the filtrate; then the filtrate was rotary evaporated at 55 °C until there was no ethanol residue and then freeze-dried to obtain the freeze-dried powder of the ethanol extract of Artemisia argyi Levl. var. argyi H. Lév.

[0037] The flavonoid and polyphenol contents in the ethanol extract of Artemisia argyi Levl. var. argyi H. Lév. prepared above were tested.

[0038] Among them, the method for determining the flavonoid content was as follows: The freeze-dried powder of the ethanol extract of Artemisia argyi Levl. var. argyi H. Lév. prepared above was dissolved in 50% ethanol. Take 200 μL of rutin standard solution or freeze-dried powder sample solution with different concentrations, add 75 μL of 0.5% NaNO2 solution, mix well, and let it stand in the dark for 5 min. Then add 150 μL of 10% Al(NO3)3·6H2O solution and let it stand for 5 min. Finally, add 500 μL of 1 mol / L NaOH solution, and measure the absorbance at λ = 510 nm. Calculate the flavonoid content in the sample according to the rutin standard curve and the absorbance of the sample solution, and the result is expressed as mg rutin / g sample.

[0039] The method for determining the polyphenol content is as follows: The freeze-dried powder of the ethanol extract of Artemisia argyi Levl. et Vant. from Qiai is dissolved in 50% ethanol. Take 500 μL of the sample and standard solutions of gallic acid with different concentrations, mix them with 250 μL of 50% Folin-Ciocalteu reagent, react for 5 min, then add 500 μL of 5% sodium carbonate solution, react in the dark for 1 h, measure the absorbance at λ = 765 nm, and calculate the polyphenol content in the sample according to the standard curve of gallic acid and the absorbance of the sample solution. The polyphenol content is expressed as mg gallic acid / g sample.

[0040] The measurement results showed that the flavonoid content in the ethanol extract of Artemisia argyi Levl. et Vant. from Qiai was 102.23 ± 0.76 mg / g, and the polyphenol content was 48.04 ± 0.45 mg / g. The results indicated that the content of bioactive substances in the ethanol extract of Artemisia argyi Levl. et Vant. from Qiai was relatively high.

[0041] Example 2 Effect of the ethanol extract of Artemisia argyi Levl. et Vant. from Qiai on the oxidative stress resistance of wild-type Caenorhabditis elegans In this example, a strong oxidant (such as hydrogen peroxide) was used to stimulate the production of a large amount of reactive oxygen species in the nematodes, which would exacerbate the oxidative stress damage of cells. Then, the effects of the ethanol extract of Artemisia argyi Levl. et Vant. from Qiai at different concentrations on the lifespan of Caenorhabditis elegans incubated in a 1 mmol / L hydrogen peroxide solution were measured.

[0042] First, the cultivation and drug addition treatment of Caenorhabditis elegans were carried out, which specifically included the following steps: Wash the nematodes in the egg-laying period from the NGM plate into a 1.5 mL centrifuge tube with phosphate buffer, remove the supernatant to wash away the bacterial solution attached to the worm body, add 1 mL of nematode lysis solution (a mixture of 5 M sodium hydroxide solution and 10% sodium hypochlorite solution) to the centrifuge tube, quickly and vigorously shake until the worm body is completely lysed, centrifuge at 10000 r / min for 1 min at room temperature, discard the supernatant, and wash the precipitate 4 times with buffer. After obtaining the eggs, place them on the NGM plate to hatch into synchronized larvae for standby; Add 300 μL of S-complete medium (0.3 mL of 1 M potassium citrate buffer, 0.3 mL of metal ion buffer, 90 μL of 1 M CaCl2, 90 μL of 1 M MgSO4, 30 μL of cholesterol) to a 24-well plate. Among them, a control group containing only DMSO and drug addition groups containing 50, 200, and 400 μg / mL of the ethanol extract of Artemisia argyi Levl. et Vant. from Qiai were set. Transfer the synchronized nematodes at the L4 stage to the S-complete medium, with about 300 nematodes in each well. The day of transfer was recorded as the 0th day, and the culture plate was placed at 20 °C for cultivation.

[0043] Then, after culturing the wild-type nematode strain N2 for 7 days according to the above-mentioned cultivation and drug addition treatment methods of Caenorhabditis elegans, the nematodes in the control group Control (containing only 0.6% DMSO) and the drug addition groups (containing 50, 200, 400 μg / mL of the ethanol extract of Artemisia argyi Levl., recorded as the 50 μg / mL-ALE, 200 μg / mL-ALE, and 400 μg / mL-ALE groups respectively) were transferred to a 96-well plate containing 100 μL of a H2O2 solution with a final concentration of 1 mmol / L per well. At room temperature, the survival rate of the nematodes was recorded every 2 hours until all the nematodes died. Finally, a survival curve was plotted using GraphPad software and the data was analyzed by log-rank (Mantel−Cox). The results are shown in Figure 1 and Table 1 respectively.

[0044] Table 1 Effects of the ethanol extract of Artemisia argyi Levl. on the lifespan of wild-type Caenorhabditis elegans under oxidative stress conditions

[0045] Note: Different letters in the same column indicate significant differences between the two groups, p <0.05.

[0046] From Figure 1 and Table 1, it can be seen that compared with the control group, the average lifespan of the nematodes treated with 50, 200, 400 μg / mL of the ethanol extract of Artemisia argyi Levl. was significantly extended by 13.21% ( p =0.0002), 21.72% ( p <0.0001), 16.11% ( p <0.0001) respectively under oxidative stress conditions. The results show that the ethanol extract of Artemisia argyi Levl. significantly enhanced the oxidative stress resistance of the nematodes, and the effect of 200 μg / mL of the ethanol extract of Artemisia argyi Levl. was the best.

[0047] Example 3 Effects of the ethanol extract of Artemisia argyi Levl. on the antioxidant enzyme activities in wild-type Caenorhabditis elegans In this example, the effects of the ethanol extract of Artemisia argyi Levl. on the SOD enzyme, CAT enzyme, and GSH level in wild-type Caenorhabditis elegans were determined.

[0048] Specifically, nematodes N2 were cultured according to the cultivation and drug addition treatment methods of Caenorhabditis elegans in Example 2. When the nematodes were cultured to the 3rd day of adulthood, the nematodes in the control group and the drug addition groups were collected and ground thoroughly on ice, and then centrifuged at 4°C for 5 minutes to obtain the supernatant. According to the operation manuals of the SOD kit, CAT kit, GSH kit, and BCA kit, the SOD enzyme activity, CAT enzyme activity, and the content of reduced glutathione GSH in the nematodes were measured. The results are shown in Figure 2 as follows.

[0049] From Figure 2It can be seen that the ethanol extract of Artemisia argyi Levl. et Vant. var. qiai (Levl.) Y. R. Ling can significantly increase the activities of SOD and CAT enzymes and the level of GSH in nematodes. Compared with the control group, the activities of SOD enzyme in nematodes treated with 50, 200, and 400 μg / mL ethanol extract of Artemisia argyi Levl. et Vant. var. qiai (Levl.) Y. R. Ling were significantly ( p <0.05) increased by 22.55%, 50.02%, and 49.14% respectively, the activities of CAT enzyme were significantly ( p <0.05) increased by 28.66%, 50.69%, and 45.61% respectively, and the contents of GSH in their bodies were significantly ( p <0.05) increased by 17.69%, 26.09%, and 25.66% respectively; and the ethanol extract of Artemisia argyi Levl. et Vant. var. qiai (Levl.) Y. R. Ling at 200 μg / mL had the best effect on increasing the antioxidant enzyme activities in nematodes. The above results indicate that the ethanol extract of Artemisia argyi Levl. et Vant. var. qiai (Levl.) Y. R. Ling enhances the antioxidant capacity in vivo by increasing the activities of antioxidant enzymes and the content of GSH in nematodes.

[0050] Example 4 Effect of Ethanol Extract of Artemisia argyi Levl. et Vant. var. qiai (Levl.) Y. R. Ling on Nuclear Localization of Transcription Factor SKN-1 This example studied the effect of the ethanol extract of Artemisia argyi Levl. et Vant. var. qiai (Levl.) Y. R. Ling on the nuclear localization of transcription factor SKN-1. Among them, SKN-1 is a transcription factor related to antioxidant defense response in Caenorhabditis elegans. When stimulated by the outside world, SKN-1 will enter the nucleus to activate antioxidant-related genes downstream of the antioxidant signaling pathway. At the same time, SKN-1 is also an important regulatory factor in the anti-aging related signaling pathway in nematodes. LD1 is a transgenic nematode strain with a GFP green fluorescent protein tag, which is used to observe and analyze the nuclear entry of transcription factor SKN-1.

[0051] Specifically, synchronized L4-stage LD1 nematodes were respectively transferred to liquid media containing 200 μg / mL ethanol extract of Artemisia argyi Levl. et Vant. var. qiai (Levl.) Y. R. Ling and 0.6% DMSO (Control group) and cultured for 48 h. Nematodes in different treatment groups were collected and fixed on agarose pads, fixed with 10 mmol / L sodium azide, and pictures were collected with a fluorescence microscope. The nuclear localization patterns of SKN-1::GFP are divided into three categories: cytoplasm, intermediate body, and nucleus. When nuclear translocation of SKN-1::GFP occurs, brighter green fluorescent spots will be observed. Cytoplasm refers to diffuse green fluorescence throughout the nematode body, intermediate body refers to some green fluorescent spots on the nematode body, and nucleus refers to the entire nematode body covered with green fluorescent spots. The proportion of each expression pattern of nematodes in different treatment groups was statistically analyzed, and the results are as Figure 3 shown.

[0052] From Figure 3It can be seen that compared with the control group, after treatment with 200 μg / mL of the ethanol extract of Artemisia argyi Lévl. Vaniot, the proportion of SKN-1 entering the nucleus of nematodes increased from 20.14% to 45.68%, and the proportion in the cytoplasm decreased from 48.68% to 24.40%, indicating that the ethanol extract of Artemisia argyi Lévl. Vaniot can promote the transcription factor SKN-1 of nematodes to enter the nucleus, and the entry of SKN-1 into the nucleus is a necessary condition for activating downstream antioxidant-related genes. The above results show that the ethanol extract of Artemisia argyi Lévl. Vaniot improves the antioxidant capacity in nematodes by promoting the entry of SKN-1 into the nucleus, and prolongs the lifespan of nematodes under oxidative stress conditions.

[0053] Example 5 Effect of Ethanol Extract of Artemisia argyi Lévl. Vaniot on Oxidative Stress Resistance of EU1 Nematodes In this example, the effect of the ethanol extract of Artemisia argyi Lévl. Vaniot on skn-1 the lifespan of the deletion mutant EU1 nematodes under oxidative stress conditions was determined.

[0054] Specifically, after culturing the skn-1 deletion mutant strain EU1 for 7 days according to the cultivation and drug addition treatment method of Caenorhabditis elegans in Example 2, the nematodes in the control group (containing only 0.6% DMSO) and the drug addition group (containing 200 μg / mL of the ethanol extract of Artemisia argyi Lévl. Vaniot) were transferred to a 96-well plate containing 100 μL of a H2O2 solution with a final concentration of 1 mmol / L per well. The survival rate of the nematodes was recorded every 2 hours at room temperature until all the nematodes died. Finally, the survival curve was plotted using GraphPad software and the data was analyzed by log-rank (Mantel−Cox). The results are shown in Figure 4 and Table 2 respectively.

[0055] Table 2 Effect of Ethanol Extract of Artemisia argyi Lévl. Vaniot on the Lifespan of EU1 Nematodes under Oxidative Stress Conditions

[0056] Note: The same letter in the second column indicates no significant difference between the two groups, p > 0.05.

[0057] From Figure 4 and Table 2, it can be seen that for the skn-1 deletion mutants treated with the ethanol extract of Artemisia argyi Lévl. Vaniot, there was no significant difference in their lifespan under oxidative stress conditions compared with the control group. Combining with the lifespan results of wild-type nematode N2 under oxidative stress conditions in Example 2, the results show that the ethanol extract of Artemisia argyi Lévl. Vaniot enhancing the oxidative stress resistance of nematodes depends on skn-1 .

[0058] Example 6 Determination of the Expression of Antioxidant-Related Genes in Nematodes by qRT-PCR In this example, the effect of the ethanol extract of Artemisia argyi Lévl. Vaniot on the skn-1 expression of wild-type nematodes in vivo and its downstream target genes was determined.

[0059] Specifically, add 300 μL of S-complete medium to each well of a 24-well plate. Set up a control group containing only 0.6% DMSO and a drug treatment group containing 200 μg / mL of ethanol extract of Artemisia argyi. Transfer synchronized L4-stage nematodes to the S-complete medium, with about 300 nematodes per well. Record the day of transfer as day 0d. Place the culture plate in an incubator at 20 °C for 3 days, and collect nematodes from different treatment groups. Extract the total RNA of nematodes from different treatment groups using the Trizol method according to the RNA extraction kit instructions. Prepare a reverse transcription system to reverse transcribe it into cDNA. Perform quantitative amplification according to the SYBR Premix EX TapⅡ instructions. The reaction system is as follows: 5 μL of 2×qPCR Mix, 3.7 μL of ddH2O, 0.4 μL of forward primer, 0.4 μL of reverse primer, and 0.5 μL of cDNA template. The reaction program is: pre-denaturation at 95 °C for 30 s, denaturation at 95 °C for 5 s, annealing at 57 °C for 30 s, extension at 72 °C for 30 s, for 40 cycles. Using act-1 as the internal reference gene, use the 2 -ΔΔCT method to calculate the relative expression level of the target gene. The primers for related genes were synthesized by Wuhan Qingke Biotechnology Co., Ltd., and the specific sequences are shown in Table 3.

[0060] Table 3 Primer sequences of related genes

[0061] The qRT-PCR results are as Figure 5 shown.

[0062] It can be seen from Figure 5 that ALE treatment significantly up-regulated the expression of skn-1 in nematodes, and its expression level was 1.53 times that of the control group. At the same time, skn-1 downstream target genes gst-4 , gcs-1 were also significantly up-regulated, and their expression levels were 1.66 and 1.92 times that of the control group respectively, indicating that the ethanol extract of Artemisia argyi activated skn-1 and its downstream target genes. This result is related to the promotion of SKN-1 nuclear entry by the ethanol extract of Artemisia argyi. In Caenorhabditis elegans, gcs-1 and gst-4 are target genes downstream of the transcription factor SKN-1, encoding γ-glutamylcysteine synthetase and glutathione S-transferase respectively, and are related to the biosynthesis and metabolism of glutathione. After SKN-1 enters the nucleus, it induces gcs-1 and gst-4 expression, which helps to maintain the redox homeostasis in nematodes. The results of this experiment not only show that the ethanol extract of Artemisia argyi activates skn-1, and also elaborates on the reason why the ethanol extract of Artemisia argyi Levl. et Vant. from Qizhou can increase the glutathione level in nematodes at the molecular level. In addition, in nematodes, sod-2 and ctl-1 encode superoxide dismutase and catalase respectively, which are related to the antioxidant reaction in nematodes. After ALE treatment, the expression levels of sod-2 , ctl-1 in nematodes were significantly increased, which were 1.46 and 1.72 times that of the control group respectively. This is consistent with the result that the ethanol extract of Artemisia argyi Levl. et Vant. from Qizhou significantly increased the enzyme activities of SOD and CAT in nematodes. The above results show that the ethanol extract of Artemisia argyi Levl. et Vant. from Qizhou up-regulates the expression of skn-1 , gst-4 , gcs-1 , sod-2 , ctl-1 genes, thus significantly increasing the enzyme activities of SOD and CAT in nematodes, increasing the GSH level in vivo, and further enhancing the antioxidant effect in vivo.

[0063] Example 7 Effect of Ethanol Extract of Artemisia argyi Levl. et Vant. from Qizhou on the Expression of GCS-1::GFP Protein In this example, the effect of the ethanol extract of Artemisia argyi Levl. et Vant. from Qizhou on the expression of the SKN-1 downstream target protein GCS-1 was determined. Among them, the transgenic nematode strain LD1171 can be used to observe and determine the expression of GCS-1 protein in nematodes, and its green fluorescent dots are mainly distributed in the head and pharynx.

[0064] Specifically, the nematode strain LD1171 at the L1 stage after synchronization was transferred to a liquid medium containing 200 μg / mL ethanol extract of Artemisia argyi Levl. et Vant. from Qizhou and 0.6% DMSO (Control group) and cultured for 72 h. Nematodes in different treatment groups were collected and fixed on agarose pads, fixed with 10 mmol / L sodium azide, and pictures were taken with a fluorescence microscope. Finally, the fluorescence intensity of nematodes in different treatment groups was analyzed using Image J software, and the results are as Figure 6 shown.

[0065] As can be seen from Figure 6 , compared with the control group, the green fluorescence in the head and pharynx of LD1171 nematodes pretreated with the ethanol extract of Artemisia argyi Levl. et Vant. from Qizhou was significantly brighter. Compared with the control group, the expression of GCS-1::GFP protein in nematodes pretreated with the ethanol extract of Artemisia argyi Levl. et Vant. from Qizhou was significantly increased by 16.97%. This result further confirmed that the ethanol extract of Artemisia argyi Levl. et Vant. from Qizhou promotes the nuclear entry of SKN-1 and activates its downstream target gene gcs-1 , thereby increasing the GSH content and enhancing the oxidative stress resistance of nematodes.

[0066] Example 8 Effect of Ethanol Extract of Artemisia argyi Levl. et Vant. from Qizhou on the Expression of GST-4::GFP Protein In this example, the effect of the ethanol extract of Artemisia argyi Levl. et Vant. on the expression of the downstream target protein GST-4 of SKN-1 was determined. Among them, the transgenic nematode strain CL2166 was used to observe and determine the expression of GST-4 protein in nematodes. In the CL2166 strain, strong fluorescent spots were mainly concentrated in the dorsal nerve cord around the nematodes.

[0067] Specifically, nematode strain CL2166 at the L1 stage after synchronization was transferred to a liquid medium containing 200 μg / mL ethanol extract of Artemisia argyi Levl. et Vant. and 0.6% DMSO (Control group) and cultured for 72 h. Nematodes in different treatment groups were collected and fixed on agarose pads, fixed with 10 mmol / L sodium azide, and pictures were taken with a fluorescence microscope. Finally, the fluorescence intensity of nematodes in different treatment groups was analyzed using Image J software. The results are as Figure 7 shown.

[0068] As can be seen from Figure 7 , in the CL2166 nematode strain pretreated with the ethanol extract of Artemisia argyi Levl. et Vant., the green fluorescence on the epidermis became significantly brighter. Compared with the control group, the expression of GST-4::GFP protein increased significantly by 14.17%. This result further verified that the ethanol extract of Artemisia argyi Levl. et Vant. activated the downstream target gene gst-4 by promoting the nuclear entry of the transcription factor SKN-1, increased the expression of GST-4 protein, thereby increasing the GSH content and antioxidant capacity in nematodes.

[0069] To sum up, the present invention first discovered that the ethanol extract of Artemisia argyi Levl. et Vant. significantly prolonged the average lifespan of wild-type Caenorhabditis elegans under oxidative stress conditions, and significantly increased the levels and / or contents of superoxide dismutase, catalase, and reduced glutathione in wild-type Caenorhabditis elegans. At the same time, it promoted the nuclear entry of the antioxidant key transcription factor SKN-1 in transgenic Caenorhabditis elegans LD1, and significantly up-regulated genes skn-1 , gst-4 , gcs-1 , sod-2 , ctl-1 related to oxidative stress resistance, and further significantly increased the expression of the resistance proteins GCS-1 and GST-4, ultimately activating the SKN-1 antioxidant signaling pathway, thereby enhancing the antioxidant effect in vivo.

[0070] It should be noted that the above examples all belong to the same inventive concept. The descriptions of each example have their own focuses. For the parts not described in detail in individual examples, reference can be made to the descriptions in other examples.

[0071] The embodiments described above merely represent the implementation modes of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. Use of the ethanol extract of Artemisia argyi Levl. var. argyi in the preparation of a drug for antioxidative stress injury, characterized in that, The preparation of the ethanol extract of Artemisia argyi Levl. var. argyi H. Lév. includes the following steps: Provide the crude powder of Artemisia argyi Levl. var. argyi H. Lév.; Mix the crude powder of Artemisia argyi Levl. var. argyi H. Lév. with an organic solvent and then perform microwave treatment. After filtration, obtain a filtrate; Remove the organic solvent from the filtrate and then perform freeze-drying to obtain the ethanol extract of Artemisia argyi Levl. var. argyi H. Lév.

2. The application according to claim 1, characterized in that, The effective concentration of the ethanol extract of Artemisia argyi Levl. var. argyi H. Lév. is 50 - 400 μg / mL.

3. The application according to claim 1, wherein During the process of mixing the crude powder of Artemisia argyi Levl. var. argyi H. Lév. with an organic solvent and then performing microwave treatment, the material-liquid ratio of the crude powder of Artemisia argyi Levl. var. argyi H. Lév. to the organic solvent is (0.5 - 1.5 g) : (70 - 90 mL).

4. The application according to claim 1, wherein During the process of mixing the crude powder of Artemisia argyi Levl. var. argyi H. Lév. with an organic solvent and then performing microwave treatment, the organic solvent includes ethanol. The microwave treatment specifically includes: under the condition of a microwave power of 300 - 500 W, treat for 3 - 7 min.

5. The application according to claim 1, wherein The ethanol extract of Artemisia argyi Levl. var. argyi H. Lév. includes flavonoids and polyphenols; Among them, the flavonoid content is 100 - 104 mg / g, and the polyphenol content is 46 - 50 mg / g.

6. The application according to claim 1, wherein The Qiai Artemisia argyi leaf ethanol extract prolongs the average lifespan of wild-type Caenorhabditis elegans under oxidative stress conditions, and the prolongation of the average lifespan of wild-type Caenorhabditis elegans under oxidative stress conditions depends on skn-1 , and by increasing the levels and / or contents of superoxide dismutase, catalase, and reduced glutathione in wild-type Caenorhabditis elegans, thereby achieving antioxidant effects in vivo.

7. The application according to claim 1, wherein The ethanol extract of Artemisia argyi Levl. et Vant. var. qiai (Levl.) Hsiao et K. M. Xu promotes the nuclear entry of the key antioxidant transcription factor SKN-1 in transgenic Caenorhabditis elegans LD1 and upregulates the genes skn-1 , gst-4 , gcs-1 , sod-2 , ctl-1 related to oxidative stress resistance. At the same time, it increases the expression of the resistance proteins GCS-1 and GST-4, ultimately activating the SKN-1 antioxidant signaling pathway, thereby enhancing the antioxidant effect in vivo.

8. A pharmaceutical composition for preventing and / or treating oxidative stress injury, characterized in that, It includes the ethanol extract of Artemisia argyi Levl. var. argyi H. Lév. described in claim 1.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition further includes a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 8, wherein The dosage form of the pharmaceutical composition includes at least one of solid preparations, semi-solid preparations, and liquid preparations.

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