Use of fructus trichosanthis extract in preparation of medicine for preventing or treating psoriasis

Abstract

The application provides a use of a fructus trichosanthis extract in preparation of a medicine for preventing or treating psoriasis, and the medicine is used for preventing or treating psoriasis. The fructus trichosanthis extract can effectively prevent or treat psoriasis, and can effectively down-regulate expression of inflammatory factors in a STAT1 signal path and inhibit STAT1 phosphorylation in an in-vitro cell, and has important scientific research and clinical application values.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to the use of Herpetospermum caudigerum seed extract in the preparation of a medicament for preventing or treating psoriasis. Background Art

[0002] Psoriasis, also known as psoriasis vulgaris, is a common autoimmune disease that affects approximately 3% of the world's population. The disease is characterized by obvious inflammatory infiltration of epidermal keratinocytes and excessive proliferation, resulting in scaly, sclerotic, erythematous plaques and other skin lesions. Currently, there are no specific drugs for psoriasis. Many patients use hormonal drugs. Although the symptoms are temporarily relieved within a certain period of time after use, the side effects are significant. Therefore, there is an urgent need to develop a non-hormonal drug that can rapidly alleviate the above symptoms.

[0003] Herpetospermum caudigerum seed, the dried mature seed of the Cucurbitaceae plant Herpetospermum caudigerum, is a traditional Tibetan medicine in China. It has been included in the "Drug Standards of the Ministry of Health of the People's Republic of China" and is widely used in the treatment of liver diseases.

[0004] At present, there has been no research report on the association between Herpetospermum caudigerum seed and psoriasis. Summary of the Invention

[0005] The present invention aims to solve the technical problems existing in the prior art at least to a certain extent. To this end, the present invention provides the use of Herpetospermum caudigerum seed extract in the preparation of medicaments and kits, and a method for down-regulating the expression of inflammatory factors in the STAT1 signaling pathway of cells in vitro and inhibiting STAT1 phosphorylation. The Herpetospermum caudigerum seed extract of the present invention can effectively prevent or treat psoriasis, and can also effectively inhibit the expression of the inflammatory factor STAT1 in the STAT1 signaling pathway in cells in vitro, which has important scientific research and clinical application value.

[0006] It should be noted that the present invention is completed based on the following findings of the inventors:

[0007] The inventors of the present invention found that after Herpetospermum caudigerum seed extract intervenes in IFN-γ-induced keratinocytes (HaCaT cells), it can down-regulate inflammatory factors such as IL-17A, ICAM-1 and CXCL9, and down-regulate IL-17A, ICAM-1, CXCL9, CXCL10 and TNF-α in the skin of psoriasis-like mice. Among them, IL-17A, ICAM-1, CXCL9, CXCL10, TNF-α and the like are inflammatory factors that are closely related to inflammation-related diseases. When the levels of IL-17A, ICAM-1, CXCL9, CXCL10, TNF-α and the like increase, inflammation-related diseases will be exacerbated. Therefore, the inventors found that Herpetospermum caudigerum seed extract has the effect of treating inflammatory diseases, especially psoriasis.

[0008] In one aspect of the invention, the use of *Pleurotus ostreatus* seed extract in the preparation of a medicament is proposed. According to embodiments of the invention, the medicament is used for the prevention or treatment of psoriasis.

[0009] In another aspect, the present invention proposes the use of *Pleurotus ostreatus* seed extract in the preparation of a kit. According to embodiments of the invention, the kit is used to downregulate the expression of inflammatory factors in the STAT1 signaling pathway of cells and inhibit STAT1 phosphorylation. According to embodiments of the invention, the inflammatory factors include IL-17A, ICAM-1, CXCL9, CXCL10, or TNF-α. The inventors have found that *Pleurotus ostreatus* seed extract can effectively inhibit the expression of the above-mentioned inflammatory factors, which is helpful for scientific research on these inflammatory factors.

[0010] In another aspect, the present invention proposes a method for downregulating the expression levels of inflammatory factors in the STAT1 signaling pathway or inhibiting STAT1 phosphorylation in in vitro cells. According to an embodiment of the present invention, the method includes: adding a *Pyracantha fortuneana* seed extract to IFN-γ-induced keratinocytes and culturing them. When the *Pyracantha fortuneana* seed extract acts on IFN-γ-induced HaCaT cells, it inhibits the expression of inflammatory factors such as IL-17A, ICAM-1, CXCL9, CXCL10, and TNF-α in HaCaT cells, which is beneficial for scientific research on these inflammatory factors.

[0011] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0012] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0013] Figure 1 A schematic diagram of Western blotting results according to an embodiment of the present invention is shown;

[0014] Figure 2 A schematic diagram of RT-PCR results according to an embodiment of the present invention is shown;

[0015] Figure 3 A photograph of the back of a mouse model of psoriasis-like disease is shown, illustrating the effect of a crude extract of Cucurbita moschata according to an embodiment of the present invention.

[0016] Figure 4 A HE slice of the mouse back is shown according to an embodiment of the present invention;

[0017] Figure 5This image shows an analysis of the skin thickness on the back of a mouse according to an embodiment of the present invention;

[0018] Figure 6 The following is a graph showing the immunohistochemical results analysis according to an embodiment of the present invention;

[0019] Figure 7 A protein analysis diagram of mouse skin tissue according to an embodiment of the present invention is shown;

[0020] Figure 8 The present invention illustrates a mouse skin RT-PCR analysis according to an embodiment of the present invention;

[0021] Figure 9 The total ion spectrum of the extracted mass spectrum of sunflower seeds according to an embodiment of the present invention is shown.

[0022] Figure label:

[0023] BLG: Melon seed extract; IMQ: Psoriasis mouse group. Detailed Implementation

[0024] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0025] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0026] Uses of Melon Seed Extract in Drug Preparation

[0027] In one aspect of the invention, the use of *Pleurotus ostreatus* seed extract in the preparation of a medicament is proposed. According to embodiments of the invention, the medicament is used for the prevention or treatment of psoriasis.

[0028] According to an embodiment of the present invention, the drug is used to downregulate the expression of inflammatory factors in the STAT1 signaling pathway and inhibit STAT1 phosphorylation. According to another embodiment of the present invention, the inflammatory factors include IL-17A, ICAM-1, CXCL9, CXCL10, or TNF-α. The inventors have found that *Pleurotus ostreatus* seed extract can effectively reduce the above-mentioned inflammatory factors, which is helpful for the research of these inflammatory factors, the research of psoriasis, and the clinical application of psoriasis.

[0029] According to embodiments of the present invention, the *Pleurotus ostreatus* seed extract comprises: elelitanol C, herpetatol C, dehydrobispinelol, elelitanol A, elelitanol, elelitanol, elelitanol B, and herpetetrol. Through research and analysis of the composition of the *Pleurotus ostreatus* seed extract, the inventors discovered that the *Pleurotus ostreatus* seed extract containing elelitanol, herpetatol, dehydrobispinelol, elelitanol A, elelitanol, elelitanol, elelitanol B, and herpetetrol has good preventive or therapeutic effects on psoriasis.

[0030] According to an embodiment of the present invention, the extract of *Pleurotus ostreatus* seeds is obtained by extraction with alcohols. Extracting *Pleurotus ostreatus* seeds with alcohols can increase the yield of active ingredients in the extract, and the resulting extract can better exert its efficacy in preventing or treating psoriasis.

[0031] According to embodiments of the present invention, the alcohol is selected from at least one of ethanol, methanol, propanol, and isopropanol. Therefore, using the above-mentioned alcohol can increase the yield of active ingredients in the *Pleurotus ostreatus* seed extract, and the extracted extract can better exert its efficacy in preventing or treating psoriasis.

[0032] According to an embodiment of the present invention, the method for obtaining the *Pleurotus ostreatus* seed extract includes: heating *Pleurotus ostreatus* seeds and an alcohol under reflux; filtering the resulting reflux liquid and collecting the filtrate; evaporating and drying the filtrate; extracting the resulting concentrate with petroleum ether to obtain the extract; evaporating and drying the extract; and then dissolving the dried product in ethanol to obtain the *Pleurotus ostreatus* seed extract. This further improves the yield of the extract, and the obtained extract can better exert its efficacy in preventing or treating psoriasis.

[0033] As used herein, the term "treatment" refers to achieving a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of the disease or its symptoms, and / or therapeutic in terms of partial or complete cure of the disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease (e.g., prevention of psoriasis) or the onset of disease in individuals susceptible to the disease but not yet diagnosed with it; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing the extract of *Pyracantha fortuneana* described herein to an individual in need.

[0034] As used herein, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The medicaments of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are foreseeable, including peritoneal, intravenous, intramuscular, subcutaneous, dermal, oral, local, nasal, and rectal administration; however, the present invention is not limited to these exemplified routes of administration.

[0035] The dosing frequency and dosage of the drug of the present invention can be determined by several relevant factors, including the type of disease to be treated, the route of administration, the patient's age, sex, weight, and severity of the disease, as well as the type of drug as the active ingredient. According to some embodiments of the present invention, the daily dose can be divided into one, two, or multiple doses in a suitable form, administered once, twice, or more times throughout the entire time period, as long as a therapeutically effective amount is achieved.

[0036] The term "therapeuticly effective amount" refers to the amount of a compound sufficient to significantly improve certain symptoms associated with a disease or condition; that is, the amount that provides a therapeutic effect for a given condition and dosing regimen. For example, in psoriasis, a drug or compound that reduces, prevents, delays, inhibits, or blocks any symptoms of the disease or condition should be considered therapeutically effective. A therapeutically effective amount of a drug or compound does not need to cure the disease or condition, but will provide treatment for the disease or condition, such that the onset of the individual's disease or condition is delayed, stopped, or prevented, or the symptoms of the disease or condition are relieved, or the duration of the disease or condition is altered, or, for example, the disease or condition becomes less severe, or recovery is accelerated.

[0037] Use of Melon Seed Extract in the Preparation Kit

[0038] In another aspect, the present invention proposes the use of *Pleurotus ostreatus* seed extract in the preparation of a kit. According to embodiments of the invention, the kit is used to downregulate the expression of inflammatory factors in the STAT1 signaling pathway of cells and to inhibit STAT1 phosphorylation.

[0039] According to embodiments of the present invention, the inflammatory factors include IL-17A, ICAM-1, CXCL9, CXCL10, or TNF-α. The inventors have discovered that extracts from *Pyracantha fortuneana* seeds can effectively inhibit the expression of the aforementioned inflammatory factors, contributing to scientific research on these factors.

[0040] According to an embodiment of the present invention, the cells are selected from IFN-γ-induced keratinocytes (also referred to herein as "HaCaT cells"). When the extract of *Potamogeton crispus* seeds is applied to IFN-γ-induced HaCaT cells, it inhibits the expression of inflammatory factors such as IL-17A, ICAM-1, CXCL9, CXCL10, and TNF-α in keratinocytes, which is helpful for scientific research on these inflammatory factors.

[0041] According to embodiments of the present invention, the *Pleurotus ostreatus* seed extract is as defined above in its use in pharmaceutical preparation. The above method can improve the yield of the active ingredient in the *Pleurotus ostreatus* seed extract, and the extracted extract can better exert its effects in inhibiting the expression of the aforementioned proteins and inhibiting STAT1 phosphorylation, for example, in proteomics studies and metabolic pathway studies.

[0042] It should be noted that the characteristics and advantages described above regarding the use of the bougainvillea seed extract in drug preparation also apply to its use in the preparation of reagent kits, and will not be repeated here.

[0043] Methods to downregulate the expression levels of inflammatory factors in the STAT1 signaling pathway in in vitro cells or inhibit STAT1 phosphorylation

[0044] In another aspect of the invention, a method is proposed to downregulate the expression level of inflammatory factors in the STAT1 signaling pathway or inhibit STAT1 phosphorylation in in vitro cells. According to an embodiment of the invention, the method includes: adding a pine nut seed extract to IFN-γ-induced keratinocytes and culturing them.

[0045] When the extract of pine melon seeds is applied to IFN-γ-induced HaCaT cells, it inhibits the expression of inflammatory factors such as IL-17A, ICAM-1, CXCL9, CXCL10, and TNF-α in HaCaT cells, which is helpful for scientific research on these inflammatory factors.

[0046] According to an embodiment of the present invention, the inflammatory factors include IL-17A, ICAM-1, CXCL9, CXCL10, and TNF-α.

[0047] According to an embodiment of the present invention, the culture time is 18 to 30 hours.

[0048] According to embodiments of the present invention, the *Pleurotus ostreatus* seed extract is as defined above in its use in the preparation of pharmaceuticals. The above method can improve the yield of the active ingredient in the *Pleurotus ostreatus* seed extract, and the extracted extract can better exert its effect of inhibiting the expression of the aforementioned proteins or inhibiting STAT1 phosphorylation.

[0049] It should be noted that the characteristics and advantages described above regarding the use of pine nut seed extract in drug preparation also apply to this method of downregulating the expression level of inflammatory factors in the STAT1 signaling pathway in in vitro cells or inhibiting STAT1 phosphorylation, and will not be repeated here.

[0050] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0051] Example 1: Effect of Melon Seed Extract on Keratinocyte HaCaT

[0052] 1. Materials and Methods

[0053] 1.1 Materials

[0054] 1.1.1 Cells

[0055] Human immortalized keratinocytes (HaCaT cells) were derived from the Tumor Biology and Innovative Drug Research Center of Chengdu University. HaCaT cells are human immortalized epidermal cells, a non-tumor-derived immortalized keratinocyte cell line from normal human skin.

[0056] 1.1.2 Preparation of crude extract stock solution

[0057] Weigh 30g of pine nut seeds, soak them in 180ml of anhydrous ethanol for 24h, reflux for 2-3h, repeat twice, filter, rotary evaporate, add 2 times the volume of petroleum ether for extraction, rotary evaporate until the petroleum ether is removed, finally add anhydrous ethanol to dissolve, and obtain 0.01% pine nut seed extract.

[0058] 1.1.3 Main Reagents

[0059] DMEM high glucose medium (WISNT INC), fetal bovine serum (WISNT INC), PBS (WISNT INC), trypsin (WISNT INC), dimethyl sulfoxide (Solarbio), and anhydrous ethanol (for testing).

[0060] 1.1.4 Major Instruments and Consumables

[0061] Cell culture incubator (Thermo Scientific), biosafety cabinet (Thermo Scientific), upright microscope (Nikon Eclipse Ni-U), high-speed refrigerated centrifuge (Eppendorf 5425R), real-time quantitative PCR instrument (BIO-RAD), cell culture dish (NEST), 24-well plate (WHB).

[0062] 1.1.5 Solution Preparation

[0063] Complete cell culture medium: DMEM high glucose medium (84%), fetal bovine serum (15%), penicillin-streptomycin solution (1%).

[0064] Cell cryopreservation solution: complete cell culture medium (90%), DMSO (10%).

[0065] 1.2 Experimental Methods

[0066] 1.2.1 HaCaT cell culture and cell resuscitation

[0067] 1.2.1.1 Cell resuscitation

[0068] 1) Before the experiment, irradiate the biosafety cabinet with ultraviolet light for 30 minutes and take the culture medium out of the 4℃ refrigerator in advance to bring it to room temperature.

[0069] 2) Remove the cryovials from -80℃, wipe the outside of the cryovials with 75% ethanol, and then place them in a clean bench to allow them to return to room temperature. During this process, prepare consumables such as cell culture dishes, pipettes, and serum pipettes, and clearly mark the information on the culture dishes with a marker.

[0070] 3) Transfer 7 ml of complete cell culture medium into a culture dish, and then use a pipette to transfer the thawed cell cryopreservation solution into the culture dish. Gently tap the edge of the culture dish to mix the cell components evenly.

[0071] 4) Place the culture dish in a cell culture incubator at 37°C and 5% CO2 for incubation.

[0072] 5) Take out the culture dish daily and place it under a microscope to observe the cell growth status. Change the cell culture medium in a timely manner. When the cell density reaches about 80%, passage the cells and freeze them.

[0073] 1.2.1.2 Cell passage culture

[0074] 1) Observe cell density and adherence growth under a microscope. Passage the cells when the density is about 80% to prevent the culture medium from becoming turbid due to excessive density, which could affect normal cell growth. Irradiate the clean bench with ultraviolet light 30 minutes before the experiment, and remove the cell culture medium, trypsin, and PBS from the 4°C freezer to allow them to return to room temperature.

[0075] 2) Use a vacuum pump to remove the old culture medium, then slowly add 5 ml of sterile PBS along the wall of the culture dish, gently shake the culture dish to wash the cell surface with PBS, remove the PBS, and repeat twice.

[0076] 3) Add 1 ml of trypsin to a culture dish, gently shake the dish until the trypsin covers the bottom, and place it in a cell culture incubator for about 8 minutes to digest. During this time, prepare a new culture dish, add 7 ml of complete culture medium, and label it. Observe under a microscope that the cells become round and detach in a quicksand-like manner.

[0077] 4) Immediately add 2 ml of complete culture medium to the culture dish, and gently shake to cover the cells to terminate the trypsin digestion reaction. Tilt the culture dish and gently pipette the cell suspension.

[0078] 5) Transfer 1.5 ml of cell suspension to a new culture dish, and transfer 7 ml of complete culture medium to the old culture dish. Gently shake the culture dish to distribute the cells evenly, and place it in a cell culture incubator for culture.

[0079] 1.2.1.3 Cell Seeding

[0080] 1) Observe the density and adherent growth status of HaCaT cells under a microscope. Before the experiment, the clean bench was irradiated with ultraviolet light for 30 minutes. The complete cell culture medium and sterile PBS solution were taken out of the 4℃ refrigerator in advance and allowed to return to room temperature.

[0081] 2) Remove the old culture medium from the culture dish, slowly add PBS along the wall, gently shake the culture dish to wash the cell surface, remove the PBS solution, and repeat twice.

[0082] 3) Add 1 ml of trypsin, cover the dish and gently shake it to cover the cells with trypsin. Place the dish in a cell culture incubator and wait for 8 minutes. Observe under a microscope.

[0083] 4) Once the cells become round and detach in a quicksand-like manner, immediately add 6 ml of complete culture medium to the culture dish to stop the digestion reaction. Tilt the culture dish and gently pipette the cell suspension.

[0084] 5) During digestion, prepare a 24-well plate, add 0.5 ml of cell culture medium to each well, and label the plate with information.

[0085] 6) Use an electric pipette to gently disperse and mix the digested cell suspension. Seed 2 drops of cell suspension into each well of a 24-well plate. Then gently tap the edges of the plate to distribute the cells evenly. Place the 24-well plate in a cell culture incubator for incubation.

[0086] 1.2.2 Cell Drug Administration

[0087] When the cell adhesion and growth density is about 80%, change the medium and add drugs.

[0088] 1) IFN-γ induction: Take out IFN-γ from the -80℃ freezer with an initial concentration of 200 μg / ml, and immediately dilute it with complete cell culture medium to a working concentration of 2 ng / ml. Use 0.5 ml per well as the model group, and use only complete culture medium as the blank control group.

[0089] 2) BLG (Bolang melon seed extract) intervention: After IFN-γ induction in step 1), the crude extract stock solution prepared in step 1.1.2 was taken out from the -20℃ freezer. The crude extract stock solution was diluted with anhydrous ethanol, and the working concentration was set to 0 and 10. -7 10 -6 10 -5 10 -4 Add 5 μl of the corresponding crude extract working solution to each well as the experimental group. Simultaneously, use complete culture medium containing the same dose of anhydrous ethanol as the blank control group. After addition, return the cells to the cell culture incubator and continue culturing for 24 h.

[0090] 1.2.3 Western Blotting

[0091] 1.2.3.1 Protein Extraction

[0092] 1) Preparation of protein lysis buffer: RIPA lysis buffer: PI: sodium glycerophosphate = 90:10:1 (volume ratio), after preparation, store in a refrigerator at 4℃ for later use.

[0093] 2) Loading buffer: 50 μl of DTT and 950 μl of bromophenol blue, mix well and store at -20℃ for later use.

[0094] 3) Remove the 24-well plate from the cell culture incubator, remove the complete cell culture medium, take out the protein lysis buffer, add 80 μl to each well, and collect the protein lysis buffer into the corresponding labeled EP tube after it becomes viscous. Then place it on ice. After all samples have been collected, add 20 μL of loading buffer to each EP tube, heat at 100°C for about 7 min until the liquid in the tube is no longer viscous, and store it in a -20°C freezer.

[0095] 1.2.3.2 Electrophoresis

[0096] 1) Preparation and operation steps of main reagents: 10% APS: Weigh 1g of ammonium persulfate, dissolve it in pure water, prepare 10ml of solution, dispense and store in a -20℃ refrigerator for later use.

[0097] Electrophoresis buffer: Tris base 30g, glycine 144g, SDS 10g, bring to a final volume of 1L, and store at room temperature for later use.

[0098] Transfer buffer: 28.8g glycine, 6g Tris base, 400ml methanol, bring to a final volume of 2L, and store at room temperature for later use.

[0099] 10×TBS solution: 88g sodium chloride, 24g Tris base, 13ml concentrated hydrochloric acid, bring to a final volume of 1L, and let stand at room temperature for later use.

[0100] 1×TBST solution: Measure 100ml of 10×TBS solution and mix with 1ml of 20% Tween, bring the volume to 1L, and let stand at room temperature for later use.

[0101] Blocking solution: Weigh 1g of skim milk powder, dissolve it in 20ml of 1×TBST solution, and let it stand at room temperature for later use.

[0102] Antibody dilution buffer: Weigh 1g of BSA, dissolve it in 20ml of 1×TBST, and store at 4℃ for later use.

[0103] 2) Antibody preparation

[0104]

[0105]

[0106] 3) Prepare 10% SDS-PAGE

[0107] Separating gel: Add the required reagents to a 50ml centrifuge tube in sequence, shake gently for 30s, then pour into a fixed glass plate, add isopropanol solution to seal, and wait for about 30 minutes. Observe the interface and the presence of a clear dividing line indicates that the gel has solidified.

[0108] Stacking gel: Discard the isopropanol sealed on the separating gel, rinse 5 times with pure water and blot dry. Add the required reagents sequentially to a 50ml centrifuge tube, gently shake for 30s, then pour into a glass plate until the liquid level is flush with the short end of the glass plate. Insert the comb horizontally and wait for about 30 minutes for the stacking gel to solidify. Remove the comb, rinse the stacking gel with distilled water, install it in the electrophoresis tank, and add electrophoresis buffer.

[0109] 4) Electrophoresis: Before loading the sample, remove it from the -20℃ freezer and boil it at 100℃ for 5 minutes. The loading volume is 5μl. Use a constant voltage of 80V for the stacking gel. After the sample enters the separating gel, switch to a constant voltage of 136V until the blue mark is about 1cm from the bottom of the gel. Then turn off the power.

[0110] 5) Transfer: Cut the filter paper and PVDF membrane in advance, and activate the PVDF membrane with methanol. Soak the membrane in pre-cooled transfer buffer. Remove the gel from the glass plate, trim off any excess, and stack them neatly in the order of "black plate-filter paper-gel-membrane-filter paper-white plate," taking care to remove air bubbles between the layers. After closing the clamps, place the membrane in the transfer electrophoresis tank, add transfer buffer, and perform transfer at a constant current of 200mA at 4°C for 2 hours.

[0111] 6) Blocking: After the transfer is complete, immerse the membrane in the blocking solution and incubate it on a shaker for 1 hour at room temperature.

[0112] 7) Antibody Incubation: After blocking, cut the PVDF membrane according to the target protein, transfer it to an incubation chamber, add the corresponding primary antibody, completely immerse the membrane, and incubate overnight at 4°C. Recover the primary antibody the next day. Wash with 1×TBST buffer for 5 minutes each time, for a total of 5 washes, replacing the 1×TBST solution each time. After washing, add the secondary antibody to the incubation chamber, completely immerse the membrane, and incubate at room temperature for 1 hour. Then wash with 1×TBST buffer for 5 minutes each time, for a total of 5 washes, replacing the 1×TBST solution each time.

[0113] 8) Development: Add ECL luminescent solution to the membrane, expose it in a protein immunoblotting instrument, save the file, and then immerse the membrane in 1×TBST solution.

[0114] 1.2.4 RT-PCR

[0115] 1) RNA Extraction: Pre-cool the centrifuge to 4°C, then remove the 24-well plate from the cell culture incubator. After aspirating the complete culture medium from the plate, add 250 μl of TRIzol lysis buffer to each well. Once the lysis buffer becomes viscous, transfer it to a labeled EP tube, add 50 μl of chloroform, vortex for 20 s, and centrifuge at 4°C, 13000 rpm, for 15 min. Transfer the colorless supernatant to an enzyme-free EP tube, add 150 μl of isopropanol, vortex to mix thoroughly, and precipitate at room temperature for 15 min. After precipitation, centrifuge at 4°C, 13000 rpm, for 10 min. A white precipitate will be visible at the bottom of the EP tube after centrifugation. Aspirate the supernatant, wash with 70% low-temperature ethanol, centrifuge at 4°C, 12000 rpm, for 10 min, aspirate the supernatant, and repeat the washing process twice. Air dry in a biosafety cabinet. After air drying, dissolve each sample in 20 μl of enzyme-free water and measure the RNA concentration using a micro spectrophotometer.

[0116] 2) Reverse transcription: Dilute the RNA concentration to 100 ng / μl and set aside. Reverse transcription kit composition: 4.5 μl enzyme-free water, 2 μl 5×All-in-one qRT SuperMix, 0.5 μl Enzyme Mix, 3 μl RNA. Mix well and place in a PCR instrument. Incubate at 50℃ for 15 min; then at 85℃ for 5 s for reverse transcription.

[0117] 3) RT-PCR: Remove the Sybergreen mix and place it on ice for later use. The reaction mixture consists of 5 μl Sybergreen mix + 0.3 μl F target primer + 0.3 μl R target primer + 5 μl template, with 3 replicates. The real-time quantitative PCR instrument parameters are set as follows: pre-denaturation 95℃, 15 min; denaturation 95℃, 15 s; annealing 60℃, 20 s; extension 72℃, 45 s; 50 cycles.

[0118] 2 Experimental Results

[0119] Using HaCaT cells as the research subject, four concentrations of *Pyracantha fortuneana* seed extract were applied to keratinocytes. The results of Western blotting after protein extraction, electrophoresis, wet transfection, incubation with primary and secondary antibodies, and development are as follows: Figure 1 As shown in the figure, analysis of protein expression bands revealed that IFN-γ induced high expression of ICAM-1 and CXCL9 proteins in keratinocytes. After treatment with the drug (melon seed extract), compared with the control group, the levels of ICAM-1 and CXCL9 proteins were reduced in samples treated with four concentrations of melon seed extract. These results indicate that melon seed extract has an inhibitory effect on the expression of ICAM-1 and CXCL9 in keratinocytes.

[0120] qRT-PCR results were analyzed using GraphPad Prism 8 software. After ANOVA analysis, the results were expressed as mean ± standard deviation (SD). The significance level was set at α = 0.05 (P < 0.05 was considered statistically significant, otherwise not statistically significant). "####" indicates the comparison between the blank control group and the model group, with P < 0.0001; "****", "***", and "**" indicate the comparison between the experimental group and the model group, corresponding to P < 0.0001, P < 0.0002, and P < 0.0015, respectively.

[0121] like Figure 2 As shown, the results indicate that IFN-γ can induce high expression of IL-17A in keratinocytes, and the level of IL-17A in keratinocytes decreased significantly after intervention with pine nut seed extract.

[0122] Example 2: Effects of Melon Seed Extract on IMQ-Induced Psoriasis-Like Mouse Model

[0123] 1. Materials

[0124] 1.1 Experimental Instruments and Consumables

[0125]

[0126]

[0127] 1.2 Immunohistochemical antibodies

[0128]

[0129] 1.3 Laboratory Animals

[0130] SPF-grade male C57BL / 6 mice were purchased from Chengdu Yaokang Biotechnology Co., Ltd. The animal room temperature was 24±1℃, and the mice were allowed free access to food.

[0131] 1.3.1 Drug Preparation

[0132] Working solution of pine nut seed extract: Take out the mother liquor of pine nut seed extract from the -20℃ freezer, dilute it 100 times with anhydrous ethanol, mix the diluted solution with urea cream evenly, and prepare a cream containing 0.01% pine nut seed extract in a total of 6g. After labeling, store it in a 4℃ freezer.

[0133] Blank matrix: Mix anhydrous ethanol and urea cream evenly to prepare 6g of cream as blank cream, mark it and store it in a refrigerator at 4℃.

[0134] 2 Experimental Methods

[0135] 2.1 Establishment of an IMQ-induced psoriasis-like mouse model

[0136] Fifteen mice were kept in a constant temperature and humidity environment for three days to acclimatize to the environment. Then, the 15 mice were randomly divided into three groups: (1) control group (n=5), (2) model group (imidaquimod emulsion group, abbreviated as IMQ) (n=5), and (3) experimental group (urea cream + pine nut seed extract group) (n=5). The back hair of the mice was removed using an electric shaver. Except for the control group, the other two groups were evenly coated with 5% IMQ on the back of the mice every day to establish an IMQ-induced psoriasis-like inflammation model for 14 days. On the 14th day, the back of the mice was photographed. Figure 3 )

[0137] 2.2 Treatment

[0138] Starting from day 7, the control group received urea cream containing anhydrous ethanol applied to the back of mice three times daily; the model group received 5% IMQ applied to the ear skin, followed by urea cream containing anhydrous ethanol applied to the back skin three times daily; the treatment group received 5% IMQ applied to the back skin, followed by 0.01% Cucurbita melon seed extract and urea cream applied to the back skin three times daily for six consecutive days. The backs of the mice were photographed after six days. Figure 3 Mice were euthanized by cervical dislocation, and skin lesion samples were collected and divided into 4 portions. One portion was fixed in formalin solution, and the other 3 portions were frozen in liquid nitrogen for later use.

[0139] 2.3H&E

[0140] Twenty-four hours after collecting the damaged samples, the formalin solution was discarded, and the samples were soaked in 70% ethanol. After paraffin embedding, H&E staining was performed, and the samples were observed and imaged under an optical microscope. Figure 4 The thickness of the ear epidermis in mice from different experimental groups was measured, and data analysis was performed using GraphPad Prism 8 software. Figure 5 ).

[0141] 2.4 Immunohistochemistry

[0142] Prepare ear tissue sections, xylene, anhydrous ethanol, PBS, and other solutions for the control group, model group, and treatment group mice, following these steps:

[0143] 1) Baking: Place the tissue slices in a 60℃ oven for 2 hours.

[0144] 2) Dewaxing: Remove the slices from the oven and immediately soak them in No. 1 100% xylene for 10 minutes, then soak them in No. 2 100% xylene for 10 minutes.

[0145] 3) Alcohol washing: Remove from xylene No. 2 and immerse in anhydrous ethanol No. 1 for 5 minutes, then immerse in anhydrous ethanol No. 2 for 5 minutes; immerse in 95% ethanol for 5 minutes; immerse in 70% ethanol for 5 minutes; immerse in 50% ethanol for 5 minutes. Place the sections on a shaker and wash with PBS for 10 minutes, changing the PBS every 5 minutes.

[0146] 4) Antigen retrieval: Place the slide in a 1000ml beaker, add 800ml of citrate antigen retrieval solution, seal with plastic wrap, heat in a microwave on medium-high heat until boiling, then reduce to medium heat and boil for 20 minutes. After boiling, allow to cool naturally to room temperature. Wash with PBS for 15 minutes, changing the PBS every 5 minutes.

[0147] 5) Inactivate endogenous peroxidase: Add reagent 1 (inactivating endogenous peroxidase blocker) from the kit to the tissue, block at room temperature for 10 min, wash with PBS for 15 min, and replace PBS every 5 min.

[0148] 6) Blocking: Add 5% BSA blocking solution, block at room temperature for 30 min, wash with PBS for 6 min, and replace PBS every 2 min.

[0149] 7) Incubation with primary antibodies: Dilute the antibodies p-STAT1 (1:100), STAT1 (1:50), IL-17A (1:500), ICAM-1 (1:600), CXCL9 (1:200), CXCL10 (1:200), and TNF-α (1:100) with 5% BSA and add them to the tissue sections. Incubate overnight at 4°C in a humidified chamber. The next day, wash with PBS for 9 minutes, changing the PBS every 3 minutes.

[0150] 8) Incubation of secondary antibody: Add reagent 2 (reaction enhancement solution) from the kit to the tissue section, incubate at room temperature for 30 min, then wash with PBS for 6 min, changing the PBS every 2 min. Add reagent 3 (enhancing enzyme-labeled goat anti-mouse / rabbit IgG polymer), incubate at room temperature for 1.5 h, then wash with PBS for 6 min, changing the PBS every 2 min.

[0151] 9) DAB chromogenic reaction: Dilute 20× DAB concentrate to 1× with DAB substrate solution, mix well, and store in the dark. Add the chromogenic reaction solution to the tissue and observe under a microscope, controlling the chromogenic reaction time as needed.

[0152] 10) Hematoxylin counterstaining: Add filtered hematoxylin staining solution to the slide, let it stand for 20 seconds, and then immediately rinse slowly with water for 5 minutes. Observe the staining under a microscope.

[0153] 11) Mounting: Immerse the slides in 50% ethanol for 3 minutes; 70% ethanol for 3 minutes; 95% ethanol for 3 minutes; anhydrous ethanol (No. 2) for 3 minutes; and 100% xylene (No. 2) for 3 minutes. Add neutral resin to the slides, cover with a coverslip, avoiding air bubbles. After the neutral resin has air-dried, observe under a microscope, take photographs, and preserve the images. Figure 6 ).

[0154] 2.5 Animal Tissue Western Blotting

[0155] 1) Mouse skin tissue protein extraction: Pre-cool the centrifuge and biosample homogenizer, and prepare sterile pipette tips, EP tubes, and grinding tubes. Remove mouse skin tissue from liquid nitrogen, transfer it to a labeled grinding tube, and add 600 μl of protein lysis buffer. Place the grinding tube containing the lysis buffer into the biosample homogenizer for low-temperature homogenization. Immediately after homogenization, centrifuge at 4°C, 13000 rpm, for 10 min; collect the supernatant, add 1 / 4 of the loading buffer, and heat at 100°C for 10 min.

[0156] 2) Electrophoresis

[0157] Same as 1.2.3.2

[0158] 3) Antibody preparation

[0159] 1 p-stst1(S727)ET1611-20 1:1000 Huaan Bio 2 GAPDH(60004-1-Ig) 1:6000 proteintech

[0160] 2.6 Animal tissue RT-PCR

[0161] 1) RNA extraction from mouse skin tissue: Pre-cool the centrifuge and biosample homogenizer, and prepare enzyme-free sterilized pipette tips, enzyme-free EP tubes, and homogenizing tubes. Remove mouse skin tissue from liquid nitrogen, transfer it to a labeled homogenizing tube, and add 900 μl of Trizol lysis buffer. Place the homogenizing tube containing the lysis buffer into the biosample homogenizer for low-temperature homogenization. Immediately after homogenization, centrifuge at 4°C, 13000 rpm, for 10 min; collect the supernatant, aliquot, and store at -80°C for later use.

[0162] 2) Add 50 μl of chloroform to the supernatant of each sample, vortex for 20 s, and centrifuge at 4°C, 13000 rpm, for 15 min. Transfer the colorless, transparent upper layer to an enzyme-free EP tube, add 150 μl of isopropanol, vortex to mix, and precipitate at room temperature for 15 min. After precipitation, centrifuge at 4°C, 13000 rpm, for 10 min. After centrifugation, a white precipitate will be visible at the bottom of the EP tube. Remove the supernatant, wash with 70% low-temperature ethanol, and centrifuge again at 4°C, 13000 rpm, for 10 min. Remove the supernatant and repeat the washing process twice. Air dry in a biosafety cabinet. Then, dissolve each sample in 50 μl of enzyme-free water and measure the RNA concentration using a micro spectrophotometer.

[0163] 3) Reverse transcription

[0164] Same as 1.2.4

[0165] 3. Statistical Analysis

[0166] The experimental results were analyzed using GraphPad Prism 8 software. After ANOVA analysis, the significance level was tested at α = 0.05 (P < 0.05 was considered statistically significant, otherwise it was considered not statistically significant). The results were indicated by "*", "**" for P < 0.0015, "***" for P < 0.0002, and "****" for P < 0.0001.

[0167] 4 Experimental Results

[0168] like Figure 3 As shown, after 6 days of continuous application of imiquimod cream, mice developed obvious psoriasis phenotypes and pathological features on their skin.

[0169] like Figure 3 , Figure 5 As shown, the extract of *Pleurotus ostreatus* seeds significantly improved the epidermal thickness and scales in an IMQ-induced psoriasis-like mouse model.

[0170] like Figure 6 As shown, the expression levels of P-SATA1, STAT1, IL-17A, ICAM-1, CXCL9, CXCL10, and TNF-α were significantly increased in the epidermis of mice with IMQ-induced psoriatic dermatitis. After treatment with Pyrus pyrifolia seed extract, the levels of P-STAT1, STAT1, IL-17A, ICAM-1, CXCL9, CXCL10, and TNF-α were significantly decreased.

[0171] like Figure 7 As shown, analysis of protein expression in mouse tissues revealed that the expression of p-STAT1 protein was reduced after treatment with melon seed extract compared to the model group.

[0172] like Figure 8 As shown, analysis of mouse tissue mRNA revealed that the level of IL-17AmRNA was significantly reduced after treatment with melon seed extract compared to the model group.

[0173] Example 3: Component Analysis of Melon Seed Extract

[0174] The extract of pine nuts obtained in step 1.1.2 of Example 1 was subjected to mass spectrometry analysis under the following conditions:

[0175] Instruments: UPLC-Q-TOF / MS (Shimadzu, Japan), liquid crystal single-frequency ultrasonic cleaning instrument (Shenzhen Jiemeng Cleaning Equipment Co., Ltd.), ultrapure water was prepared by Milli-Q pure water system (Millipore, USA).

[0176] Elution conditions: 1-10 min 25-25% B, 10-20 min 25-30% B, 20-40 min 30-45%, 40-45 min 45-15% B. Solution A is an aqueous solution containing 5% formic acid, and solution B is acetonitrile (mass spectrometry grade acetonitrile).

[0177] Flow rate: 0.4 ml / min.

[0178] Column: Shim-pack Scepter C18-120 column (2.1 mm × 50 mm i.d., 1.9 μm) (Shimadzu, Japan). The column oven temperature and autosampler temperature were set to 40 °C and 4 °C, respectively. The MS was operated in positive ion mode with an interface voltage of +4.50 kV and an interface temperature of 300 °C. 35% collision energy was used for MS / MS fragmentation, with a mass range of 100-3000 m / z and a DDA mode range of 30-1000 m / z.

[0179] MS data was analyzed using LabSolutions Insight Explore software (Shimadzu, Japan).

[0180] By analyzing mass spectrometry data ( Figure 9 Analysis revealed eight active ingredients, as shown in Table 1, which play an important role in the prevention and treatment of psoriasis.

[0181] Table 1. Active ingredients in melon seed extract

[0182] 1 Ehletianol C 11.662 +Na 579.2200 579.2167 2 herpetatol C 12.113 +Na 397.1257 397.1237 3 Dehydrobis(2-pyridyl)ol 15.823 <![CDATA[[+H-H20]]]> 341.1325 341.1367 4 Pollen Methyl 23.561 +Na 575.1888 575.1862 5 Pollen 23.970 +Na 561.2095 561.2068 6 Bordenol 25.536 +Na 559.1939 559.1910 7 Polanyl 28.803 +Na 753.2517 753.2482

[0183] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0184] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. Use of a Momordica foetida seed extract for the preparation of a medicament, characterized in that, The drug is used to prevent or treat psoriasis; The method for obtaining the bouncy melon seed extract includes: The seeds and alcohol were heated and refluxed, the resulting reflux liquid was filtered, and the filtrate was collected. The filtrate was evaporated and dried, and the resulting concentrate was extracted with petroleum ether to obtain the extract. The extract was evaporated and dried, and the dried product was then dissolved in ethanol to obtain a pine nut seed extract. The extract of *Pleurotus ostreatus* seeds includes: Ehletianol C, herpetatol C, dehydrobispineol, pleurotus ostreatus A, pleurotus ostreatus B, pleurotus ostreatus alcohol, and pleurotus ostreatus B.

2. Use according to claim 1, characterized in that, The drug is used to downregulate the expression of inflammatory factors in the STAT1 signaling pathway and inhibit STAT1 phosphorylation.

3. The use according to claim 2, characterized in that, The inflammatory factors include IL-17A, ICAM-1, CXCL9, CXCL10, or TNF-α.

4. The use according to claim 1, characterized in that, The alcohol is selected from at least one of ethanol, methanol, propanol and isopropanol.

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