Application of prunus tomentosa seed alcohol extract in preparation of medicine for treating colitis

Abstract

The invention discloses an application of a prunus tomentosa seed alcohol extract in preparation of an anti-colitis medicine. In the invention, the prunus tomentosa seed alcohol extract with the function of resisting colitis is obtained by reflux extraction of prunus tomentosa seeds with 70% ethanol, and has a remarkable inhibition effect on dextran sodium sulfate induced mouse colitis; wherein the weight loss of the mouse is slowed down, the DAI score of the mouse is reduced, the injury of the colon tissue of the mouse is relieved, the content of IL-6, IL-1beta, TNF-alpha and MDA in the colon tissue and serum of the mouse is reduced, and the activity of CAT and SOD in the colon tissue and serum of the mouse is enhanced. The prunus tomentosa seed alcohol extract inhibits the activation of ROS-mediated MAPKs and NF-kappa B pathways, and further inhibits the excessive secretion of proinflammatory mediators NO and PGE2 and proinflammatory cytokines TNF-alpha, IL-6 and IL-1beta in lipopolysaccharide-induced RAW264.7 cells. Therefore, the prunus tomentosa 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 present invention belongs to the field of biomedicine, and specifically relates to the application of the ethanol extract of Prunus tomentosa seeds in the preparation of a drug for treating colitis. Background Art

[0002] Inflammatory bowel disease (IBD) includes Crohn's disease and ulcerative colitis. It is a complex disease caused by viral or bacterial infections, ischemic injury, or autoimmune diseases, and the symptoms include rectal bleeding, abdominal pain, diarrhea, abdominal distension, malabsorption, bloody stools, and mucus in stools (Am. J. Physiology - Gastr. L. 2005, 288, G1328–G1338; Jove - J. Vis. Exp. 2010(35), e1652). In recent years, the incidence and prevalence of IBD have been increasing, and more and more people have begun to pay attention to the treatment methods and pathogenesis of IBD. Compared with traditional drug treatments, the treatment of colitis with natural products has the advantages of high safety, few side effects, and good efficacy (J. Funct. Foods 2022, 96, 105201). Natural products are widely sourced and rich in resources, and are potential resources for developing natural anti - inflammatory agents and drugs for treating ulcerative colitis, which is of great significance for the treatment of colitis.

[0003] Prunus tomentosa Thunb. belongs to the genus Prunus of the Rosaceae family and has good soil and water conservation functions and high economic value (Forests. 2022, 13(3):381). Prunus tomentosa is a common tree species that can be used as both medicine and food in the western and northern regions of China and is commonly planted as an ornamental plant in the north (Acta Phytopathologica Sinica, 2025, 1 - 4; Molecular Plant Breeding, 2025, 1 - 12). Prunus tomentosa is widely distributed in various provinces (Rural Science & Technology, 2022, (03):62 - 64) and is rich in nutritional value, containing nutrients such as amino acids, vitamins, and minerals (Brewing Industry, 2024, 51(02):136 - 137). In addition, Prunus tomentosa has important medicinal value and is a traditional Chinese medicine. The main active ingredient is amygdalin, which has the effects of tonifying qi, strengthening the spleen, treating food retention and diarrhea, treating beriberi, spermatorrhea, and constipation (J. Chromatogr. Sci. 2005, 43(7):383 - 387; China Medical University, 2024); the dried roots, leaves, and seeds of Prunus tomentosa are used in traditional Chinese medicine for removing dampness, dispelling wind, relieving pain, and treating frostbite (Ind. Crop. Prod. 2018, 111:590 - 596).

[0004] As a fruit, cherry is usually eaten fresh. In folk culture, cherry is not only made into jam, juice and candied fruit, but also often used to make fruit wine, thus generating a large amount of cherry seed waste. However, cherry seeds have important medicinal value. Cherry seeds are also called Prunus mume seeds, which have the effect of moistening the intestines and promoting diuresis. They are flat in nature, bitter and sweet in taste, and have the effects of moistening the lungs and smoothing the intestines, promoting qi and promoting diuresis, and can treat large intestinal qi stagnation, abdominal distension and constipation, abdominal edema, quadriplegia and inflammatory diseases (Journal of Beihua University, 2010, 11(05):408-411). Therefore, it is urgent to develop and utilize cherry seeds. Summary of the invention

[0005] The purpose of the present invention is to provide the use of the alcohol extract of cherry seeds in the preparation of anti-colitis drugs, to explore new uses of the alcohol extract of cherry seeds, and to provide a new choice for the preparation of anti-colitis drugs.

[0006] The present invention also found that the alcohol extract of cherry seeds (EE) can alleviate the symptoms of acute colitis in mice caused by dextran sulfate sodium (DSS), including slowing down the weight loss of mice, reducing the DAI score of mice, reducing the damage of colon tissue of mice, down-regulating 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. The chemical components of the alcohol extract of cherry seeds were identified. The alcohol extract of cherry seeds can inhibit ROS-mediated activation of MAPKs (by inhibiting the phosphorylation of ERK, p38 and JNK) and NF-κB pathways (by blocking the nuclear translocation of NF-κB and the phosphorylation and degradation of IκBα), inhibit the transcription of LPS-induced proinflammatory cytokine (TNF-α, IL-6 and IL-1β) genes and the expression of genes and proteins that synthesize proinflammatory mediator enzymes (iNOS and COX-2); thereby inhibiting the excessive secretion of proinflammatory mediators (NO and PGE2) and proinflammatory cytokines (TNF-α, IL-6 and IL-1β) in RAW264.7 cells induced by lipopolysaccharide (LPS).

[0007] The technical solution adopted by the present invention is as follows:

[0008] The alcohol extract of cherry seeds was prepared by the following method:

[0009] Fresh cherry seeds were crushed, and the crushed raw materials were mixed with 70% ethanol at a solid-liquid ratio of 1:3 to 1:8, with the unit of g / mL, and extracted in an extractor for 2 to 4 hours, and the extraction was repeated twice. The two extracts were combined, filtered, and rotary evaporated to obtain an extract, which was then vacuum freeze-dried to obtain an alcohol extract of cherry seeds.

[0010] Through experimental research, the present invention has found that the ethanol extract (EE) of Prunus tomentosa Thunb. seeds can alleviate the symptoms of acute colitis in mice caused by dextran sulfate sodium (DSS), including slowing down the weight loss of mice, reducing the DAI score of mice, alleviating the damage of mouse colon tissue, downregulating the contents of IL-6, IL-1β, TNF-α and MDA in mouse colon tissue and serum, and enhancing the activities of CAT and SOD in mouse colon tissue and serum. And the chemical components of the ethanol extract of Prunus tomentosa Thunb. seeds were identified. The ethanol extract of Prunus tomentosa Thunb. seeds can inhibit the activation of ROS-mediated MAPKs (by inhibiting the phosphorylation of ERK, p38 and JNK) and NF-κB pathway (by blocking the nuclear translocation of NF-κB and the phosphorylation and degradation of IκBα), inhibit the transcription of genes of LPS-induced pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) and the expression of genes and proteins of pro-inflammatory mediator enzymes (iNOS and COX-2); and further inhibit the excessive secretion of pro-inflammatory mediators (NO and PGE2) and pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) in LPS-induced RAW264.7 cells.

[0011] By adopting the above technical scheme, the present invention has first discovered the new use of the ethanol extract of Prunus tomentosa Thunb. seeds in the treatment of colitis, provided a new option for the preparation of anti-colitis drugs, and has important application value in the pharmaceutical industry. In the present invention, the ethanol extract of Prunus tomentosa Thunb. seeds with anti-colitis is obtained by extracting Prunus tomentosa Thunb. seeds with 70% ethanol, and has a significant inhibitory effect on dextran sulfate sodium (DSS)-induced colitis in mice. The ethanol extract of Prunus tomentosa Thunb. seeds has significant anti-inflammatory effects both in vivo and in vitro and can be used to treat colitis. Brief Description of the Drawings

[0012] Figure 1 is the total ion chromatogram of the ethanol extract of Prunus tomentosa Thunb. seeds (A: EE positive ion mode; B: EE negative ion mode);

[0013] Figure 2 is the effect of the ethanol extract of Prunus tomentosa Thunb. seeds on the body weight (A) and DAI score (B) of mice;

[0014] Figure 3 is the effect of the ethanol extract of Prunus tomentosa Thunb. seeds on the colon length;

[0015] Figure 4 is the H&E staining image of mouse colon tissue;

[0016] Figure 5 is the effect of the ethanol extract of Prunus tomentosa Thunb. seeds on the contents of IL-6, IL-1β and TNF-α in colon tissue fluid and serum;

[0017] Figure 6Effect of ethanol extract of Prunus tomentosa Thunb. seeds on the activities of CAT and SOD and the content of MDA in colon tissue fluid and serum.

[0018] Figure 7 Cytotoxicity of ethanol extract (EE) of Prunus tomentosa Thunb. seeds against mouse macrophage RAW264.7;

[0019] Figure 8 Inhibitory effects of ethanol extract of Prunus tomentosa Thunb. seeds on the cell morphology of LPS-induced RAW264.7 cells and on pro-inflammatory mediators (NO and PGE2) and pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β);

[0020] Figure 9 Inhibitory effect of ethanol extract of Prunus tomentosa Thunb. seeds on the release of ROS in LPS-induced RAW264.7 cells;

[0021] Figure 10 Inhibitory effects of ethanol extract of Prunus tomentosa Thunb. seeds on the mRNA expressions of COX-2, TNF-α, iNOS, IL-6 and IL-1β in LPS-induced RAW264.7 cells;

[0022] Figure 11 Inhibitory effects of ethanol extract of Prunus tomentosa Thunb. seeds on the protein expressions of iNOS and COX-2 in LPS-induced RAW264.7 cells;

[0023] Figure 12 Inhibitory effects of ethanol extract of Prunus tomentosa Thunb. seeds on the phosphorylation of MPAKs (p38, ERK and JNK) induced by LPS;

[0024] Figure 13 Inhibitory effect of ethanol extract of Prunus tomentosa Thunb. seeds on LPS-induced NF-κB activity;

[0025] Figure 14 Inhibitory effect of ethanol extract of Prunus tomentosa Thunb. seeds on LPS-induced nuclear translocation of NF-κB p65. Detailed implementation manners

[0026] Examples of the present invention: Fresh Prunus tomentosa Thunb. seeds (harvesting place: Hezhang County, Bijie City, Guizhou Province; identified by Professor Hu Guoxiong of Guizhou University) were crushed. The crushed raw materials were mixed with 70% ethanol at a material-liquid ratio of 1:7. The mixed materials were extracted in an extractor for 2 h and the extraction was repeated twice. The two extraction liquids were combined, filtered, and rotary evaporated to obtain an extract, which was then freeze-dried under vacuum to obtain a dry powder of ethanol extract of Prunus tomentosa Thunb. seeds at 0.95% (based on the fresh weight of Prunus tomentosa Thunb. seeds), and stored in a desiccator. Chemical components of the ethanol extract of Prunus tomentosa Thunb. seeds: The chemical components of the ethanol extract of Prunus tomentosa Thunb. seeds were detected and identified by UHPLC-Q-Orbitriap MS. The UHPLC chromatographic conditions used in this experiment are as follows in the table:

[0027] Table 1 Liquid Chromatography Conditions

[0028]

[0029] The remaining gradient elution conditions, ion source parameters, and mass spectrometry scan parameters are shown in Tables 2 to 4 specifically.

[0030] Table 2 Gradient Elution Conditions

[0031]

[0032]

[0033] Table 3 Ion Source Parameter Settings

[0034]

[0035] Table 4 Mass Spectrometry Scan Parameter Settings

[0036]

[0037]

[0038] The chemical constituents of the ethanol extract from Prunus tomentosa Thunb. seeds are as Figure 1 (A: EE positive ion mode; B: EE negative ion mode) and shown in Table 5. A total of 42 components were identified.

[0039] Table 5 Chemical Constituents of the Ethanol Extract from Prunus tomentosa Thunb. Seeds

[0040]

[0041]

[0042]

[0043] Pharmacological Example 1: Effect of the Ethanol Extract from Prunus tomentosa Thunb. Seeds on Dextran Sulfate Sodium (DSS)-induced Colitis in Mice (In Vivo Anti-inflammatory Experiment)

[0044] Male C57BL / 6 mice (20 ± 2 g, 6 - 8 weeks old) were divided into four groups: model group, blank group, positive group, and administration group, with 10 male mice in each group. After 7 days of stabilization, the experiment was conducted:

[0045] DSS group: Freely drank 4% DSS solution and was intragastrically administered normal saline (0.3 mL) daily;

[0046] Blank group: Freely drank pure water and was intragastrically administered normal saline (0.3 mL) daily;

[0047] Positive group: Freely drank 4% DSS solution and was intragastrically administered with mesalazine solution (0.3 mL, 200 mg / kg) daily.

[0048] Administration group: Freely drank 4% DSS solution and was intragastrically administered with the ethanol extract solution of Prunus tomentosa Thunb. seeds (0.3 mL, 400 mg / kg) daily.

[0049] The experimental period was 7 days. The mice were allowed to eat freely. The body weight changes of the mice, the hardness and softness of the feces, and the degree of bloody stool were recorded daily to obtain the disease activity index (DAI). After being sacrificed under ether anesthesia, blood was collected from the eyeballs, and the mice were dissected and the colon part was taken out. Finally, the colon length of the mice was measured, and the histopathological sections of the colon tissue of the mice stained with H&E were observed, and the inflammatory factors (IL-6, IL-1β, and TNF-α) and antioxidant parameters (CAT, SOD, and MDA) in the colon tissue and blood samples of the mice were measured.

[0050] As Figure 2 shown, intragastric administration of the ethanol extract (EE) of Prunus tomentosa Thunb. seeds could reduce the degree of body weight loss and the increase in DAI score induced by dextran sulfate sodium (DSS) in mice. Furthermore, it could be known that EE could relieve the symptoms of acute colitis. As Figure 3 shown, compared with the blank group, the colon length of the mice in the model group was significantly reduced (p < 0.05). After intragastric treatment with EE (400 mg / kg), the colon length of the mice was significantly increased compared with the model group (p < 0.05), and its effect was equivalent to that of mesalazine (200 mg / kg) in the positive group. As Figure 4 shown, the colon tissue of normal mice was intact. Affected by dextran sulfate sodium (DSS), severe colon damage occurred in the model group, with abnormal changes in the morphology of epithelial cells, disappearance of the crypt structure, disappearance of goblet cells, and morphological changes of inflammatory infiltration. After treatment with EE, the colon damage caused by dextran sulfate sodium (DSS) was significantly reduced. Figure 5 and 6 shown, compared with the model group, treatment with EE could effectively reduce the contents of IL-6, IL-1β, TNF-α, and MDA in the colon tissue and serum of mice and enhance the activities of CAT and SOD. Thus, it could be known that EE could effectively relieve acute colitis in mice induced by dextran sulfate sodium (DSS).

[0051] Pharmacological example 2: Toxicity of the ethanol extract of Prunus tomentosa Thunb. seeds to RAW264.7 cells

[0052] RAW264.7 cells were cultured in DMEM medium containing penicillin (100 U / mL), streptomycin (100 μg / mL), fetal bovine serum (10%), and glutamine (2 mM). The cytotoxic effect of the ethanol extract of Prunus tomentosa Thunb. seeds on the RAW264.7 cell line was evaluated by the MTT assay. The ethanol extract of Prunus tomentosa Thunb. seeds dissolved in DMSO was serially diluted two-fold with the medium (the maximum final concentration of DMSO was 0.05%). After the cells (2×10 4 cells / well) in the 96-well plates were cultured for 24 h, the diluted ethanol extract solution of Prunus tomentosa Thunb. seeds was added to a final concentration of 0, 15.625, 31.25, 62.5, 125, 250, and 500 μg / mL, and then cultured for 24 h. MTT solution (10 μL, 5 mg / mL) was added. After incubation for 4 h, the supernatant was removed, and 150 μL DMSO was added to dissolve the formazan crystals. The optical density at 490 nm was measured by a VarioskanLux microplate reader (Thermo Fisher Scientific, USA).

[0053] As Figure 7 shown, compared with the untreated group, EE did not show cytotoxicity to RAW264.7 cells at 0 - 250 μg / mL. When the concentration was greater than 250 μg / mL, compared with the untreated group, the cell viability of the EE group was significantly decreased (p < 0.001), and it was toxic to the cells. Therefore, the non-toxic doses of 62.5, 125, and 250 μg / mL were selected for subsequent experiments.

[0054] Pharmacological Example 3: The ethanol extract of Prunus tomentosa Thunb. seeds inhibited the morphological changes and the release of pro-inflammatory mediators and cytokines in LPS-induced RAW264.7 cells

[0055] RAW264.7 cells were seeded in 96-well plates (2×10 4 cells / well) and cultured for 24 h. After pretreatment with fresh medium containing different doses (0, 62.5, 125, and 250 μg / mL) of the ethanol extract of Prunus tomentosa Thunb. seeds for 2 h, lipopolysaccharide (LPS, 1 μg / mL) was added and incubated for another 24 h. The morphological changes of RAW264.7 cells were recorded using a Leica DMi8 inverted microscope (Leica Microsystems, Germany). Dexamethasone (DXM, 20 μg / mL) was used as a positive control drug. After collecting the cell supernatant, the NO released by RAW264.7 cells was measured using a NO detection kit (Beyotime, Shanghai). In addition, according to the manufacturer's instructions, the levels of PGE2, IL-1β, IL-6, and TNF-α were evaluated using their respective ELISA kits.

[0056] As Figure 8As shown in Figure A, the cells in the control group were round with a smooth surface. The volume of RAW264.7 cells treated with LPS became larger and their shape became irregular, while the cell morphological changes in the ethanol extract (EE) of Prunus tomentosa Thunb. seed-treated group were smaller. As Figure 8 As shown in Figures B - F, compared with the control group, the induction by LPS alone significantly increased the levels of NO, PGE2, TNF-α, IL-1β, and IL-6. When the cells were pretreated with EE (62.5, 125, and 250 μg / mL), the release of NO, PGE2, TNF-α, IL-1β, and IL-6 was significantly inhibited compared with the LPS group (p < 0.05). In particular, the inhibitory effect of EE at a dose of 250 μg / mL on NO, PGE2, TNF-α, IL-6, and IL-1β was better than that of DXM (20 μg / mL). The above results indicate that EE inhibits the release of pro-inflammatory mediators and cytokines in LPS-induced RAW264.7 cells in a dose-dependent manner.

[0057] Pharmacological Example 4: EE of Prunus tomentosa Thunb. seeds inhibits the release of ROS in LPS-induced RAW264.7 cells

[0058] A blank group, an LPS group, and a sample group were set up. 1 mL of RAW264.7 cells (6×10 5 cells / mL) were inoculated into a 6-well plate and incubated in a CO2 incubator at 37 °C for 24 h. After that, the old supernatant was aspirated and discarded. 1 mL of DMEM medium was added to the blank group and the LPS group, and the prepared sample solution was added to the sample group. Then, they were continued to be incubated in a CO2 incubator at 37 °C for 2 h. After 2 h of incubation, the original supernatant was retained. 1 mL of DMEM medium was added to the blank group, and 1 mL of LPS solution was added to the LPS and sample groups, so that the final sample concentrations in the sample group were 62.5, 125, and 250 μg / mL, and the final LPS concentration in the sample group and the LPS group was 1 μg / mL. They were incubated in a CO2 incubator at 37 °C for 24 h. After 24 h, the old liquid was discarded, and the cells were washed twice with PBS solution for 5 min each time. Referring to the ROS detection kit, DCFH-DA was diluted with DMEM medium (1:1000) to prepare the dilution solution. 700 μL of the dilution solution was added to each well and incubated at 37 °C for 20 min. Then, the old supernatant was aspirated, and the cells were washed three times with DMEM medium and photographed for record. As Figure 9 shown, there were basically no green fluorescent cells in the blank group. Compared with the blank group, the amount of green fluorescence in RAW264.7 cells in the LPS group increased significantly. Compared with the LPS group, EE (125 μg / mL and 250 μg / mL) could effectively reduce the number of cells with green fluorescence and inhibit the production of ROS.

[0059] Pharmacological Example 5: The ethanol extract of Prunus tomentosa Thunb. seeds inhibited the expression of COX-2, iNOS, TNF-α, IL-6 and IL-1β genes induced by LPS

[0060] RAW264.7 cells were seeded in 6-well plates (6×10 5 cells / well) and cultured for 24 h. Subsequently, they were pretreated with the ethanol extract of Prunus tomentosa Thunb. seeds for 2 h, and then treated with LPS (1 μg / mL) for 24 h. Total RNA of the cells was extracted using Total RNA Kit I (Omega Bio-Tek, Norcross, GA, USA), and then reverse transcribed into single-stranded cDNA according to the RT EasyTMII kit (Fujian Biological Technology, Chengdu, China). The qRT-PCR reaction was performed using the Real Time PCR EasyTM-SYBR Green I kit (Fujian Biological Technology, Chengdu, China) on a CFX ConnectTM Real-Time System (Bio-Rad, CA, USA) instrument. The primer sequences were as follows:

[0061] Table 6 Quantitative Real-time PCR primer sequences

[0062]

[0063] The qRT-PCR results were analyzed using Bio-Rad CFX Maestro 1.0 software. Using GAPDH as a reference gene, the mRNA levels of COX-2, iNOS, TNF-α, IL-6 and IL-1β were analyzed. As shown, after induction with LPS alone, compared with the blank group, the mRNA expression levels of COX-2, iNOS, TNF-α, IL-6 and IL-1β increased significantly. The ethanol extract of Prunus tomentosa Thunb. seeds significantly inhibited the mRNA expression levels of COX-2, iNOS, TNF-α, IL-6 and IL-1β induced by LPS in a dose-dependent manner. The above data indicate that the ethanol extract of Prunus tomentosa Thunb. seeds reduces the secretion of pro-inflammatory mediators and cytokines by inhibiting the expression of COX-2, iNOS, TNF-α, IL-6 and IL-1β genes induced by LPS. Figure 10

[0064] Pharmacological Example 6: The ethanol extract of Prunus tomentosa Thunb. seeds inhibited the expression of iNOS and COX-2 proteins induced by LPS

[0065] RAW264.7 cells were seeded in 6-well plates (6×10 5 ​​After culturing (at a density of [[[number of cells]]] cells / well) for 24 h, the cells were pretreated with ethanol extracts of Prunus tomentosa Thunb. seeds at different concentrations (0, 62.5, 125, and 250 μg / mL) for 2 h, and then treated with 1 μg / mL of LPS for 24 h. Subsequently, total proteins were extracted using RIPA lysis buffer. The protein concentration was quantified using an enhanced BCA protein assay kit. Proteins (20 - 40 μg) were separated by 8% SDS-PAGE electrophoresis and transferred to a PVDF membrane. After blocking with 5% skim milk for 1 h, the membrane was incubated with the primary antibody overnight at 4 °C, washed 3 times with TBST, and then exposed to the HRP-labeled secondary antibody at room temperature for 1 h. Imaging was performed using a ChemiDoc touch imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and Image Lab 6.0 (Bio-Rad, CA, USA) was used to analyze the intensity of protein bands.

[0066] Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are the key enzymes for synthesizing NO and PGE2, respectively (J. Ethnopharmacol. 2013, 147, 208–214). To investigate whether the ethanol extracts of Prunus tomentosa Thunb. seeds attenuate the release of pro-inflammatory mediators by regulating the expression of their catalytic enzymes, the protein expression levels of iNOS and COX-2 were detected by Western blotting. As Figure 11 shown, compared with the blank group, LPS stimulation alone significantly promoted the protein expression of iNOS and COX-2. However, after pretreatment with the ethanol extracts of Prunus tomentosa Thunb. seeds, the protein levels decreased significantly in a dose-dependent manner compared with treatment with LPS alone. The results indicate that the ethanol extracts of Prunus tomentosa Thunb. seeds inhibit the expression of iNOS and COX-2 at the translational level.

[0067] Pharmacological Example 7: Ethanol extracts of Prunus tomentosa Thunb. seeds inhibited the phosphorylation levels of LPS-induced MAPKs

[0068] After 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, and then regulate the expression of pro-inflammatory genes (TNF-α, IL-6, and IL-1β) by affecting the activation of AP-1 transcription factors (Food Chem. Toxicol. 2021, 147, 111915). Therefore, the protein levels of p38, p-p38, ERK, p-ERK, JNK, and p-JNK were measured by Western blotting ( Figure 12)。After stimulation with LPS alone, the phosphorylation levels of p38, ERK, and JNK were significantly increased compared to the control group. The ethanol extract of Prunus tomentosa Thunb. seeds significantly inhibited the phosphorylation levels of p38, ERK, and JNK induced by LPS in a dose-dependent manner. The above results indicate that the ethanol extract of Prunus tomentosa Thunb. seeds can effectively inhibit the phosphorylation of MAPKs (p38, ERK, and JNK) induced by LPS, thereby inhibiting the activation of the MAPK pathway. Pharmacological example 8: The ethanol extract of Prunus tomentosa Thunb. seeds inhibits LPS-induced NF-κB activity

[0069] NF-κB is a transcription factor that regulates the transcription of genes for pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) and pro-inflammatory enzymes (iNOS and COX-2) (Oncogene, 1999, 18, 6853–6866). Under normal circumstances, NF-κB is a heterodimer composed of p50 and p65 subunits and exists in an inactive form in the cytoplasm in association with the κB inhibitor (IκB). After LPS stimulation, IκB is phosphorylated and rapidly degraded, and then the subunits of NF-κB can freely translocate to the nucleus and activate the transcription of pro-inflammatory genes (Nat. Immunol. 2002, 3(1), 20–26). Therefore, Western blotting was used to evaluate the effects of the ethanol extract of Prunus tomentosa Thunb. seeds on LPS-induced nuclear translocation of NF-κB and phosphorylation and degradation of IκBα. As Figure 13 shown, after LPS induction, the phosphorylation and degradation levels of IκBα were significantly increased; the ethanol extract of Prunus tomentosa Thunb. seeds significantly inhibited the phosphorylation and degradation of IκBα induced by LPS in a concentration-dependent manner. In addition, after induction with LPS 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 after LPS induction, NF-κB p65 translocated to the nucleus. However, at doses of 62.5, 125, and 250 μg / mL of the ethanol extract of Prunus tomentosa Thunb. seeds, the level of NF-κB p65 in the cytoplasm was significantly upregulated, while the level of NF-κB p65 in the nucleus was downregulated, indicating that the ethanol extract of Prunus tomentosa Thunb. seeds inhibited LPS-induced nuclear translocation of p65.

[0070] Pharmacological example 9: The ethanol extract of Prunus tomentosa Thunb. seeds inhibits LPS-induced nuclear translocation of NF-κB p65

[0071] RAW264.7 cells (6×10 5Cells (number of cells per well) were seeded into a 6-well plate with cover slips and incubated for 24 h. Subsequently, they were pretreated with the ethanol extract of Prunus tomentosa Thunb. seeds (250 μg / mL) for 2 h, and then treated with LPS (1 μg / mL) for 24 h. After washing 3 times with PBS, they were fixed with 4% paraformaldehyde solution for 15 min and then permeabilized with 0.3% Triton X-100 for 5 min. Then, the cells were blocked in 5% BSA for 1 h, and after washing 3 times with PBS, they were incubated with the primary antibody NF-κB p65 overnight at 4 °C. After washing 3 times with PBS, Alexa Fluor 488-labeled secondary antibody was added and incubated for 1 h. After the cells were stained with DAPI for 5 min, they were observed under a Leica TCS SP8 laser confocal scanning microscope (Leica Microsystems, Germany).

[0072] As Figure 14 shown, NF-κB p65 (green) in untreated RAW264.7 cells appeared in the cytoplasm; however, after LPS induction, the green fluorescence was mainly concentrated in the nucleus. After treatment with the ethanol extract of Prunus tomentosa Thunb. seeds (250 μg / mL), the green fluorescence was mainly distributed in the cytoplasm, indicating that the ethanol extract of Prunus tomentosa Thunb. seeds inhibited the nuclear translocation of NF-κB p65 induced by LPS.

[0073] Based on the above pharmacological examples, the ethanol extract of Prunus tomentosa Thunb. seeds can relieve the symptoms of acute colitis in mice caused by dextran sulfate sodium (DSS), including slowing down the weight loss of mice, reducing the DAI score of mice, alleviating the damage of mouse colon tissue, downregulating the contents of IL-6, IL-1β, TNF-α and MDA in mouse colon tissue and serum, and enhancing the activities of CAT and SOD in mouse colon tissue and serum; the ethanol extract of Prunus tomentosa Thunb. seeds inhibited the production of pro-inflammatory mediators (NO and PGE2) and cytokines (TNF-α, IL-6 and IL-1β) induced by LPS at non-toxic concentrations; inhibited the production of ROS in LPS-induced RAW264.7 cells; the ethanol extract of Prunus tomentosa Thunb. seeds inhibited the transcription of pro-inflammatory cytokine genes and the expression of genes and proteins of pro-inflammatory mediator enzymes (iNOS and COX-2); relevant mechanism studies showed that the ethanol extract of Prunus tomentosa Thunb. seeds not only inhibited the nuclear translocation of NF-κB by reducing the phosphorylation and degradation of IκBα, but also inhibited the phosphorylation of MPAKs (ERK, p38 and JNK); therefore, the ethanol extract of Prunus tomentosa Thunb. seeds has significant anti-inflammatory effects both in vivo and in vitro and can be used to treat colitis.

Claims

1. Use of the ethanol extract of Prunus tomentosa Thunb. seeds in the preparation of a medicament for treating colitis.

2. The application according to claim 1, wherein: The ethanol extract of Prunus tomentosa Thunb. seeds is prepared by the following method: Crush fresh Prunus tomentosa Thunb. seeds, mix the crushed raw materials with 70% ethanol at a material-liquid ratio of 1:3 - 1:8, in g / mL, extract in an extractor for 2 - 4 h, repeat twice, combine the two extraction liquids, filter, rotary evaporate to obtain an extract, and then vacuum freeze-dry to obtain the ethanol extract of Prunus tomentosa Thunb. seeds.

3. The application according to claim 1, wherein: The ethanol extract of Prunus tomentosa Thunb. seeds can relieve the symptoms of acute colitis in mice caused by dextran sulfate sodium (DSS), including slowing down the weight loss of mice, reducing the DAI score of mice, alleviating the damage of mouse colon tissues, down-regulating the contents of IL-6, IL-1β, TNF-α and MDA in mouse colon tissues and serum, and increasing the activities of CAT and SOD in colon tissues and serum.

4. The application according to claim 1, characterized in that: The ethanol extract of Prunus tomentosa Thunb. seeds inhibits the activation of the ROS-mediated MAPKs and NF-κB pathways, blocks the phosphorylation of ERK, p38 and JNK, the nuclear translocation of NF-κB and the phosphorylation and degradation of IκBα by inhibiting their phosphorylation; inhibits the transcription of pro-inflammatory cytokine TNF-α, IL-6 and IL-1β genes and the expression of genes and proteins of pro-inflammatory mediator enzymes iNOS and COX-2 induced by LPS; and further inhibits the excessive secretion of pro-inflammatory mediators NO and PGE2 and pro-inflammatory cytokines TNF-α, IL-6 and IL-1β in RAW264.7 cells induced by lipopolysaccharide (LPS).

5. Use of the ethanol extract of Prunus tomentosa Thunb. seeds according to claim 1 in the preparation of a drug for treating colitis, characterized in that, The ethanol extract of Prunus tomentosa Thunb. seeds and a pharmaceutically acceptable carrier are made into tablets, capsules or ointment dosage forms of drugs.

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