Anti-tumor active component separated from sheep small intestine mucosa and method and application thereof

Abstract

The invention discloses an anti-tumor active component separated from sheep small intestine mucosa and a method and application thereof, and the method specifically comprises the following steps: cleaning waste sheep small intestine after animal slaughtering with normal saline, removing connective tissue and intestinal contents, scraping off the mucosa, pre-freezing the pretreated sheep small intestine mucosa, and freeze-drying the pre-frozen sheep small intestine mucosa to obtain the anti-tumor active component separated from the sheep small intestine mucosa. And grinding the freeze-dried product in a mortar to obtain sheep small intestine mucosa powder. Adding a phosphate buffer solution into the pretreated sheep small intestine mucosa powder in proportion, and performing low-temperature stirring extraction for 2-4 hours; and filtering the extracting solution with gauze or a screen, centrifuging, taking the supernatant, and freeze-drying to obtain the crude extract of the small intestinal mucosa of the sheep, wherein the yield is 36.11%. The crude extract is re-dissolved with deionized water and centrifuged, gel chromatography is used for desalination, purification is carried out, the component SI-2 with the antitumor activity is obtained, the half inhibition concentration (IC50) of the component to human colon cancer cells 29 (HT-29) is 0.786 + / -0.051 mg / mL, and the half inhibition concentration (IC50) of the component to human colon cancer cells 116 (HCT-116) is 0.095 + / -0.041 mg / mL.

Description

Technical Field

[0001] The invention relates to the development and application field of an anti-tumor component, and in particular to a method for separating and purifying an anti-tumor active component from sheep small intestinal mucosa and its application. Background Art

[0002] At present, the three major conventional treatments for malignant tumors are still surgical treatment, chemotherapy, and radiotherapy. Among them, chemotherapy is one of the main treatment methods for malignant tumors and is widely used. However, while chemotherapy kills cancer cells and achieves significant therapeutic effects, it also damages the body's normal cells, which reduces the quality of life of patients. In recent years, through a large number of clinical and basic experimental studies, it has been found that the combination of biological drugs and chemotherapy drugs for tumor internal medicine treatment can reduce the production of drug toxicity and side effects caused by chemotherapy to a certain extent. Since the 1940s, nearly 70 commonly used anti-tumor drugs have been put into clinical use, and there are more than 300 new anti-tumor drugs in the clinical trial stage. There are many anti-tumor drugs, but ideal anti-tumor drugs are rare. However, most anti-tumor drugs have the disadvantages of poor selectivity, strong toxic side effects, and easy to produce drug resistance. Therefore, the development of low-toxicity and high-homology drugs to prevent or treat early mild cancer is of great significance to human development.

[0003] Xinjiang is a major animal husbandry region in my country, with a large number of sheep slaughtered each year. However, most of the byproducts of sheep, such as sheep intestines, sheep brains, and sheep stomachs, are discarded and not used, resulting in a huge waste of resources. However, if these organs are used as raw materials to extract active substances that can be used for treatment, not only can the rational use of biological resources be achieved, but also the drugs of biological origin have high efficacy, small toxic side effects, high homology, low biosafety risks, are not easy to produce drug resistance, and have high yields. Using "Sheep Intestine Extraction" as the keyword, 48 related patents were retrieved, of which 7 patents used sheep intestine as raw material to extract active ingredients. Chinese patent applications CN114794311A and CN108260707A are methods for preparing sheep intestinal mucosal protein powder, specifically, adding water to fresh sheep intestinal membrane to adjust pH (glacial acetic acid and sodium hydroxide), adding enzymes, and then enzymolysis, extracting gauze filtration, decompression concentration, spray drying, and obtaining enzymolysis sheep intestinal membrane protein products. This patent process is cumbersome and complicated, and the cost required for extraction after adding enzymes is greatly increased, and it takes a long time. In summary, most of the patents that use sheep small intestine as raw material for extraction target products are heparin, heparin sodium and protein powder, all of which require enzymatic hydrolysis, which is time-consuming and costly. Searching with the keyword "anti-tumor component extraction" found CN113842401A, CN108553490A, CN103751225A, CN104800294B, CN103933125B, CN103006761B, CN101559084B, CN111135161A, CN112999208A, CN109232701B, CN113384604B, CN107029028B, CN102516344B, CN114042112A, CN106366169A, CN112321670A Patents CN106237265B, CN110713525A, CN104306410B, CN101972385B, CN111743972B, CN103272216B, CN115671157A, CN113116972B, CN105641000A, CN103230417B, and CN110507749B are all macromolecules or small molecules with anti-tumor activity extracted from plants or fungal organisms such as chamagu, imitation chestnut leaves, Cordyceps militaris, mountain root beans, sweet potatoes, and gray tree fungus; there are very few anti-tumor active components extracted from animals as raw materials. Patents CN103054911B, CN105326869A, and CN101856368B are anti-tumor components obtained by enzymatic protein hydrolysis of crocodiles. Summary of the invention

[0004] The purpose of the present invention is to provide an anti-tumor active component and method for isolating it from the mucosa of sheep small intestine. Most of the research objects of anti-tumor component extraction are plants, and the separated components are mostly secondary metabolites such as alkaloids, flavonoids, terpenes, organic acids, lignin, etc. The extraction object selected by the present invention is sheep small intestine. This raw material has not been systematically extracted and developed, and has a high homology. There is no potential threat and toxicity when applied to the fields of food, medicine, and health care products. In terms of social benefits, the extraction and separation of anti-tumor active components from the sheep small intestinal mucosa realizes the reuse of waste resources, and the recovery and treatment of by-products of mutton, sheep intestines, is conducive to resource reuse and environmental protection (direct incineration of by-products greatly pollutes the environment), fully enhances the inherent value of the resource, implements the social concept of sustainable development of resources, and actively responds to the call for green development and harmonious coexistence between man and nature; in terms of extraction and separation, the use of inexpensive phosphate buffer for extraction is compared with other methods. The reagents used are non-toxic, easy to purchase and inexpensive, maintain pH stability during the extraction process, and avoid denaturation or decreased activity of the extract due to pH changes. Low-temperature extraction effectively maintains the stable configuration and strong activity of the substance, and realizes the simultaneous enrichment of anti-tumor active components and desalting through one-step gel column chromatography, which greatly improves the efficiency of separation and purification. The process steps are simple, reproducible, and pollution-free, which is suitable for large-scale batch production by enterprises, and achieves the separation and purification of anti-tumor active ingredients with the most streamlined steps.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The invention discloses an antitumor active component separated from sheep small intestinal mucosa. The antitumor active component comprises, by mass percentage, a total sugar content of 5% to 30%, a protein content of 20% to 50%, a uronic acid content of 1% to 10%, and a molecular weight of 45 kDa to 75 kDa.

[0007] A method for separating antitumor active components from sheep small intestinal mucosa, the method is carried out according to the following steps:

[0008] 1) Wash the fresh sheep small intestine, scrape off the mucosa and freeze-dry it to obtain sheep small intestine mucosa freeze-dried powder;

[0009] 2) adding sodium chloride solution to the sheep small intestinal mucosa freeze-dried powder for full contact, then adding phosphate buffer and phenylmethylsulfonyl fluoride for extraction, filtering, centrifuging, and directly freeze-drying the supernatant to obtain a white powder, thereby obtaining a crude extract of sheep small intestinal mucosa;

[0010] 3) Based on the anti-tumor activity, the sample is loaded onto a G25 dextran gel column, and the anti-tumor active components are obtained after collection.

[0011] Preferably, in step 2), the extraction temperature is 4°C-15°C, the extraction time is 2-4 hours, the number of extractions is 2-4 times, and the stirring rate is 200r / min.

[0012] Preferably, in step 2), the pH of the phosphate buffer is 7.0-9.0, and the concentration is 0.01-0.1 mol / L.

[0013] Preferably, in step 3), the sample loading amount of the G25 dextran gel column is 300-800 mL, the eluent is ultrapure water, and the elution flow rate is 30-60 mL / min to obtain the SI-2 component.

[0014] Preferably, the SI-2 component is dialyzed with cold distilled water at a temperature of 4°C-15°C, and the dialyzation time is 48 hours through a dialysis bag of 1000-4000Da. The solution inside the dialysis bag is freeze-dried to obtain SI-2 freeze-dried powder, which is the anti-tumor active component.

[0015] The present invention also provides the use of the above anti-tumor active component or the anti-tumor active component obtained by the above method in the preparation of food, health care products or medicines.

[0016] Preferably, the drug is a drug for treating colon cancer, gastric cancer or lung cancer.

[0017] Preferably, the food or health product is an essential amino acid supplement for the human body.

[0018] The present invention further provides the use of the above anti-tumor active components in the preparation of medicines for inhibiting human intestinal cancer cells HCT-116, human gastric cancer cells HGC27, human intestinal cancer cells HT-29 or human lung cancer cells A549.

[0019] Specifically, the method for separating and purifying anti-tumor active components from sheep small intestinal mucosa of the present invention is carried out according to the following steps:

[0020] a. Wash the fresh sheep small intestine with clean water, remove the contents and digestion residues, scrape off the mucosa, and freeze-dry for 24 hours to obtain the sheep small intestine mucosa dry powder;

[0021] b. Grind the dry powder obtained in step a into a mortar, add 0.05M-0.45M sodium chloride solution at a low temperature of 4°C-15°C, stir for 5 minutes to allow the sheep small intestinal mucosa powder to fully contact the solution, then add 20mM phosphate buffer with a pH of 7.0-9.0 and 0.05mM phenylmethylsulfonyl fluoride, stir and extract for 2 hours, filter with gauze, and repeat the above steps for 2 times;

[0022] c. Collect the three extraction filtrates obtained in step b, centrifuge them together with the filtrate from the last extraction, and freeze-dry the supernatant to obtain a crude extract of sheep small intestinal mucosa;

[0023] d. Dissolve and centrifuge the crude extract of sheep small intestinal mucosa in step c, load the supernatant onto a G25 dextran gel column, the loading volume is 300-800 mL, the eluent is ultrapure water, the elution flow rate is 30-60 mL / min, and the absorption at ultraviolet 220 nm and 280 nm is detected, combined with conductivity and differential real-time values, to obtain two components, SI-1 and SI-2;

[0024] e. Directly freeze-dry the SI-1 component in step d to obtain SI-1 freeze-dried powder; dialyze with cold distilled water at 4° C., replace the dialysis solution once every 2 hours in a SI-2 dialysis bag (1000-4000Da), and dialysis for 48 hours, and then freeze-dry the solution inside the dialysis bag to obtain SI-2 freeze-dried powder;

[0025] f. The two components SI-1 and SI-2 obtained in step e are subjected to total sugar content, protein content, uronic acid content determination and anti-tumor activity screening experiments to obtain component SI-2 with anti-tumor activity.

[0026] The invention discloses a method for separating and purifying antitumor active components from sheep small intestinal mucosa and its application. The method takes sheep small intestinal mucosa as the research object. First, the fresh sheep small intestine is washed, the intestinal contents and digestion residues are removed, the mucosa is scraped off, and the sheep small intestinal mucosa freeze-dried powder is obtained. After grinding in a mortar, phosphate buffer is added for extraction, gauze filtering and centrifugation are performed, and the supernatant is freeze-dried. The sheep small intestinal mucosa extract is obtained by low-temperature stirring extraction, gauze filtering, centrifugation, dialyzing the supernatant, and freeze-drying to obtain white powder, and the sheep small intestinal mucosa extract is obtained, which is stored in a -20°C refrigerator for standby use; guided by the antitumor activity, the G25 dextran gel column is loaded, and the antitumor active components in the sheep small intestinal mucosa are obtained after collection. The detection and activity evaluation results show that the sheep small intestinal mucosa antitumor component SI-2 obtained by the method has a yield of 11.27%, a total sugar content of 8.64%, a protein content of 28.83%, and a uronic acid content of 1.48%. The anti-tumor cell inhibition experiment showed that the half-inhibitory concentration IC50 values ​​of SI-2 component for human colon cancer cells HT-29 and human colon cancer cells HCT-116 were 0.786±0.051 mg / mL and 0.095±0.041 mg / mL, respectively.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] Most of the research objects of anti-tumor component extraction are plants, and the separated components are mostly secondary metabolites such as alkaloids, flavonoids, terpenes, organic acids, lignin, etc., while the extraction object selected by the present invention is sheep intestine, which has not been systematically extracted and developed, and has high homology, and there is no potential threat and toxicity in the fields of food, medicine, and health care products. In terms of social benefits, the extraction and separation of anti-tumor active components from sheep intestine realizes the reuse of waste resources, fully enhances the self-value of the resource, implements the social concept of sustainable development of resources (direct incineration of waste sheep intestines is extremely destructive to the environment), and actively responds to the call for green development and harmonious coexistence between man and nature; in terms of extraction and separation, the extraction is carried out using a low-cost phosphate buffer solution to maintain pH stability during the extraction process, avoid the problem of denaturation or decreased activity of the extract due to pH changes, and the low-temperature extraction effectively maintains the stable configuration and strong activity of the substance, and realizes the enrichment of anti-tumor active components through one-step G25 polysaccharide gel column chromatography. The process is simple, reproducible, pollution-free, and suitable for large-scale mass production by enterprises. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The flowchart of the method for separating anti-tumor active components from sheep small intestinal mucosa of the present invention is as follows;

[0030] Figure 2 The chromatogram of the crude extract of sheep small intestinal mucosa separated by G25 dextran gel chromatography in the present invention; the SI-2 sample collected according to this figure is the target component of the anti-tumor activity of sheep small intestinal mucosa;

[0031] Figure 3 This is a 15% SDS-polyacrylamide gel silver-stained electrophoresis diagram of the anti-tumor active component SI-2 of the invention. The sample on the left is the SI-2 component of the dextran gel G25, and the sample on the right is the protein standard;

[0032] Figure 4 This is a phase diagram of the gel separation liquid of the anti-tumor active component SI-2 of the present invention (ultraviolet detector). The ultraviolet peaks at different times represent proteins of different molecular weights, indicating that SI-2 contains a variety of different proteins, and these proteins synergistically play an anti-tumor role;

[0033] Figure 5 This is the full-wavelength ultraviolet scan of SI-2 component. The presence of absorption peaks at 240nm-280nm indicates the presence of proteins and nucleic acids;

[0034] Figure 6 This is the infrared spectrum of the SI-2 component. The functional groups in the component are obtained based on the infrared wavenumber, and the composition of SI-2 is further determined. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0036] Example 1

[0037] like Figure 1 As shown, a method for isolating anti-tumor active components from sheep small intestinal mucosa, the method comprising the following steps:

[0038] a. Wash the fresh sheep small intestine with clean water, remove the contents and digestion residues, scrape off the mucosa, and freeze-dry for 24 hours to obtain the sheep small intestine mucosa dry powder;

[0039] b. Grind the dry powder obtained in step a into a mortar, add 0.15 M sodium chloride solution at 4°C, stir for 5 minutes to allow the sheep small intestinal mucosa powder to fully contact the solution, then add 20 mM phosphate buffer with a pH of 8.0 and 0.05 mM phenylmethylsulfonyl fluoride, stir and extract for 2 hours, filter with gauze, and repeat the above steps for 2 more times;

[0040] c. Collect the three extraction filtrates obtained in step b, combine them with the residue from the last extraction, and centrifuge them at room temperature for 10 minutes at a speed of 1440 rpm / min. The supernatant is freeze-dried to obtain a crude extract of sheep small intestinal mucosa;

[0041] d. The sheep intestinal mucosa crude extract in step c was dissolved and centrifuged, and the supernatant was loaded onto a G25 dextran gel column balanced with ultrapure water, with a column volume of 5.3 L (column inner diameter 100 mm, height 65 cm), a loading amount of 700 mL, an eluent of ultrapure water, and an elution flow rate of 50 mL / min. The absorption at ultraviolet 220 nm and 280 nm was detected, and the conductivity and differential real-time values ​​were combined to obtain two components, SI-1 and SI-2. The chromatogram of the sheep intestinal mucosa crude extract separated by G25 dextran gel chromatography is shown in FIG. Figure 2 As shown; the SI-2 sample collected according to this figure is the target component with anti-tumor activity in sheep small intestine;

[0042] e. Directly freeze-dry the SI-1 component in step d to obtain SI-1 freeze-dried powder; dialyze with cold distilled water at 4° C., replace the dialysis solution once every 2 hours in a SI-2 dialysis bag (1000-4000Da), and dialysis for 48 hours, and then freeze-dry the solution inside the dialysis bag to obtain SI-2 freeze-dried powder;

[0043] f. The two components SI-1 and SI-2 obtained in step e are subjected to total sugar content, protein content, uronic acid content determination and anti-tumor activity screening experiments to obtain component SI-2 with anti-tumor activity.

[0044] The liquid phase diagram of the anti-tumor active component SI-2 gel separation (ultraviolet detector) is as follows Figure 4As shown, the UV peaks at different times represent proteins of different molecular weights, indicating that SI-2 contains a variety of different proteins that synergistically exert anti-tumor effects.

[0045] Example 2

[0046] like Figure 1 As shown, a method for isolating anti-tumor active components from sheep small intestinal mucosa, the method comprising the following steps:

[0047] a. Wash the fresh sheep small intestine with clean water, remove the contents and digestion residues, scrape off the mucosa, and freeze-dry for 24 hours to obtain the sheep small intestine mucosa dry powder;

[0048] b. Grind the dry powder obtained in step a into a mortar, add 0.45 M sodium chloride solution at 7°C, stir for 5 minutes to allow the sheep small intestine powder to fully contact with the solution, then add 50 mM phosphate buffer with a pH of 7.8 and 0.05 mM phenylmethylsulfonyl fluoride, stir and extract for 2 hours, filter with gauze, and repeat the above steps for 2 times;

[0049] c. Collect the three extraction filtrates obtained in step b, combine them with the residue from the last extraction, and centrifuge them at room temperature for 10 minutes at a speed of 1440 rpm / min. The supernatant is freeze-dried to obtain a crude extract of sheep small intestinal mucosa;

[0050] d. Dissolve and centrifuge the crude extract of sheep small intestinal mucosa in step c, load the supernatant onto a G25 dextran gel column balanced with ultrapure water, with a column volume of 5.3 L (column inner diameter 100 mm, height 65 cm), a loading amount of 600 mL, an eluent of ultrapure water, and an elution flow rate of 30 mL / min, and obtain two components, SI-1 and SI-2, by detecting the absorption at ultraviolet 220 nm and 280 nm, combined with conductivity and differential real-time values;

[0051] e. The SI-1 component in step d was directly freeze-dried to obtain SI-1 freeze-dried powder, and dialyzed with cold distilled water at 4°C. The dialysis solution was changed once every 2 hours in a SI-2 dialysis bag (1000-4000Da). The dialysis time was 48 hours, and the dialysis solution was replaced 14 times. The solution inside the dialysis bag was freeze-dried to obtain SI-2 freeze-dried powder;

[0052] f. The two components SI-1 and SI-2 obtained in step e are subjected to total sugar content, protein content, uronic acid content determination and anti-tumor activity screening experiments to obtain component SI-2 with anti-tumor activity.

[0053] Example 3

[0054] like Figure 1 As shown, a method for isolating anti-tumor active components from sheep small intestinal mucosa, the method comprising the following steps:

[0055] a. Wash the fresh sheep small intestine with clean water, remove the contents and digestion residues, scrape off the mucosa, and freeze-dry for 24 hours to obtain the sheep small intestine mucosa dry powder;

[0056] b. Grind the dry powder obtained in step a into a mortar, add 0.25 M sodium chloride solution at 15° C., stir for 5 minutes to allow the sheep small intestinal mucosa powder to fully contact the solution, then add 50 mM phosphate buffer with a pH of 7.0 and 0.05 mM phenylmethylsulfonyl fluoride, stir and extract for 2 hours, filter with gauze, and repeat the above steps for 2 more times;

[0057] c. Collect the three extraction filtrates obtained in step b, combine them with the residue from the last extraction, and centrifuge them at room temperature for 10 minutes at a speed of 1440 rpm / min. The supernatant is freeze-dried to obtain a crude extract of sheep small intestinal mucosa;

[0058] d. Dissolve and centrifuge the crude extract of sheep small intestinal mucosa in step c, load the supernatant onto a G25 dextran gel column balanced with ultrapure water, with a column volume of 5.3 L (column inner diameter 100 mm, height 65 cm), a loading amount of 400 mL, an eluent of ultrapure water, and an elution flow rate of 20 mL / min, and obtain two components, SI-1 and SI-2, by detecting the absorption at ultraviolet 220 nm and 280 nm, combined with conductivity and differential real-time values;

[0059] e. The SI-1 component in step d was directly freeze-dried to obtain SI-1 freeze-dried powder, which was dialyzed with cold distilled water at 4°C. The dialysis solution was changed once every 2 hours in a SI-2 dialysis bag (1000-4000Da) for 48 hours. The solution inside the dialysis bag was then freeze-dried to obtain SI-2 freeze-dried powder;

[0060] f. The two components SI-1 and SI-2 obtained in step e are subjected to total sugar content, protein content, uronic acid content determination and anti-tumor activity screening experiments to obtain component SI-2 with anti-tumor activity.

[0061] Example 4

[0062] like Figure 1 As shown, a method for isolating anti-tumor active components from sheep small intestinal mucosa, the method comprising the following steps:

[0063] a. Wash the fresh sheep small intestinal mucosa with water, remove the contents and digestion residues, scrape the mucosa, and freeze-dry for 24 hours to obtain the sheep small intestinal mucosa dry powder;

[0064] b. Grind the dry powder obtained in step a into a mortar, add 0.55 M sodium chloride solution at 9°C, stir for 5 minutes to allow the sheep small intestinal mucosa powder to fully contact with the solution, then add 30 mM phosphate buffer with a pH of 7.4 and 0.05 mM phenylmethylsulfonyl fluoride, stir and extract for 2 hours, filter with gauze, and repeat the above steps for 2 times;

[0065] c. Collect the three extraction filtrates obtained in step b, combine them with the residue from the last extraction, and centrifuge them at room temperature for 10 minutes at a speed of 1440 rpm / min. The supernatant is freeze-dried to obtain a crude extract of sheep small intestinal mucosa;

[0066] d. Dissolve and centrifuge the crude extract of sheep small intestinal mucosa in step c, load the supernatant onto a G25 dextran gel column balanced with ultrapure water, with a column volume of 5.3 L (column inner diameter 100 mm, height 65 cm), a loading amount of 500 mL, an eluent of ultrapure water, and an elution flow rate of 50 mL / min, and obtain two components, SI-1 and SI-2, by detecting the absorption at ultraviolet 220 nm and 280 nm, combined with conductivity and differential real-time values;

[0067] e. The SI-1 component in step d was directly freeze-dried to obtain SI-1 freeze-dried powder, which was dialyzed with cold distilled water at 4°C. The dialysis solution was changed once every 2 hours in a SI-2 dialysis bag (1000-4000Da) for 48 hours. The solution inside the dialysis bag was then freeze-dried to obtain SI-2 freeze-dried powder;

[0068] f. The two components SI-1 and SI-2 obtained in step e are subjected to total sugar content, protein content, uronic acid content determination and anti-tumor activity screening experiments to obtain component SI-2 with anti-tumor activity.

[0069] Example 5

[0070] like Figure 1 As shown, a method for isolating anti-tumor active components from sheep small intestinal mucosa, the method comprising the following steps:

[0071] a. Wash the fresh sheep small intestine with clean water, remove the contents and digestion residues, scrape off the mucosa, and freeze-dry for 24 hours to obtain the sheep small intestine mucosa dry powder;

[0072] b. Grind the dry powder obtained in step a into a mortar, add 0.35 M sodium chloride solution at 8°C, stir for 5 minutes to allow the sheep small intestinal mucosa powder to fully contact with the solution, then add 35 mM phosphate buffer with a pH of 7.6 and 0.05 mM phenylmethylsulfonyl fluoride, stir and extract for 2 hours, filter with gauze, and repeat the above steps for 2 times;

[0073] c. Collect the three extraction filtrates obtained in step b, combine them with the residue from the last extraction, and centrifuge them at room temperature for 10 minutes at a speed of 1440 rpm / min. The supernatant is freeze-dried to obtain a crude extract of sheep small intestinal mucosa;

[0074] d. Dissolve and centrifuge the crude extract of sheep small intestinal mucosa in step c, load the supernatant onto a G25 dextran gel column balanced with ultrapure water, with a column volume of 5.3 L (column inner diameter 100 mm, height 65 cm), a loading amount of 300 mL, an eluent of ultrapure water, and an elution flow rate of 100 mL / min, and obtain two components, SI-1 and SI-2, by detecting the absorption at ultraviolet 220 nm and 280 nm, combined with conductivity and differential real-time values;

[0075] e. The SI-1 component in step d was directly freeze-dried to obtain SI-1 freeze-dried powder, which was dialyzed with cold distilled water at 4°C. The dialysis solution was changed once every 2 hours in a SI-2 dialysis bag (1000-4000Da) for 48 hours. The solution inside the dialysis bag was then freeze-dried to obtain SI-2 freeze-dried powder;

[0076] f. The two components SI-1 and SI-2 obtained in step e are subjected to total sugar content, protein content, uronic acid content determination and anti-tumor activity screening experiments to obtain component SI-2 with anti-tumor activity.

[0077] Example 6

[0078] The anti-tumor component SI-2 obtained in Examples 1-5 was subjected to component composition determination, and the average value of the 5 groups of data obtained was taken as the data in Table 1.

[0079] Experimental methods:

[0080] Protein content determination:

[0081] The protein content of SI-2 component was determined by 2,2-biquinoline-4,4-dicarboxylic acid disodium (BCA) method: accurately measure 2 mg / mL standard bovine serum protein solution and prepare 25-2000 μg / mL standard solution. Prepare the working solution according to the instructions of the BCA protein content determination kit. The BCA reagent is divided into reagent A and reagent B. For every 50 times the volume of reagent A, add 1 times the volume of reagent B, shake well and set aside. Measure 25 μL of standard solution of different concentrations into a 96-well plate, add 200 μL of BCA working solution respectively, oscillate slightly, mix thoroughly, incubate at 37°C in a constant temperature box for 30 min, and measure the absorbance of the solution at 562 nm using an enzyme marker. Draw a standard curve with the mass concentration of bovine serum albumin standard as the abscissa and the absorbance value corresponding to each concentration as the ordinate; accurately weigh 1.0 mg of sample, add 1 mL of distilled water to dissolve it into a 1 mg / mL sample solution, take 25 μL into a 96-well plate, and measure the absorbance value of the sample solution using the above-mentioned standard curve operation method; calculation shows that the average protein content in the SI-2 component is 29.01% ± 0.03.

[0082] Determination of total sugar content:

[0083] The total sugar content of SI-2 component was determined by anthrone-sulfuric acid method: 10 mg of glucose standard was accurately weighed, dissolved in distilled water, and fixed to 50 mL. The final concentration of the standard was 0.2 mg / mL. 0, 0.4, 0.8, 1.2, 1.6, and 2.0 mL were accurately pipetted into different test tubes; distilled water was added to make the volume of each tube reach 2 mL; 50 μL of the standard was accurately pipetted into a 1.5 mL microcentrifuge tube, and then 200 μL of 0.2% anthrone-sulfuric acid was added. After shaking, it was boiled in a 90°C water bath for 8 minutes. After the end, 200 μL of the solution was transferred to a 96-well plate and the absorbance was measured at 620 nm using an enzyme reader. The mass concentration of the glucose standard solution was used as the horizontal axis, the absorbance value corresponding to each concentration was used as the vertical axis, and distilled water was used as the blank control to prepare a standard curve. A sample solution with a concentration of 1 mg / mL was prepared. The absorbance was measured by the same standard curve operation method as above, and the total sugar content was calculated. The average total sugar content of SI-2 component was calculated to be 8.18% ± 0.02;

[0084] Determination of uronic acid content:

[0085] The total sugar content of SI-2 component was determined by sodium tetraborate method: 10.00 mg of galacturonic acid standard was accurately weighed, fully dissolved in distilled water, fixed to volume in a 10 mL volumetric flask, a 1 mg / mL solution was prepared, and the standard solution was diluted to 0.2 mg / mL. 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mL were placed in a 15 ml stoppered dry glass test tube, and water was added to 1 mL. 6.0 mL of sodium tetraborate sulfuric acid solution was added in sequence and mixed. Heat in a boiling water bath for 15 min, take out and cool to room temperature, add 80 μL of 0.15% m-hydroxybiphenyl solution, mix, stand at room temperature for 30 min, and measure the absorbance at 525 nm with a UV spectrophotometer. The standard curve was prepared with the mass concentration of the standard solution as the horizontal axis, the absorbance as the vertical axis, and distilled water as the blank control. Weigh 10.00 mg of sample accurately, dissolve it in distilled water, and dilute to 10 mL to make the sample solution concentration 1 mg / mL. Measure the absorbance using the same standard curve operation method as above. Calculation shows that the average uronic acid content in SI-2 fraction is 2.12% ± 0.01.

[0086] Table 1 Chemical properties of antitumor active components of sheep small intestine

[0087]

[0088] From Table 1, we can get:

[0089] The anti-tumor active component SI-2 contains protein, polysaccharide, and uronic acid. It is a type of anti-tumor active component containing biological macromolecules with high homology and good solubility. It can be used in the fields of food, health products, and medicine.

[0090] Example 7

[0091] The molecular weight of the anti-tumor active components obtained in Examples 1-5 was estimated by electrophoresis. The positions of the electrophoresis bands in the five examples were basically the same, indicating that they were all proteins of the same molecular weight. The electrophoresis pattern with clear bands was selected as Figure 3 .

[0092] Experimental methods

[0093] The molecular weight of SI-2 was estimated by sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE). A 4% stacking gel and a 15% separation gel were used. 10 μL SI-2 sample and 3.5 μL molecular weight marker were added to different lanes of the gel under reducing conditions for analysis. The stacking gel was run at 75V and the separation gel was run at 130V until the sample reached the bottom of the separation gel. Sensitive silver staining was used, and the electrophoresis gel was added to the fixative and fixed on a shaker (55 rpm / min) for 30 minutes, then the fixative was replaced, and fixed a total of 3 times. After fixation, the gel was washed with water twice, 10 minutes each time. Prepare 0.8mmol / L sodium thiosulfate sensitizer, wash twice with water after 1 minute, add 0.012mol / L silver nitrate solution and stain on a shaker (55rpm / min) in the dark. After staining for 1 hour, rinse the gel with water for 10 seconds, add color developer to develop color, soak the color developer for 30 minutes, observe the bands appear clear, stop color development, add terminator to stop color development, and take electrophoresis photos. The molecular weight of SI-2 component is estimated by comparing with the molecular weight marker bands. The electrophoresis diagram is shown in the figure. Figure 3 shown.

[0094] Figure 3 The molecular weight of SI-2 was shown to be between 50 kDa and 70 kDa.

[0095] Example 8

[0096] The active component SI-2 obtained in Example 1-5 was subjected to full-wavelength ultraviolet scanning:

[0097] Experimental methods:

[0098] Accurately weigh 1 mg of SI-2 sample, add 1 mL of ultrapure water, mix well, filter with a 0.45 micron filter, and perform full-wavelength UV scanning on the transparent solution. Set the wavelength to 190nm-600nm for UV scanning. Since the 190-200nm region is the fingerprint spectrum region, it has no characteristics and high and messy absorbance, and the 400nm-600nm region is flat and has no absorption, the UV absorption at 200nm-400nm is selected for plotting, as shown in Figure 1. Figure 5 The samples obtained in Examples 1-5 have the same ultraviolet full wavelength scans. The absorption peaks at 240nm-280nm in the figure indicate that the SI-2 sample is absorbed by proteins and nucleic acids, because the benzene ring structures such as tyrosine, tryptophan, and phenylalanine in protein molecules have the maximum absorption peak at the ultraviolet wavelength of 280nm.

[0099] Example 9

[0100] The active component SI-2 obtained in Example 1-5 was subjected to Fourier transform-infrared scanning:

[0101] Experimental methods:

[0102] Accurately weigh 0.01 mg of SI-2 sample, add 10 mg of potassium bromide powder dried to constant weight, grind in an agate mortar until no larger particles appear, place in a tablet pressing mold, set the pressure to 9 MPa and keep for 5 minutes to obtain a transparent thin sample sheet, and perform Fourier transform-infrared scanning in the scanning range of 400-4000 cm -1 ; Background scanning times: 32 times; Resolution: 4.0cm -1 ; Detector; DTGS, the spectra obtained by infrared scanning of the samples obtained in Examples 1-5 are basically the same. The functional group information provided by the spectra was integrated and analyzed, and the results are shown in Table 2.

[0103] Table 2 Infrared wave numbers corresponding to functional groups

[0104]

[0105] Figure 6 For infrared spectrum. 3000cm -1 ~3500cm -1 The broad and strong absorption peaks in the region are caused by the stretching vibrations of OH and NH, indicating that both -NH2 and -OH exist in SI-2. The wave number is 2930.34 cm -1 The absorption peak near 1650cm is -CH3 stretching vibration. -1 and 1538cm -1 The double peak absorption indicates that the groups connected to -C=O are asymmetric, and the -CH3 stretching vibration indicates the presence of acetylamino. -1 It is -CH bending vibration. Fingerprint region for fine structure identification is 900cm -1 ~1280cm -1 , 1241cm -1 and 1096cm -1 Indicates the presence of a pyranose ring. 857cm -1 Indicates the presence of α-glycosidic bonds. In summary, the infrared data showed that SI-2 fraction contained proteins and polysaccharides.

[0106] Example 10

[0107] The active component SI-2 obtained in Examples 1-5 was tested for its tumor cell growth inhibition activity:

[0108] Experimental methods:

[0109] The cytotoxic activity of the samples was determined by MTT colorimetry with doxorubicin as a positive control; the cells in the logarithmic growth phase were digested and resuspended, and then inoculated into a 96-well plate at a corresponding cell density, 100 μL of cell solution was added to each well, and after culturing in a cell culture incubator at 37°C, 5% CO2 and 95% humidity for 24 hours, different concentrations of the tested monomer compound were added, 20 μL per well, the supernatant was discarded, and 100 μL of different concentrations of the compound (prepared with a culture medium containing a mycoplasma remover and filtered with a 0.45 μm filter membrane) was added;

[0110] Set up 3-6 duplicate wells; set up a cell-free background group, a solvent control group and a positive control group; continue to culture at a temperature of 37°C and 5% CO2 for 48 hours, then discard the supernatant, add 100 μL / well of thiazolyl blue (MTT) solution, and continue to culture for 2-4 hours; discard the supernatant, add 150 μL of dimethyl sulfoxide (DMSO) to each well, and oscillate at a low speed on a shaker for 10 minutes to fully dissolve the crystals; after the formazan is dissolved, use an enzyme marker to detect the light absorption value (OD) of each well at 570nm; then calculate the inhibition rate of the compound on the growth of different cancer cells and the half inhibition concentration IC 50 value;

[0111] Calculation formula:

[0112] Cell viability percentage % = (compound OD-blank OD / control group OD-blank OD) × 100%

[0113] Cell inhibition rate % = 1-cell viability % = [1-(compound OD-blank OD / control group OD-blank OD)] × 100%, IC 50 .

[0114] The inhibition rates of the five SI-2 components obtained in Examples 1-5 on different cancer cells obtained by MTT colorimetry were averaged, and the data are shown in Table 3 below:

[0115] Table 3 Average inhibition rates of SI-2 components on different cancer cells

[0116]

[0117] Embodiment 11

[0118] Amino acid analysis of the active component SI-2 obtained in Example 1-5:

[0119] Experimental methods:

[0120] Sample acid hydrolysis: 8 mg of sample was placed in a 20 ml screw cap (with built-in septum) headspace bottle, 10 ml of 0.1% phenol and 6 mol / l hydrochloric acid aqueous solution were added, the cap was tightened, the mixture was shaken and mixed, and the reaction was carried out at 110°C for 24 h. After the reaction was completed, the reaction solution was transferred to a 100 ml distillation bottle, and the reaction solution was evaporated to near dryness under reduced pressure at 75°C to remove the residual hydrochloric acid in the reaction solution. Then, 12 ml of 0.2 mol / l hydrochloric acid aqueous solution was used to dissolve the residue three times, and the residue was transferred to a 20 ml glass stoppered graduated test tube, and then the volume was made up to 20 ml with pure water for derivatization;

[0121] Phenyl isothiocyanate derivatization: Take 200 μl of the sample solution after acid hydrolysis, place it in a 1.5 ml centrifuge tube, add 100 μl of 1 mol / l triethylamine acetonitrile solution and 100 μl of 0.2 mol / l phenyl isothiocyanate acetonitrile solution, mix well, react at room temperature for 1 hour, then add 400 μl of n-hexane, tighten the lid and shake vigorously for 5-10 seconds, let it stand to separate, take 200 μl of the lower layer solution and mix it with 800 μl of water, filter it with a 0.22 μm needle filter, and wait for analysis;

[0122] HPLC amino acid composition analysis: According to the instruction manual of Diamonsil AAA amino acid analyzer, the derivatized amino acid mixed standard and sample were analyzed by HPLC respectively, and the types of amino acids contained in the sample were determined by comparing the peak elution time of the spectrum obtained from the sample with the peak elution time of different amino acids in the mixed standard; the relative molar ratio of amino acids contained in the sample was determined by comparing the peak area ratio of each peak in the sample spectrum; the results are shown in Table 4 below:

[0123] Table 4 Amino acid analysis of SI-2 components

[0124] Amino acid name Molar Ratio Aspartic acid 11.86％ Glutamate 9.39％ Serine 5.92％ Glycine 13.63％ Histidine 1.60％ Arginine 0.77％ Threonine 2.10％ Alanine 10.61％ Proline 4.19％ Tyrosine 3.15％ Valine 9.24％ Methionine 1.71％ Cystine 0.25％ Isoleucine 3.43％ Leucine 6.22％ Phenylalanine 4.06％ Lysine 11.87％ Tyrosine 3.15％

[0125] It can be seen from Table 4 that the content of essential amino acids is 38.63%, which can be used as food or health care products to supplement essential amino acids for the human body; the highest content of SI-2 components is glycine, which is a semi-essential amino acid, which can improve the growth and proliferation of small intestinal epithelial cells and the ability to resist oxidative stress in newborn mammals, and promote the development of small intestinal mucosa. At the same time, adding glycine to the diet can improve the growth performance of newborn and weaned mammals, promote intestinal development and health, indicating that glycine has the ability to repair the intestinal mucosal barrier, and the intestinal mucosal barrier is related to intestinal diseases, so SI-2 components have the potential to prevent or early treat intestinal diseases.

[0126] The sheep intestinal mucosal glycoprotein obtained by the invention can be applied to the research and development of medicines, health products and foods, and has great reference value for the development and utilization of sheep small intestine resources.

[0127] The present invention discloses a method for separating antitumor active components from sheep intestinal mucosa and its application. The method uses phosphate buffer to extract sheep intestinal mucosa freeze-dried powder to obtain a sheep intestinal mucosa crude extract; after freeze drying, the sample is loaded on a dextran gel G25 column for desalting and purification to obtain two components, and antitumor activity screening experiments are performed separately to obtain a component with antitumor activity SI-2, with a yield of 11.27%, and a molecular weight of the component between 50kDa and 70kDa. The component contains protein, polysaccharide and uronic acid through component detection, and its antitumor activity is evaluated. The inhibitory activity of the sample on HT-29 and HCT-116 cells is detected, and the results show that IC 50 The separation and preparation method of the present invention is simpler, greatly saves manpower and material resources, obtains components with good anti-tumor activity, and provides a scientific basis for the subsequent application of related drugs based on sheep small intestine.

[0128] The process parameters (such as temperature, time, etc.) of the present invention can realize the method by taking upper and lower limits and interval values, and the embodiments are not listed one by one here.

[0129] Any contents not described in detail in the present invention can be based on the conventional technical knowledge in the art.

[0130] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention is described in detail with reference to the embodiments, it should be understood by those skilled in the art that any modification or equivalent replacement of the technical solutions of the present invention does not depart from the spirit and scope of the technical solutions of the present invention and should be included in the scope of the claims of the present invention.

Claims

1. An anti-tumor active component isolated from sheep small intestinal mucosa, characterized in that: The anti-tumor active component comprises a total sugar content of 5% to 30%, a protein content of 20% to 50%, a uronic acid content of 1% to 10%, and a molecular weight of 50 kDa to 70 kDa.

2. A method for isolating antitumor active components from sheep small intestinal mucosa, the method comprising the following steps: 1) Wash the fresh sheep small intestine, scrape off the mucosa and freeze-dry it to obtain sheep small intestine mucosa freeze-dried powder; 2) adding sodium chloride solution to the sheep small intestinal mucosa freeze-dried powder to fully contact it, then adding phosphate buffer and phenylmethylsulfonyl fluoride for extraction, filtering with gauze, taking the supernatant after centrifugation and directly freeze-drying to obtain a white powder, thereby obtaining a crude extract of sheep small intestinal mucosa; 3) Based on the anti-tumor activity, the sample is loaded onto a G25 dextran gel column, and the anti-tumor active components are obtained after collection.

3. The method for separating antitumor active components from sheep small intestinal mucosa according to claim 2, characterized in that: In step 2), the extraction temperature is 4° C.-15° C., the extraction time is 2-4 hours, and the extraction times are 2-4 times.

4. The method for separating antitumor active components from sheep small intestinal mucosa according to claim 2, characterized in that: In step 2), the pH of the phosphate buffer is 7.0-9.0, and the concentration is 0.01-0.1 mol / L.

5. The method for separating antitumor active components from sheep small intestinal mucosa according to claim 2, characterized in that: In step 3), the sample loading amount of the G25 dextran gel column is 300-800 mL, the eluent is ultrapure water, and the elution flow rate is 30-60 mL / min to obtain the SI-2 component.

6. The method for separating antitumor active components from sheep small intestinal mucosa according to claim 5, characterized in that: The SI-2 component is dialyzed with cold distilled water at a temperature of 4°C-15°C, and desalted by dialyzing through a 1000-4000Da dialysis bag. The solution inside the dialysis bag is then freeze-dried to obtain SI-2 freeze-dried powder, which is the anti-tumor active component.

7. Use of the antitumor active component according to claim 1 or the antitumor active component obtained by the method according to any one of claims 2 to 6 in the preparation of medicines, foods or health products.

8. The use according to claim 7, characterized in that: The medicine is a medicine for treating colon cancer, gastric cancer or lung cancer.

9. The use according to claim 7, characterized in that: The food or health product is an essential amino acid supplement for the human body.

10. Use of the antitumor active component of claim 1 or the antitumor active component obtained by the method of any one of claims 2 to 6 in the preparation of drugs for inhibiting human intestinal cancer cells HCT-116, human gastric cancer cells HGC27, human intestinal cancer cells HT-29 or human lung cancer cells A549.

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