Application of honeysuckle polysaccharide in preparation of medicine for treating ulcerative colitis and inflammation-related colorectal cancer

Abstract

The invention relates to the technical field of ulcerative colitis related medicines, in particular to application of honeysuckle polysaccharide in preparation of medicines for treating ulcerative colitis and inflammation related colorectal cancer. According to the technical scheme, it is found that the honeysuckle flower polysaccharide WLJP-025P has an obvious improvement effect on ulcerative colitis induced by dextran sodium sulfate and has an obvious treatment effect on a mouse with colorectal cancer induced by azoxymethane / dextran sodium sulfate (AOM / DSS). The treatment effect of the WLJP-025P is better than that of the honeysuckle aqueous extract and the honeysuckle crude polysaccharide, the WLJP-025P is expected to be developed into a candidate medicinal substance for ulcerative colitis and related colorectal cancer, the technical problem that in the prior art, a medicine which is small in side effect and high in specificity and is used for treating ulcerative colitis is lacked can be solved, and the WLJP-025P has ideal application and popularization prospects.

Description

Technical Field

[0001] The invention relates to the technical field of ulcerative colitis-related drugs, and in particular to use of honeysuckle polysaccharide in preparing drugs for treating ulcerative colitis and inflammation-related colorectal cancer. Background Art

[0002] In recent years, the incidence of ulcerative colitis (UC) has increased year by year and is listed as one of the intractable diseases in the world. Data from the past decade show that the incidence of UC in my country has increased rapidly (an average annual increase of 14.9%), and the number of patients has exceeded 200 million. UC is a chronic nonspecific inflammatory disease of the intestine with serious complications. The lesions mainly extend from the rectum to the proximal colon. It is characterized by being difficult to cure and prone to cancer. There is no effective cure. There are various treatments for ulcerative colitis, aiming to control inflammation, relieve symptoms, prevent recurrence, and improve the quality of life. Drug treatment mainly includes anti-inflammatory drugs (e.g., aminosalicylic acid drugs, corticosteroids), immunomodulators (e.g., azathioprine) and biological agents (e.g., anti-TNF-α antibodies, interleukin inhibitors). For patients who are ineffective with drug treatment and have serious complications (such as massive bleeding, perforation, and increased risk of cancer), surgical treatment is required.

[0003] The risk of canceration of UC increases significantly when the disease lasts for a long time or the range of lesions is wide. Studies have shown that the risk of canceration in UC patients is 1.7 times that of the normal population, while the rate of colorectal canceration in UC patients in my country is 0.8%. Since the patient rate of UC is much higher than that of colorectal cancer, UC-related colorectal cancer accounts for 10%-15% of the total number of colorectal cancers. Worldwide, colorectal cancer (CRC) is the third most common malignant tumor in the world and the second most common in terms of mortality. In recent years, the incidence and mortality of colorectal cancer in my country have continued to rise. The 2020 China Cancer Statistics Report shows that the incidence and mortality of colorectal cancer rank second and fifth, respectively, with approximately 555,000 new cases and approximately 286,000 deaths. At present, the treatment of colorectal cancer is mainly surgical resection, and the risks and benefits of treatment are seriously unequal in clinical practice: surgery has high risks, great trauma to the human body, and is prone to postoperative complications; radiotherapy and chemotherapy are prone to drug resistance, adverse reactions and toxic effects.

[0004] In general, the treatment of UC is relatively difficult, mainly due to its complex etiology, easy relapse, and long course of disease. Existing drugs and treatments have limited therapeutic effects on UC and have certain side effects. There is an urgent need for further development and research of drugs that can be used to treat UC to meet the needs of medical practice. Summary of the invention

[0005] The purpose of the present invention is to use a honeysuckle polysaccharide in the preparation of a drug for treating ulcerative colitis and / or inflammation-related colorectal cancer, so as to solve the technical problem of the lack of drugs with small side effects and high specificity for treating ulcerative colitis in the prior art. There is no effective cure for ulcerative colitis and inflammation-related colorectal cancer. This solution has found a new active substance that can improve ulcerative colitis and inflammation-related colorectal cancer.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] A use of honeysuckle polysaccharide in preparing a medicine for treating ulcerative colitis and / or inflammation-related colorectal cancer.

[0008] Furthermore, the honeysuckle polysaccharide is used to improve weight loss, increased disease activity index, colon atrophy and spleen enlargement caused by ulcerative colitis.

[0009] Furthermore, the honeysuckle polysaccharide is used to improve inflammation caused by ulcerative colitis, to upregulate the expression of intestinal barrier proteins, and to repair the intestinal mechanical barrier.

[0010] Furthermore, the honeysuckle polysaccharide is used to improve the elevated disease activity index, colon atrophy and spleen enlargement caused by colorectal cancer, to repair colon tissue pathological damage caused by colorectal cancer, and to inhibit the progression of colon tumors.

[0011] Furthermore, the monosaccharide composition of the honeysuckle polysaccharide is: galacturonic acid 53.4% ​​to 80.2%, glucose 8% to 12%, galactose 11.7% to 17.5%, arabinose 6.6% to 9.8%, and mannose 0.3% to 0.5%.

[0012] Furthermore, the honeysuckle polysaccharide is composed of α-(1→4)-D-GalpA connected in sequence to form a main chain; the side chains of the honeysuckle polysaccharide include β-(1→4)-linked galactan, α-(1→5)-linked and (1→3,5)-linked arabinan / arabinogalactan, and α-(1→4)-linked glucan.

[0013] Furthermore, the relative average molecular weight of honeysuckle polysaccharide is 10-40 kDa; preferably, the relative average molecular weight of honeysuckle polysaccharide obtained by high performance liquid chromatography based on TSKgelG4000PWxl gel exclusion chromatography column is 23±4 kDa.

[0014] Furthermore, the honeysuckle polysaccharide is prepared by the following method:

[0015] S1: After removing fat-soluble substances from honeysuckle, water is used as a solvent for decoction and extraction, and the supernatant is taken; after concentration, protein removal, and alcohol precipitation, the precipitate is collected; and after freeze-drying, the total sugar of honeysuckle is obtained;

[0016] S2: Load the aqueous solution of total sugars from honeysuckle onto a DEAE-cellulose chromatography column, and use distilled water and 0.25 mol / L NaCl solution to elute the DEAE-cellulose chromatography column in sequence, and take the portion corresponding to the total sugar content peak and the uronic acid peak of the elution curve from the components eluted with the 0.25 mol / L NaCl solution; and then concentrate, dialyze and freeze-dry to obtain the honeysuckle polysaccharide WLJP-025 component;

[0017] S3: The honeysuckle polysaccharide WLJP-025 component was dissolved in sodium chloride solution, loaded on a Sepharose CL-6B chromatography column, eluted with a 0.1 mol / L NaCl solution, and the eluate corresponding to the maximum total sugar content peak and uronic acid peak of the elution curve was taken; after concentration, dialyzation and freeze-drying, the honeysuckle polysaccharide WLJP-025p was obtained.

[0018] Further, in S2, the DEAE-cellulose chromatography column was eluted with 2 column volumes of distilled water and 0.25 mol / L NaCl solution, and the elution flow rate was 10 mL / min; in S3, 0.1 mol / L NaCl solution was used for elution at a flow rate of 0.1 mL / min.

[0019] Furthermore, the dosage of the honeysuckle polysaccharide for the ulcerative colitis mouse model or the colorectal cancer mouse model is 30-60 mg / kg.

[0020] The technical principle and beneficial effects of this technical solution are:

[0021] Honeysuckle is a traditional Chinese herbal medicine with a long history. It is cold in nature, sweet in taste, and has the effects of clearing away heat and detoxifying, cooling and dispersing wind-heat. It enters the lung, stomach and large intestine meridians. It was first recorded in "Famous Doctors' Records". It is non-toxic and listed as a top-grade product. This technical solution found that a honeysuckle polysaccharide WLJP-025P has a significant improvement effect on ulcerative colitis (UC) induced by dextran sulfate sodium (DSS), and in animal experiments on colorectal cancer (CRC) in mice, it was found that it has a significant inhibitory effect on mice with colorectal cancer induced by azoxymethane / dextran sulfate sodium (AOM / DSS). There is no effective cure for ulcerative colitis and inflammation-related colorectal cancer. This technical solution discloses an active substance that can improve ulcerative colitis and inflammation-related colorectal cancer. WLJP-025P is expected to be developed as a candidate medicinal substance for ulcerative colitis and related colorectal cancer. Ulcerative colitis has a higher risk of cancer. Studies have shown that the risk of cancer in patients with ulcerative colitis is 1.7 times that of the normal population, and 10-15% of colon cancer patients originate from ulcerative colitis. Polysaccharides are a highly effective, low-toxic active substance that has a therapeutic effect on ulcerative colitis and also show excellent therapeutic potential in colorectal cancer.

[0022] The existing technology of research on honeysuckle mainly focuses on active ingredients with anti-inflammatory effects such as chlorogenic acid, rutin, quercetin, luteolin, etc., and the utilization value of honeysuckle polysaccharides has not been deeply developed. Whether honeysuckle polysaccharides have the effect of treating ulcerative colitis and inflammation-related colorectal cancer cannot be known through the existing technology. Polysaccharides are a class of high-molecular compounds composed of multiple monosaccharide molecules connected by glycosidic bonds. They have the characteristics of low toxicity and good tolerance, and the probability of causing obvious adverse reactions is lower than other active ingredients. This technical solution verifies through experiments that WLJP-025P, a specific polysaccharide component, has a very significant effect on the treatment of ulcerative colitis and inflammation-related colorectal cancer. Its effect is better than that of general honeysuckle water extracts and honeysuckle crude polysaccharides. It is a high-molecular compound isolated from honeysuckle, which has the effect of treating ulcerative colitis and colorectal cancer and is highly safe. The successful development of this program is of certain significance for overcoming the problems of ulcerative colitis and inflammation-related colorectal cancer, as well as improving the comprehensive utilization effect of honeysuckle. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the elution curve of the total sugar from Flos Lonicerae separated and purified by DEAE cellulose column chromatography in Example 1 (490 nm: total sugar content; 525 nm: uronic acid content).

[0024] Figure 2This is the elution curve of the honeysuckle polysaccharide WLJP-025 of Example 1 on a Sepharose CL-6B chromatography column (490 nm: total sugar content; 525 nm: uronic acid content; Vo: external water volume; Vt: total volume).

[0025] Figure 3 This is the step of extracting and separating honeysuckle polysaccharides in Example 1.

[0026] Figure 4 This is the test result of honeysuckle polysaccharide WLJP-025p in Example 2 on TSKgel G4000PWxl molecular sieve.

[0027] Figure 5 The monosaccharide composition detection results of the honeysuckle polysaccharide WLJP-025p of Example 2 (Man: mannose; Rha: rhamnose; GlcA: glucuronic acid; GalA: galacturonic acid; Glc: glucose; Gal: galactose; Xyl,: xylose; Ara: arabinose; Fuc: fucose).

[0028] Figure 6 This is the NMR detection spectrum of the honeysuckle polysaccharide WLJP-025p of Example 2.

[0029] Figure 7 This is the UC animal experiment modeling and drug administration process of Example 3.

[0030] Figure 8 These are the research results of Example 3 on the effects of WLJP025p on the changes in body weight and DAI scores of UC mice (*P<0.05, **P<0.01, ***P<0.001 vs. Control group; #P<0.05, ###P<0.001 vs. UC group).

[0031] Fig. 9 These are the research results of Example 3 on the effect of WLJP025p on the colon length of UC mice (***P<0.001 vs. Control group; #P<0.05, ##P<0.01 vs. UC group).

[0032] Fig.10 These are the research results of Example 3 on the effect of WLJP025p on spleen enlargement in UC mice (***P<0.001 vs. Control group; ##P<0.01, ###P<0.001 vs. UC group).

[0033] Fig.11These are the research results of Example 3 on the effects of WLJP025p on inflammatory factors in UC mice (***P<0.001 vs. Control group; #P<0.05, ###P<0.001 vs. UC group).

[0034] Fig.12 These are the research results of Example 3 on the effect of WLJP025p on colon tissue lesions in UC mice.

[0035] Fig.13 These are the immunofluorescence staining images of AJ (adherence junction) proteins beta Catenin and E-cadherin in the colon tissue of each group of mice in Example 3.

[0036] Fig.14 These are the immunofluorescence staining images of TJ (tight junction) proteins ZO-1 and Occludin in the colon tissue of each group of mice in Example 3.

[0037] Fig.15 These are the research results of Example 3, in which the expression levels of intestinal barrier proteins in the colon tissues of each group of mice were detected by western blotting (*P<0.05, ***P<0.001 vs. Control group; ##P<0.01, ###P<0.001 vs. UC group).

[0038] Fig.16 This is the CRC animal experiment modeling and drug administration process of Example 4.

[0039] Fig.17 The results of the study on the effect of WLJP025p on tumorigenesis in CRC mice in Example 4 (***P<0.001 vs. Control group; ##P<0.01 vs. CRC group).

[0040] Fig.18 These are the research results of Example 4 on the effect of WLJP025p on the disease activity index score (DAI) of CRC mice (***P<0.001 vs. Control group; ###P<0.001 vs. CRC group).

[0041] Fig.19 These are the research results of Example 4 on the effect of WLJP025p on the colon length of CRC mice (***P<0.001 vs. Control group; #P<0.05 vs. CRC group).

[0042] Fig. 20These are the research results of Example 4 on the effect of WLJP-025p on spleen enlargement in CRC mice (***P<0.001 vs. Control group; ###P<0.001 vs. CRC group).

[0043] Fig.21 These are the research results of Example 4 on the effect of WLJP025p on colon tissue lesions in CRC mice.

[0044] Fig. 22 These are the research results of the effects of crude honeysuckle polysaccharide, honeysuckle water extract and WLJP025p in comparative example 1 on the changes in DAI scores of UC mice (*P<0.05, **P<0.01, ***P<0.001 vs. Control group; #P<0.05, ###P<0.001 vs. UC group).

[0045] Fig.23 These are the research results of the effects of crude honeysuckle polysaccharide, honeysuckle water extract and WLJP025p in comparative example 1 on the colon length of UC mice (***P<0.001 vs. Control group; ##P<0.01 vs. UC group; &P<0.05 vs. WLJP-025p group).

[0046] Fig.24 These are the research results of the effects of crude honeysuckle polysaccharide, honeysuckle water extract and WLJP025p in comparative example 1 on spleen enlargement in UC mice (***P<0.001vs.Control group; ###P<0.001vs.UC group; &&P<0.01, &&&P<0.001vs.WLJP-025p group).

[0047] Fig.25 These are the research results on the effects of crude honeysuckle polysaccharide, honeysuckle water extract and WLJP025p in comparative example 1 on colon tissue lesions in UC mice (***P<0.001vs.Control group; ##P<0.01vs.UC group; &P<0.05, &&&P<0.001vs.WLJP-025p group). DETAILED DESCRIPTION

[0048] The following is further described in detail through specific implementation methods:

[0049] Example 1: Preparation of Lonicera japonica polysaccharides (LJP)

[0050] The preparation method of honeysuckle polysaccharide (abbreviated as: WLJP-025p) used in this scheme can be found in the doctoral dissertation "Separation and purification of honeysuckle polysaccharide and its protective effect on allergic rhinitis / dermatitis" (Bai Xinyu, Yanbian University). This scheme mainly explores the new uses of honeysuckle polysaccharide to fully explore the application direction of honeysuckle polysaccharide and increase new treatment methods for thyroid cancer. Specifically, the preparation method of honeysuckle polysaccharide is as follows:

[0051] (1) Preparation of total sugar from honeysuckle

[0052] Add ethanol to dried honeysuckle (dried flower buds or flowers with initial opening of Lonicera japonica Thunb.; Lonicerae japonicae flos), reflux extract for 4 hours to remove fat-soluble substances; then add 10L of distilled water to 500g of the residue, boil and extract twice at 100°C (4 hours / time), combine the supernatants of the two boilings and concentrate to about 700mL, centrifuge for 15min (4500rpm / min, 15min), collect the supernatant, and treat with 15% trichloroacetic acid at 4°C for 4 hours to remove protein. After neutralization, add 3 times the volume of anhydrous ethanol to precipitate polysaccharides. The next day, centrifuge the alcohol precipitate (4500rpm / min, 10min), collect the precipitate and freeze-dry it in vacuum to obtain light brown honeysuckle total sugar powder (WLJP, yield of about 5.1%, w / w). Extracting total honeysuckle polysaccharides by water extraction and alcohol precipitation is a conventional form in the prior art. By appropriately adjusting the process parameters according to the above process, light brown total honeysuckle sugar powder can also be prepared.

[0053] The phenol-sulfuric acid method is used to detect the sugar content of total sugar: concentrated sulfuric acid can hydrolyze and dehydrate polysaccharides to generate substances such as furfural or hydroxymethylfurfural. These substances can react with phenol to generate brown products. The product has an obvious absorption peak at 490nm, and the absorbance change at this wavelength is proportional to the sugar content within a certain range.

[0054] Standard curve: Take 1 mL of each mixed standard solution of glucose and galacturonic acid of different concentrations (0, 10, 20, 30, 40, 50, 60 μg / mL) in a test tube, add 0.5 mL of 6% phenol solution (w / w) and 2.5 mL of concentrated sulfuric acid to each test tube, shake and mix immediately, boil in a boiling water bath for 10 min, and after cooling, measure the absorbance of each test tube at 490 nm. Draw a standard curve with the standard concentration C (μg / mL) as the horizontal axis and the absorbance at 490 nm as the vertical axis.

[0055] Detection of total sugar content: Take 1 mL of 50 μg / mL honeysuckle total sugar solution, 0.5 mL of 6% phenol solution (w / w) and 2.5 mL of concentrated sulfuric acid, and perform the other operations as above. Measure the absorbance of the total sugar solution, substitute the absorbance into the standard curve equation to calculate the concentration, and divide this concentration by the sample concentration to obtain the total sugar content.

[0056] After the dried honeysuckle is boiled in hot water, concentrated, protein removed, ethanol precipitated, and freeze-dried in vacuum, the honeysuckle water-extracted total sugar WLJP is obtained, with a yield of about 5.1% (w / w). The purity of WLJP is about 93.7% when analyzed by phenol-sulfuric acid method.

[0057] (2) Separation and purification of total sugars from honeysuckle

[0058] Purification of Total Sugar from Honeysuckle by DEAE-cellulose

[0059] First, pretreat the DEAE-cellulose (Shanghai Hengxin Chemical Reagent Co., Ltd., CAS: 9013-34-7) column filler. First, soak the DEAE-cellulose in distilled water (3 to 5 days); after wringing out the water, soak the cellulose in 0.5 mol / L NaOH for 1 hour, and wash it with distilled water until it is neutral; finally, soak the cellulose in 0.5 mol / L hydrochloric acid for 1 hour, wash it with distilled water until it is neutral, and set aside. The above-mentioned DEAE-cellulose treatment processes are all conventional methods in the prior art. After completing the above-mentioned pretreatment, DEAE-cellulose is loaded into the column. After the treated DEAE-cellulose is debubbled, it is slowly loaded into a chromatography column (50×7.5 cm, Cl-type), and washed with 2 times the column volume of distilled water, 2M NaCl solution, and distilled water, respectively, and set aside.

[0060] After completing the above preparations, DEAE-cellulose is used to purify the total sugar of honeysuckle. Weigh the total sugar of honeysuckle and dissolve it in distilled water (about 40mg / mL). After centrifugation (4500rpm / min, 15min), all the supernatants are loaded on the treated DEAE-cellulose chromatography column, and gradient elution is performed with 2 column volumes of distilled water, 0.25mol / L NaCl solution and 0.5mol / L NaCl solution, respectively, at a flow rate of 10mL / min. In a specific embodiment, when collecting the eluate of the gradient elution, it is collected in order of elution time and about 130mL is used as one tube, and the sugar content and uronic acid content in each eluate are detected by phenol-sulfuric acid method and m-hydroxybiphenyl method, respectively. Among them, the parts corresponding to the total sugar content peak and uronic acid peak of the elution curve are taken, and the components in tubes 36-55 are used to prepare WLJP-025. In actual operation, the collected components for preparing WLJP-025 are not strictly limited to tubes 36-55. It is only necessary to collect the eluent in the collection tube corresponding to the total sugar content peak and the uronic acid peak according to the elution curve. The components corresponding to the total sugar content peak and the uronic acid peak of the elution curve obtained by eluting with 0.25 mol / L NaCl solution are sequentially subjected to conventional concentration, dialysis and vacuum freeze-drying steps to obtain an acidic sugar component WLJP-025.

[0061] like Figure 1 As shown, the total sugar WLJP of honeysuckle was loaded on DEAE-cellulose column chromatography. From the elution curve, it can be seen that a major polysaccharide component WLJP-025 was obtained by elution with 0.25 mol / L sodium chloride solution. The distribution peaks of sugar content and uronic acid content in its elution curve were consistent, indicating that the component had good charge uniformity.

[0062] Further purification of WLJP-025 by Sepharose CL-6B

[0063] 40 mg of WLJP-025 component was weighed and dissolved in 1 mL of 0.15 mol / L NaCl. After centrifugation (12000 rpm / min, 3 min), the supernatant was loaded on a Sepharose CL-6B molecular sieve column (GE Healthcare, CAS: 62610-50-8, 60 cm×1.0 cm), and eluted with 0.1 mol / L NaCl solution at a flow rate of 0.1 mL / min. One portion (tube) was collected approximately every 12 min, and the sugar content and uronic acid content in each eluate were detected. The elution curve was plotted with the number of collected portions as the abscissa and the absorbance of each portion as the ordinate. The eluate portion corresponding to the maximum total sugar content peak and uronic acid peak of the elution curve was taken for the preparation of WLJP-025p, wherein the components in tubes 10-35 were used to prepare WLJP-025p. In actual operation, the collected components for preparing WLJP-025p are not strictly limited to tubes 10-35. It is only necessary to collect the eluate in the collection tubes corresponding to the total sugar content peak and the uronic acid peak according to the elution curve. After the obtained components are sequentially subjected to conventional concentration, dialysis and vacuum freeze-drying steps, a component WLJP-025p with good uniformity is obtained, and the yield is about 1.9% (w / w).

[0064] After the honeysuckle polysaccharide component WLJP-025 was separated and purified by Sepharose CL-6B column chromatography, the elution curve was as follows: Figure 2 As shown in the figure, WLJP-025 showed two elution peaks, one large and one small, in Sepharose CL-6B column chromatography, and the total sugar distribution was basically consistent with the uronic acid distribution. As shown in the figure, we collected the relatively rich polysaccharide portion WLJP-025p.

[0065] In addition, the overall process of separation and purification of total sugars from honeysuckle can be seen Figure 3 From the total sugar WLJP of honeysuckle, WLJP-N (fraction collected by elution with distilled water), WLJP-025 (fraction collected by elution with 0.25 mol / L NaCl solution), and WLJP-05 (fraction collected by elution with 0.5 mol / L NaCl solution) can be obtained by DEAE-cellulose chromatography. WLJP-025 is then chromatographed by Sepharose CL-6B molecular sieve to obtain WLJP-025p.

[0066] Example 2: Characterization of polysaccharide WLJP-025p

[0067] (1) Homogeneity and molecular weight detection of WLJP-025p

[0068] A Shimadzu HPLC system (Shimadzu, Japan) and a Schambeck RI2000A differential refractometer (Schambeck SFD GmbH, Germany) were used, connected in series with a TSKgel G4000 PWxl gel exclusion chromatography column (7.8×300mm, TOSOH, Japan), with a detection temperature of 35°C and a flow rate of 0.5mL / min. 20μL of 5mg / mL WLJP-025p solution was taken for detection. The standard products were a series of dextran with known molecular weights (2000kDa, 800kDa, 500kDa, 100kDa, 10kDa). The logarithm of the molecular weight of each standard product was used as the ordinate and the corresponding retention time was used as the abscissa to draw a standard curve for molecular weight calculation. The molecular weight can be calculated based on the regression equation of the standard curve and the retention time of WLJP-025p.

[0069] The molecular weight and homogeneity of WLJP-025p were analyzed by HPLC. Figure 4 As shown, WLJP-025p mainly presents one elution peak, indicating that its uniformity is good, which is consistent with the results of Sepharose CL-6B chromatography column. According to the molecular weight standard curve and the elution time of WLJP-025p, the relative average molecular weight of WLJP-025p is calculated to be 23±4kDa. If a different measurement method is used (for example, changing the chromatographic column, TSKgel G3000 is also one of the commonly used chromatographic columns for molecular weight detection), the measured molecular weight values ​​will show certain differences, but are generally within the range of 10-40kDa.

[0070] (2) Detection of monosaccharide composition of WLJP-025p

[0071] Hydrolysis of polysaccharides

[0072] Weigh 2 mg of honeysuckle polysaccharide WLJP-025p, put it into an acid-resistant and high-temperature resistant glass vial, add 1 mL of 2 mol / L hydrochloric acid methanol, seal it and react at 80°C for 16 hours; after blowing dry, add 1 mL of 2 mol / L trifluoroacetic acid solution, seal it and react at 120°C for 1 hour, remove the trifluoroacetic acid and evaporate it to dryness.

[0073] Derivatization of hydrolysates

[0074] First, add 0.5mL of 0.5mol / L PMP and 0.5mL of 0.3mol / L NaOH solution to the hydrolyzate evaporated in the "polysaccharide hydrolysis" experiment, mix thoroughly and react at 70°C for 30min. Then, add 0.3mol / L HCl solution to the reaction solution for neutralization; finally, extract and remove free PMP with chloroform, filter with 0.22μm filter membrane, and wait for HPLC monitoring.

[0075] HPLC on-machine monitoring

[0076] A Waters e2695 high performance liquid chromatography system and a Waters 2489 UV detector (detection wavelength of 245 nm) were used, and the brand and model of the chromatographic column was Dikma Platisil ODS (4.6×250 mm, 5 μm). The mobile phase was acetonitrile: PBS (0.1 mol / L, pH 7.0) = 18:81 (V / V), the flow rate was 1.0 mL / min, the injection volume was 20 μL, and the column temperature was 35°C.

[0077] Test results

[0078] The monosaccharide composition of honeysuckle polysaccharide component WLJP-025p is as follows Figure 5 As shown in Table 1, WLJP-025p is mainly composed of GalA, Ara, Glc, Gal and Man. After multiple tests, the content of several polysaccharides ranges from GalA (galacturonic acid): 53.4% ​​to 80.2% (66.8±13.4%); Ara (arabinose): 6.6% to 9.8% (8.2±1.6%); Gal (galactose): 11.7% to 17.5% (14.6±2.9%); Glc (glucose): 8%-12% (10±2%); Man (mannose): 0.3% to 0.5% (0.4±0.2%). Since WLJP-025p contains more GalA residues, it is speculated that WLJP-025p is a pectin polysaccharide. In addition, WLJP-025p contains some neutral monosaccharide residues, such as Gal, Ara and Glc residues, and the content of Gal residues is higher than that of Ara residues, indicating that WLJP-025p may contain sugar structural units of galactan, arabinan, glucan and / or arabinogalactan. Therefore, WLJP-025p may be a pectin polysaccharide rich in HG domains, containing neutral sugar side chains of different structural types.

[0079] Table 1: Monosaccharide composition of WLJP-025p

[0080]

[0081] (3) Nuclear magnetic resonance detection of WLJP-025p

[0082] WLJP-025p (20 mg) was dissolved in D2O (1 mL, 99.8%) and stirred at room temperature overnight. One-dimensional NMR spectra were obtained on a Bruker AV600 spectrometer ( 1 H NMR, 13 C NMR) and two-dimensional NMR spectroscopy (HSQC, HMBC).

[0083] The fine structure of WLJP-025p was analyzed by one-dimensional and two-dimensional NMR spectroscopy. Figure 6 As shown, the chemical shifts of each peak are listed in Table 2. The NMR spectrum of the HG-type domain-rich pectin polysaccharide WLJP-025p shows that the GalA residues in WLJP-025p are partially methylated, with chemical shifts at 99.75 / 5.01ppm, 70.81 / 3.97ppm, 71.03 / 4.15ppm, 79.27 / 4.47ppm, 73.38 / 3.74ppm and 173.88 / -ppm, corresponding to the C1 / H1 to C6 / H6 groups in the methylated GalA. The C-2 or C-3 of the GalA residue is easily partially acetylated in natural pectin polysaccharides. In the WLJP-025p NMR results, the chemical shift of -CH3COO in the O-2 or O-3 of the GalA residue is 22.95ppm, indicating that WLJP-025p is also partially acetylated. In addition, for Gal monosaccharide residues, the chemical shifts at 103.30 / 4.61ppm and 102.32 / 4.65ppm represent the (1→4)-linked and terminal β-D-Galp residues, respectively; for Ara monosaccharide residues, the chemical shifts at 107.46 / 5.11ppm, 110.03 / 5.09ppm and 110.13 / 5.16ppm represent the (1→3,5)-linked, (1→5)-linked, and terminal α-Araf residues; for Glc monosaccharide residues, the chemical shifts at 99.04 / 5.10ppm and 99.37 / 4.86ppm correspond to the (1→4)-linked, (1→5)-linked and terminal α-Glcp residues, respectively. In summary, WLJP-025p is mainly composed of α-(1→4)-D-GalpA connected in sequence as the main chain, and the side chains are mainly composed of β-(1→4)-galactan, α-(1→5)-linked and (1→3,5)-linked arabinan / arabinogalactan, and α-(1→4)-linked glucan.

[0084] Table 2: WLJP-025p 13 C and 1 Chemical shifts in H NMR

[0085]

[0086] Example 3: Study on the effect of honeysuckle polysaccharide in UC mouse model

[0087] The ulcerative colitis mouse model (UC model) was established by administering 1.5% DSS solution (Dextran Sulfate Sodium). DSS was administered to mice by adding it to drinking water at a concentration of 1.5%. DSS was added to drinking water during days 0-5, 8-12, 15-18, and 22-26, and DSS was not added to drinking water at other times. The mice were randomly divided into a control group (DSS and WLJP025p were replaced with normal saline throughout the process), a UC model group (DSS solution was administered to establish a UC model, and normal saline was administered by gavage), a WLJP-L group, and a WLJP-H group (DSS was administered to establish a UC model, and WLJP025p 30 / 60 mg / kg was administered by gavage for treatment. The WLJP-L group and the WLJP-H group were gavaged once a day from the 5th day to the 28th day, and the UC group was given normal saline), with 10 mice in each group. The UC modeling and WLJP025p treatment process are as follows. Figure 7 The weight, stool viscosity, and rectal blood in the mice were recorded every day.

[0088] The experimental results are detailed in Figure 8 , Fig. 9 and Fig.10 , by evaluating the weight of mice, the disease activity index (DAI) of mice, the atrophy of the colon of mice, and the enlargement of the spleen of mice. Among them, the Disease Activity Index (DAI) is an important tool for evaluating the severity of inflammatory bowel diseases (such as ulcerative colitis and Crohn's disease). In mouse models, the DAI score is often used to evaluate the severity of the DSS (dextran sulfate sodium)-induced colitis model. The DAI score takes into account multiple indicators such as weight changes, stool characteristics, fecal occult blood, and activity. From the experimental results, it can be concluded that WLJP-025p improved the weight loss, increased disease activity index, colon atrophy, and splenomegaly caused by DSS-induced UC mice. From these basic indicators, it is clear that WLJP-025p has a therapeutic effect on ulcerative colitis.

[0089] The production of cytokines is essential in the inflammatory response, and the expression levels of major pro-inflammatory factors in UC were determined by ELISA. Fig.11 It can be seen that WLJP025p can inhibit the increase of proinflammatory factors in mice induced by DSS, and the inhibitory effect increases with the increase of dose. It is further confirmed that WLJP-025p can inhibit inflammation and treat UC mice.

[0090] H&E staining results showed that the colon tissue structure of the UC group treated with DSS was severely damaged, characterized by disordered glandular arrangement of the intestinal epithelium, damaged goblet cells, inflammatory cell infiltration in the lamina propria, and loss of crypts. The WLJP025p-administered group significantly improved the damage to the colon tissue structure, colon inflammatory cell infiltration, destruction of mucosal epithelial cell integrity, and reduction of goblet cells. Fig.12 As shown, WLJP025p can effectively alleviate DSS-induced colon tissue lesions in mice.

[0091] The expression of intestinal barrier proteins Occludin, betaCatenin, E-cadherin and ZO-1 in colon tissue was detected by immunofluorescence and protein immunoblotting. The results showed that ( Fig.13 , Fig.14 , Fig.15 ), compared with the blank group, the protein expression of beta Catenin, E-cadherin, ZO-1 and Occludin in the UC group was significantly decreased, and WLJP025p significantly upregulated the related proteins. This indicates that WLJP025p treats UC by repairing the intestinal mechanical barrier.

[0092] Among them, the intestinal mechanical barrier is an important part of the intestinal defense system. Its main function is to physically block harmful substances from entering the body while allowing the absorption of necessary nutrients and water. Intestinal barrier proteins play a vital role in maintaining the tight junctions and overall barrier function of intestinal epithelial cells. These proteins not only prevent the invasion of harmful substances and pathogens, but also ensure the selective absorption of nutrients and water. The main intestinal barrier proteins include: tight junction proteins and adherens junction proteins. Tight junction proteins are located at the top of adjacent epithelial cells, forming a continuous barrier to prevent large molecules and pathogens from entering the body through the intercellular gap. The main tight junction proteins include ZO-1 (Zonula Occludens-1) and Occludin. Adhesion junction proteins are located below the tight junctions and maintain the structural stability of epithelial cells through intercellular adhesion. The main adhesion junction proteins include beta Catenin and E-cadherin. Mechanical barrier repair is an important process in the treatment of UC.

[0093] Example 4: Study on the effect of honeysuckle polysaccharide in CRC mouse model

[0094] Thirty 6-8 week old C57BL / 6J male mice (10 mice in each group) were selected and divided into Control, CRC and WLJP025p groups. Fig.16As shown. The colorectal cancer (CRC) mouse model was established by administering dextran sulfate sodium (DSS) and azoxymethane (AOM). Specifically, for the CRC model, mice were fed with drinking water containing 1.5% DSS in weeks 1, 3, 6, and 9, and were fed with ordinary drinking water at other times. AOM was administered by a single intraperitoneal injection at the end of the first week. The dosage of WLJP025p was 60 mg / kg, and the frequency of administration was once a day (week 3-week 11). The CRC mouse model induced by the combination of AOM and DSS is a widely used experimental model of inflammation-related colorectal cancer. This method can simulate the pathogenesis of human inflammation-related colorectal cancer, including inflammation, chronic atypical hyperplasia, and tumor formation. During the entire modeling period, changes in mouse weight, fecal occult blood, viscosity, etc. were recorded.

[0095] In ulcerative colitis mice, we found that WLJP025p had a good therapeutic effect, so we used honeysuckle polysaccharide in CRC mice to observe whether WLJP025p also has an improvement effect on colorectal cancer. Figure 17-20 It can be seen that by evaluating the number of colon tumors in CRC mice, the disease activity index (DAI) of mice, the atrophy of the colon of mice, and the degree of spleen enlargement in mice, we can draw the same conclusion as used to treat UC mice. WLJP025p improves tumor formation, increased disease activity index, colon atrophy, and spleen enlargement caused by AOM / DSS-induced CRC mice. From these basic indicators, it is clear that WLJP025p has an improvement effect on CRC mice.

[0096] WLJP025p has a certain repair effect on AOM / DSS-induced colon tissue pathological damage in CRC mice. Fig.21 The results showed that the tissue structure of the CRC group treated with AOM / DSS was severely damaged, such as disordered glandular arrangement of the intestinal epithelium, loss of crypts, damaged goblet cells, and infiltration of inflammatory cells. WLJP-025p significantly improved the damage to the colon tissue structure. Consistent with the UC animal experiment, it showed that WLJP-025p may effectively repair the intestinal barrier damage in mice and effectively improve colon tissue lesions, thereby improving colorectal cancer.

[0097] Comparative Example 1

[0098] The document "Research on the Mechanism of Honeysuckle in Preventing and Treating Ulcerative Colitis, Luo Mengxiong, Master's Thesis of Shandong University of Traditional Chinese Medicine, 2019" mentions the application of honeysuckle water extract in the treatment of ulcerative colitis. In this document, honeysuckle water extract is obtained according to the following method: weigh dry honeysuckle, crush the medicinal materials with a multifunctional Chinese medicine grinder, put them into a round-bottom flask, add pure water, reflux extraction, and after multiple refluxes, combine and concentrate the extracts. The concentrate is freeze-dried into powder by a freeze dryer to obtain honeysuckle water extract freeze-dried powder. In this scheme, the researchers focused on the following active ingredients: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, loganin, deoxyloganin, rutin, and isochlorogenic acid, and did not pay attention to the polysaccharide component at all. Although honeysuckle water extract has a certain effect in treating ulcerative colitis, it is not known from the results of this study whether the polysaccharide component of honeysuckle polysaccharide also has the function of treating ulcerative colitis. And according to the records in this document, those skilled in the art will start with active substances such as chlorogenic acid to further explore the effective ingredients that have the effect of treating ulcerative colitis. It is difficult to imagine whether honeysuckle polysaccharides with a larger molecular weight have the effect of treating ulcerative colitis, and it is even more difficult to speculate whether honeysuckle polysaccharides have the effect of treating colorectal cancer.

[0099] Before the present patent study, the inventors also conducted extensive explorations on different honeysuckle-related products. For example, the effects of honeysuckle water extract and honeysuckle crude polysaccharide on ulcerative colitis (UC) were compared. The effects of honeysuckle water extract (LJ-W) and honeysuckle crude polysaccharide (LJP) were evaluated by measuring mouse body weight, mouse disease activity index (DAI), mouse colon atrophy, mouse spleen enlargement and pathological indicators.

[0100] Honeysuckle crude polysaccharide was prepared by the following method: first, 95% ethanol was refluxed for 4 hours (3 times) to remove hydrophobic compounds, and then the residue (500 g) was extracted twice with hot distilled water (10 L, 100 ° C) (4 hours each time). The extract was filtered, combined and concentrated to 700 mL, and treated with 15% trichloroacetic acid at 4 ° C for 4 hours to remove protein. After neutralization, 75% anhydrous ethanol was added to precipitate the polysaccharide, which was collected and vacuum dried to obtain honeysuckle polysaccharide (LJP, yield 5.1%, w / w).

[0101] The honeysuckle water extract is prepared by the following method: weigh 500 g of dried honeysuckle, grind the medicinal materials with a multifunctional Chinese medicine grinder, put them into a round-bottom flask, add 20 times the amount of pure water, reflux and extract at 100° C. for 2 hours, filter, repeat the above steps twice for the residue, combine the filtrate, concentrate under reduced pressure, and freeze-dry the concentrate into powder by a freeze dryer to obtain honeysuckle water extract freeze-dried powder (LJP, yield 20%, w / w).

[0102] The recommended dosage of honeysuckle tea is about 5g, and the mouse experimental dosage of honeysuckle water extract, honeysuckle crude polysaccharide and WLJP-025p is calculated according to the recommended dosage. According to the water extract experience yield of 20%, the clinical equivalent dose of honeysuckle water extract is 20mg / kg, and the clinical equivalent dose is converted to a mouse dose of about 250mg / kg. The yield of honeysuckle crude polysaccharide is 5.1%, and the clinical equivalent dose of crude polysaccharide converted from honeysuckle is about 5mg / kg, which is a mouse dose of about 60mg / kg. Therefore, 250mg / kg of honeysuckle water extract, 60mg / kg of honeysuckle crude polysaccharide and 60mg / kg of WLJP-025p were selected for relevant experimental research, and the experimental method was the same as Example 2.

[0103] The study found that WLJP-025p had a more significant effect on the improvement of the main symptoms and pathological indicators of UC and CRC mice than honeysuckle water extract and honeysuckle crude polysaccharide. More specifically, WLJP-025p had a more significant effect on the disease activity index score (DAI) of UC mice than honeysuckle water extract and honeysuckle crude polysaccharide. Fig. 22 WLJP-025p improved the disease activity index (DAI) of UC mice induced by DSS, and its effect was better than that of honeysuckle water extract and honeysuckle crude polysaccharide.

[0104] WLJP025p compared the effects of honeysuckle water extract and honeysuckle crude polysaccharide on colon atrophy in UC mice. Fig.23 It can be seen that WLJP025p improved the colon atrophy of UC mice induced by DSS, and the effect was better than that of honeysuckle water extract (LJ-W) and honeysuckle crude polysaccharide (LJP).

[0105] WLJP025p compared the effects of honeysuckle water extract and honeysuckle crude polysaccharide on spleen enlargement in UC mice. Fig.24 It can be seen that WLJP025p improved the spleen enlargement induced by DSS in UC mice, and the effect was better than that of honeysuckle water extract (LJ-W) and honeysuckle crude polysaccharide (LJP).

[0106] WLJP025p compared the effects of honeysuckle water extract and honeysuckle crude polysaccharide on pathological damage in UC mice. Fig.25 WLJP025p improved the pathological damage of UC mice induced by DSS, and the effect was better than that of honeysuckle water extract and honeysuckle crude polysaccharide.

[0107] From the prior art, we only know that honeysuckle water extract has a certain effect on UC, but the ingredients in the water extract are complex, including chlorogenic acid, loganin and rutin, which are known to have good anti-inflammatory and antibacterial effects. Whether there is a dominant component in the water extract that has a therapeutic effect on UC is still unknown through the prior art. Honeysuckle crude polysaccharide is a product obtained by removing fat-soluble components, removing protein components and undergoing alcohol precipitation treatment on the basis of honeysuckle water extract. There is no significant improvement in the therapeutic effect of honeysuckle crude polysaccharide on UC compared to honeysuckle water extract. The honeysuckle polysaccharide component WLJP-025p provided in this scheme is a substance purified and separated from honeysuckle crude polysaccharide. WLJP-025p shows a very excellent and outstanding effect on the treatment of UC. It is a dominant component in honeysuckle that has a therapeutic effect on UC, which was not predicted by the inventor before the study (before this, there was no prior art report on which specific polysaccharide in the crude polysaccharide has the effect of treating UC). Polysaccharides are a class of high-molecular compounds composed of multiple monosaccharide molecules connected by glycosidic bonds. They have low toxicity and good tolerance. Compared with other active ingredients of honeysuckle, their safety is more ideal, and compared with other effective ingredients of honeysuckle, they have greater clinical application value.

[0108] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A use of honeysuckle polysaccharide in the preparation of a drug for treating ulcerative colitis and / or inflammation-related colorectal cancer.

2. The use of a honeysuckle polysaccharide according to claim 1 in the preparation of a medicament for treating ulcerative colitis and / or inflammation-related colorectal cancer, characterized in that: The honeysuckle polysaccharide is used for improving weight loss, increased disease activity index, colon atrophy and spleen enlargement caused by ulcerative colitis.

3. The use of a honeysuckle polysaccharide according to claim 2 in the preparation of a medicament for treating ulcerative colitis and / or inflammation-related colorectal cancer, characterized in that: The honeysuckle polysaccharide is used for improving inflammation caused by ulcerative colitis, for up-regulating the expression of intestinal barrier proteins, and for repairing the intestinal mechanical barrier.

4. The use of a honeysuckle polysaccharide according to claim 1 in the preparation of a medicament for treating ulcerative colitis and / or inflammation-related colorectal cancer, characterized in that: The honeysuckle polysaccharide is used for improving the elevated disease activity index, colon atrophy and spleen enlargement caused by colorectal cancer, for repairing colon tissue pathological damage caused by colorectal cancer, and for inhibiting the progression of colon tumors.

5. Use of a honeysuckle polysaccharide according to any one of claims 1 to 4 in the preparation of a medicament for treating ulcerative colitis and / or inflammation-related colorectal cancer, characterized in that: The monosaccharide composition of the honeysuckle polysaccharide is: 53.4% ​​to 80.2% of galacturonic acid, 8% to 12% of glucose, 11.7% to 17.5% of galactose, 6.6% to 9.8% of arabinose, and 0.3% to 0.5% of mannose.

6. Use of a honeysuckle polysaccharide according to claim 5 in the preparation of a medicament for treating ulcerative colitis and / or inflammation-related colorectal cancer, characterized in that: The honeysuckle polysaccharide is composed of α-(1→4)-D-GalpA connected in sequence to form a main chain; the side chains of the honeysuckle polysaccharide include β-(1→4)-linked galactan, α-(1→5)-linked and (1→3,5)-linked arabinan / arabinogalactan, and α-(1→4)-linked glucan.

7. Use of a honeysuckle polysaccharide according to claim 6 in the preparation of a medicament for treating ulcerative colitis and / or inflammation-related colorectal cancer, characterized in that: The relative average molecular weight of honeysuckle polysaccharide is 10-40 kDa; preferably, the relative average molecular weight of honeysuckle polysaccharide obtained by high performance liquid chromatography based on TSKgel G4000 PWxl gel exclusion chromatography column is 23±4 kDa.

8. Use of a honeysuckle polysaccharide according to claim 7 in the preparation of a medicament for treating ulcerative colitis and / or inflammation-related colorectal cancer, characterized in that: The honeysuckle polysaccharide is prepared by the following method: S1: After removing fat-soluble substances from honeysuckle, water is used as a solvent for decoction and extraction, and the supernatant is taken; after concentration, protein removal, and alcohol precipitation, the precipitate is collected; and after freeze-drying, the total sugar of honeysuckle is obtained; S2: Load the aqueous solution of total sugars from honeysuckle onto a DEAE-cellulose chromatography column, and use distilled water and 0.25 mol / L NaCl solution to elute the DEAE-cellulose chromatography column in sequence, and take the portion corresponding to the total sugar content peak and the uronic acid peak of the elution curve from the components eluted with the 0.25 mol / L NaCl solution; and then concentrate, dialyze and freeze-dry to obtain the honeysuckle polysaccharide WLJP-025 component; S3: The honeysuckle polysaccharide WLJP-025 component was dissolved in sodium chloride solution, loaded on a Sepharose CL-6B chromatography column, eluted with a 0.1 mol / L NaCl solution, and the eluate corresponding to the maximum total sugar content peak and uronic acid peak of the elution curve was taken; after concentration, dialyzation and freeze-drying, the honeysuckle polysaccharide WLJP-025p was obtained.

9. Use of a honeysuckle polysaccharide according to claim 8 in the preparation of a medicament for treating ulcerative colitis and / or inflammation-related colorectal cancer, characterized in that: In S2, the DEAE-cellulose chromatography column was eluted with 2 column volumes of distilled water and 0.25 mol / L NaCl solution, and the elution flow rate was 10 mL / min; in S3, 0.1 mol / L NaCl solution was used for elution at a flow rate of 0.1 mL / min.

10. Use of a honeysuckle polysaccharide according to any one of claims 1 to 4 in the preparation of a medicament for treating ulcerative colitis and / or inflammation-related colorectal cancer, characterized in that: The dosage of the honeysuckle polysaccharide for the ulcerative colitis mouse model or the colorectal cancer mouse model is 30-60 mg / kg.

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