Application of waxberry seed alcohol extract in preparation of medicine for treating colitis

Abstract

The invention discloses application of a waxberry seed alcohol extract in preparation of a medicine for treating colitis. The waxberry seed alcohol extract inhibits the generation of LPS-induced proinflammatory mediators (NO) and proinflammatory cytokines (TNF-alpha, IL-6 and IL-1beta) under the condition of no cytotoxicity; the waxberry seed alcohol extract inhibits the expression of the protein of LPS-induced pro-inflammatory mediator enzymes (iNOS and COX-2); the waxberry seed alcohol extract not only inhibits nuclear transfer of NF-kappa B by reducing phosphorylation and degradation of I kappa B alpha, but also inhibits phosphorylation of MPAKs (ERK, p38 and JNK); animal experiments prove that the waxberry seed alcohol extract shortens mouse colon, reduces mouse DAI score, alleviates mouse colon tissue damage, reduces the content of IL-6, IL-1beta, TNF-alpha and MDA in mouse colon tissue and serum, and enhances the activity of CAT and SOD in mouse colon tissue and serum. Therefore, the waxberry seed alcohol extract has a remarkable anti-inflammatory effect in vivo and in vitro, and can be used for treating colitis.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to application of Myrica rubra seed alcohol extract in preparation of a drug for treating colitis. BACKGROUND

[0002] Ulcerative colitis (UC) refers to inflammatory lesions such as damage, swelling, ulceration, erosion and bleeding of the colonic mucosal barrier, which can cause persistent mucosal inflammation of varying degrees from the rectum to the entire colon (Gastroenterol. Rep. 2014, 2, 178-192). Currently, drugs for treating colitis mainly include several categories: immunosuppressants (tacrolimus, cyclosporine, methotrexate, 6-mercaptopurine, azathioprine), corticosteroids (prednisolone, beclometasone dipropionate), aminosalicylic acids (sulfasalazine tablets, olsalazine, mesalazine), biological agents (infliximab, adalimumab, golimumab), microbial agents (Escherichia coli), and severe patients need to undergo colonic resection surgery (Am. J. Gastroenterol. 2011, 106, 601-616; Inflammopharmacology 2022, 30, 397-434). The improvement of living standards has led more people to pay attention to intestinal health, and the development of natural products for preventing and treating colitis is of great significance and gradually becomes a research hotspot (Front. Pharmacol. 2022, 13, 806148). Compared with traditional drug therapy, natural product therapy for colitis has the advantages of high safety, few side effects and good efficacy (J. Funct. Foods 2022, 96, 105201), and its treatment mechanism may include: regulating intestinal flora (Int. J. Biol. Macromol. 2021, 166, 1035-1045), promoting mucosal healing, maintaining intestinal immune homeostasis (Food Funct 2020, 11, 10690-10699), inhibiting the NF-κB signaling pathway (Chem. Biodivers. 2021, 18, e2100130), reducing oxidative stress (J. Food Sci. 2020, 85, 2596-2607), etc. Natural products are widely sourced and abundant in resources, and are potential resources for developing natural anti-inflammatory agents and drugs for treating ulcerative colitis, and are of great significance for the treatment of colitis.

[0003] Morella rubra Lour., English name China Waxmyrtle or China Bayberry, is a evergreen tree of Myricaceae Morella Lour. and mainly distributes in Guizhou, Sichuan, Yunnan, Zhejiang, Guangxi, Fujian, Jiangsu and Taiwan of China. The seed, pulp and whole fruit of M. rubra are used in traditional Chinese medicine. The fruit can relieve thirst, aid digestion, and has the effects of diuresis, anti-sugar, anti-diarrhea and liver protection (J. Nat. Med. 2014, 68, 521-529; Molecules 2016, 21, 1148; J. Agric. Food Chem. 2011, 59, 5312-5317). The leaf and seed of M. rubra can be used to treat skin diseases, enterogastritis, sprains and fractures, and stomach pain (J. Ethnobiol. Ethnomed. 2015, 11, 32; Molecules 2016, 21, 1148). The seed of M. rubra can also be used to treat red and swollen pain, gum inflammation and sweaty feet. As an astringent, it can help to stop bleeding and promote tissue regeneration in wounds (Food Sci. Technol. 2007, (10), 75-78).

[0004] Food processing by-products (such as inedible or difficult-to-eat parts) are often discarded as food waste. According to statistics, about 864 million tons of food waste are generated annually during food production, and the disposal of food waste has caused huge economic losses and environmental pollution, which is not conducive to the sustainable development of food (Trends. Food Sci. Tech. 2023, 143, 104316; J. Environ. Manage. 2019, 233, 352-370). Plant food waste generally includes processing scraps, peels, stems, seeds, husks, bran, and residues after oil or juice extraction or protein and starch extraction. After processing into various products, M. rubra seeds are often discarded as food waste. Current research on M. rubra mainly focuses on the pulp, bark and leaves, and there is little research on the chemical composition and biological activity of the seeds. There is no related report on the anti-colitis effect of M. rubra seed ethanol extract, which may hinder its development and utilization. SUMMARY

[0005] The purpose of the present application is to provide the use of M. rubra seed ethanol extract in the preparation of a drug for treating colitis, to open up new uses of M. rubra seed ethanol extract, and to provide a new choice for the preparation of a drug for treating colitis.

[0006] The present application also finds that the alcohol extract of the waxberry seeds inhibits the production of LPS-induced proinflammatory mediators (NO) and proinflammatory cytokines (TNF-α, IL-6 and IL-1β); the alcohol extract of the waxberry seeds inhibits the protein expression of LPS-induced proinflammatory mediator enzymes (iNOS and COX-2); the related mechanism research shows that the alcohol extract of the waxberry seeds not only inhibits the nuclear transfer of NF-κB by reducing the phosphorylation and degradation of IκBα, but also inhibits the phosphorylation of MPAKs (ERK, p38 and JNK); the alcohol extract of the waxberry seeds improves the dextran sulfate sodium (DSS)-induced colonitis in mice, including inhibiting the colon shortening of the mice, reducing the DAI score of the mice, reducing the colon tissue damage of the mice, reducing the content of IL-6, IL-1β, TNF-α and MDA in the colon tissue and serum of the mice, and enhancing the CAT and SOD activities in the colon tissue and serum of the mice; and the chemical components of the alcohol extract of the waxberry seeds are identified.

[0007] The technical scheme adopted by the present application is as follows:

[0008] The alcohol extract of the waxberry seeds is prepared by the following method:

[0009] Fresh waxberry seeds are crushed, the crushed raw materials are mixed with 70% ethanol at a material-liquid ratio of 1:3-1:8 g / mL, and extracted in an extractor for 2-4 h, repeated twice, the two extraction solutions are combined, filtered, and rotary evaporated to obtain an extract, and then vacuum freeze-dried to obtain 9.35% (based on the fresh weight of the waxberry seeds) dry powder of the alcohol extract of the waxberry seeds.

[0010] The present application finds through experimental research that the alcohol extract of the waxberry seeds significantly inhibits the production of LPS-induced proinflammatory mediators (NO) and proinflammatory cytokines (TNF-α, IL-6 and IL-1β) without cytotoxicity; the alcohol extract of the waxberry seeds inhibits the protein expression of LPS-induced proinflammatory mediator enzymes (iNOS and COX-2); the related mechanism research shows that the alcohol extract of the waxberry seeds not only inhibits the nuclear transfer of NF-κB by reducing the phosphorylation and degradation of IκBα, but also inhibits the phosphorylation of MPAKs (ERK, p38 and JNK); the alcohol extract of the waxberry seeds can alleviate the acute colon inflammation symptoms caused by dextran sulfate sodium in mice, including inhibiting the colon shortening of the mice, reducing the DAI score of the mice, reducing the colon tissue damage of the mice, reducing the content of IL-6, IL-1β, TNF-α and MDA in the colon tissue and serum of the mice, and enhancing the CAT and SOD activities in the colon tissue and serum of the mice.

[0011] By adopting the above technical scheme, the present application first finds the new use of the alcohol extract of the waxberry seeds in treating inflammation-related diseases, provides a new choice for preparing a drug for treating colonitis, and has important application value in the pharmaceutical industry. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 Total ion chromatogram of the methanol extract of Myrica rubra seeds (MEE), A: positive ion mode, B: negative ion mode;

[0013] Figure 2 Cytotoxicity of the methanol extract of Myrica rubra seeds (MEE) on mouse macrophage RAW264.7 cells;

[0014] Figure 3 Inhibitory effect of the methanol extract of Myrica rubra seeds on cell morphology and proinflammatory mediators (NO) and proinflammatory cytokines (TNF-a, IL-6 and IL-1b) in LPS-induced RAW264.7 cell line;

[0015] Figure 4 Inhibitory effect of the methanol extract of Myrica rubra seeds on iNOS and COX-2 protein expression in LPS-induced RAW264.7 cells;

[0016] Figure 5 Inhibitory effect of the methanol extract of Myrica rubra seeds on LPS-induced phosphorylation of MAPAKs (p38, ERK and JNK);

[0017] Figure 6 Inhibitory effect of the methanol extract of Myrica rubra seeds on LPS-induced NF-κΒ activity;

[0018] Figure 7 Inhibitory effect of the methanol extract of Myrica rubra seeds on LPS-induced NF-κΒ p65 nuclear translocation;

[0019] Figure 8 Effect of the methanol extract of Myrica rubra seeds on improving DSS-induced colon shortening;

[0020] Figure 9 The methanol extract of Myrica rubra seeds reduced the DAI index of mice;

[0021] Figure 10 The methanol extract of Myrica rubra seeds improved DSS-induced pathological damage of the colon;

[0022] Figure 11 Inhibitory effect of the methanol extract of Myrica rubra seeds on proinflammatory mediators (NO) and proinflammatory cytokines (TNF-a, IL-6 and IL-1b) in DSS-induced mouse serum and tissues;

[0023] Figure 12 Effect of the methanol extract of Myrica rubra seeds on CAT, SOD and MDA in DSS-induced mouse serum and tissues. DETAILED DESCRIPTION

[0024] Embodiment of the application: fresh waxberry seeds (collection site: Guiyang City, Guizhou Province; identified by Professor Hu Guoxiong of Guizhou University) were crushed, the crushed raw materials were mixed with 70% ethanol at a solid-liquid ratio of 1:7, the mixture was extracted in an extractor for 2 h, repeated twice, and the two extraction solutions were combined, filtered, and rotary evaporated to obtain an extract, which was then vacuum freeze-dried to obtain 9.35% (based on the fresh weight of waxberry seeds) dry powder of waxberry seed ethanol extract, which was stored in a desiccator. Chemical composition of the waxberry seed ethanol extract of this embodiment: the chemical composition of the waxberry seed ethanol extract was detected and identified by UHPLC-Q-Orbitriap MS. The UHPLC chromatographic conditions used in this experiment are as follows:

[0025] Table 1 Liquid chromatography conditions

[0026]

[0027] The remaining gradient elution conditions, ion source parameters and mass spectrometry scanning parameters are as shown in Tables 2 to 4.

[0028] Table 2 Gradient elution conditions

[0029]

[0030] Table 3 Ion source parameter settings

[0031]

[0032]

[0033] Table 4 Mass spectrometry scanning parameter settings

[0034]

[0035] The chemical composition of the waxberry seed ethanol extract is as shown in Figure 1 and Table 5, and a total of 57 components were identified.

[0036] Table 5 Chemical composition of waxberry seed ethanol extract

[0037]

[0038]

[0039]

[0040]

[0041] Pharmacological Example 1: Toxicity of waxberry seed ethanol extract to RAW264.7 cells

[0042] RAW264.7 cells were cultured in DMEM medium containing penicillin (100 U / mL), streptomycin (100 pg / mL), fetal bovine serum (10%) and glutamine (2 mM). The cytotoxic effect of the Myrica rubra seed ethanol extract on the RAW264.7 cell line was evaluated by the MTT assay. The Myrica rubra seed ethanol extract dissolved in DMSO was serially diluted by half (the maximum final concentration of DMSO was 0.05%). After the cells (2 x 10 4 cells / well) in a 96-well plate were incubated for 24 h, the diluted Myrica rubra seed ethanol extract solution was added to a final concentration of 0, 7.8125, 15.625, 31.25, 62.5, 125 and 250 pg / mL and incubated for 24 h. After the MTT solution (10 pL, 5 mg / mL) was added and incubated for 4 h, the supernatant was removed and 150 pL of DMSO was added to dissolve the formazan crystals. The optical density at 490 nm was measured by a Varioskan Lux microplate reader (Thermo Fisher Scientific, USA).

[0043] As shown in Figure 2 , the Myrica rubra seed ethanol extract (MEE) at a concentration range of 7.8125-125 pg / mL did not exhibit significant cytotoxicity to RAW264.7 cells compared to the control (p > 0.05). Therefore, the Myrica rubra seed ethanol extract concentrations (31.25, 62.5 and 125 pg / mL) that were not cytotoxic to RAW264.7 cells were selected for subsequent testing.

[0044] Pharmacological Example 2: Myrica rubra seed ethanol extract inhibited LPS-induced RAW264.7 cell morphological changes and the release of pro-inflammatory mediators and cytokines

[0045] RAW264.7 cells were seeded in a 96-well plate (2 x 10 4 cells / well) and incubated for 24 h. After pre-treatment with fresh medium containing different doses (0, 31.25, 62.5 and 125 pg / mL) of Myrica rubra seed ethanol extract for 2 h, lipopolysaccharide (LPS, 1 pg / mL) was added and incubated for another 24 h. The changes in RAW264.7 cell morphology were recorded using a Leica DMi8 inverted microscope (Leica Microsystems, Germany). Dexamethasone (DXM, 20 pg / mL) was used as a positive control drug. After the cell supernatant was collected, the NO released by RAW264.7 cells was measured using a NO assay kit (Bi Yun Tian, Shanghai). In addition, the levels of IL-1 b, IL-6 and TNF-a were evaluated using the respective ELISA kits according to the manufacturer’s instructions.

[0046] As shown in Figure 3As shown in FIG. A, the cells of the control group were round and smooth. The RAW264.7 cells treated with LPS were larger in size and irregular in shape, while the cells of the groups treated with the Myrica rubra seed ethanol extract showed less change in morphology. As shown in FIG. B-E, compared with the control group, the induction of LPS alone significantly increased the levels of NO, TNF-a, IL-1b, and IL-6. When the cells were pretreated with MEE (31.25, 62.5, and 125 pg / mL), the release of NO, TNF-a, IL-1b, and IL-6 was significantly inhibited compared with the LPS group (a-f, p<0.05). The above results indicate that MEE inhibited the release of proinflammatory mediators and cytokines in LPS-induced RAW264.7 cells in a dose-dependent manner. Figure 3 As shown in FIG. A, the cells of the control group were round and smooth. The RAW264.7 cells treated with LPS were larger in size and irregular in shape, while the cells of the groups treated with the Myrica rubra seed ethanol extract showed less change in morphology. As shown in FIG. B-E, compared with the control group, the induction of LPS alone significantly increased the levels of NO, TNF-a, IL-1b, and IL-6. When the cells were pretreated with MEE (31.25, 62.5, and 125 pg / mL), the release of NO, TNF-a, IL-1b, and IL-6 was significantly inhibited compared with the LPS group (a-f, p<0.05). The above results indicate that MEE inhibited the release of proinflammatory mediators and cytokines in LPS-induced RAW264.7 cells in a dose-dependent manner. 5 RAW264.7 cells were seeded in 6-well plates (6 x 10

[0047] Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are key enzymes for the synthesis of NO and PGE2, respectively (J. Ethnopharmacol. 2013, 147, 208-214). To investigate whether the Myrica rubra seed ethanol extract attenuated the release of proinflammatory mediators by regulating the expression of enzymes that catalyze their synthesis, the protein expression levels of iNOS and COX-2 were detected by Western blotting. As shown in FIG. A, compared with the blank group, LPS alone significantly promoted the protein expression of iNOS and COX-2. However, after pretreatment with the Myrica rubra seed ethanol extract, the protein levels were significantly decreased in a dose-dependent manner compared with treatment with LPS alone. The results indicate that the Myrica rubra seed ethanol extract significantly inhibited the release of NO by inhibiting the expression of iNOS at the translation level. Figure 4 Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are key enzymes for the synthesis of NO and PGE2, respectively (J. Ethnopharmacol. 2013, 147, 208-214). To investigate whether the Myrica rubra seed ethanol extract attenuated the release of proinflammatory mediators by regulating the expression of enzymes that catalyze their synthesis, the protein expression levels of iNOS and COX-2 were detected by Western blotting. As shown in FIG. A, compared with the blank group, LPS alone significantly promoted the protein expression of iNOS and COX-2. However, after pretreatment with the Myrica rubra seed ethanol extract, the protein levels were significantly decreased in a dose-dependent manner compared with treatment with LPS alone. The results indicate that the Myrica rubra seed ethanol extract significantly inhibited the release of NO by inhibiting the expression of iNOS at the translation level.

[0048] Pharmacological Example 4: Myrica nucipersida seed ethanol extract inhibited the phosphorylation level of LPS-induced MAPKs

[0049] Upon LPS stimulation, mitogen-activated protein kinases (MAPKs) such as c-Jun N-terminal kinase (JNK), p38 and extracellular signal-regulated kinase (ERK) are phosphorylated and activated, subsequently modulating the expression of proinflammatory genes (TNF-a, IL-6 and IL-1 b) by affecting the activation of AP-1 transcription factor (Food Chem. Toxicol. 2021, 147, 111915). Therefore, the protein levels of p38, p-p38, ERK, p-ERK, JNK and p-JNK were measured using western blotting. Figure 5 Upon LPS stimulation alone, the phosphorylation levels of p38, ERK and JNK were significantly increased relative to the control group. Myrica nucipersida seed ethanol extract significantly inhibited the phosphorylation levels of LPS-induced p38, ERK and JNK in a dose-dependent manner. The above results indicate that Myrica nucipersida seed ethanol extract can effectively inhibit the phosphorylation of LPS-induced MAPKs (p38, ERK and JNK), thereby inhibiting the activation of the MAPKs pathway.

[0050] Pharmacological Example 5: Myrica nucipersida seed ethanol extract inhibited LPS-induced NF-κB activity

[0051] NF-κB is a transcription factor that regulates the transcription of genes for proinflammatory cytokines (TNF-a, IL-6 and IL-1 b) and proinflammatory enzymes (iNOS and COX-2) (Oncogene, 1999, 18, 6853-6866). Normally, NF-κB is a heterodimer composed of p50 and p65 subunits, which exists in an inactive form in the cytoplasm bound to an inhibitor of κB (IκB). Upon LPS stimulation, IκB is phosphorylated and rapidly degraded, then the subunits of NF-κB can freely translocate to the nucleus and activate the transcription of proinflammatory genes (Nat. Immunol. 2002, 3(1), 20-26). Therefore, the effects of Myrica nucipersida seed ethanol extract on LPS-induced nuclear translocation of NF-κB and phosphorylation and degradation of IκB a were evaluated using western blotting. As shown in Figure 5, upon LPS stimulation, the phosphorylation and degradation of IκB a were significantly increased relative to the control group. Myrica nucipersida seed ethanol extract significantly inhibited the phosphorylation and degradation of IκB a in a dose-dependent manner. Figure 6As shown, after LPS induction, the phosphorylation and degradation levels of IκBα were significantly increased; the ethanol extract of bayberry seeds significantly inhibited LPS-induced IκBα phosphorylation and degradation in a concentration-dependent manner. Furthermore, after LPS induction alone, the level of the NF-κB p65 subunit in the nucleus was significantly increased, while its level in the cytoplasm was significantly decreased, indicating that NF-κB p65 translocated to the nucleus after LPS induction. However, at a high dose of 125 μg / mL, the ethanol extract of bayberry seeds significantly upregulated the level of NF-κB p65 in the cytoplasm while downregulating the level of NF-κB p65 in the nucleus, indicating that the ethanol extract of bayberry seeds inhibited the LPS-induced nuclear translocation of p65.

[0052] Pharmacological Example 6: Bayberry seed ethanol extract inhibits LPS-induced nuclear translocation of NF-κB p65

[0053] RAW264.7 cells (6×10 5 Cells were seeded into 6-well plates with coverslips (100 μg / well) and incubated for 24 hours. Subsequently, cells were pretreated with an alcohol extract of bayberry seeds (125 μg / mL) for 2 hours and then treated with LPS (1 μg / mL) for 24 hours. After washing three times with PBS, the cells were fixed with 4% paraformaldehyde solution for 15 minutes and permeabilized with 0.3% Triton X-100 for 5 minutes. Then, the cells were blocked in 5% BSA for 1 hour, washed three times with PBS, and incubated with the primary antibody NF-κB p65 at 4°C overnight. After washing three times with PBS, a secondary antibody labeled with Alexa Fluor 488 was added and incubated for 1 hour. The cells were stained with DAPI for 5 minutes and observed using a Leica TCS SP8 laser confocal scanning microscope (Leica Microsystems, Germany).

[0054] like Figure 7 As shown, NF-κB p65 (green) in untreated RAW264.7 cells appears in the cytoplasm; however, after LPS induction, green fluorescence is mainly concentrated in the cell nucleus. After treatment with Bayberry seed ethanol extract (125 μg / mL), green fluorescence is mainly distributed in the cytoplasm, indicating that Bayberry seed ethanol extract inhibits LPS-induced NF-κB p65 nuclear translocation.

[0055] Pharmacological Example 7: Effect of Bayberry Seed Alcohol Extract on Colitis Induced by Dextran Sulfate Sodium (In Vivo Anti-inflammatory Experiment)

[0056] Male C57BL / 6 mice 40, body weight 20±2g, were randomly divided into 4 groups (10 each): pure water control group, positive control group (mesalamine 200mg / kg), model group (4% DSS), Yangmei seed alcohol extract group (400mg / kg). Continuous administration for 7d, daily record of mice body weight, stool hardness and blood stool. At the end of the test, the eye ball blood, colon photography, preparation of colon sections and HE staining.

[0057] As shown in Figure 8 , Yangmei seed alcohol extract has a significant improvement effect on the pathology of 4% DSS induced colitis mouse model, as follows: compared with the model group, Yangmei seed alcohol extract can effectively inhibit the shortening of mouse colon; as shown in Figure 9 , Yangmei seed alcohol extract reduces the DAI index of mice, and can relieve the symptoms of diarrhea, blood stool and weight loss of colitis mice; HE staining results show that Figure 10 , the colon structure of the blank group mice is complete, the morphology is clear, the colon of the model group (4% DSS) mice has serious inflammation symptoms: edema occurs in the upper layer of the mucosa, neutrophils in the mucosa appear serious infiltration, cup-shaped cells and U-shaped crypt structure deformation, after Yangmei seed alcohol extract treatment, the pathological damage of mouse colon is effectively relieved; in addition, as shown in Figure 11 , Yangmei seed alcohol extract (400mg / kg) can inhibit the production of inflammatory factors (IL-6, IL-1β and TNF-α) in the serum and tissue of colitis mice, and the inhibition is equivalent to or better than the positive control mesalamine (200mg / kg) (p<0.01); on the other hand, as shown in Figure 12 , Yangmei seed alcohol extract can effectively improve the activity of catalase (CAT) and superoxide dismutase (SOD) in the serum and tissue of mice, and has a significant inhibitory effect on the production of malondialdehyde (MDA). Therefore, Yangmei seed alcohol extract can effectively relieve DSS induced colitis in mice, and has a significant anti-inflammatory effect in vivo.

[0058] In summary, the myrica rubra seed ethanol extract inhibited the production of proinflammatory mediators (NO) and proinflammatory cytokines (TNF-α, IL-6 and IL-1β) induced by LPS at non-toxic concentrations; the myrica rubra seed ethanol extract inhibited the protein expression of LPS-induced proinflammatory mediators enzymes (iNOS and COX-2); the related mechanism studies showed that the myrica rubra seed ethanol extract not only inhibited the nuclear transfer of NF-κB by reducing the phosphorylation and degradation of IκBα, but also inhibited the phosphorylation of MPAKs (ERK, p38 and JNK); improved dextran sulfate sodium-induced colitis, including inhibiting the shortening of the colon, reducing the DAI score, reducing the damage of the colon tissue, reducing the content of IL-6, IL-1β, TNF-α and MDA in the colon tissue and serum of mice and enhancing the activity of CAT and SOD in the colon tissue and serum of mice. Therefore, the myrica rubra seed ethanol extract has a significant anti-inflammatory effect in vivo and in vitro, and can be used for the treatment of colitis.

Claims

1. Use of the alcohol extract of Myrica rubra seeds in the preparation of a drug for treating colitis.

2. Use according to claim 1, characterized in that: The alcohol extract of Myrica rubra seeds is prepared by the following method: Fresh Myrica rubra seeds are crushed, and the crushed raw material is mixed with 70% ethanol at a ratio of 1:3-1:8 (g / mL) and extracted in an extractor for 2-4 h, repeated twice, and the two extraction solutions are combined, filtered, and rotary evaporated to obtain an extract, which is then vacuum freeze-dried to obtain the alcohol extract of Myrica rubra seeds.

3. Use according to claim 1, characterized in that: The alcohol extract of Myrica rubra seeds is used in the preparation of a drug for improving the over-secretion of pro-inflammatory mediators NO and pro-inflammatory cytokines TNF-α, IL-6 and IL-1β in LPS-induced RAW264.7 cells.

4. Use according to claim 1, characterized in that: The alcohol extract of Myrica rubra seeds is used in the preparation of a drug for inhibiting the LPS-activated MAPK and NF-κB pathways.

5. Use according to claim 4, characterized in that: The alcohol extract of Myrica rubra seeds inhibits the activation of LPS-induced MAPKs by inhibiting the phosphorylation of ERK, p38 and JNK, and inhibits the LPS-induced activation of NF-κB by blocking the nuclear translocation of NF-κB and the phosphorylation and degradation of IκBα.

6. Use according to claim 1, characterized in that: The alcohol extract of Myrica rubra seeds can alleviate the symptoms of acute colitis in mice caused by dextran sulfate sodium, including inhibiting the shortening of the colon, reducing the DAI score, reducing the damage to the colon tissue, reducing the content of IL-6, IL-1β, TNF-α and MDA in the colon tissue and serum, and enhancing the activities of CAT and SOD in the colon tissue and serum.

7. The use of the alcohol extract of the Myrica rubra seed according to claim 1 in the preparation of a drug for treating colitis, characterized in that, The alcohol extract of Myrica rubra seeds and a pharmaceutically acceptable carrier are formulated into tablets, capsules, and ointment dosage forms.

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