Use of ampelopsis japonica stem extract in preparation of anti-colitis drugs

Abstract

The application discloses application of Vitis amurensis stem alcohol extract in preparation of anti-colitis drugs. In the application, the Vitis amurensis stem alcohol extract improves LPS-induced M1 polarization of RAW264.7 cells, inhibits expression of proinflammatory cytokines TNF-alpha, IL-6 and IL-1beta by down-regulating proinflammatory cytokine-related genes; inhibits expression of proinflammatory mediators NO and PGE2 by down-regulating transcription and translation levels of proinflammatory mediator-related synthetases (iNOS and COX-2); the Vitis amurensis stem alcohol extract inhibits activation of the NF-kappa B pathway by reducing phosphorylation and degradation of IKBalpha and nuclear transfer of NF-kappa B p65, and inhibits activation of the MPAK pathway by reducing phosphorylation of ERK, p38 and JNK; in addition, the Vitis amurensis stem alcohol extract improves colitis of mice caused by dextran sulfate sodium, slows down weight loss of the mice, reduces DAI scores of the mice, reduces colon tissue damage of the mice and reduces IL-6, IL-1beta and TNF-alpha levels in colon tissue and serum of the mice. Therefore, the Vitis amurensis stem alcohol extract has significant anti-inflammatory effects in vivo and in vitro, and can be used for treating colitis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of wild grape stem alcohol extract in the preparation of anti-colitis drugs. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of immune-mediated diseases characterized by chronic, relapsing intestinal inflammation, including ulcerative colitis and Crohn's disease. Symptoms include colorectal bleeding, abdominal pain, diarrhea, bloating, malabsorption, bloody stools, and mucus in stools (Lancet 2017, 390(10114), 2769–2778; N. Engl.J. Med. 2020, 383(27), 2652–2664; Nat. Rev. Gastroenterol. Hepatol. 2022, 19(12), 753–767). In recent years, the global incidence of IBD has continued to rise, becoming a significant public health issue (Am.Fam. Physician. 2007, 76(9), 1323–1330; J. Crohns Colitis 2022, 16(1), 2–17). Currently, 5-aminosalicylic acid, glucocorticoids, and biologics remain the main treatments for IBD, but long-term use can easily lead to adverse reactions and drug resistance (Int. Immunopharmacol. 2024, 128, 111570; Front. Cell.Infect. Microbiol. 2023, 13, 1268041). In contrast, natural products and plant extracts, due to their advantages such as multiple targets and low toxicity, have gradually become a hot topic in IBD prevention and treatment research (Nutrients 2024, 16(7), 934).

[0003] The Miao ethnic medicinal herb wild grape refers to Ampelopsis delavayana Planch. of the genus Ampelopsis in the family Vitaceae, which mainly grows in valley forests, hillside shrubs or forests in regions including Fujian, Guangdong, Guangxi, Hainan, Sichuan, Guizhou and Yunnan (Flora of China. Beijing: Science Press, 1998, 48(2), 43). The Miao medicinal name of wild grape is "ghab jongx zend gheid dlub", it is cold in nature, astringent in taste, and acts on the heat meridian, with the effects of activating blood circulation to dredge collaterals, stopping bleeding and promoting tissue regeneration, and detoxifying and reducing swelling, and is mainly used to treat diseases such as rheumatic arthralgia, traumatic ecchymoma and swelling, traumatic hemorrhage, scalds, and sores and abscesses (Chinese Materia Medica · Miao Medicine Volume. Guiyang: Guizhou Science and Technology Publishing House, 2005: 502-503). In addition, as a commonly used medicinal material of ethnic minorities such as the Miao ethnic group, the water decoction or wine infusion of wild grape stems (used raw, 15g-60g) can be used for the treatment of diseases such as diarrhea and dysentery (Primary Color Atlas of Medicinal Plants of Sichuan: Volume 1. Chengdu: Sichuan Science and Technology Publishing House, 2021: 552-553; Yunnan Provincial Standard for Chinese Medicinal Materials: 2005 Edition. Volume 2, Yi Ethnic Medicine. Kunming: Yunnan Science and Technology Publishing House, 2007; Guizhou Herbal Medicine: Episode 1. Guiyang: Guizhou People's Publishing House, 1970: 896; Home Herbal Medicine. Guiyang: Guizhou Science and Technology Publishing House, 2004:226). At present, there are no relevant reports on the anti-colitis effect of wild grape stems, nor are there any reports related to the chemical components of wild grape stems, which may hinder their development and utilization. Contents of the Invention

[0004] The objective of the present invention is to provide the application of wild grape stem ethanol extract in the preparation of anti-colitis medicaments, which develops a new use of the wild grape stem ethanol extract and provides a new choice for the preparation of anti-colitis medicaments.

[0005] This invention also discovered that the ethanol extract of wild grape stems improved M1 polarization in RAW264.7 cells, significantly inhibited the production of LPS-induced pro-inflammatory mediators (NO, PGE2) and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β); inhibited the transcription of LPS-induced pro-inflammatory cytokine genes and the expression of genes and proteins synthesizing pro-inflammatory mediator enzymes (iNOS and COX-2); related mechanistic studies showed that the ethanol extract of wild grape stems not only inhibited the nuclear translocation of NF-κB p65 by reducing the phosphorylation and degradation of IκBα, but also inhibited the phosphorylation of MPAKs (ERK, p38, and JNK); the ethanol extract of wild grape stems improved dextran sulfate-induced colitis in mice, including alleviating weight loss in mice, reducing DAI scores in mice, reducing colonic tissue damage in mice, and downregulating the expression levels of IL-6, IL-1β, and TNF-α in colonic tissue and serum of mice; in addition, the chemical components of the ethanol extract of wild grape stems were identified.

[0006] The technical solution adopted in this invention is as follows:

[0007] Wild grape stem alcohol extract was prepared and screened using the following methods:

[0008] Fresh roots, stems, and leaves of wild grapes were washed, crushed, and precipitated. 70% ethanol or pure water was added at a material-to-liquid ratio of 1:3 (m / v), and the mixture was refluxed for 2 hours, then filtered. The residue was extracted again using the same method. The two filtrates were combined, filtered, concentrated by rotary evaporation, and then freeze-dried to obtain ethanol and water extracts of the roots, stems, and leaves of wild grapes, respectively. Through MTT assays and comparisons of the inhibitory effects on LPS-induced NO production, the ethanol extract of the wild grape stems showed the best anti-inflammatory activity and was therefore selected for further research.

[0009] This invention, through experimental research, discovered that wild grape stem ethanol extract, without cytotoxicity, improved LPS-induced M1 polarization in RAW264.7 cells, inhibited the expression of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β by downregulating pro-inflammatory cytokine-related genes, and inhibited the expression of pro-inflammatory mediators NO and PGE2 by downregulating the transcription and translation levels of pro-inflammatory mediator-related synthases (iNOS and COX-2). Wild grape stem ethanol extract inhibited NF-κB pathway activation by reducing IκBα phosphorylation and degradation and NF-κB p65 nuclear translocation, and also inhibited MPAK pathway activation by reducing ERK, p38, and JNK phosphorylation. Furthermore, wild grape stem ethanol extract ameliorated dextran sulfate-induced colitis in mice, slowed weight loss, reduced DAI scores, alleviated colonic tissue damage, and reduced the levels of IL-6, IL-1β, and TNF-α in mouse colonic tissue and serum.

[0010] By adopting the above technical solution, this invention has for the first time discovered a new use of wild grape stem alcohol extract in the treatment of inflammation-related diseases, providing a new option for the preparation of anti-colitis drugs, and has important application value in the pharmaceutical industry. Attached Figure Description

[0011] Figure 1 Screening of anti-inflammatory active components of *Vitis vinifera*. A: MTT plot of the effects of *Vitis vinifera* root aqueous extract (RWE), root alcohol extract (RWE), stem aqueous extract (SWE), stem alcohol extract (SEE), leaf aqueous extract (LWE), and leaf alcohol extract (LEE) on RAW264.7 cell viability; B: Inhibition of LPS-induced NO production by *Vitis vinifera* root aqueous extract (RWE), root alcohol extract (RWE), stem aqueous extract (SWE), stem alcohol extract (SEE), leaf aqueous extract (LWE), and leaf alcohol extract (LEE);

[0012] Figure 2 Total ion spectrum of wild grape stem alcohol extract (A: positive ion mode; B: negative ion mode).

[0013] Figure 3 The inhibitory effects of wild grape stem ethanol extract on LPS-induced cell morphology, pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) and their mRNA expression in the RAW264.7 cell line were investigated.

[0014] Figure 4 The inhibitory effects of wild grape stem alcohol extract on the protein and mRNA of pro-inflammatory mediators (NO and PGE2) as well as iNOS and COX-2 induced by LPS in RAW264.7 cells;

[0015] Figure 5 The effects of wild grape stem alcohol extract on LPS-induced NF-κB pathway-related proteins and NF-κB p65 nuclear translocation;

[0016] Figure 6 The inhibitory effect of wild grape stem alcohol extract on LPS-induced phosphorylation of MPAKs (p38, ERK and JNK) proteins;

[0017] Figure 7 The effects of wild grape stem alcohol extract on body weight and DAI score in DSS-induced colitis mice;

[0018] Figure 8 The effect of wild grape stem alcohol extract on colon length in DSS-induced colitis mice;

[0019] Figure 9 H&E staining image of colon tissue from DSS-induced colitis mice;

[0020] Figure 10The effects of wild grape stem alcohol extract on the levels of IL-6, IL-1β and TNF-α in colon tissue and serum of mice with DSS-induced colitis. Detailed Implementation

[0021] Example of this invention: Fresh roots, stems, and leaves of wild grapes (harvested in Nanming District, Guiyang City, Guizhou Province; identified by Professor Hu Guoxiong of Guizhou University) were washed, dried, and pulverized. The pulverized raw materials were mixed with 70% ethanol or pure water at a material-to-liquid ratio of 1:3. The mixture was extracted in an extractor for 2 hours, repeated twice, and the two extracts were combined, filtered, rotary evaporated, and then freeze-dried under vacuum to obtain alcohol and water extracts of wild grape roots, stems, and leaves, which were stored in a desiccator. Through MTT assays and comparisons of the inhibition results of LPS-induced NO production, the wild grape stem alcohol extract showed the best anti-inflammatory activity and was therefore selected for further research. The yield of the wild grape stem alcohol extract dry powder was 2.19% (based on the fresh weight of the wild grape stems). Chemical composition of the wild grape stem alcohol extract in this example: The chemical components of the wild grape stem alcohol extract were detected and identified by UHPLC-Q-Orbitriap MS. The UHPLC chromatographic conditions used in this experiment are as follows:

[0022] Analysis was performed using a Dionex Ultimate 3000 RSLC ultra-high performance liquid chromatography system with a Thermo Fisher Hypersil GOLD aQ column (100 mm × 2.1 mm, 1.9 μm). The mobile phase consisted of 0.1% formic acid acetonitrile (A) and 0.1% formic acid aqueous solution (B), with an injection volume of 5 μL, a flow rate of 0.3 mL / min, and a column temperature of 40 ℃. The gradient elution program was as follows: 0–2 min, 5% A; 2–42 min, 5%–95% A; 42–47 min, 95% A; 47–47.1 min, 95%–5% A; 47.1–50 min, 5% A. Mass spectrometry analysis was performed using a Q-Orbitrap mass spectrometer equipped with a heated electrospray ionization source (HESI-II, Thermo Fisher Scientific). The MS parameters were set as follows: RF lens amplitude (60); spray voltage -2.5 kV (negative ion mode) / +3.0 kV (positive ion mode); vaporizer temperature 350 ℃; capillary temperature 320 ℃; sheath gas (N2) flow rate 35 arb; auxiliary gas flow rate 10 arb; purge gas flow rate 0 arb. Full MS / dd-MS was used. 2 Data acquisition mode, parameter settings are as follows: stepped normalized collision energy is 20, 40, and 60 eV; MS 1 The resolution is 70,000, MS 2Resolution: 17,500; Automatic gain control (AGC) target value: MS 1 For 1e6, MS 2 2e5; Maximum injection time: MS 1 For 100 ms, MS 2 The scan time was 50 ms; the scan range was 100–1500 m / z. Thermo Fisher Scientific Xcalibur 4.1 software was used to analyze and process the mass spectrometry data. Analysis of MS... 1 Fragment information was used to determine the molecular formula of the compound, and MS was used to... 2 The chemical components in the wild grape stem alcohol extract were identified by comparing the fragments with the mzVault database, with a maximum allowable relative mass error of 10 ppm.

[0023] Chemical components of wild grape stem alcohol extract, such as Figure 2 (A: Positive ion mode; B: Negative ion mode) and as shown in Table 1, a total of 62 compounds were identified. Their chemical composition mainly includes phenols, flavonoids, terpenes, organic acids, and amino acids.

[0024] Phenolic compounds: 4-Hydroxybenzoic acid, Syringic acid, 3,4-Dihydroxybenzaldehyde, Orsellinic acid, 4-Methoxybenzaldehyde, Homoveratrumic acid, o-Veratraldehyde, 1,6-Bis-O-(3,4,5-trihydroxybenzoyl)hexopyranose, Ethyl gallate, Ellagic acid, Benzoic acid Components include: methylparaben, methylcinnamate, trans-cinnamaldehyde, shogaol, polydatin, and cis-resveratrol; flavonoids: tiliroside, (-)-gallocatechin, epigallocatechin, procyanidin B1, procyanidin B2, (+)-catechin hydrate / ciandanol, luteolin, myricetin, and myricitrin. Gallate, Quercitrin, Morin, Kaempferol; Terpenes: Bilobalide, Arglabin, Pulegone, Curcumol, (-)-Caryophyllene oxide, Sarracenin, Asperuloside, Dehydrodiisoeugenol, Atractyloside A;Organic acids: D-(-)-Quinic acid, Citric acid, D-Saccharic acid (D-gluconic acid), (15Z)-9,12,13-Trihydroxy-15-octadecenoic acid, Indole-3-acrylic acid, BMK methyl glycidate; Amino acids: L-Valine, D-Alanine methyl ester, L-Norleucine, L-Phenylalanine, DL-Tryptophan, Pipecolic acid, L-Pyroglutamic acid. acid); others: α,α-trehalose, D-mannose 6-phosphate, adenosine, choline, nicotinamide, 4-indolecarbaldehyde.

[0025] Table 1 Chemical components of alcohol extract from wild grape stems

[0026] NO. RT [min] Identification Formula <![CDATA[MS 1 (mass accuracy, ppm)]]> <![CDATA[MS 2 fragment ions]]> 1 0.815 Choline <![CDATA[C5H 13 NO]]> <![CDATA[104.10746[M-H] + (-4.5)]]> 87.04462, 60.08165, 58.06596 2 0.863 α,α-Trehalose α,α-Trehalose <![CDATA[C 12 H 22 O 11 ]]> <![CDATA[341.10858[M-H] - (-2.0)]]> 179.05518, 89.02306, 59.01248 3 0.967 L-Valine <![CDATA[C5H 11 NO2]]> <![CDATA[118.08646[M-H] + (-1.7)]]> 100.02465,72.08151, 59.07376 4 0.98 Pipecolic acid <![CDATA[C6H 11 NO2]]> <![CDATA[130.08640[M-H] + (-1.1)]]> 84.08148, 70.06584, 56.05032 5 1.033 D-Alanine methyl ester (D-alanine methyl ester) <![CDATA[C4H9NO2]]> <![CDATA[102.05537[M-H] + (-4.1)]]> 86.06059, 60.08160, 58.06878 6 1.034 D-(-)-Quinic acid <![CDATA[C7H 12 O6]]> <![CDATA[191.05519[M-H] - (-4.8)]]> 171.02885, 93.03328, 85.02811 7 1.191 Nicotinamide <![CDATA[C6H6N2O]]> <![CDATA[123.05554[M-H] + (-2.0)]]> 105.03735, 96.04481, 80.05012 8 1.285 D-Saccharic acid <![CDATA[C6H 10 O8]]> <![CDATA[209.02954[M-H] - (-3.6)]]> 191.01889, 133.01309, 85.02811 9 1.346 Adenosine <![CDATA[C 10 H 13 N5O4]]> <![CDATA[268.10382[M-H] + (-0.8)]]> 136.06183, 97.02924, 73.02909 10 1.441 D-Mannose 6-phosphate <![CDATA[C6H 13 O9P]]> <![CDATA[259.01865[M-H] - (-14.6)]]> 138.96921, 96.96828, 78.95769 11 1.455 L-Norleucine (L-leucine) <![CDATA[C6H 13 NO2]]> <![CDATA[132.10193[M-H] + (-0.2)]]> 86.09707, 69.07060, 57.05824 12 1.611 L-Pyroglutamic acid (L-pyroglutamic acid) <![CDATA[C5H7NO3]]> <![CDATA[130.04996[M-H] + (-0.7)]]> 102.05553, 84.04507, 56.05030 13 1.611 D-(-)-Quinic acid <![CDATA[C7H 12 O6]]> <![CDATA[191.05521[M-H] - (-4.7)]]> 127.03876, 111.00748, 85.02811 14 2.369 Citric acid <![CDATA[C6H8O7]]> <![CDATA[191.01889[M-H] - (-4.4)]]> 111.00748, 87.00739, 85.02810 15 2.849 L-Phenylalanine (L-phenylalanine) <![CDATA[C9H 11 NO2]]> <![CDATA[166.08592[M-H] + (-2.0)]]> 120.08108, 103.05456, 74.02428 16 3.214 Tiliroside <![CDATA[C 30 H 26 O 13 ]]> <![CDATA[595.14392[M-H] + (-1.2)]]> 287.05429, 139.03877, 127.03899 17 3.661 (-)-Gallocatechin gallatechin <![CDATA[C 15 H 14 O7]]> <![CDATA[305.06628[M-H] - (-1.3)]]> 167.03389, 137.02315, 125.02316 18 3.71 4-Hydroxybenzoic acid (p-hydroxybenzoic acid) <![CDATA[C7H6O3]]> <![CDATA[139.03893[M-H] + (-0.3)]]> 121.03948, 111.04427, 65.03933 19 3.744 Epigallocatechin (-) - Epigallocatechin <![CDATA[C 15 H 14 O7]]> <![CDATA[305.06650[M-H] - (-0.6)]]> 167.03398, 137.02317, 125.02317 20 4.272 o-Veratraldehyde <![CDATA[C9H 10 O3]]> <![CDATA[167.07010[M-H] + (-1.0)]]> 151.03876, 123.04414, 78.04704 21 4.423 DL-Tryptophan <![CDATA[C 11 H 12 N2O2]]> <![CDATA[205.09650[M-H] + (-3.2)]]> 188.07037, 146.05994, 118.06529 22 4.568 Indole-3-acrylic acid <![CDATA[C 11 H9NO2]]> <![CDATA[188.07037[M-H] + (-1.3)]]> 146.05995, 144.08061, 118.06531 23 4.741 4-Indolecarbaldehyde <![CDATA[C9H7NO]]> <![CDATA[146.05992[M-H] + (-0.8)]]> 118.06528, 100.04776, 91.05466 24 8.022 Syringic acid <![CDATA[C9H 10 O5]]> <![CDATA[199.05986[M-H] + (-1.2)]]> 155.07004, 140.04668, 95.04958 25 8.561 Procyanidin B2 <![CDATA[C 30 H 26 O 12 ]]> <![CDATA[579.14874[M-H] + (-1.7)]]> 409.09067, 139.03883, 127.03902 26 8.726 3,4-Dihydroxybenzaldehyde <![CDATA[C7H6O3]]> <![CDATA[139.03900[M-H] + (-0.2)]]> 121.02867, 111.05583, 65.03935 27 8.85 Procyanidin B1 <![CDATA[C 30 H 26 O 12 ]]> <![CDATA[577.13568[M-H] - (-0.9)]]> 407.07654, 289.07147, 125.02314 28 9.296 Benzoic acid <![CDATA[C7H6O2]]> <![CDATA[123.04435[M-H] + (-2.4)]]> 105.03381, 95.04961, 67.05499 29 9.298 (+)-Catechin hydrate (+)-Catechins <![CDATA[C 15 H 14 O6]]> <![CDATA[289.07169[M-H] - (-0.2)]]> 245.08156, 123.04391, 109.02821 30 9.531 Orsellinic acid <![CDATA[C8H8O4]]> <![CDATA[169.04938[M-H] + (-0.9)]]> 151.03886, 141.05449, 123.04417 31 9.627 1,6-Bis-O-(3,4,5-trihydroxybenzoyl)hexopyranose <![CDATA[C 20 H 20 O 14 ]]> <![CDATA[483.07727[M-H] - (-1.6)]]> 271.04578, 169.01328, 125.02322 32 9.656 Bilobalide (Ginkgolide) <![CDATA[C 15 H 18 O8]]> <![CDATA[325.09348[M-H] - (-1.8)]]> 163.03906, 145.02835, 119.04900 33 9.784 Cianidanol catechin <![CDATA[C 15 H 14 O6]]> <![CDATA[291.08618[M-H] + (-0.5)]]> 147.04395, 139.03894, 123.04420 34 9.91 4-Methoxybenzaldehyde <![CDATA[C8H8O2]]> <![CDATA[137.05977[M-H] + (-0.5)]]> 122.03637, 94.04179, 81.07043 35 9.929 BMK methyl glycidate (BMK methyl glycerate) <![CDATA[C 11 H 12 O3]]> <![CDATA[193.08585[M-H] + (-0.4)]]> 161.05952, 133.06474, 105.07021 36 10.282 Asperuloside <![CDATA[C 18 H 22 O 11 ]]> <![CDATA[413.10895[M-H] - (-0.1)]]> 147.04405, 119.04898, 59.01250 37 10.284 Sarracenin dehydrated monoglycoside <![CDATA[C 11 H 14 O5]]> <![CDATA[227.09186[M-H] + (-2.0)]]> 181.04933, 151.03888, 139.03885 38 10.348 Homoveratrumic acid <![CDATA[C 10 H 12 O4]]> <![CDATA[197.08080[M-H] + (-0.2)]]> 151.03896, 125.05975, 73.02908 39 11.25 (+)-Catechin hydrate (+)-Catechins <![CDATA[C 15 H 14 O6]]> <![CDATA[289.07162[M-H] - (-0.5)]]> 245.08148, 123.04389, 109.02821 40 11.412 Luteolin <![CDATA[C 15 H 10 O6]]> <![CDATA[287.05457[M-H] + (-1.5)]]> 203.03348, 167.07010, 68.99434 41 11.612 Methylparaben <![CDATA[C8H8O3]]> <![CDATA[153.05431[M-H] + (-2.0)]]> 121.02860, 111.04430, 79.01848 42 12.171 Methyl cinnamate <![CDATA[C 10 H 10 O2]]> <![CDATA[163.07529[M-H] + (-3.9)]]> 131.04926, 103.05467, 91.05481 43 12.221 Ethyl gallate <![CDATA[C9H 10 O5]]> <![CDATA[197.04478[M-H] - (-3.9)]]> 169.01326, 139.00247, 125.02320 44 12.315 Methyl cinnamate <![CDATA[C 10 H 10 O2]]> <![CDATA[163.07512[M-H] + (-1.4)]]> 145.10135, 131.04921, 103.05466 45 12.527 trans-Cinnamaldehyde trans-cinnamaldehyde <![CDATA[C9H8O]]> <![CDATA[133.06477[M-H] + (-0.2)]]> 118.04150, 105.07020, 79.05482 46 13.294 Polydatin <![CDATA[C 20 H 22 O8]]> <![CDATA[389.12625[M-H] - (-5.3)]]> 227.07085, 185.05995, 143.04909 47 13.3 cis-Resveratrol <![CDATA[C 14 H 12 O3]]> <![CDATA[229.08546[M-H] + (-2.0)]]> 135.04395, 107.04943, 91.05469 48 13.412 Shogaol (gingerol) <![CDATA[C 17 H 24 O3]]> <![CDATA[277.17880[M-H] + (-2.7)]]> 191.10638, 137.05974, 109.10143 49 13.49 Myricetin <![CDATA[C 15 H 10 O8]]> <![CDATA[319.04425[M-H] + (-1.9)]]> 273.03882, 217.04919, 153.01805 50 13.515 Myricitrin <![CDATA[C 21 H 20 O 12 ]]> <![CDATA[463.08795[M-H] - (-0.5)]]> 316.02228, 271.02469, 151.00255 51 13.552 (-)-Epicatechin gallate (-)-Epicatechin gallate <![CDATA[C 22 H 18 O 10 ]]> <![CDATA[441.08157[M-H] - (-2.6)]]> 289.07153, 169.01323, 125.02318 52 13.783 Ellagic acid <![CDATA[C 14 H6O8]]> <![CDATA[300.99872[M-H] - (-0.9)]]> 229.01360, 201.01866, 173.02356 53 13.913 Arglabin <![CDATA[C 15 H 18 O3]]> <![CDATA[229.12180[M-H] + (-2.2)]]> 201.12704, 187.11151, 173.13220 54 14.774 Pulegone <![CDATA[C 10 H 16 O]]> <![CDATA[153.12733[M-H] + (-0.4)]]> 135.11676, 107.08581, 81.07047 55 14.822 Quercitrin <![CDATA[C 21 H20O 11 ]]> <![CDATA[447.10016[M-H] - (-15.4)]]> 301.03510, 300.02734, 271.02466 56 14.823 Morin <![CDATA[C 15 H10O7]]> <![CDATA[303.04926[M-H] + (-2.2)]]> 247.06024, 153.01805, 137.02327 57 15.009 Dehydrodiisoeugenol <![CDATA[C20H 22 O4]]> <![CDATA[327.15799[M-H] + (-3.4)]]> 163.07510, 137.05965, 133.06474 58 15.753 Kaempferol <![CDATA[C 15 H 10 O6]]> <![CDATA[287.05457[M-H] + (-1.6)]]> 153.01807, 121.02855, 68.99784 59 16.616 Curcumol <![CDATA[C 15 H 24 O2]]> <![CDATA[219.17389[M-H] + (-2.1)]]> 133.10110, 105.07020, 81.07047 60 16.67 Atractyloside A <![CDATA[C 21 H 36 O 10 ]]> <![CDATA[447.22330[M-H] - (-0.6)]]> 315.18124, 161.04446, 101.02305 61 17.481 (-)-Caryophyllene oxide (-)-Caryophyllene oxide <![CDATA[C 15 H 24 O]]> <![CDATA[221.18962[M-H] + (-1.7)]]> 203.17911, 135.11671, 81.07047 62 21.019 (15Z)-9,12,13-Trihydroxy-15-octadecenoic acid <![CDATA[C 18 H 34 O5]]> <![CDATA[329.23297[M-H] - (-1.1)]]> 229.14375, 211.13312, 171.10168

[0027] Pharmacological Example 1: Toxicity of Wild Grape Stem Alcohol Extract on RAW264.7 Cells

[0028] The toxic effects of water and alcohol extracts of *Vitis vinifera*, stems, and leaves on RAW264.7 cells were detected using the MTT assay. RAW264.7 cells in the logarithmic growth phase were collected and their concentration adjusted to 2 × 10⁻⁶ cells / mL. 5 Cells were seeded at 1 / mL in 96-well plates (sample group, solvent control group, and blank control group, 3 replicates each), and cultured for 24 h. After discarding the old solution, half-diluted aqueous and alcoholic extracts of wild grape, stems, and leaves (1000–3.91 μg / mL, 9 concentrations) were added, with DMSO as the solvent control, and cultured for another 24 h. MTT (final concentration 5 mg / mL) was added to each well and incubated in the dark for 4 h. The supernatant was discarded, and formazan was dissolved in DMSO. The plates were shaken for 10 min, and the OD value (A) was measured at 490 nm. Cell viability (%) = (A) / (Mt / mL) * ... 样品组 -A 空白对照组 ) / (A 溶剂对照组 -A空白对照组 )×100.

[0029] As shown in Figure 1A, compared with the untreated group, neither the aqueous extract (WE) nor the ethanolic extract (EE) from any part of the stem showed cytotoxicity against RAW264.7 cells at concentrations ranging from 3.91 to 250 μg / mL. Even at a concentration of 1000 μg / mL, neither WE nor EE from the stems showed cytotoxicity. To ensure comparative analysis of WE and EE in subsequent anti-inflammatory screening experiments, non-toxic concentrations of 31.25, 62.5, 125, and 250 μg / mL of WE and EE were selected for subsequent experiments.

[0030] Lipopolysaccharide (LPS) induced the release of the inflammatory mediator nitric oxide (NO) from RAW264.7 macrophages. Therefore, NO production was used as a primary screening indicator to evaluate the anti-inflammatory potential of extracts from different parts of *Vitis vinifera*. Log-phase RAW264.7 cells were harvested and adjusted to 3 × 10⁻⁶ cells. 5 Inoculate 100 μL of DMEM, DXM, or drug solution into 96-well plates and incubate for 24 h. Include blank control, model (LPS), positive control (dexamethasone, DXM), and different drug concentration groups, with 5 replicates per group. First, add DMEM, DXM, or drug solution and incubate for 2 h. Then, add LPS to the model, positive control, and drug groups to a final concentration of 1 μg / mL (DMEM for the blank control), and 20 μg / mL for the positive control group, and continue incubation for another 24 h. Collect the supernatant, freeze at -80 ℃, and use a NO kit to detect the NO content in the supernatant of each group.

[0031] As shown in 1B, compared with the blank control group, the NO level in the cell supernatant was significantly increased after LPS stimulation (p < 0.001), indicating that the in vitro inflammation model was successfully established; the positive control dexamethasone significantly inhibited LPS-induced NO release. Different parts of the WE and EE showed varying degrees of inhibitory effects on LPS-stimulated NO production, with the most significant inhibitory effect observed in the ethanol extract of wild grape stem (Stem EE, SEE). Based on this, subsequent studies selected wild grape stem EE as the research subject to further explore its anti-inflammatory effects and potential mechanisms.

[0032] Pharmacological Example 2: The ethanol extract of wild grape stems inhibited LPS-induced morphological changes in RAW264.7 cells and the expression and release of pro-inflammatory cytokines mRNA.

[0033] RAW264.7 cells were seeded in 96-well plates (2 × 10⁻⁶ cells / well). 4RAW264.7 cells were cultured for 24 h per well. Pretreatment with fresh medium containing different doses (0, 62.5, 125, and 250 μg / mL) of wild grape stem extract for 2 h was followed by incubation with lipopolysaccharide (LPS, 1 μg / mL) for another 24 h. Morphological changes in RAW264.7 cells were recorded using an inverted microscope (Leica Microsystems, Germany). Dexamethasone (DXM, 20 μg / mL) was used as a positive control. After collecting the cell supernatant, the NO level secreted by RAW264.7 cells was measured using a NO detection kit (Beyotime, Shanghai). Furthermore, the levels of IL-1β, IL-6, and TNF-α in the cell supernatant were assessed using an ELISA kit. Total RNA was then extracted from the cells using the EZNA® Total RNA Kit I (OmegaBio-Tek, Norcross, GA, USA), followed by RT Easy RNA analysis. TM The II kit (Fujibio, Chengdu, China) reverse transcribed RNA into single-stranded cDNA. qRT-PCR was performed using the Real-Time PCR Easy™-SYBR Green Ikit (Fujibio, Chengdu, China) via CFX Connect. TMThe reaction was performed using a real-time quantitative PCR detection system (Bio-Rad, CA, USA) to detect the expression levels of IL-1β, IL-6, and TNF-α mRNA. The primer sequences are as follows: TNF-α (forward, 5′-CCT GTA GCC CAC GTC GTA G-3′; reverse, 5′-GGG AGT AGA CAA GGTACA ACC C-3′), IL-1β (forward, 5′-CCT GTG TCT TTC CCG TGG AC-3′; reverse, 5′-CAT CTC GGA GCC TGT AGT GC-3′), IL-6 (forward, 5′-ACA ACC ACG GCC TTC CCT ACTT-3′; reverse, 5′-TTT CTC ATT TCC ACG ATT TCC C-3′), iNOS (forward, 5′-TCGGGT TGA AGT GGT ATG C-3′; reverse, 5′-GAG GCC AGT GTG TGG GTC T-3′), COX-2(forward, 5′-TGA CTG CCC AAC TCC CAT-3′; reverse, 5′-GAA CCC AGG TCC TCG CTT-3′), and GAPDH (forward, 5′-AGC CTC GTC CCG TAG ACA AAA-3′; reverse, 5′-GAGGCA ACA ATC TCC ACT TT-3′).

[0034] Lipopolysaccharide (LPS) can induce pro-inflammatory M1-like phenotype changes in RAW264.7 macrophages, such as increased cell volume, enhanced adhesion, and dendritic protrusion formation. Figure 3 As shown in Figure A, the unstimulated group cells were generally round or oval with clear boundaries; after LPS stimulation, the cell morphology became significantly irregular, with dendritic extensions and pseudopodia structures, and the cell outlines enlarged (indicated by white arrows). Compared with the LPS model group, pretreatment with wild grape stem alcohol extract significantly alleviated the above-mentioned abnormal morphological characteristics. Figure 3As shown in Figures B–G, compared with the control group, LPS stimulation significantly increased the secretion and mRNA expression levels of TNF-α, IL-1β, and IL-6 in RAW264.7 cells. Pretreatment with *Vitis vinifera* stem extract (62.5, 125, and 250 μg / mL) resulted in a dose-dependent decrease in the secretion and mRNA expression of these pro-inflammatory cytokines (p < 0.05). The inhibitory effect of 250 μg / mL *Vitis vinifera* stem extract on IL-6 and IL-1β was comparable to or even stronger than that of DXM (20 μg / mL). These results indicate that *Vitis vinifera* stem extract exerts its anti-inflammatory effect by inhibiting the expression of TNF-α, IL-1β, and IL-6 genes and reducing the production of pro-inflammatory cytokines.

[0035] Pharmacological Example 3: Wild grape stem alcohol extract inhibited LPS-induced pro-inflammatory mediators (NO and PGE2) and their synthesis-related enzymes iNOS and COX-2 protein and mRNA levels.

[0036] RAW264.7 cells were cultured at a rate of 6 × 10⁻⁶. 5Cells were seeded per well in 6-well plates and cultured for 24 h. Cells were then pretreated with different concentrations of volatile oil (0, 62.5, 125, and 250 μg / mL) for 2 h, followed by stimulation with LPS (1 μg / mL) for 24 h. After cell collection, total protein was extracted using RIPA lysis buffer, and protein concentration was determined using an enhanced BCA protein quantification kit. 20 μg of protein from each sample was separated by 10% SDS-PAGE and transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk powder for 1 h, incubated with primary antibody overnight at 4 °C, washed three times with TBST, and then incubated with HRP-labeled secondary antibody at room temperature for 1 h. Protein bands were imaged using a ChemiDoc Touch chemiluminescence imaging system (Bio-Rad, CA, USA), and grayscale analysis was performed using Image Lab 6.0 software. In addition, the levels of NO and PGE2 in cell supernatant were detected by ELISA, and the mRNA expression levels of iNOS and COX-2 in cells were detected by RT-qPCR, with GAPDH as an internal control. The primer sequences are as follows: iNOS (forward, 5′-TCG GGT TGA AGT GGTATG C-3′; reverse, 5′-GAG GCC AGT GTG TGG GTC T-3′), COX-2 (forward, 5′-TGA CTGCCC AAC TCC CAT-3′; reverse, 5′-GAA CCC AGG TCC TCG CTT-3′) and GAPDH (forward, 5′-AGC CTC GTC CCG TAG ACA AAA-3′; reverse, 5′-GAG GCA ACA ATC TCC ACT TT-3′).

[0037] Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are key enzymes in the synthesis of NO and PGE2, respectively. Figure 4 As shown in A and B, compared with the control group, LPS stimulation significantly increased the release of NO and PGE2 in RAW264.7 cells (p < 0.05), while treatment with wild grape stem ethanol extract dose-dependently inhibited their production. Further examination of iNOS and COX-2 expression revealed ( Figure 4 C–F), LPS significantly upregulated the mRNA and protein expression levels of both, while pretreatment with wild grape stem ethanol extract dose-dependently downregulated their expression. The results indicate that wild grape stem ethanol extract exerts its anti-inflammatory effect by inhibiting the expression of iNOS and COX-2 and reducing the production of NO and PGE2.

[0038] Pharmacological Example 4: Wild grape stem alcohol extract inhibited LPS-induced NF-κB signaling pathway activation.

[0039] RAW264.7 cells were cultured at a rate of 6 × 10⁻⁶. 5 Cells were seeded per well in 6-well plates and cultured for 24 h. Cells were then pretreated with different concentrations of wild grape stem ethanol extract (0, 62.5, 125, and 250 μg / mL) for 2 h, followed by stimulation with LPS (1 μg / mL) for 24 h. After cell collection, total protein was extracted using RIPA lysis buffer, and protein concentration was determined using an enhanced BCA protein quantification kit. 20 μg of protein from each sample was separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk powder for 1 h, incubated with primary antibody overnight at 4 °C, washed three times with TBST, and then incubated with HRP-labeled secondary antibody at room temperature for 1 h. Protein bands were imaged using a ChemiDocTouch chemiluminescent gel imaging system (Bio-Rad, CA, USA), and grayscale analysis was performed using Image Lab 6.0 software.

[0040] RAW264.7 cells were cultured at a rate of 6 × 10⁻⁶. 5 Cells were seeded per well in 6-well plates containing cell spreaders and cultured for 24 h. The cells were then divided into a control group, an LPS group, and a wild grape stem extract group (250 μg / mL). The wild grape stem extract group was pretreated for 2 h, while the control and LPS groups were added with an equal volume of culture medium. Subsequently, the control group continued to receive culture medium, while the LPS and wild grape stem extract groups were stimulated with LPS (final concentration 1 μg / mL) for 24 h. After treatment, the culture medium was discarded, and the cells were washed three times with PBS (5 min each time), fixed with 4% paraformaldehyde for 15 min, washed three more times with PBS, and then blocked with 5% goat serum at room temperature for 1 h. After discarding the blocking solution, NF-κB p65 primary antibody was added, and the cells were incubated overnight at 4 °C. The next day, the cells were washed three times with PBS, and then incubated with fluorescently labeled secondary antibody (1:500) in the dark for 1.5 h. The cells were then washed five times with PBS, stained with DAPI in the dark for 5 min, and washed five more times. Finally, the slides were mounted with mounting medium containing an anti-fluorescence quencher, and the intracellular localization and nuclear translocation of NF-κB p65 were observed under a laser confocal microscope.

[0041] Western blot and immunofluorescence techniques were used to detect the effect of wild grape stem ethanol extract on LPS-induced NF-κB signaling pathway activation, and to observe IκBα phosphorylation and NF-κB p65 nuclear translocation. Figure 5 Western blot results showed ( Figure 5 A) Compared with the control group, LPS treatment significantly increased IκBα phosphorylation level, while pretreatment with ethanol extract decreased IκBα phosphorylation level in a dose-dependent manner. Nuclear / cytoplasmic protein separation results showed ( Figure 5 B), LPS treatment increased p65 expression in the nucleus and decreased p65 expression in the cytoplasm; pretreatment with the ethanol extract decreased p65 expression in the nucleus, and p65 levels in the cytoplasm were concentration-dependent. Immunofluorescence results showed ( Figure 5 (C) Under unstimulated conditions, p65 signaling is mainly distributed in the cytoplasm, with weak intranuclear fluorescence. After LPS stimulation, p65 fluorescence significantly accumulates in the nucleus, with increased intranuclear fluorescence intensity and relatively weakened cytoplasmic signal. Pretreatment with *Vitis vinifera* stem ethanol extract (250 μg / mL) significantly reduced LPS-induced intranuclear fluorescence, while cytoplasmic signaling was relatively abundant, indicating weakened overall nuclear translocation. In conclusion, *Vitis vinifera* stem ethanol extract inhibits IκBα phosphorylation and degradation, thereby blocking NF-κB p65 nuclear translocation and ultimately inhibiting the activation of the NF-κB signaling pathway.

[0042] Pharmacological Example 5: Wild grape stem alcohol extract inhibited LPS-induced activation of the MAPK signaling pathway.

[0043] RAW264.7 cells were cultured at a rate of 6 × 10⁻⁶. 5 Cells were seeded per well in 6-well plates and cultured for 24 h. Cells were then pretreated with different concentrations of wild grape stem ethanol extract (0, 62.5, 125, and 250 μg / mL) for 2 h, followed by stimulation with LPS (1 μg / mL) for 24 h. After cell collection, total protein was extracted using RIPA lysis buffer, and protein concentration was determined using an enhanced BCA protein quantification kit. 20 μg of protein from each sample was separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk powder for 1 h, incubated with primary antibody overnight at 4 °C, washed three times with TBST, and then incubated with HRP-labeled secondary antibody at room temperature for 1 h. Protein bands were imaged using a ChemiDocTouch chemiluminescent gel imaging system (Bio-Rad, CA, USA), and grayscale analysis was performed using Image Lab 6.0 software.

[0044] Western blot was used to detect the protein expression levels of p38, p-p38, ERK, p-ERK, JNK, and p-JNK. Figure 6Compared with the blank control group, LPS stimulation alone significantly increased the phosphorylation levels of p38, ERK, and JNK, while the expression of their corresponding total proteins remained unchanged. Pretreatment with wild grape stem ethanol extract significantly reduced LPS-induced phosphorylation levels of p38, ERK, and JNK in a dose-dependent manner. Among different dose groups, the medium and high dose groups showed more significant inhibitory effects on the phosphorylation of key proteins in the MAPK pathway, with the high dose group exhibiting the most significant effect. These results indicate that wild grape stem ethanol extract can effectively inhibit LPS-induced phosphorylation of MAPKs (p38, ERK, and JNK), thereby inhibiting the activation of the MAPK pathway.

[0045] Pharmacological Example 6: Wild grape stem alcohol extract improved DSS-induced colitis symptoms in mice.

[0046] Effects of wild grape stem ethanol extract on body weight changes and DAI in DSS-induced colitis mice. Animal experiments were approved by the Experimental Animal Ethics Committee of Guizhou University (Ethics No.: EAF-GZU-2024-E024). Male C57BL / 6 mice were acclimatized under SPF conditions for 7 days and then randomly divided into four groups (n=10 per group): a blank control group (Control), a model group (Model), a positive control group (5-ASA, 200 mg / kg), and low-dose, medium-dose, and high-dose (400 mg / kg) wild grape stem ethanol extract groups. Starting from day 8, the model group, positive control group, and each wild grape stem ethanol extract group freely drank drinking water containing 3.5% DSS to establish an acute colitis model, while the control group drank purified water. Concurrently, each group received daily oral gavage administration of: 100, 200, and 400 mg / kg of wild grape stem ethanol extract (0.2 mL) to each dose group; mesalazine 200 mg / kg (0.2 mL) to the positive control group; and an equal volume of physiological saline (0.2 mL) to the control and model groups. Changes in body weight, stool characteristics, and hematochezia were recorded daily during the experiment, and scores were calculated according to the Disease Activity Index (DAI) scoring criteria.

[0047] The effect of wild grape stem alcohol extract on DSS-induced changes in mouse body weight is shown in the figure. Figure 7A. Except for the blank control group, the body weight of mice in all groups showed a decreasing trend with the extension of DSS treatment time. By day 7, the body weight of the model group decreased to 18.10 ± 1.05 g, significantly lower than that of the control group (25.06 ± 0.94 g, p < 0.001). After administration of wild grape stem ethanol extract (100, 200, 400 mg / kg) and mesalazine (200 mg / kg), the body weights of the groups on day 7 were 18.43 ± 0.74 g, 19.54 ± 1.10 g, 20.52 ± 0.81 g, and 20.11 ± 0.58 g, respectively. Compared with the model group, the body weight loss was improved in the 200 mg / kg, 400 mg / kg ethanol extract, and mesalazine groups (p < 0.001), with the 400 mg / kg ethanol extract level being similar to that of the mesalazine group. DAI score results are as follows. Figure 7 As shown in B. On day 7, the DAI in the model group was significantly increased (p < 0.001). Compared with the model group, the DAI in all dose groups of the alcohol extract and the mesalazine group was significantly decreased (p < 0.01).

[0048] Effects of wild grape stem alcohol extract on colon length in DSS-induced colitis mice. After drug administration, mice were anesthetized with ether, and blood was collected from their eyes into EP tubes. After standing at room temperature for 1–2 h, serum was obtained by centrifugation at 3000 rpm for 10 min and stored at −20 ℃ for later use. Subsequently, mice were euthanized by cervical dislocation, and colon tissue was quickly removed and photographed to record morphological changes and length. Some tissue was fixed in 4% paraformaldehyde for later use.

[0049] like Figure 8 As shown, there were significant differences in colon length among the groups of mice. The colon length in the blank control group was 7.92 ± 0.36 cm, while it was significantly shortened to 4.70 ± 0.54 cm in the DSS model group (p < 0.001). Compared with the model group, the colon lengths after treatment with wild grape stem ethanol extract (100, 200, 400 mg / kg) and mesalazine (5-ASA, 200 mg / kg) were 5.32 ± 0.43 cm, 6.38 ± 0.65 cm, 7.91 ± 0.34 cm, and 7.27 ± 0.32 cm, respectively (p < 0.05). Among them, the colon length in the high-dose ethanol extract group was close to that of the blank control group and higher than that in the mesalazine group.

[0050] Effects of wild grape stem ethanol extract on colonic pathological damage in DSS-induced colitis mice. Colonic tissue was fixed with 4% paraformaldehyde, followed by graded alcohol dehydration and paraffin embedding after 24 h. The paraffin-embedded tissue was then cut into 4 μm thick slices using an embedding machine and floated on a flattener in 40 °C warm water to smooth the tissue. The tissue sections were collected and baked in a 60 °C oven. After dewaxing, the sections were rinsed with distilled water and stained with hematoxylin for 5 min, followed by differentiation with differentiation solution and inverse blue solution treatment, and then rinsed with distilled water. The sections were then successively immersed in 85% and 95% ethanol for 5 min each, and stained with eosin for 5 min each. After dehydration and xylene treatment, the sections were mounted with neutral resin. Finally, the analysis was performed using microscopic observation and image acquisition.

[0051] H&E staining results showed ( Figure 9 In the blank control group, the colonic mucosa structure was intact, the crypts were regularly arranged, the epithelium was continuous, and the goblet cells were evenly distributed. In the model group, the colonic tissue showed significant damage, with disrupted epithelial structure, disordered or even absent crypts, reduced or absent goblet cells, and mucosal edema, suggesting DSS-induced acute colitis-like pathological changes. Compared with the model group, treatment with wild grape stem ethanol extract (100, 200, 400 mg / kg) and 5-ASA (200 mg / kg) reduced the degree of colonic tissue damage. In the low and medium dose ethanol extract groups, some crypt structures and goblet cells remained, while the high dose ethanol extract group and the 5-ASA group showed more significant recovery of the colonic mucosa structure, better epithelial continuity, relatively intact crypt structures, and an increased number of goblet cells.

[0052] Effects of wild grape stem ethanol extract on the release of inflammatory factors in serum and tissues of mice with DSS-induced colitis. Mouse colon tissue was collected, gently rinsed with PBS to remove contents, blotted dry, and weighed. It was then placed in a pre-chilled homogenization tube, and RIPA lysis buffer containing a protease inhibitor was added. Homogenization was performed at a tissue mass to lysis buffer volume ratio of 1:10. The homogenate was lysed at 4 ℃ for 30 min, centrifuged at 4 ℃, 12,000 × g for 15 min, and the supernatant was collected as the tissue protein extract, stored at −80 ℃ for later use. Protein concentration was determined using a BCA protein quantification kit. Working solutions were prepared according to the manufacturer's instructions. Samples and standards were added to 96-well plates, incubated at 37 ℃ for 30 min, and absorbance was measured at 562 nm. Protein concentration was calculated based on a standard curve. The secretion of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α in colon tissue protein solution and serum was detected using an ELISA kit.

[0053] like Figure 10As shown, compared with the blank control group, the levels of IL-6, IL-1β, and TNF-α in the colon tissue and serum of mice in the DSS model group were significantly increased (p < 0.05). After intervention with the wild grape stem ethanol extract, the expression of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α in tissues and serum decreased in a concentration-dependent manner (p < 0.05). The high-dose ethanol extract group (400 mg / kg) was similar to the positive control 5-ASA (200 mg / kg) in reducing multiple pro-inflammatory factors in serum and colon tissue.

Claims

1. Application of wild grape stem alcohol extract in the preparation of drugs for treating colitis.

2. The application according to claim 1, characterized in that: The wild grape stem alcohol extract was prepared by the following method: Fresh wild grape stems were washed, crushed, and 70% ethanol was added at a material-to-liquid ratio of 1:3 m / v. The mixture was refluxed for 2 hours and then filtered. The residue was extracted again using the same method. The two filtrates were combined, filtered, concentrated by rotary evaporation, and then freeze-dried to obtain the wild grape stem ethanol extract.

3. The application according to claim 1, characterized in that: The ethanol extract of wild grape stem improved the M1 polarization of RAW264.7 cells induced by lipopolysaccharide (LPS), inhibited the transcription of LPS-induced pro-inflammatory cytokines TNF-α, IL-6, and IL-1β genes, and the expression of genes and proteins of pro-inflammatory mediators iNOS and COX-2, thereby inhibiting the excessive secretion of pro-inflammatory mediators NO and PGE2 and pro-inflammatory cytokines TNF-α, IL-6, and IL-1β.

4. The application according to claim 1, characterized in that: The application of wild grape stem alcohol extract in inhibiting LPS-induced MAPK pathway activation by reducing ERK, p38 and JNK phosphorylation; and inhibiting LPS-induced NF-κB pathway activation by reducing IκBα phosphorylation and degradation and inhibiting NF-κB p65 nuclear translocation.

5. The application according to claim 1, characterized in that: Application of wild grape stem alcohol extract in the preparation of drugs that improve symptoms of acute colitis induced by dextran sulfate sodium DSS in mice, including slowing down weight loss in mice, reducing mice DAI scores, alleviating colonic tissue damage in mice, and inhibiting the levels of inflammatory factors IL-1β, IL-6 and TNF-α in tissues and serum induced by dextran sulfate.

6. The application of the wild grape stem alcohol extract according to claim 1 in the preparation of an anti-colitis drug, characterized in that, A drug made by combining an alcoholic extract of wild grape stems with a pharmaceutically acceptable carrier into tablets or capsules.

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