Use of 4-acetylamino butyric acid and 3,4-dihydroxyphenyl acetic acid for the preparation of products for the promotion of intestinal motility

Abstract

The present application discloses a kind of 4-acetylamino butyric acid and 3,4-dihydroxyphenylacetic acid in Guizhou Kaili Hong acid soup, which can enhance intestinal microecological balance, promote intestinal motility, and improve constipation or inflammatory bowel disease. The principle is that 4-acetylamino butyric acid or 3,4-dihydroxyphenylacetic acid can significantly increase the expression of C-Kit in the intestine, and significantly inhibit the expression of macrophage inflammatory factors.

Description

Technical Field

[0001] This invention relates to the field of nutritional supplement technology, specifically to the application of 4-acetaminophen and 3,4-dihydroxyphenylacetic acid in the preparation of products that promote intestinal motility. Background Technology

[0002] Miao red sour soup is a traditional fermented food of the Miao ethnic group in Guizhou Province, made from tomatoes, red chilies, litsea cubeba, and ginger through a secondary fermentation process. The sour soup retains the original lycopene and capsaicin, while also developing organic acids such as acetic acid and lactic acid through fermentation. It is reported that the earliest sour soup was made from the tail end of brewing wine, and since then, several varieties of sour soup have emerged. Researchers used Kaili red sour soup produced in Kaili City, Qiandongnan Prefecture, Guizhou Province as experimental material, analyzing the content of various components using organic acids (lactic acid, acetic acid, citric acid) and minerals (calcium, phosphorus, iron, zinc) as indicators. The experimental results showed that lactic acid had the highest content among the organic acids in the sour soup, followed by acetic acid; calcium, phosphorus, and iron were the most abundant minerals. Comparison with other sour soup products showed that the nutritional components of Guizhou Kaili red sour soup are superior to other sour soup products.

[0003] Lycopene and capsaicin in Guizhou sour soup have cholesterol-lowering, lipid-improving, and inflammation-reducing effects, while various organic acids in it have antioxidant properties. In a rat model of non-alcoholic fatty liver disease (NAFLD), a sour soup diet can inhibit the progression of NAFLD and alter the proteome and lipidome of hepatitis cells. However, the molecular mechanism by which sour soup inhibits inflammatory progression remains unclear. Summary of the Invention

[0004] This invention was made to solve the above-mentioned problems, and its purpose is to explore the mechanism by which sour soup inhibits the progression of inflammation. Based on this, this invention provides the application of 4-acetaminophen and 3,4-dihydroxyphenylacetic acid in the preparation of products that promote intestinal motility.

[0005] In the application of 4-acetaminophen and 3,4-dihydroxyphenylacetic acid provided in this invention in the preparation of products that promote intestinal motility, the following feature may also be provided: wherein the source of 4-acetaminophen and 3,4-dihydroxyphenylacetic acid is Guizhou red sour soup.

[0006] In the application of 4-acetaminophen and 3,4-dihydroxyphenylacetic acid provided in this invention in the preparation of products that promote intestinal motility, the following characteristics may also be present: wherein the content of 4-acetaminophen is 3.04-3.41 mg / g, and the content of 3,4-dihydroxyphenylacetic acid is 1.04-1.27 mg / g.

[0007] Compared with the prior art, the present invention has the following advantages:

[0008] In this invention, 4-acetaminophen and 3,4-dihydroxyphenylacetic acid can significantly increase the expression level of C-Kit in the intestine and significantly inhibit the expression of macrophage inflammatory factors. Therefore, they can enhance the balance of intestinal microecology, promote intestinal motility, and improve constipation or inflammatory bowel disease, thus making them suitable for the preparation of products that promote intestinal motility. Attached Figure Description

[0009] Figure 1 This is a graph showing the effect of Guizhou Kaili red sour soup on intestinal motility in C57BL / 6 mice in an embodiment of the present invention. Figure 1 A is a photograph showing the effect of gavage treatment with Guizhou Kaili red sour soup on the small intestine of constipated C57BL / 6 mice; Figure 1 Figure B shows the effect of gavage treatment with Guizhou Kaili red sour soup on the small intestinal propulsion rate of constipated C57BL / 6 mice. Figure 1 Figure C shows the effect of gavage treatment with Guizhou Kaili red sour soup on fecal water content in constipated C57BL / 6 mice. Figure 1 Figure D shows the effect of gavage treatment with Guizhou Kaili red sour soup on the small intestinal propulsion rate of C57BL / 6 mice with inflammatory bowel disease; Figure 1 Figure E shows the effect of gavage treatment with Guizhou Kaili red sour soup on fecal water content in C57BL / 6 mice with inflammatory bowel disease.

[0010] Figure 2 The figure shows the effect of gavage administration of Guizhou Kaili red sour soup on the gut microbiota of constipated C57BL / 6 mice. Figure 2 A is a phylogenetic tree diagram showing the effect of Guizhou Kaili red sour soup gavage treatment on the intestinal microbiota of constipated C57BL / 6 mice; Figure 2 Figure B shows the LEFSe analysis results of the effect of gavage treatment with Guizhou Kaili red sour soup on the intestinal microbiota of constipated C57BL / 6 mice;

[0011] Figure 3 This is a graph showing the detection results of components in Guizhou Kaili Red Sour Soup in an embodiment of the present invention, wherein... Figure 3 A shows the serum mass spectrometry results of C57BL / 6 mice 2 hours after being administered Guizhou Kaili Red Sour Soup via gavage. Figure 3 B represents the molecular structural formulas of 3,4-dihydroxyphenylacetic acid and 4-acetaminophen.

[0012] Figure 4 The figure shows the effects of 4-acetaminophen and 3,4-dihydroxyphenylacetic acid on intestinal motility in C57BL / 6 mice. Figure 4Figure A shows the effects of 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) on the small intestinal propulsion rate of constipated C57BL / 6 mice. Figure 4 Figure B shows the effect of gavage treatment with 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in Kaili, Guizhou, on fecal water content in constipated C57BL / 6 mice. Figure 4 Figure C shows the effect of 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) on the small intestinal propulsion rate of C57BL / 6 mice with inflammatory bowel disease. Figure 4 Figure D shows the effects of 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) on fecal water content in C57BL / 6 mice with inflammatory bowel disease.

[0013] Figure 5 The figure shows the effects of 4-acetaminophen and 3,4-dihydroxyphenylacetic acid on C-KIT and inflammatory factors. Figure 5 A is an IHC image showing the effects of 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) on C-KIT+ cells in constipated C57BL / 6 mice. Figure 5 Figure B shows the effects of 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) on the protein expression levels of C-KIT and inflammatory factors. Figure 5 C represents the effect of 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) on the expression levels of C-KIT and inflammatory factors at the mRNA level. Figure 5 Figure D shows the effects of 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) on the mRNA expression levels of C-KIT and the inflammatory factor IL-6. Figure 5 E is a graph showing the effects of 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) on the expression levels of C-KIT and the inflammatory factor TNF-α at the mRNA level. Detailed Implementation

[0014] To facilitate understanding of the technical means, creative features, objectives, and effects of this invention, the following embodiments, in conjunction with the accompanying drawings, specifically illustrate the application of 4-acetaminophen and 3,4-dihydroxyphenylacetic acid in the preparation of products promoting intestinal motility. In the following embodiments, Guizhou Red Sour Soup is a commercially available product (purchased from Guizhou Lianghuanzhai Catering and Entertainment Management Co., Ltd.). Reagents and materials not specified were obtained through general commercial channels, and experimental operations and conditions not described were performed in accordance with conventional procedures and conditions in the art.

[0015] The inflammatory bowel disease-induced C57BL / 6 mouse model, constipation C57BL / 6 mouse model, and control C57BL / 6 mouse model of this invention were obtained through the following steps:

[0016] Inflammatory bowel disease-induced C57BL / 6 mouse model:

[0017] Experimental principle: Dextran sulfate sodium (DSS) can damage the intestinal epithelial barrier and induce inflammatory bowel disease.

[0018] a. Allow C57BL / 6 mice free access to an aqueous solution containing 1-5% DSS for 5-7 days.

[0019] b. Upon observation, symptoms such as weight loss, diarrhea, and bloody stools were observed.

[0020] c. The drug can be administered repeatedly, such as 7 days of oral administration followed by 14 days of water, to simulate chronic inflammation.

[0021] Constipation C57BL / 6 mouse model:

[0022] Experimental principle: Loperamide (Lop), an opioid receptor agonist, can inhibit intestinal peristalsis.

[0023] a. Mice were administered Lop (5 mg / kg / d) by gavage for 3 consecutive days.

[0024] b. Observation revealed symptoms such as reduced number of stool particles, hard stool, and prolonged intestinal transit time.

[0025] Control C57BL / 6 mouse model:

[0026] a. Mice were administered phosphate-buffered saline (PBS) (1x) by gavage for 3 consecutive days.

[0027] b. Upon observation, no obvious symptoms were observed.

[0028] Example 1

[0029] This embodiment examines the effect of Kaili red sour soup from Guizhou on intestinal motility.

[0030] The experimental method in this embodiment is as follows:

[0031] 1.1 Treatment with Kaili Red Sour Soup via Gavage

[0032] The original Kaili red sour soup liquid was diluted 5 times with sterile double-distilled water.

[0033] The mouse model was treated by gavage with 100 μL of diluted Kaili red sour soup (KSS) daily for 5 days.

[0034] 1.2 Determination of small intestinal propulsion rate

[0035] 1.2.1 Administration of markers via gavage

[0036] After the mice were fixed, they were administered activated charcoal suspension (0.2 mL / 10 g) by gavage according to their body weight.

[0037] Record time: Start timing from the completion of gavage.

[0038] 1.2.2 Execution and Sampling

[0039] Time point: 30 minutes after gavage.

[0040] Mice were euthanized by cervical dislocation, and the small intestine from the pylorus to the ileocecal junction was quickly removed by laparotomy and placed in ice-cold saline.

[0041] 1.2.3 Measurement Data

[0042] Gently straighten the small intestine to avoid stretching and deformation.

[0043] Measure the total length of the small intestine (Ltotal) and the distance from the front edge of the charcoal powder to the pylorus (Lcharcoal).

[0044] Calculate the small intestinal propulsion rate: Propulsion rate (%) = (Lchar / Ltotal) × 100%

[0045] 1.3 Determination of water content in mouse feces

[0046] 1.3.1 Fecal sample collection

[0047] The natural defecation method is suitable for short-term experiments: place mice alone in a clean cage (lined with filter paper), observe for 30-60 minutes, and collect fresh fecal particles.

[0048] 1.3.2 Weigh the wet weight W0

[0049] After collection, the fresh feces were quickly weighed using an electronic balance.

[0050] Record the initial wet weight W0 of each fecal pellet, in mg.

[0051] 1.3.3 Drying feces

[0052] Oven drying method: Place feces in aluminum foil or weighing dish and dry in an oven at 65°C for 6 hours until constant weight is achieved, i.e., the difference between two weighings is < 0.1 mg.

[0053] 1.3.4 Weigh the dry weight W1

[0054] Remove the dried feces, cool it to room temperature, and weigh it as dry weight W1.

[0055] 1.3.5 Calculate water content

[0056] Moisture content (%) = (W0 − W1) / W0 × 100%

[0057] Figure 1 This is a graph showing the effect of Guizhou Kaili red sour soup on intestinal motility in C57BL / 6 mice in an embodiment of the present invention. Figure 1 A is a photograph showing the effect of gavage treatment with Guizhou Kaili red sour soup on the small intestine of constipated C57BL / 6 mice; Figure 1 Figure B shows the effect of gavage treatment with Guizhou Kaili red sour soup on the small intestinal propulsion rate of constipated C57BL / 6 mice. Figure 1 Figure C shows the effect of gavage treatment with Guizhou Kaili red sour soup on fecal water content in constipated C57BL / 6 mice. Figure 1 Figure D shows the effect of gavage treatment with Guizhou Kaili red sour soup on the small intestinal propulsion rate of C57BL / 6 mice with inflammatory bowel disease; Figure 1 Figure E shows the effect of gavage administration of Guizhou Kaili red sour soup on the fecal water content of C57BL / 6 mice with inflammatory bowel disease.

[0058] like Figure 1 As shown in A and B, gavage treatment with Guizhou Kaili red sour soup significantly improved the small intestinal propulsion rate in constipated C57BL / 6 mice. Figure 1 As shown in Figure C, gavage treatment with Guizhou Kaili red sour soup significantly increased the fecal water content of a constipated C57BL / 6 mouse model. Figure 1 As shown in Figure D, gavage treatment with Guizhou Kaili red sour soup significantly improved the small intestinal propulsion rate in a C57BL / 6 mouse model of inflammatory bowel disease. Figure 1 As shown in E, gavage treatment with Guizhou Kaili red sour soup can significantly increase the fecal water content of C57BL / 6 mouse model of inflammatory bowel disease.

[0059] Experimental results showed that gavage treatment with Guizhou Kaili red sour soup significantly promoted intestinal motility in C57BL / 6 mice.

[0060] Example 2

[0061] This embodiment examines the effects of Kaili red sour soup from Guizhou on intestinal microbiota.

[0062] The experimental method in this embodiment is as follows:

[0063] 2.1 Intestinal flora detection

[0064] 2.1.1 Collect intestinal contents of mouse model

[0065] 2.1.2 DNA extraction (using the QIAamp DNA Stool Mini Kit) and detection of DNA concentration (Qubit) and purity (A260 / A280 ≈ 1.8-2.0).

[0066] 2.1.2 PCR amplification of the 16S rRNA gene

[0067] (1) Primer selection (taking the amplification of the V3-V4 region as an example):

[0068] Forward primer: 341F (5′-CCTACGGGNGGCWGCAG-3′)

[0069] Reverse primer: 805R (5′-GACTACHVGGGTATCTAATCC-3′)

[0070] (2) Prepare the reaction system (25 μL):

[0071] 2× Taq Master Mix 12.5 μL

[0072] 1 μL each of primers (10 μM)

[0073] DNA template 10-50 ng

[0074] Add ddH2O to a final volume of 25 μL.

[0075] (3) Set up the amplification program:

[0076] 95℃ for 3 min

[0077] 95℃ 30 sec, 55℃ 30 sec, 72℃ 45 sec (30 cycles)

[0078] 72℃ for 10 min

[0079] (4). PCR products were obtained after the PCR program was completed.

[0080] 2.1.3 Library Construction and Sequencing

[0081] (1) Purification of PCR products: PCR products were purified using AMPure XP magnetic beads to remove primers, unreacted nucleotides and other impurities from the PCR reaction.

[0082] (2) Add adapter and index: Use the Illumina Nextera XT Index Kit to add adapter and index sequence to the purified PCR product.

[0083] (3) Library quantification: Qubit + Agilent Bioanalyzer (detecting fragment size).

[0084] Specifically, to ensure library quality and sequencing accuracy, quantification and quality assessment were performed after library construction. The concentration of the DNA library was determined using a Qubit real-time quantitation system to ensure sufficient DNA for subsequent sequencing. Simultaneously, fragment size analysis of the library was performed using an Agilent Bioanalyzer to assess the size distribution of DNA fragments and ensure that the fragment sizes met the requirements of the sequencing platform.

[0085] (4) Sequencing platform: Illumina MiSeq / NovaSeq (paired-end 250 / 300 bp).

[0086] Specifically, the constructed libraries are sequenced using Illumina MiSeq or NovaSeq sequencing platforms. These platforms employ paired-end sequencing technology, reading sequence information from both ends of the DNA fragment, typically with a sequencing length of 250 or 300 base pairs (bp).

[0087] 2.2 Diversity Analysis

[0088] 2.2.1 Alpha diversity (within a single sample)

[0089] The gut microbiota of constipated C57BL / 6 mice treated with Guizhou Kaili red sour soup by gavage was analyzed for Chao1 richness, Shannon diversity, and Simpson dominance.

[0090] The gut microbiota of untreated constipated C57BL / 6 mice was analyzed for richness (Chao1), diversity (Shannon), and dominance (Simpson).

[0091] 2.2.2 β-diversity (differences between samples)

[0092] Based on the Bray-Curtis / UniFrac distance, the differences in gut microbiota between constipated C57BL / 6 mice treated with Guizhou Kaili red sour soup via gavage and untreated constipated C57BL / 6 mice were analyzed.

[0093] Figure 2The figure shows the effect of gavage administration of Guizhou Kaili red sour soup on the gut microbiota of constipated C57BL / 6 mice. Figure 2 A is a phylogenetic tree diagram showing the effect of Guizhou Kaili red sour soup gavage treatment on the intestinal microbiota of constipated C57BL / 6 mice; Figure 2 Figure B shows the LEFSe analysis results of the effect of gavage treatment with Guizhou Kaili red sour soup on the intestinal microbiota of constipated C57BL / 6 mice.

[0094] like Figure 2 As shown, 16sDNA sequencing results indicate that gavage treatment with Guizhou Kaili red sour soup can significantly improve the diversity of gut microbiota in a constipated C57BL / 6 mouse model and alter the abundance of various gut microbiota, meaning that Guizhou Kaili red sour soup can enhance the balance of gut microbiota.

[0095] Example 3

[0096] This embodiment examines 4-acetaminophen and 3,4-dihydroxyphenylacetic acid as substances that promote intestinal motility in Kaili Red Sour Soup from Guizhou.

[0097] The experimental method in this embodiment is as follows:

[0098] 3.1 Mass spectrometry detection of mouse plasma metabolomics

[0099] 3.1.1 Sample Collection and Preservation

[0100] Blood collection: Two hours after gavage administration of diluted Kaili red sour soup, blood was collected from the orbital region of mice, allowed to stand at room temperature for 30 minutes, and then centrifuged to obtain plasma.

[0101] 3.1.2 Metabolite Extraction

[0102] Metabolite extraction was performed by mixing methanol and plasma in a 9:1 ratio.

[0103] 3.1.3 Determination by liquid chromatography-mass spectrometry (LC-MS)

[0104] Figure 3 This is a graph showing the detection results of components in Kaili Red Sour Soup from Guizhou Province, as described in an embodiment of the present invention. Among them, Figure 3 A shows the serum mass spectrometry results of C57BL / 6 mice 2 hours after being administered Guizhou Kaili Red Sour Soup via gavage. Figure 3 B represents the molecular structural formulas of 3,4-dihydroxyphenylacetic acid and 4-acetaminophen.

[0105] like Figure 3As shown in Figure A, serum mass spectrometry results revealed significantly increased relative abundances of 4-acetaminophen and 3,4-dihydroxyphenylacetic acid, indicating that Guizhou Kaili red sour soup is rich in 4-acetaminophen and 3,4-dihydroxyphenylacetic acid, suggesting that these two substances are key components in promoting intestinal motility. Figure 3 Figure B shows the molecular structural formulas of 3,4-dihydroxyphenylacetic acid and 4-acetaminophen.

[0106] Specifically, the content of 4-acetaminophen in Guizhou red sour soup is 3.04-3.41 mg / g, and the content of 3,4-dihydroxyphenylacetic acid is 1.04-1.27 mg / g.

[0107] Example 4

[0108] This example investigates the effects of 4-acetaminophen and 3,4-dihydroxyphenylacetic acid on intestinal motility.

[0109] The experimental method in this embodiment is as follows:

[0110] 4.1 Gavage treatment with 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC)

[0111] Use 4-acetaminophen (ac-GABA) (50 mg / kg) −1 d −1 ) and 3,4-dihydroxyphenylacetic acid (DOPAC) (50 mg / kg) −1 d −1 C57BL / 6 mice with inflammatory bowel disease and C57BL / 6 mice with constipation were administered the medication by gavage for 3 consecutive days. Small intestinal propulsion rate and fecal water content were then measured. 4-Acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were purchased from MedChemExpress.

[0112] Figure 4 The figure shows the effects of 4-acetaminophen and 3,4-dihydroxyphenylacetic acid on intestinal motility in C57BL / 6 mice. Figure 4 Figure A shows the effects of 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) on the small intestinal propulsion rate of constipated C57BL / 6 mice. Figure 4 Figure B shows the effect of gavage treatment with 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in Kaili, Guizhou, on fecal water content in constipated C57BL / 6 mice. Figure 4Figure C shows the effect of 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) on the small intestinal propulsion rate of C57BL / 6 mice with inflammatory bowel disease. Figure 4 Figure D shows the effects of 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) on fecal water content in C57BL / 6 mice with inflammatory bowel disease.

[0113] like Figure 4 As shown in Figure A, both 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) can improve the small intestinal propulsion rate in a constipated C57BL / 6 mouse model. Figure 4 As shown in Figure B, both 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) significantly increased fecal water content in a constipated C57BL / 6 mouse model. Figure 4 C,4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) can both significantly improve the small intestinal propulsion rate in the C57BL / 6 mouse model of inflammatory bowel disease. Figure 4 As shown in Figure D, both 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) significantly increased fecal water content in the C57BL / 6 mouse model of inflammatory bowel disease.

[0114] Experimental results showed that both 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) could promote intestinal motility in C57BL / 6 mice.

[0115] Example 5

[0116] This embodiment investigates how 4-acetaminophen and 3,4-dihydroxyphenylacetic acid enhance the vitality of Cajal cells, the rhythmic cells of the gut, by inhibiting inflammatory activation of macrophages.

[0117] This embodiment uses an experiment to analyze mRNA expression levels in Cajal cells. The experimental method is as follows:

[0118] 5.1 Total RNA extraction from cells using the Trizol method (050403A)

[0119] 5.1.1 Instruments and Materials: High-speed / refrigerated centrifuge (Eppendorf, Germany, 5415D)

[0120] 5.1.2 Reagents:

[0121] (1) Trizol; (2) DEPC water; (3) Chloroform; (4) Isopropanol; (5) Ethanol.

[0122] 5.1.3 Experimental Procedure

[0123] (1) Cell collection:

[0124] Cajal cells were loaded at 2 × 10 6 / mL were inoculated into the culture medium. After 24 hours of cell inoculation, the cells were carefully transferred into a 15ml centrifuge tube, centrifuged at 1500 rpm for 10 minutes to precipitate the suspended cells, and the supernatant was discarded.

[0125] (2) Add 1 ml of PBS to a centrifuge tube, resuspend the cells and transfer them to a 1.5 ml tube. Centrifuge at 1500 rpm for 5 min and discard the supernatant.

[0126] (3) Discard the supernatant, add 2 ml Earle's solution, gently blow the cells with a pipette, and centrifuge at 2000 rpm for 5 min. In the following steps, RNase contamination should be avoided.

[0127] (4) Pre-cool the low-temperature centrifuge to 4°C;

[0128] (5) Add 1 ml of Trizol and gently transfer the cells and Trizol to a 1.5 ml centrifuge tube using a 1 ml pipette;

[0129] (6) Shake the centrifuge tube vigorously by hand until no white cell clumps are visible, about 50 times, and let it sit at room temperature for 10 minutes.

[0130] (7) Add 1 / 5 volume (0.2 ml) of chloroform, invert and mix 10 times, place at room temperature for 5 min, (purpose: to separate RNA, DNA and protein) centrifuge at 12000g for 15 min at 4℃.

[0131] (8) Carefully aspirate about 500 μl of the upper aqueous phase into another 1.5 ml centrifuge tube (carefully avoid aspirating impurities between the aqueous and oil phases), (9) Add an equal volume of isopropanol, invert to mix, and incubate at room temperature for 10 min.

[0132] (10) Centrifuge at 4℃, 12000g for 10min;

[0133] (11) Carefully remove the centrifuge tube, carefully observe the size and position of the precipitate, and carefully pour away the supernatant;

[0134] (12) Add 1 ml of pre-cooled 75% ethanol to wash the RNA precipitate;

[0135] (13) Carefully discard the supernatant (the precipitate will be suspended after adding ethanol), and carefully aspirate the remaining supernatant with a pipette;

[0136] (14) Open the centrifuge tube and place it on a clean bench to air dry naturally (about 10-20 minutes; avoid over-drying, which may cause it to be difficult to dissolve).

[0137] (15) Depending on the size of the precipitate, redissolve it in 30-60 μl of DEPC water (a 50℃ water bath can aid dissolution);

[0138] (16) Dispense into 3-4 tubes, with 2 μl in one tube for concentration determination;

[0139] (17) Seal the opening with sealing film and store in a low-temperature refrigerator.

[0140] 5.2 RNA Concentration Measurement

[0141] 5.2.1 Instrument: Nanodrop spectrophotometer 2000-2000C (Thermo Scientific)

[0142] 5.2.2 The specific steps for RNA concentration determination are as follows:

[0143] (1) Open the software and select to detect RNA concentration;

[0144] (2) Lift the sample arm and add DEPC water to the detection base;

[0145] (3) Lower the sample arm and click blank in the software to perform blank detection;

[0146] (4) After completing the blank calibration, lift the sample arm and wipe the DEPC on the upper and lower bases clean with clean lint-free paper. Then add the sample to the detection base, lower the sample arm, and click measure to detect the sample.

[0147] (5) When testing the next sample after testing one sample, wipe the sample on the upper and lower bases with clean lint-free paper. This will prevent the sample from being left on the base.

[0148] (6) Export the measured values ​​after the test is completed.

[0149] 5.3 Reverse transcription to generate cDNA (050406A)

[0150] 5.3.1 Instrument: PCR instrument (Eppendorf, Mastercycler personal)

[0151] 5.3.2 Reagent: PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect RealTime) (Takara catalog number: RR047)

[0152] 5.3.3 The specific steps of the reverse transcription to generate cDNA experiment are as follows:

[0153] (1) Genomic DNA removal reaction: Prepare the reaction mixture on ice according to the components shown in Table 1. To ensure the accuracy of the reaction mixture preparation, prepare the Master Mix by the amount of reaction number + 2 before each reaction, and then dispense it into each reaction tube. Finally, add the RNA sample. The amount of RNA sample added is 1 μg (the amount of RNA added depends on the RNA concentration).

[0154] Table 1. Composition of the reaction solution for removing genomic DNA

[0155]

[0156] Mix thoroughly and let stand at room temperature for 5 minutes.

[0157] (2) Reverse transcription reaction

[0158] Prepare the reaction solutions on ice according to the components shown in Table 2. To ensure the accuracy of the reaction solution preparation, prepare the Master Mix by the amount of reaction number + 2 before each reaction, and then dispense 10 μl into each reaction tube. Gently mix and immediately proceed with the reverse transcription reaction.

[0159] Table 2. Composition and conditions of the reverse transcription reaction solution

[0160]

[0161] After the reaction was complete, the mixture was placed at 4°C for real-time PCR.

[0162] 5.4 Real-Time PCR

[0163] 5.4.1 Instrument: Biosystems 7500Fast Real-Time PCR System

[0164] 5.4.2 Reagent: TB Premix Ex Taq™ (Tli RNaseH Plus) (Takara catalog number: RR420)

[0165] The primer sequences for the mouse IL-1β, TNF-α, IL-6, and GAPDH genes used in this embodiment are shown in Table 3.

[0166] Table 3 Gene Primer Sequences

[0167]

[0168] The primers mentioned above were synthesized by Shanghai Sangon Biotech and used in subsequent experiments.

[0169] 5.4.3 The experimental steps are as follows:

[0170] (1) Prepare the PCR reaction solution according to the components in Table 4 (the reaction solution is prepared on ice), and prepare a 20 μl system.

[0171] The 2× real-time quantitative PCR amplification premix solution (Hieff® qPCR SYBR Green MasterMix) used was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0172] Table 4 PCR reaction solution preparation system:

[0173]

[0174] (2) Perform Real-Time PCR reaction

[0175] Two-step PCR amplification standard procedure:

[0176] Stage 1: Pre-variation

[0177] Number of Cycles:1 95℃ 30 seconds

[0178] Stage 2: PCR reaction

[0179] Number of Cycles: 40

[0180] 95℃ for 3 seconds

[0181] 60℃ for 30 seconds

[0182] Stage 3: Melt Curve

[0183] (3) Result calculation: Real-time PCR data were analyzed using the 2-ΔΔCt method.

[0184] 5.5 Immunohistochemical staining (IHC)

[0185] Mice were euthanized and colon samples were collected and then embedded in paraffin for sectioning.

[0186] 5.5.1 Dewaxing and Hydration

[0187] (1) Soak the paraffin slices in xylene I for 10 minutes, and then soak them in xylene II for 10 minutes to remove the paraffin.

[0188] (2) Wash the sections with anhydrous ethanol for 5 min to remove xylene;

[0189] (3) Immerse the slices in 95%, 80%, and 70% ethanol in sequence, immersing for 3 minutes at each concentration, gradually reducing the ethanol concentration to help the slices adapt to the hydration process.

[0190] (4) Rinse the slices with distilled water to completely remove ethanol.

[0191] 5.5.2 Antigen retrieval

[0192] 5.5.2.1 Thermal repair method, which is relatively common, involves the following steps:

[0193] (1) Immerse the slide in antigen retrieval solution (such as 0.01M citrate buffer, pH 6.0) to ensure full contact with the retrieval solution;

[0194] (2) Place the slices in a microwave heating device, heat to boiling, and maintain for 5-10 minutes to ensure that the antigen is repaired;

[0195] (3) After heating, allow the slices to cool naturally to room temperature to ensure a stable repair process.

[0196] 5.5.2.2 Enzyme repair method: Applicable to certain specific antigens, such as trypsin digestion.

[0197] 5.5.3 Blocking endogenous peroxidase

[0198] (1) Add 3% hydrogen peroxide (H2O2) solution to the slices and incubate at room temperature for 10-15 min to inhibit the activity of endogenous peroxidase (this step is required for the horseradish peroxidase (HRP) system).

[0199] (2) Wash the slides three times with phosphate-buffered saline (PBS) for 5 minutes each time to remove excess hydrogen peroxide.

[0200] 5.5.4 Closure

[0201] Add 5-10% normal sheep serum to the slide and block at room temperature for 30 minutes to reduce non-specific binding.

[0202] 5.5.5 Primary Antibody Incubation

[0203] Add diluted primary antibody C-KIT to the slide, and incubate the slide at 4°C overnight, or at 37°C for 1-2 hours;

[0204] Rinse the slides three times with PBS for five minutes each time to remove unbound primary antibody.

[0205] 5.5.6 Secondary Antibiotic Incubation

[0206] Add HRP-labeled secondary antibody to the slide and incubate at room temperature for 30-60 minutes;

[0207] Rinse the slides three times with PBS for five minutes each time to remove unbound secondary antibody.

[0208] 5.5.7 Color Development (HRP System)

[0209] (1) DAB color development: Add DAB working solution to the slide and control the color development time under a microscope. The color development time is usually 1-5 min. Stop the reaction when a clear brown or black reaction is observed in the target area.

[0210] (2) Rinse the slices with distilled water to stop the colorimetric reaction.

[0211] 5.5.8 Counterstaining and Mounting

[0212] (1) Immerse the slices in hematoxylin solution for 1-2 min to counterstain cell nuclei;

[0213] (2) Immerse the slices in 1% hydrochloric acid alcohol;

[0214] (3) Use running water or alkaline solution to blue the cell nuclei.

[0215] (4) Dehydration and transparency: The sections are soaked in a series of ethanol solutions (70%, 80%, 95%, 100%) to gradually dehydrate them, and then defatted with xylene to make the sections transparent.

[0216] (5) Seal the film with neutral resin.

[0217] 5.5.9 Microscopic observation

[0218] The processed slices were observed and analyzed using a microscope (Leica Versa 8 system), the results were recorded, and images were acquired.

[0219] 5.6 Western Blot assays (WB)

[0220] Mice were euthanized and colon samples were collected and then embedded in paraffin for sectioning.

[0221] 5.6.1 Sample Preparation

[0222] Add RIPA buffer (containing protease inhibitors) to the sample and lyse it on ice. The lysis time is usually 30 minutes.

[0223] Centrifuge at 4℃, 12,000–14,000 rpm for 10 min, and collect the supernatant after centrifugation;

[0224] The amount of protein loaded is determined based on the measured concentration using either the BCA or Bradford method.

[0225] 5.6.2 SDS-PAGE electrophoresis

[0226] 5.6.2.1 Glue application:

[0227] Separating gel (lower layer, containing 8% acrylamide) and stacking gel (upper layer, containing 5% acrylamide).

[0228] 5.6.2.2 Sample loading:

[0229] Load 20-50 μg of protein into each well (adjust according to protein concentration), and add pre-stained protein marker to the control marker well.

[0230] 5.6.2.3 Electrophoresis:

[0231] At the start of electrophoresis, set a constant voltage of 80V (for the stacking gel). After the sample enters the separating gel, adjust the voltage to 120V until the bromophenol blue reaches the bottom of the gel.

[0232] 5.6.3 Film transfer, taking wet transfer as an example

[0233] 5.6.3.1 Membrane treatment:

[0234] Soak the PVDF membrane in methanol for 1 minute to ensure it is fully wetted, then equilibrate the PVDF membrane with transfer buffer.

[0235] NC membranes can be directly immersed without methanol treatment.

[0236] 5.6.3.2 Transfer assembly (“sandwich” structure):

[0237] Assemble the membrane transfer device in the following order: negative electrode → sponge pad → filter paper → gel → membrane → filter paper → sponge pad → positive electrode, taking care to avoid the generation of air bubbles.

[0238] 5.6.3.3 Transfer conditions: constant current 300mA, time 2.5h.

[0239] 5.6.4 Closure

[0240] After the transfer was complete, the membrane was dissolved in 5% skim milk in TBST buffer (containing Tween-20) to prepare the blocking solution. The membrane was then placed in the blocking solution and blocked at room temperature for 1 hour, with gentle shaking on a shaker throughout the blocking process.

[0241] 5.6.5 Primary Antibody Incubation

[0242] Dilute the primary antibody to the preset concentration using blocking buffer. The primary antibody concentrations are shown in Table 5. Place the membrane in the primary antibody solution and incubate overnight at 4°C on a shaker. After incubation, wash the membrane three times with TBST buffer for 10 minutes each time.

[0243] Table 5. Primary antibodies corresponding to each protein

[0244]

[0245] 5.6.6 Secondary Antibiotic Incubation

[0246] The HRP-labeled secondary antibody (purchased from CST, anti-rabbit / mouse IgG) was diluted with blocking buffer at a ratio of 1:5000. The membrane was immersed in the secondary antibody solution and incubated at room temperature for 1 hour. After incubation, the membrane was washed three times with TBST buffer for 10 minutes each time.

[0247] 5.6.7 Development (ECL method)

[0248] 5.6.7.1 ECL reaction:

[0249] Prepare the ECL chemiluminescence reagent by mixing equal volumes of solution A and solution B. Add the ECL mixture dropwise onto the membrane and allow it to react for 1 minute, allowing the proteins on the membrane to react with peroxidase and generate a detectable chemiluminescent signal.

[0250] 5.6.7.2 Imaging:

[0251] Developed using a chemiluminescence analyzer by Bio-Rad Laboratories, the exposure time is adjusted according to the signal strength to ensure the clearest signal is captured.

[0252] Figure 5 The figure shows the effects of 4-acetaminophen and 3,4-dihydroxyphenylacetic acid on C-KIT and inflammatory factors. Figure 5 A is an IHC image showing the effects of 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) on C-KIT+ cells in constipated C57BL / 6 mice. Figure 5 Figure B shows the effects of 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) on the protein expression levels of C-KIT and inflammatory factors. Figure 5 C represents the effect of 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) on the expression levels of C-KIT and inflammatory factors at the mRNA level. Figure 5 Figure D shows the effects of 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) on the mRNA expression levels of C-KIT and the inflammatory factor IL-6. Figure 5 E is a graph showing the effects of 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) on the expression levels of C-KIT and the inflammatory factor TNF-α at the mRNA level.

[0253] like Figure 5 As shown in Figure A, both 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) can increase the number of C-KIT+ cells in the intestine of constipated C57BL / 6 mice. Figure 5 As shown in Figure B, both 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) increased the expression of C-KIT in the intestines of constipated C57BL / 6 mice and decreased the expression of the inflammatory factor IL-1β. Figure 5 As shown in C to E, the results of real-time quantitative PCR showed that both 4-acetaminophen (ac-GABA) and 3,4-dihydroxyphenylacetic acid (DOPAC) could significantly reduce the expression levels of inflammatory factors IL-1β, IL-6 and TNF-α.

[0254] Experimental results showed that gavage treatment with 4-acetaminophen or 3,4-dihydroxyphenylacetic acid significantly increased the expression level of C-Kit in the intestine of constipated C57BL / 6 mouse models and significantly inhibited the expression of macrophage inflammatory factors. That is, 4-acetaminophen or 3,4-dihydroxyphenylacetic acid promotes intestinal motility in C57BL / 6 mice by enhancing the activity of Cajal cells (intestinal rhythm cells) and inhibiting macrophage inflammatory activation.

[0255] Based on the experimental results of Examples 1-5 above, it can be concluded that 4-acetaminophen and 3,4-dihydroxyphenylacetic acid, components derived from Kaili red sour soup in Guizhou, can enhance the balance of intestinal microecology, promote intestinal motility, and improve constipation or inflammatory bowel disease. The mechanism is that 4-acetaminophen or 3,4-dihydroxyphenylacetic acid can significantly increase the expression level of C-Kit in the intestine and significantly inhibit the expression of inflammatory factors in macrophages.

[0256] Based on this, 4-acetaminophen and 3,4-dihydroxyphenylacetic acid can improve constipation or inflammatory bowel disease, and can be used to prepare products that promote intestinal motility.

[0257] Those skilled in the art should understand that this invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to this invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

The application of 1,4-acetaminophen and 3,4-dihydroxyphenylacetic acid in the preparation of products promoting intestinal motility, wherein the 4-acetaminophen and 3,4-dihydroxyphenylacetic acid are derived from Guizhou red sour soup, to explore the mechanism by which sour soup inhibits the progression of inflammation. in, The 4-acetaminophen and 3,4-dihydroxyphenylacetic acid can increase the expression of C-Kit in the intestine and inhibit the expression of macrophage inflammatory factors, thereby promoting intestinal motility.

2. The application of 4-acetaminophen and 3,4-dihydroxyphenylacetic acid according to claim 1 in the preparation of products promoting intestinal motility, characterized in that: in, In the Guizhou Red Sour Soup, the content of 4-acetaminophen is 3.04-3.41 mg / g, and the content of 3,4-dihydroxyphenylacetic acid is 1.04-1.27 mg / g.

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