Application of garlic polysaccharide in preparation of medicine for preventing and / or treating inflammatory colon tumor

Abstract

The invention relates to the technical field of biological medicines, in particular to application of garlic polysaccharide in preparation of a medicine for preventing and / or inflammatory colonoma. The invention provides an application of garlic polysaccharide in preparation of a medicine for preventing and / or inflammatory colon tumor, the garlic polysaccharide is mainly formed by connecting fructose and glucose through a beta-configuration glucosidic bond, and the garlic polysaccharide can resist gastrointestinal tract enzyme in digestion simulation to be utilized by intestinal flora glycolysis, so that the total sugar content is continuously reduced. The anti-tumor activity of the garlic polysaccharide is proved by constructing an azomethane (AOM) / dextran sodium sulfate (DSS) induced inflammatory coloma mouse model; meanwhile, a basis is provided for research and development of garlic polysaccharide medicines for preventing and / or treating inflammatory colonoma.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of garlic polysaccharide in the preparation of drugs for the prevention and / or inflammatory colonoma. Background Technology

[0002] Colorectal cancer is one of the most life-threatening malignant tumors of the digestive system. Statistics show that the incidence of colorectal cancer in my country is gradually increasing, currently ranking second in incidence and fourth in mortality among malignant tumors. Inflammatory colorectal cancer is a special subtype, accounting for approximately 5% of all colorectal cancer cases. The infiltration of numerous pro-inflammatory mediators and immune cells, along with elevated levels of oxidative stress, constitutes the unique tumor microenvironment of inflammatory colorectal cancer. Currently, chemotherapy, surgery, and immunotherapy are the main means of inhibiting tumor development, but they face unavoidable adverse reactions and drug resistance. Recent studies have shown that intervention using plant-derived active substances is a highly effective treatment strategy with manageable side effects.

[0003] Garlic is a classic plant used both as food and medicine, rich in active ingredients such as polysaccharides, proteins, polyphenols, vitamins, minerals, and sulfur-containing compounds. It boasts significant nutritional value and a wide range of physiological functions, earning it the reputation of a "natural treasure trove of medicines." Polysaccharides are the most abundant active substances in garlic, primarily composed of linear or non-linear fructans. Currently, garlic products on the market are mainly food-based, such as garlic slices, garlic paste, and garlic oil obtained from the initial processing of garlic. However, cases of isolating and extracting active ingredients from garlic and applying these effective components to functional foods or pharmaceuticals are very rare.

[0004] Currently, the application of garlic as a drug against colorectal tumors mainly focuses on allicin, but allicin has a low molecular weight and unstable structure, while there is no research on garlic polysaccharides. Summary of the Invention

[0005] To address the current problems in the treatment of inflammatory colonoma, this invention provides the application of garlic polysaccharide in the preparation of drugs for the prevention and / or treatment of inflammatory colonoma. Garlic polysaccharide is mainly composed of fructose and glucose linked by β-glycosidic bonds. In digestion simulations, it can resist gastrointestinal enzymes and be fermented and utilized by intestinal flora, leading to a continuous decrease in total sugar content. The antitumor activity of garlic polysaccharide was demonstrated by constructing an azomethane (AOM) / dextrose sulfate (DSS)-induced inflammatory colonoma mouse model; simultaneously, it provides a basis for the development of garlic polysaccharide drugs for the prevention and / or treatment of inflammatory colonoma.

[0006] The technical solution of this application is as follows: Application of garlic polysaccharides in the preparation of drugs for the prevention and / or treatment of inflammatory colonomas.

[0007] Total sugar and reducing sugar analysis showed that the reducing sugar content of garlic polysaccharide increased slightly during the gastric and intestinal digestion simulation stages, while the total sugar content decreased significantly in the fermentation broth during the intestinal flora fermentation stage. The reducing sugar content initially increased significantly, followed by a decreasing trend. Garlic polysaccharide alleviated intestinal inflammation by inhibiting pro-inflammatory cytokines, chemokines, and immune cell infiltration, and reduced intestinal mucosal damage by upregulating the expression of Claudin-1, Occludin, and ZO-1. Abnormal cell proliferation in the intestine was also inhibited by garlic polysaccharide, manifested by a significant downregulation of Ki67. Garlic polysaccharide promoted the proliferation of beneficial bacteria such as Lactobacillus and Bacteroides, and inhibited the overcolonization of pathogenic bacteria such as Akkermansia, thereby preventing the occurrence of colonomas. The molecular mechanism by which garlic polysaccharide inhibits inflammatory colonomas involves the regulation of the PI3K / Akt / NF-κB signaling pathway.

[0008] The garlic polysaccharide mentioned above is prepared by the following method: (1) After water extraction of garlic powder, protein was removed, alcohol precipitation was performed, and freeze-drying was carried out to obtain crude garlic polysaccharide; (2) The crude polysaccharide was eluted in a chromatographic column, the water-washed fraction was collected, combined, dialyzed, and freeze-dried to obtain garlic polysaccharide.

[0009] As a specific implementation of this application, the conditions for water extraction in step (1) include: temperature of 70℃-90℃ and time of 2h-5h.

[0010] As a specific embodiment of this application, step (1) deproteinization uses 10 vol.%-40 vol.% Sevag reagent.

[0011] As a specific embodiment of this application, the Sevag reagent comprises: chloroform: n-butanol = 4:1 (v / v).

[0012] As a specific implementation of this application, the dialysis in step (2) uses a dialysis membrane of 200Da-700Da and the time is 60h-90h.

[0013] As a specific embodiment of this application, the garlic polysaccharide does not degrade in the gastrointestinal tract.

[0014] As a specific embodiment of this application, the garlic polysaccharide can be effectively utilized in the intestinal flora.

[0015] Beneficial effects of the present invention The digestibility and utilization characteristics of garlic polysaccharides in the gastrointestinal tract were studied using in vitro digestion and microbial fermentation models. Results showed that garlic polysaccharides were almost undegraded in simulated gastrointestinal digestion, with minimal changes in reducing sugar content. However, they were effectively utilized by gut microbiota, resulting in a continuous decrease in total sugar content. This invention visually demonstrates the utilization of garlic polysaccharides by gut microbiota using an in vitro fermentation model. A mouse model of AOM / DSS-induced inflammatory colonoma was constructed, confirming that garlic polysaccharides effectively alleviated the occurrence of inflammatory colonoma by targeting pro-inflammatory and pro-tumor signaling pathways, inhibiting pro-inflammatory factors and cell proliferation, and restoring intestinal barrier and gut microbiota homeostasis. Therefore, garlic polysaccharides have the potential to prevent and / or treat tumor diseases by improving gut microbiota. These findings provide a theoretical basis for subsequent research on the preventive and / or therapeutic effects of garlic polysaccharides and their products on inflammatory colonoma, and have significant practical value and guiding significance. Attached Figure Description

[0016] Figure 1 The structural characteristics of garlic polysaccharides include: (A) molecular weight; (B) Fourier transform infrared spectrum; (C) ultraviolet-visible spectrum; (D) monosaccharide composition; (1) glucose, (2) fructose; Figure 2 This describes the hydrolysis of garlic polysaccharides during in vitro digestion. Figure 3 This study examines the utilization of garlic polysaccharides during in vitro fermentation; (A) changes in total sugars; and (B) changes in reducing sugars. Data are expressed as mean ± standard deviation; different letters indicate significant differences. P <0.05); Figure 4 The study investigated the effects of garlic polysaccharides on tumorigenesis in AOM / DSS-induced model mice. The results included: (A) intervention regimen in the AOM / DSS mouse model; (B) DAI scores for each group during the experiment; (C) body weight changes throughout the cycle; (D) mean colon length; (E) representative longitudinal anatomical photographs of the colon; (F) number of colonic tumors at the end of the experiment; (G) tumor size; (H) spleen coefficient; (I) liver coefficient; and (J) mouse survival rate. Data are expressed as mean ± standard deviation. Different letters indicate significant differences (…). P <0.05); Figure 5 The effects of garlic polysaccharides on pathological damage to the colon; Figure 6The effects of garlic polysaccharide on cytokines (AE), inflammatory enzymes (FG), chemokines (HI), immune cells (JK), and oxidative stress (LO) in AOM / DSS mice were investigated. The results showed that (A) TNF-α; (B) IL-6; (C) IFN-γ; (D) IL-1β; (E) IL-10; (F) iNOS; (G) COX-2; (H) CXCL1; (I) MCP-1; (J) CD68; (K) CD3; (L) CAT; (M) SOD; (N) MPO; and (O) MDA were statistically significant. Data are presented as mean ± standard deviation. Different letters indicate significant differences. P <0.05); Figure 7 The effect of garlic polysaccharide on the intestinal barrier in mice; (A) Immunohistochemical staining of MUC2, Claudin-1, Occludin and ZO-1 expression in the colon; (B) Mean optical density of MUC2 detected by immunohistochemistry; (C) Serum LPS level; (DF) Mean optical density of Claudin-1, Occludin and ZO-1; Data are expressed as mean ± standard deviation; different letters indicate significant differences ( ). P <0.05); Figure 8 The effect of garlic polysaccharides on gut microbiota diversity in AOM / DSS mice; (A) Venn diagram of ASV; (B) dilution curve; (CF) α diversity index; (G) β diversity analysis; data are presented as mean ± standard deviation; different letters indicate significant differences ( P <0.05); Figure 9 The effect of garlic polysaccharide on the gut microbiota composition of AOM / DSS mice; (AB) relative abundance at the phylum (A) and genus (B) levels; (CD) differential analysis of gut microbiota based on LEfSe, including clade plot (C) and LDA score bar chart (D). Figure 10 The effects of garlic polysaccharide on intestinal gene expression in AOM / DSS mice were investigated. The results included: (A) PCA score plot; (B) Venn diagram of differentially expressed genes in different datasets; (C) Volcano plot of differentially expressed genes between GP-1 and the model group; (D) Heatmap of differentially expressed genes; (E) GO enrichment analysis of differentially expressed genes between GP-1 and the model group; and (F) KEGG enrichment analysis of differentially expressed genes between the model and control groups, and between GP-1 and the model dataset. Figure 11The effects of garlic polysaccharide on the expression of inflammation and tumor-related proteins in the PI3K / Akt / NF-κB signaling pathway were investigated. (A) Representative images of protein bands; (B) Quantitative analysis of p-PI3K / PI3K; (C) Quantitative analysis of p-Akt / Akt; (D) Quantitative analysis of p-p65 / p65; (E) Quantitative analysis of p-IκB-α / IκB-α. Data are expressed as mean ± standard deviation. Different letters indicate significant differences (P < 0.05). Detailed Implementation

[0017] Example 1 Extraction and purification of garlic polysaccharides Garlic powder was extracted twice with deionized water (80°C, 3 hours), then deproteinized with 20% Sevag reagent (chloroform: n-butanol = 4:1), precipitated with ethanol for 24 hours, and lyophilized to obtain crude garlic polysaccharide. 5 g of the crude polysaccharide was then dissolved in 80 mL of deionized water and transferred to a DEAE-52 cellulose column, eluted at a flow rate of 4 mL / min. The washed fractions were collected, combined, and dialyzed against deionized water (500 Da) for 72 hours, followed by lyophilization to obtain garlic polysaccharide, named GP-1.

[0018] Structural characterization of garlic polysaccharides, results are shown in […]. Figure 1 .

[0019] 1. Molecular weight distribution The molecular weight distribution of GP-1 was determined using high performance liquid chromatography combined with Shodex OHpak SB 8.2, 802.5 and 803 HQ gel chromatography columns.

[0020] from Figure 1 As can be seen from A, the test results show that the molecular weight is 3537 Da.

[0021] 2. Infrared spectroscopy analysis Place 10 mg of GP-1 on the ATR accessory of a Nicolet IS10 infrared spectrometer and incubate at 4000-400 cm⁻¹. -1 Collect scan data within the range.

[0022] from Figure 1 As can be seen from B, the test results show that GP-1 contains β-glycosidic bonds and has a typical carbohydrate skeleton.

[0023] 3. Ultraviolet spectroscopy analysis A GP-1 solution of 5 mg / mL was prepared, and the UV-Vis spectrum of the polysaccharide was collected using a UV-2600i spectrophotometer.

[0024] from Figure 1As can be seen from C, the test results show that GP-1 contains almost no protein or nucleic acid and has high purity.

[0025] 4. Monosaccharide composition Take 10 mg of polysaccharide in a test tube, add 2 mL of 2 mol / L trifluoroacetic acid, hydrolyze at 80 °C for 2 h, then add deionized water and rotary evaporate to neutral. After removing impurities with an LC-18 column, the monosaccharide composition is detected by ICS-5000 high performance anion exchange chromatography.

[0026] from Figure 1 As can be seen from D, the test results show that GP-1 is mainly composed of fructose and glucose, with a mass ratio of 93.58:6.42.

[0027] Experimental results: Garlic polysaccharide is composed of fructose and glucose, with a molecular weight of 3537 Da. Garlic polysaccharide is linked by β-glycosidic bonds, possesses the basic framework of fructan, and contains almost no nucleic acids or proteins.

[0028] Example 2: In vitro digestion and fermentation of garlic polysaccharides 1. In vitro digestion of garlic polysaccharides 20 mL of GP-1 (5 mg / mL) solution was mixed with an equal volume of artificial gastric fluid, and the pH was adjusted to 2.0. The mixture was then incubated in a shaker at 37°C and 100 rpm. At 0, 0.5, 1, 2, and 4 h of reaction, 5 mL of the solution was transferred, the pH was adjusted to 7.0, and the enzyme was inactivated by boiling in water for 10 min. After 4 h of gastric digestion, the remaining digestate was mixed with artificial intestinal fluid at a 1:1 ratio and reacted at 37°C and pH 7.0. Samples were removed at the designated time points (0, 0.5, 1, 2, 4, and 6 h) and the enzyme was inactivated by boiling in water for subsequent analysis.

[0029] from Figure 2 The test results show that the hydrolysis rate of garlic polysaccharides in simulated gastric juice increases slightly with prolonged digestion time, reaching only 7.96% after 4 hours. This indicates that garlic polysaccharides remain relatively intact during simulated gastric digestion. from Figure 2 The test results show that the hydrolysis rate of garlic polysaccharide was only 10.99% after 6 hours of digestion in artificial intestinal fluid, reflecting that garlic polysaccharide is difficult to be degraded and utilized by gastrointestinal digestive enzymes.

[0030] 2. In vitro fermentation of garlic polysaccharides Fresh fecal samples were collected from 6 healthy volunteers, weighed, homogenized with sterile phosphate buffer, and filtered through 4 layers of sterile gauze to obtain a 10% (w / v) fecal bacterial seed culture for inoculation.

[0031] Preparation of basal culture medium: Mix distilled water (1 L), yeast extract (2 g), peptone (2 g), sodium bicarbonate (2 g), cysteine ​​hydrochloride (0.5 g), bile salts (0.5 g), NaCl (0.1 g), KH2PO4 (0.04 g), K2HPO4 (0.04 g), heme chloride (0.02 g), CaCl2 (0.01 g), MgSO47H2O (0.01 g), resazurite (0.01 g), vitamin K1 (10 μL), and Tween 80 (2 mL) thoroughly. Adjust the pH of the culture medium to 6.8-7.2 with dilute hydrochloric acid, sterilize at 121℃ for 15 min, and set aside.

[0032] Garlic polysaccharide was used as the sole carbon source, with 1% added to the basal culture medium. 9 ml of culture medium and 1 ml of fecal inoculum were added to anaerobic fermentation tubes, mixed thoroughly, and placed in an anaerobic gas generator. Anaerobic fermentation was carried out in a 37℃ incubator. A carbon-free control group and an inulin group were set up as a positive control group. Samples were taken at 0, 6, 12, 24, and 48 hours after fermentation, centrifuged, and the supernatant was used for the determination of total sugar and reducing sugar.

[0033] 3. Determination of total sugar content Take 2 mL of sample solution into a test tube, add 1 mL of 5% phenol solution, then add 5 mL of concentrated sulfuric acid, boil in a water bath for 20 min, cool to room temperature, and then measure the absorbance at 490 nm.

[0034] 4. Determination of reducing sugar content DNS reagent preparation: Dissolve 182g of anhydrous sodium potassium tartrate in water, then add 6.3g of 3,5-dinitrosalicylic acid, 6.3g of sodium hydroxide, 21g of sodium hydroxide and 5g of phenol. After dissolving, add water to make up to 1L and store in the dark for one week.

[0035] Take 1 mL of sample solution into a test tube, add 2 mL of DNS reagent, boil in a water bath for 5 min, then add 2 mL of water, and measure the absorbance at 540 nm.

[0036] from Figure 3 The test results show that during the in vitro fermentation of garlic polysaccharides by gut microbiota, the total sugar content continuously decreased, while the reducing sugar content showed a trend of first increasing and then decreasing, indicating that the gut microbiota decomposed and utilized the garlic polysaccharides.

[0037] Example 3: Garlic polysaccharides inhibit inflammatory colonomas 1. Mouse model construction and processing Sixty ICR mice were randomly divided into four groups: control group, model group, aspirin group (25 mg / kg), and garlic polysaccharide group (GP-1, 350 mg / kg). An inflammatory colonoma model was established using a combination of AOM and DSS treatment. Starting from day 8, all mice were administered 0.2 mL of aspirin daily via gavage, with aspirin used as a positive control. The control and model groups received saline instead. Tumor severity was assessed weekly, including weight loss, stool consistency, and intestinal bleeding; scores were recorded, and the Disease Activity Index (DAI) was calculated. At the end of week 12, mice were sacrificed after a 12-hour fast, blood was collected from the eyes, and serum was separated. The colon was flushed with saline, followed by longitudinal dissection to observe the tumor.

[0038] See results Figure 4 The study included: (A) the intervention regimen of the AOM / DSS mouse model; (B) the DAI scores of each group during DSS treatment; (C) the weight change throughout the cycle; (D) the average length of the colon; (E) representative photographs of the longitudinal anatomy of the colon; (F) the number of tumors in the colon at the end of the experiment; (G) the tumor size; (H) the spleen coefficient; (I) the liver coefficient; and (J) the survival rate of mice during the study. It can be seen that GP-1 supplementation can effectively restore the weight of mice with tumors, reduce intestinal bleeding and diarrhea, inhibit the growth of tumors in the colon, and thus improve the survival rate of mice.

[0039] 2. Pathological observation After fixing the colon with 4% paraformaldehyde for 24 hours, the tissue was dehydrated and embedded in paraffin, then sectioned into 4 μm sections using a microtome. The specimen sections were stained with H&E and AB-PAS, respectively, and then observed using an Olympus IX73 microscope.

[0040] See results Figure 5 As can be seen, GP-1 effectively reduced colonic damage in tumor-bearing mice.

[0041] 3. Measurement of inflammation, immunity, and antioxidant indicators According to the ELISA kit instructions, the levels of LPS, catalase (CAT), superoxide dismutase (SOD), myeloperoxidase (MPO), and malondialdehyde (MDA) in mouse serum were detected. At the same time, the levels of inflammatory mediators (TNF-α, IL-6, IFN-γ, IL-1β, IL-10, iNOS, COX-2, CXCL1, MCP-1) and immune cell markers (CD68, CD3) in the colon were analyzed.

[0042] See results Figure 6 It can be seen that GP-1 treatment significantly inhibited the abnormal infiltration of immune cells and pro-inflammatory mediators in the colon and improved the body's oxidative stress.

[0043] 4. Immunohistochemical analysis Paraffin sections were dewaxed and hydrated in xylene and graded ethanol. Antigens were repaired using citrate buffer, followed by endogenous enzyme inactivation and blocking. Primary antibodies (anti-Muc2, anti-Claudin-1, anti-Occludin, and anti-ZO-1) were added to slides and incubated overnight at 4°C, while secondary antibodies were incubated at room temperature for 60 minutes. Subsequently, DAB chromogenic solution and hematoxylin were added to the sections, and the sections were observed under an optical microscope. The average optical density (AOD) was measured using ImageJ software.

[0044] See results Figure 7 It can be seen that GP-1 treatment upregulated the expression of mucin and tight junction proteins in the intestine, enhanced the integrity of the intestinal barrier, and inhibited abnormal cell proliferation.

[0045] 5. 16S sequencing Mouse feces were collected and stored at -80°C. Total DNA was extracted using the EZNA fecal DNA kit, and its purity and concentration were determined by agarose gel electrophoresis and UV spectrophotometry. The V3-V4 region of 16S rDNA was amplified using universal primers (341F / 805R) and sequenced using an Illumina Novaseq PE250 platform.

[0046] See results Figure 8 , Figure 9 It can be seen that GP-1 can restore the intestinal flora balance in colonoma mice by inhibiting the proliferation of opportunistic pathogens and increasing the abundance of beneficial bacteria.

[0047] 6. Transcriptome sequencing Total RNA was extracted from the colon using Trizol reagent, and a PE library was constructed using high-quality RNA with a RIN > 7.0. Subsequently, using the cut mRNA fragments as templates, first and second strand cDNAs were synthesized using reverse transcriptase. The ligated cDNA fragments were amplified by PCR to obtain a library of 300 ± 50 bp in length. Paired-end sequencing (2 × 150 bp) was performed on an Illumina Novaseq 6000 platform.

[0048] See results Figure 10 It can be seen that GP-1 treatment can inhibit the development of inflammatory colonomas by suppressing the abnormal expression of tumor and inflammation-related signaling pathways in the mouse colon.

[0049] 7. Western Blotting Analysis Protein was extracted from 20 mg of colon by adding 200 μL of RIPA buffer containing a protease inhibitor (1 mM), and then quantified using a BCA kit. 20 μg of protein was separated on a 10% SDS-PAGE gel, then transferred to a membrane and blocked. The membrane was incubated overnight at 4°C with primary antibody, and then incubated for 1.5 hours at room temperature with HRP-conjugated goat anti-rabbit secondary antibody. Protein bands were observed using a BIO-RADChemiDoc MP imaging system.

[0050] See results Figure 11 It can be seen that GP-1 gavage treatment can effectively inhibit the overexpression of the PI3K / Akt / NFκB signaling pathway in the colon of tumor-bearing mice, thereby inhibiting the occurrence and development of tumors.

Claims

1. Use of garlic polysaccharide in the preparation of a drug for preventing and / or treating inflammatory colon tumor.

2. Use according to claim 1, characterized in that, The garlic polysaccharide is prepared by the following method: (1) After water extraction of garlic powder, deproteinization, alcohol precipitation, freeze-drying, garlic crude polysaccharide is obtained; (2) The crude polysaccharide is eluted in a chromatographic column, the water-washed components are collected, combined, then dialyzed, and freeze-dried to obtain garlic polysaccharide.

3. Use according to claim 2, characterized in that, The water extraction conditions in step (1) include: temperature of 70-90℃, time of 2-5h.

4. Use according to claim 2 or 3, characterized in that, The deproteinization in step (1) uses Sevag reagent of 10-40vol.%.

5. Use according to claim 4, characterized in that, The Sevag reagent includes: chloroform: n-butanol = 4:1 (v / v).

6. Use according to any one of claims 2 to 4, characterized in that, The dialysis in step (1) uses dialysis membrane of 200-700Da, time of 60-90h.

7. The use according to claim 1, characterized in that, The garlic polysaccharide is not degraded in the gastrointestinal tract.

8. The use according to claim 1, characterized in that, The garlic polysaccharide can be effectively utilized in the intestinal flora.

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