Anti-tumor active component separated from sheep placenta as well as method and application of anti-tumor active component

Abstract

The invention discloses an anti-tumor active component separated from sheep placenta and a method and application thereof, and the method specifically comprises the following steps: firstly, cleaning waste sheep placenta after animal slaughtering with normal saline to remove bloody fat and connective tissue; pre-freezing, mincing and freeze-drying the pretreated sheep placenta, and grinding the freeze-dried product in a mortar to obtain sheep placenta powder. Adding a phosphate extracting solution into the sheep placenta powder in proportion, stirring and extracting at low temperature for 2-4 hours, filtering the extracting solution through a vibrating screen, centrifuging, taking supernatant, and freeze-drying to obtain a sheep placenta crude extract; and redissolving the crude extract with deionized water, centrifuging, taking the supernatant, and carrying out Sephadex-G-25 gel chromatographic column chromatography to obtain the anti-tumor active component. The method has the advantages of simple preparation process, low cost, high efficiency, good repeatability, stable property and the like. The obtained active component is good in anti-tumor activity, can be used in the field of medicine and food health care, and is suitable for industrial popularization in medicine and food health care.

Description

Technical Field

[0001] The invention relates to the development and application field of sheep placenta, and in particular to an anti-tumor active component separated from sheep placenta and a method and application thereof. Background Art

[0002] Placenta is a commonly used Chinese medicine. It is a transitional organ for mother-child exchange of substances during pregnancy in metatherians and eutherians, formed by the embryonic membrane and the maternal endometrium. It is composed of the amnion, chorionic villus and basal decidua. Human placenta is also called "placenta". The Compendium of Materia Medica states that it has the effects of "calming the mind, nourishing blood, replenishing qi, benefiting essence, detoxifying and replenishing blood". It has a miraculous effect on "fatigue, emaciation and weakness". "Those who take it for a long time will have sharp ears and eyes, black hair and beard, and prolong life, which is like taking away the power of nature". Although human placenta extracts have high medicinal value, human placenta is prohibited from being bought and sold, and can only be donated, and the source is limited.

[0003] Sheep placenta has a similar nutritional structure and pharmacological effects to human placenta, and can be used as the preferred substitute for animal placenta. Xinjiang has rich sheep placenta resources due to its unique geographical and cultural advantages. After a large amount of data retrieval, the active substances extracted from sheep placenta as raw materials mainly include: small molecule peptides such as antioxidant, anti-aging and immunomodulatory peptides, as well as a few enzyme compounds such as superoxide dismutase, lactate dehydrogenase and alkaline phosphatase. Relevant patents were searched with the keyword "sheep placenta extraction", and a total of 39 valid and under-examination related patents were obtained. At present, the products using sheep placenta as a peptide source are mainly oral liquids, and most of the patents are focused on research on anti-aging, immunomodulatory peptides, etc. Among them, patent publication number CN 112057409 A uses fresh sheep placenta raw materials and taurine as an extraction solvent to extract sheep placenta extract with anti-aging function. Patent Publication No. CN 109943614A uses acidic protease, neutral protease and alkaline protease to enzymatically hydrolyze boiled sheep placenta to prepare sheep placenta active peptides, and uses it as an active additive for cosmetics; Patent Publication No. CN111972661 A uses sheep placenta as raw material for enzymatic hydrolysis, and mixes the extract after enzymatic hydrolysis with wolfberry extract to prepare sheep placenta wolfberry product; Patent Publication No. CN 114592024 A uses sheep placenta as raw material and adopts enzymatic hydrolysis to prepare sheep placenta antioxidant polypeptides; Patent Publication No. CN 110613128 A obtains high-purity sheep placenta powder by low-temperature enzymatic hydrolysis, multiple filtration and freeze-drying to remove odor; Patent Publication No. CN 106434803 A uses sheep placenta scraps as raw materials, enzymolysis by papain, and then separation and purification by macroporous adsorption resin, ultrafiltration membrane, dextran gel chromatography and semi-preparative RP-HPLC reverse phase high performance liquid chromatography, and freeze-drying after collection to obtain sheep placenta polypeptide with high antioxidant activity; Patent Publication No. CN115300443A uses combined enzyme preparation enzymolysis method and stress yeast fermentation to obtain sheep placenta with high antioxidant capacity for cosmetic products; Patent Publication No. CN114832017 A extracts sheep placenta by ultrasound-assisted homogenization, and then ultrafiltration segmentation of the filtrate, and it is found that the sheep placenta extract with a molecular weight greater than 50kDa has a significant effect of inhibiting the proliferation of colon cancer cells, and has good safety, and can be used to prepare anti-tumor drugs; Patent Publication No. CN

[0004] 114886925A found that the sheep placenta extract extracted by ultrasound-assisted homogenization has a significant therapeutic effect on the mouse cachexia model; the extraction methods of sheep placenta are mainly concentrated on homogenization, buffer extraction, ultrasound and enzymatic hydrolysis. The common problems are that the extraction method is not suitable for large-scale production, the enzymatic preparation of small molecule peptides is difficult to control, the process is unstable and organic solvent pollution. At present, the pharmacological effects of sheep placenta extracts are mainly reported in anti-oxidation, anti-fatigue and immunity, while there are fewer reports on anti-tumor. Therefore, finding a process suitable for industrial extraction of active compounds from sheep placenta and developing the medicinal value of sheep placenta in anti-tumor is of great significance for the production and application of sheep placenta products, and is also of great significance for making full use of Xinjiang's animal husbandry resources. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-tumor active component and method for extracting and separating from sheep placenta. The preparation process of the present invention is simple, and the reagents used are common reagents that are easy to purchase and non-toxic. Compared with the currently patented extraction method, a new pre-treatment method and mixed phosphate buffer solution extraction, column chromatography separation and purification are used for the first time, which greatly reduces the extraction cost while improving production safety. The prepared anti-tumor site has good activity and stable properties. The entire extraction process is short in time, low in cost, and highly repeatable, and is suitable for large-scale industrial production. The anti-tumor active site of sheep placenta obtained by the method of the present invention has been confirmed by in vitro anti-tumor activity experiments to have excellent inhibitory effects on the growth of cancer cells. The IC of the F2 component against HCT-116 cancer cells is 50 The value is 183±34ug / ml, and the IC of F3 component against HCT-116 cancer cells 50 The value is 95±11ug / ml.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] The present invention provides two anti-tumor active components separated from sheep placenta, wherein the molecular weight of the anti-tumor active component F2 is distributed between 10-20 kDa (such as about 15 kDa) and 50-70 kDa, wherein: the protein mass content is 18.7%-19.8%, the sugar mass content is 4.95%-56%, and the uronic acid mass content is 5.9%-6.6%. The molecular weight of the anti-tumor active component F3 is distributed between about 15 kDa, 35 kDa, 50 kDa, 70 kDa and greater than 130 kDa, wherein: the protein mass content is 23.7%-24.4%, the sugar mass content is 5.12%-5.82%, and the uronic acid mass content is 11.2%-12.4%.

[0008] The present invention also provides a method for separating anti-tumor active components from sheep placenta, the method comprising the following steps:

[0009] 1) washing the sheep placenta, removing blood stains, mincing and freeze-drying, and grinding to obtain sheep placenta powder;

[0010] 2) mixing sheep placenta powder and phosphate extract at a solid-liquid ratio of 1:20-1:50 g / mL, extracting, filtering, and freeze-drying to obtain a sheep placenta crude extract;

[0011] 3) The crude extract of sheep placenta was separated and purified by Sephadex-G-25 gel column to obtain the anti-tumor active components.

[0012] Preferably, the phosphate extract comprises: 0.5%-3% sodium chloride, 10-100 mM phosphate, and 0.5-1.0 mM serine protease inhibitor.

[0013] Preferably, the serine protease inhibitor is phenylmethylsulfonyl fluoride.

[0014] Preferably, in step 2), the extraction temperature is 4°C-10°C, the extraction time is 1-3h, and the extraction times are 2-5 times.

[0015] Preferably, in step 3), the separation conditions of the Sephadex-G-25 gel column are:

[0016] The crude extract of sheep placenta was dissolved in purified water, and the supernatant was taken as the loading solution by centrifugation. The purified water was used to wash and balance the Sephadex-G-25 gel column. Purified water was used as the eluent, and the flow rate was 3-5 mL / min. The protein absorption of the eluent was detected at 280 nm and 220 nm, and the eluent was collected by ultraviolet absorption. The salt-containing part F2 component was dialyzed for 48 hours with a 3.5 kDa dialysis bag and then freeze-dried. The salt-free components F1 and F3 were directly freeze-dried to obtain 3 components, which were named F1-F3, among which F2-F3 were anti-tumor active components.

[0017] The present invention further provides the use of the above anti-tumor active component in the preparation of medicines for inhibiting the growth of tumor cells.

[0018] Preferably, the drug is a drug for treating colon cancer, gastric cancer or lung cancer.

[0019] The present invention also provides the use of the above anti-tumor active components 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.

[0020] Specifically, the method for preparing the effective fraction with anti-tumor activity in sheep placenta of the present invention is carried out according to the following steps:

[0021] Extraction of Sheep Placenta Extract:

[0022] a. Take fresh sheep placenta, remove blood, fat and connective tissue with physiological saline, freeze-dry at -80°C and 25 MPa for 48 hours to obtain freeze-dried powder, and grind the freeze-dried powder in a mortar to obtain sheep placenta powder;

[0023] b. Add the sheep placenta powder obtained in step a to a phosphate extract with a pH of 7.4 (wherein the extract contains 0.5%-3% sodium chloride, 0.01-0.1M phosphate (the phosphate is a conventional phosphate used to prepare phosphate buffer, such as Na2HPO4, KH2PO4), and 0.5-1.0mM phenylmethylsulfonyl fluoride) at a material-liquid ratio of 1:20-1:50 g / mL at 4°C-10°C, stir and extract for 2h, repeat 3 times, filter with gauze, centrifuge and combine the extracts;

[0024] c. freeze-drying the extract obtained in step b at a temperature of -80°C and a pressure of 25 MPa for 48 hours to obtain a crude sheep placenta extract;

[0025] Preparation of anti-tumor active fractions from sheep placenta:

[0026] d. Dissolve the crude extract of sheep placenta obtained in step c with purified water and centrifuge at 6000rpm for 10min. Take the supernatant and load it onto a Sephadex-G-25 gel chromatography column at a flow rate of 4mL / min, and elute with purified water. The eluate is tested for protein absorption at 280nm and 220nm, and the eluate is collected. The salt-containing part F2 component is dialyzed for 48h with a 3.5kDa dialysis bag and then freeze-dried. The salt-free components F1 and F3 are freeze-dried separately to obtain three components, which are named F1-F3 respectively.

[0027] e. The protein content of the three protein components obtained in step d was determined by the BCA method, the neutral sugar was determined by the phenol-sulfuric acid method, the uronic acid content was determined by the m-hydroxybiphenyl colorimetric method, and the inhibition rate of F1-F3 components on tumor cells was determined by the MTT method.

[0028] The invention discloses an anti-tumor active component separated from sheep placenta, wherein the obtained active parts are F2 and F3 components obtained by Sephadex-G-25 gel column chromatography; in vitro anti-tumor activity experiments confirm that the sheep placenta active components F2 and F3 extracted by this method have the effect of inhibiting the growth of different cancer cells, thus providing a scientific basis for the use of sheep placenta extract in treating cancer.

[0029] The present invention firstly washes the sheep placenta with physiological saline to remove blood stains, minces it, freeze-dries it, and grinds it in a mortar to obtain sheep placenta powder; the sheep placenta powder is stirred and extracted for 2 hours in a temperature range of 4°C-10°C with a pH=7.4 phosphate extract (0.5%-3% sodium chloride, 0.01-0.1M phosphate, 0.5-1.0mM phenylmethylsulfonyl fluoride), extracted 3 times, filtered with gauze, combined with the filtrate, and freeze-dried to obtain a sheep placenta crude extract; the sheep placenta crude extract is separated and purified by Sephadex-G-25 gel column to obtain an anti-tumor active fraction. The sheep placenta extract extracted by the present invention has an extraction rate of 43.2±0.6%; the anti-tumor active fraction obtained by Sephadex-G-25 gel column chromatography is 4.53±1.2% and 3.23±0.8%.

[0030] The present invention can take advantage of animal husbandry resources (especially in Xinjiang), realize the full and reasonable reuse of animal placenta resources, improve the added value of sheep placenta, avoid the extreme waste of the resource, and turn waste into treasure. Compared with other patents, the present invention has "three new features": 1. The sample pretreatment method is new: the extraction samples selected by other patents are mostly fresh placental tissue homogenates and then directly added to the extract or ultrasonic-assisted extraction, while the present invention uses the placenta minced and freeze-dried powder extraction. First, the insoluble matter and blood stains of the placenta are completely cleaned and removed, and the impurities do not affect the subsequent extraction; secondly, the placenta powder after freeze-drying is more conducive to the long-term preservation of the raw materials. In addition, the specific surface area of ​​the powder in contact with the extract is large, the protein can better enter the extract, stretch and disperse in the extract, and the extraction rate is higher than the homogenate; 2. The sample extraction method is new: the extraction methods of other patents are relatively complicated, the extraction cost is long, and it is only suitable for laboratory production. The extraction solvent selected by the present invention is a phosphate buffer solution with low cost and mild conditions, which avoids the use of organic reagents, ensures the safety of the extraction process, and the process is simple, convenient and easy to operate, and is suitable for industrial mass production; 3. New application research: Sheep placenta extract is a type of extract with multiple biological activities. The main application research aspects reported in previous studies are anti-oxidation, anti-aging, anti-fatigue and immune effects, and the present invention finds that it has obvious effects in anti-tumor. The present invention uses sheep placenta as raw material and combines it with modern biopharmaceutical technology to extract and prepare effective parts with anti-tumor activity from sheep placenta, filling the gap in extraction, separation and purification using sheep placenta as raw material, and providing strong theoretical support and basis for the medical and biological utilization of sheep placenta extract.

[0031] The invention has simple process, low cost, high safety, good repeatability, stable process, and is suitable for application in the fields of medicine and food and health care products. The extracted and prepared active fraction has a good in vitro inhibitory effect on cancer cell growth, can be applied to cancer prevention or early treatment of mild cases, and has broad prospects and far-reaching value in the field of medicine and health.

[0032] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0033] The method of the present invention is guided by protein content and activity, and uses phosphate buffer to extract biomacromolecules. Compared with the traditional homogenate extraction method, organic buffer extraction and freeze-thaw method, it not only has mild extraction conditions and high extraction rate, but also uses low-priced reagents, which greatly saves the reagent cost of industrial production, and does not need to consider the corrosiveness of the reagents and the inconvenience of storage and transportation. The most important thing is that the potential safety hazard is small. Sephadex-G-25 gel column chromatography is used to perform preliminary separation and purification of the crude extract of sheep placenta to obtain highly active anti-tumor components. The active components of sheep placenta obtained by the present invention have the effect of inhibiting the growth of various cancer cells in vitro, and can be used to develop anti-tumor related drugs, anti-tumor related health products and foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The present invention is a flow chart of the method for separating anti-tumor active components from sheep placenta;

[0035] Figure 2 The elution curve of the anti-tumor active component of the present invention by Sephadex-G-25 column chromatography;

[0036] Figure 3 The SDS-PAGE gel electrophoresis diagram of the anti-tumor active components of the present invention;

[0037] Figure 4 HPLC analysis chart of the anti-tumor active component of the present invention;

[0038] Figure 5 The ultraviolet spectrum analysis diagram of the anti-tumor active component of the present invention;

[0039] Figure 6 The infrared spectrum analysis diagram of the anti-tumor active component of the present invention. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0041] Example 1

[0042] like Figure 1 As shown, a method for isolating anti-tumor active components from sheep placenta comprises the following steps:

[0043] Extraction of Sheep Placenta Extract:

[0044] a. Take fresh sheep placenta, remove blood, fat and connective tissue with physiological saline, freeze-dry at -80°C and 25 MPa for 48 hours to obtain freeze-dried powder, and grind the freeze-dried powder with a mortar to obtain sheep placenta powder;

[0045] b. Add the sheep placenta powder obtained in step a to a phosphate buffer solution (0.5% sodium chloride, 0.05M phosphate, 0.5mM phenylmethylsulfonyl fluoride) at pH 7.4 at a solid-liquid ratio of 1:20 g / mL at 4°C, stir and extract for 2 hours, repeat 3 times, filter the combined extracts through gauze;

[0046] c. freeze-drying the extract obtained in step b at -80°C and 25 MPa for 48 hours to obtain a crude sheep placenta extract, with an extraction rate of 42.7%;

[0047] Preparation of anti-tumor active fractions from sheep placenta:

[0048] d. Dissolve the sheep placenta crude extract obtained in step c with purified water and centrifuge at 6000 rpm for 10 min. Take the supernatant and load it onto a Sephadex-G-25 gel chromatography column at a flow rate of 4 mL / min, and elute with purified water. Detect the protein absorption of the eluate at 280 nm and 220 nm. Figure 2 As shown, the eluate was collected according to the peak time, namely F1, F2 and F3, the salt-containing fraction F2 was dialyzed for 48 h with a 3.5 kDa dialysis bag and then freeze-dried, and the salt-free fractions F1 and F3 were freeze-dried to obtain three fractions, named F1, F2 and F3;

[0049] e. The three components obtained in step d were determined by BCA method for protein content, phenol-sulfuric acid method for neutral sugar, and m-hydroxybiphenyl colorimetric method for uronic acid content. The results are shown in Table 1, and the yields of F1, F2 and F3 components were 30.89%, 4.53% and 3.23% respectively.

[0050] Table 1 Example 1 Column chromatography component content determination

[0051]

[0052] Example 2

[0053] like Figure 1 As shown, a method for isolating anti-tumor active components from sheep placenta comprises the following steps:

[0054] Extraction of Sheep Placenta Extract:

[0055] a. Take fresh sheep placenta, remove blood, fat and connective tissue with physiological saline, freeze-dry at -80°C and 25 MPa for 48 hours to obtain freeze-dried powder, and grind the freeze-dried powder with a mortar to obtain sheep placenta powder;

[0056] b. Add the sheep placenta powder obtained in step a to a phosphate buffer solution (0.9% sodium chloride, 0.02M phosphate, 0.5mM phenylmethylsulfonyl fluoride) at pH 7.4 at a solid-liquid ratio of 1:30 g / mL at 7°C, stir and extract for 2 hours, repeat 3 times, filter the combined extracts through gauze;

[0057] c. freeze-drying the extract obtained in step b at -80°C and 25 MPa for 48 hours to obtain a crude sheep placenta extract, with an extraction rate of 43.8%;

[0058] Preparation of anti-tumor active fractions from sheep placenta:

[0059] d. Dissolve the sheep placenta crude extract obtained in step c with purified water and centrifuge at 6000 rpm for 10 min. Take the supernatant and load it onto a Sephadex-G-25 gel chromatography column at a flow rate of 4 mL / min, and elute with purified water. Detect the protein absorption of the eluate at 280 nm and 220 nm. Figure 2 As shown, the eluate was collected according to the peak time, namely F1, F2 and F3, the salt-containing fraction F2 was dialyzed for 48 h with a 3.5 kDa dialysis bag and then freeze-dried, and the salt-free fractions F1 and F3 were freeze-dried to obtain three fractions, named F1, F2 and F3;

[0060] e. The three components obtained in step d were determined by BCA method for protein content, phenol-sulfuric acid method for neutral sugar, and m-hydroxybiphenyl colorimetric method for uronic acid content. The results are shown in Table 2, and the yields of F1, F2 and F3 components are 30.52%, 4.32% and 3.56%.

[0061] Table 2 Example 2 Column chromatography component content determination

[0062]

[0063] Example 3

[0064] like Figure 1 As shown, a method for isolating anti-tumor active components from sheep placenta comprises the following steps:

[0065] a. Take fresh sheep placenta, remove blood, fat and connective tissue with physiological saline, freeze-dry at -80°C and 25 MPa for 48 hours to obtain freeze-dried powder, and grind the freeze-dried powder with a mortar to obtain sheep placenta powder;

[0066] b. Add the sheep placenta powder obtained in step a to a phosphate buffer solution (0.5% sodium chloride, 0.1 M phosphate, 1 mM phenylmethylsulfonyl fluoride) at pH 7.4 at a solid-liquid ratio of 1:50 g / mL at 10°C, stir and extract for 2 h, repeat 3 times, filter the combined extracts through gauze;

[0067] c. freeze-drying the extract obtained in step b at -80°C and 25 MPa for 48 hours to obtain a crude sheep placenta extract, with an extraction rate of 44.2%;

[0068] Preparation of anti-tumor active fractions from sheep placenta:

[0069] d. Dissolve the sheep placenta crude extract obtained in step c with purified water and centrifuge at 6000 rpm for 10 min. Take the supernatant and load it onto a Sephadex-G-25 gel chromatography column at a flow rate of 4 mL / min, and elute with purified water. Detect the protein absorption of the eluate at 280 nm and 220 nm. Figure 2 As shown, the eluate was collected according to the peak time, namely F1, F2 and F3, the salt-containing fraction F2 was dialyzed for 48 h with a 3.5 kDa dialysis bag and then freeze-dried, and the salt-free fractions F1 and F3 were freeze-dried to obtain three fractions, named F1, F2 and F3 respectively;

[0070] e. The three components obtained in step d were determined by BCA method for protein content, phenol-sulfuric acid method for neutral sugar, and m-hydroxybiphenyl colorimetric method for uronic acid content. The results are shown in Table 3, and the yields of F1, F2 and F3 components are 31.21%, 4.42% and 3.46% respectively.

[0071] Table 3 Example 3 Column chromatography component content determination

[0072]

[0073] Example 4

[0074] like Figure 1 As shown, a method for isolating anti-tumor active components from sheep placenta comprises the following steps:

[0075] a. Take fresh sheep placenta, remove blood, fat and connective tissue with physiological saline, freeze-dry at -80°C and 25 MPa for 48 hours to obtain freeze-dried powder, and grind the freeze-dried powder with a mortar to obtain sheep placenta powder;

[0076] b. Add the sheep placenta powder obtained in step a to a phosphate buffer solution (0.9% sodium chloride, 0.1 M phosphate, 0.5 mM phenylmethylsulfonyl fluoride) at pH 7.4 at a solid-liquid ratio of 1:50 g / mL at 10°C, stir and extract for 2 h, repeat 3 times, filter the combined extracts through gauze;

[0077] c. freeze-drying the extract obtained in step b at -80°C and 25 MPa for 48 hours to obtain a crude sheep placenta extract, with an extraction rate of 43.2%;

[0078] Preparation of anti-tumor active fractions from sheep placenta:

[0079] d. Dissolve the sheep placenta crude extract obtained in step c with purified water and centrifuge at 6000 rpm for 10 min. Take the supernatant and load it onto a Sephadex-G-25 gel chromatography column at a flow rate of 4 mL / min, and elute with purified water. Detect the protein absorption of the eluate at 280 nm and 220 nm. Figure 2 As shown, the eluate was collected according to the peak time, namely F1, F2 and F3, the salt-containing fraction F2 was dialyzed for 48 h with a 3.5 kDa dialysis bag and then freeze-dried, and the salt-free fractions F1 and F3 were freeze-dried to obtain three fractions, named F1, F2 and F3 respectively;

[0080] e. The three components obtained in step d were determined by BCA method for protein content, phenol-sulfuric acid method for neutral sugar, and m-hydroxybiphenyl colorimetric method for uronic acid content. The results are shown in Table 4, and the yields of F1, F2 and F3 components are 30.66%, 4.32% and 3.64% respectively.

[0081] Table 4 Example 4 Column chromatography component content determination

[0082]

[0083] Example 5

[0084] like Figure 1 As shown, a method for isolating anti-tumor active components from sheep placenta comprises the following steps:

[0085] a. Take fresh sheep placenta, remove blood, fat and connective tissue with physiological saline, freeze-dry at -80°C and 25 MPa for 48 hours to obtain freeze-dried powder, and grind the freeze-dried powder with a mortar to obtain sheep placenta powder;

[0086] b. Add the sheep placenta powder obtained in step a to a phosphate buffer solution (0.9% sodium chloride, 0.1 M phosphate, 1 mM phenylmethylsulfonyl fluoride) at pH 7.4 at a solid-liquid ratio of 1:50 g / mL at 10°C, stir and extract for 2 h, repeat 3 times, filter the combined extracts through gauze;

[0087] c. freeze-drying the extract obtained in step b at -80°C and 25 MPa for 48 hours to obtain a crude sheep placenta extract, with an extraction rate of 44.8%;

[0088] Preparation of anti-tumor active fractions from sheep placenta:

[0089] d. Dissolve the sheep placenta crude extract obtained in step c with purified water and centrifuge at 6000 rpm for 10 min. Take the supernatant and load it onto a Sephadex-G-25 gel chromatography column at a flow rate of 4 mL / min, and elute with purified water. Detect the protein absorption of the eluate at 280 nm and 220 nm. Figure 2 As shown, the eluate was collected according to the peak time, namely F1, F2 and F3, the salt-containing fraction F2 was dialyzed for 48 h with a 3.5 kDa dialysis bag and then freeze-dried, and the salt-free fractions F1 and F3 were freeze-dried to obtain three fractions, named F1, F2 and F3;

[0090] e. The three components obtained in step d were determined by BCA method for protein content, phenol-sulfuric acid method for neutral sugar, and m-hydroxybiphenyl colorimetric method for uronic acid content. The results are shown in Table 5, and the yields of F1, F2 and F3 components are 31.42%, 4.35% and 3.64% respectively.

[0091] Table 5 Example 5 Column chromatography component content determination

[0092]

[0093] Example 6

[0094] In vitro antitumor activity experiment of Sephadex-G-25 gel column chromatography fractions of sheep placenta extract:

[0095] The human cancer cell lines used in the following examples include human colorectal cancer cells HCT-116 and HT-29, human gastric cancer cells HGC-27, and human lung cancer cells A549. The specific method is: using the MTT method, the cells in the logarithmic growth phase are digested and resuspended, and then inoculated into a 96-well plate at a corresponding cell density, that is, 100 μL of cell solution is added to each well, and cultured in a cell culture incubator at 37°C, 5% CO2, and 95% humidity for 24 hours. 20 μL per well, discard the supernatant, and add 100 μL of different concentrations of compounds (prepared with a culture medium containing a mycoplasma remover, filtered with a 0.45 μm filter membrane).

[0096] Set up 3 to 6 duplicate wells; set up a cell-free background group, a solvent control group and a positive control group (doxorubicin); continue to culture at 37°C and 5% CO2 for 48 hours, discard the supernatant, add 100μL / well of thiazolyl blue (MTT) solution, and continue to culture for 2 to 4 hours; discard the supernatant, add 150μL DMSO to each well, and shake 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.

[0097] Calculation formula:

[0098] Cell viability percentage % = (compound OD-blank OD / control group OD-blank OD) × 100%;

[0099] Cell inhibition rate %=1-cell viability %=[1-(compound OD-blank OD / control group OD-blank OD)]×100%.

[0100] The results show that the inhibition rates of the crude sheep placenta extract extracted from sheep placenta and the F2-F3 components prepared by Sephadex-G-25 gel column chromatography in the above Examples 1-5 on cancer cells at the same concentration are shown in Table 6; the crude sheep placenta extract has a certain inhibitory activity on gastric cancer cells HGC-27; the F2 and F3 components of Sephadex-G-25 gel column chromatography have good inhibitory activity on a variety of cancer cells; compared with the crude sheep placenta extract, the F2 and F3 components of Sephadex-G-25 gel column chromatography provided by the present invention can significantly inhibit the proliferation of a variety of cancer cells, and have no inhibitory effect on normal cells, and have good safety. Compared with CN 114832017 A, the best anti-tumor activity of its sheep placenta extract is IC against HCT-116 cancer cells. 50 The value was 555.38±254.73μg / ml, while the IC of our F2 fraction against HCT-116 cancer cells 50 The value is 183±34ug / ml, and the IC of F3 component against HCT-116 cancer cells 50 The value was 95±11μg / ml, and our effective components F2 and F3 showed the inhibitory activity of A549 tumor cells that was not shown in CN114832017 A. The IC of the ultrafiltration fraction extract in CN 114848676 A against HCT116 tumor cells was 50 The value was 2118.37±56.83μg / ml, and the IC 50 The value is 14870.17 μg / ml. The same IC in CN 114848676 A 50 At the maximum concentration, as shown in Table 6, our active site exhibited antitumor activity exceeding that of CN 114848676 A at a concentration of 2 mg / ml, and the F2 and F3 components showed low toxicity in the normal cell (human normal liver cell L-02) proliferation experiment (as shown in Table 7).

[0101] Table 6 Inhibition rate of different cancer cells at the same concentration of sheep placenta extracts from different parts

[0102] cancer cell HT-29 HCT-116 HGC-27 A549 Example 1F2 61.82±1.33％ 91.41±0.96％ 91.79±1.32％ 90.02±0.96％ Example 1F3 52.72±1.21％ 80.04±1.45％ 93.62±0.78％ 77.52±0.85％ Example 2F2 64.47±1.12％ 92.11±1.66％ 90.72±1.02％ 91.78±0.56％ Example 2F3 54.74±1.01％ 82.86±0.94％ 94.58±0.79％ 79.32±0.41％ Example 3F2 60.12±1.63％ 91.51±1.43％ 90.69±1.25％ 89.42±0.96％ Example 3F3 53.25±0.87％ 81.54±0.81％ 92.26±1.16％ 78.25±0.35％ Example 4F2 61.59±1.21％ 90.14±1.73％ 91.57±1.32％ 90.45±0.46％ Example 4F3 52.43±1.25％ 80.89±1.62％ 93.13±1.67％ 79.25±0.68％ Example 5F2 60.85±1.33％ 91.41±0.96％ 91.79±1.32％ 89.72±0.86％ Example 5F3 53.54±1.36％ 81.77±1.44％ 92.62±1.78％ 78.45±0.58％

[0103] Note: All samples were at a concentration of 2 mg / ml.

[0104] Table 7 Effects of F2 and F3 components on proliferation of normal cells (human normal liver cells L-O2)

[0105]

[0106] The anti-tumor active part of the sheep placenta extract obtained by the invention can effectively inhibit the proliferation of various cancer cells and has good safety. It can be applied to medical research and development and has great reference value for the development and utilization of sheep placenta resources.

[0107] Example 7

[0108] SDS-PAGE, HPLC, UV spectrum, IR spectrum and amino acid composition analysis of anti-tumor active components:

[0109] SDS-PAGE analysis: Sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) was used to estimate the molecular weight of F2 and F3 components. The electrophoresis gel was 1.0 mm × 15 cm. A 12% separation gel and a 6% stacking gel were used for denaturing non-reducing gel electrophoresis analysis. The stacking gel and separation gel were run at 75 V until the sample reached the bottom of the separation gel. After the electrophoresis gel was added with the fixative, the fixative was replaced every 30 minutes for a total of 3 times. After the fixation was completed, sensitized with 0.8 mmol / L sodium thiosulfate for 1 minute, washed with water twice, and 0.012 mol / L silver nitrate solution was added for 1 hour of light-proof silver staining. After the staining was completed, the color developer was added and soaked until the bands appeared clearly, then the terminator was added to terminate the color development and the electrophoresis photos were taken.

[0110] HPLC analysis: sample concentration was 3 mg / ml, injection volume was 50 μL, analytical columns were G5000PW (XL), G3000PW (XL) and G2000SW (XL) in series, mobile phase was 10% ethanol-water, containing 0.1% formic acid, flow rate was 0.6 ml / min, and analysis was monitored at 220 nm and 280 nm.

[0111] Ultraviolet spectrum analysis: Prepare solutions of F2 and F3 components with a concentration of 1 mg / mL respectively, and measure their ultraviolet absorption spectra in the range of 200-400 nm.

[0112] Infrared spectrum analysis: Take a small amount of F2 and F3 components, mix and grind with an appropriate amount of potassium bromide at a ratio of 1:200, and use the tableting method to measure the 4000-400cm -1 Infrared absorption spectrum in the range, resolution: 4.0cm -1 .

[0113] Amino acid analysis: Take a small amount of F2 and F3 components, and use 0.1% phenol and 6M HCl to react at 110°C for 24 hours for acid hydrolysis of the sample. Then add the sample acid hydrolysis solution, 1M triethylamine acetonitrile solution and 0.2M phenyl isothiocyanate acetonitrile solution in a ratio of 2:1:1, mix well and react at room temperature for 1 hour. After the reaction is completed, add n-hexane solution in a ratio of 1:1, shake and separate the layers, take the lower layer solution and mix it with water, and filter it through a 0.22μm needle filter. According to the instruction manual of Diamonsil AAA amino acid analysis, perform HPLC analysis on the derivatized amino acid mixed standard and sample, compare the peak time of the sample spectrum with the peak time of different amino acids in the mixed standard to determine the type of amino acids contained in the sample; compare the peak area ratio of each peak in the sample spectrum to determine the relative molar ratio of the amino acids contained in the sample.

[0114] The results of SDS-PAGE analysis were as follows Figure 3 As shown in Figure 1, the molecular weight of the anti-tumor active component F2 is distributed between about 15 kDa and 50-70 kDa, and the molecular weight of the anti-tumor active component F3 is distributed between about 15 kDa, 35 kDa, 50 kDa, 70 kDa and greater than 130 kDa. The HPLC analysis results are shown in Figure 1. Figure 4 As shown in Figure 2, the volume-displacement column coupled with HPLC analysis also demonstrated that the molecular weights of the proteins contained in the two anti-tumor components F2 and F3 were significantly different. Figure 5 As shown in the figure, the F2 component has absorption at 260-280nm, which is mainly due to the characteristic absorption caused by the conformational change of aromatic amino acids in the protein. The three-dimensional conformational change of the protein and the presence of a small amount of nucleic acid cause it to have a certain degree of blue shift. The F3 component has two absorption peaks at 240nm and 280nm, proving that in addition to the protein, there is also a certain amount of nucleic acid. The infrared analysis results are shown in the figure. Figure 6 As shown, the infrared spectra of tumor components F2 and F3 showed evidence of the presence of proteins (1700-1500 cm -1 Protein secondary structure characteristic region), amide I band (1624cm -1 ), amide II band (1532cm -1 )Amide III band (1085cm -1 ), in addition at 3430cm -1 There is also -NH stretching vibration at 1700-1600cm -1 In addition to proteins, there are also peaks at 1110cm in the infrared analysis spectrum. -1The CO stretching vibration of sugar (CO vibration of carbohydrate) was observed, which is consistent with the sugar content determination in the previous Examples 1-5. The results of amino acid analysis are shown in Table 8. The amino acids with higher content in the F2 component are glycine, leucine, glutamic acid and alanine, and the amino acids with higher content in the F3 component are leucine and glycine. Both F2 and F3 components contain essential amino acids for the human body, and can supplement essential amino acids for the human body while providing drug treatment. (Note: There is no obvious difference in SDS-PAGE, HPLC, UV spectrum, infrared spectrum and amino acid composition analysis of the 5 embodiments, so the representative ones are selected for chart reference)

[0115] Table 8 Amino acid analysis of F2 and F3 components

[0116]

[0117] Note: * indicates essential amino acids

[0118] 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.

[0119] Any contents not described in detail in the present invention can be based on the conventional technical knowledge in the art.

[0120] 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 F2 isolated from sheep placenta, characterized in that: The molecular weight of the anti-tumor active component F2 is distributed between 10-20 kDa and 50-70 kDa, wherein the mass content of protein is 18.7%-19.8%, the mass content of sugar is 4.95%-56%, and the mass content of uronic acid is 5.9%-6.6%.

2. An anti-tumor active component F3 isolated from sheep placenta, characterized in that: The molecular weight of the anti-tumor active component F3 is distributed at 15kDa, 35kDa, 50kDa, 70kDa and greater than 130kDa, wherein the mass content of protein is 23.7%-24.4%, the mass content of sugar is 5.12%-5.82%, and the mass content of uronic acid is 11.2%-12.4%.

3. A method for separating antitumor active components from sheep placenta, the method comprising the following steps: 1) washing the sheep placenta, removing blood stains, mincing and freeze-drying, and grinding to obtain sheep placenta powder; 2) mixing sheep placenta powder and phosphate extract at a solid-liquid ratio of 1:20-1:50 g / mL, extracting, filtering, and freeze-drying to obtain a sheep placenta crude extract; 3) The crude extract of sheep placenta was separated and purified by Sephadex-G-25 gel column to obtain the anti-tumor active components.

4. The method according to claim 3, characterized in that The concentrations of the components in the phosphate extract are 0.5%-3% sodium chloride, 10-100mM phosphate, and 0.5-1.0mM serine protease inhibitor.

5. The method according to claim 4, characterized in that The serine protease inhibitor is phenylmethylsulfonyl fluoride.

6. The method according to claim 3, characterized in that In step 2), the extraction temperature is 4°C-10°C, the extraction time is 1-3h, the number of extractions is 2-5 times, and the stirring rate is 100-300rpm / min.

7. The method according to claim 3, characterized in that In step 3), the separation conditions of the Sephadex-G-25 gel column are: The crude extract of sheep placenta was dissolved in purified water, and the supernatant was taken as the loading solution by centrifugation. The purified water was used to wash and balance the Sephadex-G-25 gel column, and the purified water was used as the eluent. The flow rate was 3-5 mL / min. The protein absorption of the eluent was detected at 280 nm and 220 nm, and the eluent was collected by ultraviolet absorption. The salt-containing part F2 component was dialyzed and desalted with a dialysis bag and then freeze-dried. The salt-free components F1 and F3 were freeze-dried separately to obtain 3 components, which were named F1, F2, and F3, respectively. Among them, F2-F3 were anti-tumor active components.

8. Use of the antitumor active component according to claim 1 or 2 or the antitumor active component obtained by the method according to any one of claims 3 to 7 in the preparation of a drug for inhibiting the growth of tumor cells.

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

10. Use of the antitumor active component according to claim 1 or 2 or the antitumor active component obtained by the method according to any one of claims 3 to 7 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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