A method for extracting crocodile bone peptide

Abstract

The present invention provides a method for extracting crocodile bone peptides. The present invention separates two kinds of active short peptides from crocodile bones for the first time. The active short peptides prepared by the separation method have high purity, and activity analysis shows that the active short peptides have good anti-tumor effects, which expands the economic value of crocodile bones and provides a positive reference for the treatment of tumors.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and more specifically to a method for extracting crocodile bone peptide. Background Art

[0002] Crocodiles are reptiles of the Crocodilian and Alligatoridae families, with a total of 8 genera and 23 species. Crocodiles have long trunks, well-developed muscles, flat heads, skin on the head close to the skull, and the skull is firmly connected and cannot move. The teeth are conical and grow in the tooth sockets, with more than 25 teeth on each side. The tongue is short and flat and cannot protrude. The eyes are small and slightly protruding. The tail is thick and slightly flat. The limbs are short and thick. The external nostrils are located on the back of the snout with valves on them. There are armor-like scales.

[0003] All of the crocodile's body is a treasure. For example, crocodile meat is rich in high-quality protein and essential amino acids, unsaturated fatty acids, vitamins and various trace elements. Crocodile liver has the functions of nourishing the brain, generating new blood and removing moisture. Eating crocodile liver can detoxify and improve eyesight, nourish blood and benefit the kidneys. It has obvious therapeutic effects on those with poor liver function and poor eyesight, and can also inhibit the formation of stones. Crocodile bones can prevent and treat rheumatic bone pain. Bone powder contains a large amount of active calcium and phosphorus and other substances, which can be used to prevent and treat osteoporosis in the elderly and rickets in children. Crocodile tail glue can not only nourish the liver and kidneys, replenish qi and strengthen the foundation, and enhance cell vitality, but also protect the skin and beautify the skin, increase skin elasticity, reduce and delay the appearance of wrinkles, and have satisfactory effects on allergies. However, the current research on crocodile bones is not systematic, and there are few studies at home and abroad, and there are even fewer studies on crocodile bone active short peptides. Summary of the invention

[0004] In view of the technical problems existing in the prior art, the present invention provides a method for extracting crocodile bone peptides. The present invention separates two kinds of active short peptides from crocodile bones for the first time. The active short peptides prepared by the separation method have high purity, and activity analysis shows that the active short peptides have good anti-tumor effects, which expands the economic value of crocodile bones and provides a positive reference for the treatment of tumors.

[0005] Specifically, the present invention first provides a crocodile bone peptide, characterized in that the sequence of the peptide can be selected from one or more of those shown in SEQ ID NO.1-2.

[0006] Another aspect of the present invention provides a method for extracting crocodile bone peptide, characterized in that it comprises the following steps:

[0007] (1) taking crocodile bones, removing the remaining meat and remaining fat, and using a grinder to obtain crocodile bone mince;

[0008] (2) adding 4-6 times the mass of water to the obtained crocodile bone powder and adjusting the pH thereof to 7.0-7.4;

[0009] (3) adding 50-70% (v / v) composite protease, stirring continuously for enzymolysis at 60-70° C. for 12-20 hours, centrifuging, and taking the supernatant to obtain crocodile bone protein solution; wherein the composite protease is composed of papain and alkaline protease in a dosage ratio of (1-5): (2-9);

[0010] (4) After enzymatic hydrolysis, the product is inactivated and cooled;

[0011] (5) centrifuging the cooled enzymatic hydrolysate at 1500-2200 g for 5-15 min, taking the supernatant; and filtering the obtained supernatant with an ultrafiltration membrane to retain polypeptide molecules with a molecular weight between 4000-6000 Da;

[0012] (6) The retained polypeptide molecules are sequenced by Obitrap Fusion Lumos mass spectrometer and analyzed by high performance liquid chromatography to identify the purity.

[0013] Preferably, the method for extracting crocodile bone peptide comprises the following steps:

[0014] (1) taking crocodile bones, removing the remaining meat and remaining fat, and using a grinder to obtain crocodile bone mince;

[0015] (2) adding 4 times the amount of water to the obtained crocodile bone powder and adjusting the pH thereof to 7.0;

[0016] (3) adding 50% (v / v) composite protease, stirring continuously for enzymolysis at 60° C. for 12 h, and centrifuging the supernatant to obtain crocodile bone protein solution; wherein the composite protease is composed of papain and alkaline protease, and the dosage ratio of the two is 1:2;

[0017] (4) After enzymatic hydrolysis, the enzyme was inactivated at 100°C and then cooled to room temperature;

[0018] (5) centrifuging the cooled enzymatic hydrolysate at 1500 g for 5 min, taking the supernatant; and filtering the obtained supernatant with an ultrafiltration membrane to retain polypeptide molecules with a molecular weight between 4000 and 6000 Da;

[0019] (6) The retained polypeptide molecules are sequenced by Obitrap Fusion Lumos mass spectrometer and analyzed by high performance liquid chromatography to identify the purity.

[0020] Preferably, the method for extracting crocodile bone peptide comprises the following steps:

[0021] (1) taking crocodile bones, removing the remaining meat and remaining fat, and using a grinder to obtain crocodile bone mince;

[0022] (2) adding 5 times the amount of water to the obtained crocodile bone powder and adjusting the pH thereof to 7.2;

[0023] (3) adding 60% (v / v) composite protease, stirring continuously at 65° C. for 18 h, and centrifuging the supernatant to obtain crocodile bone protein solution; wherein the composite protease is composed of papain and alkaline protease, and the usage ratio of the two is 3:4;

[0024] (4) After enzymatic hydrolysis, the enzyme was inactivated at 100°C and then cooled to room temperature;

[0025] (5) centrifuging the cooled enzymatic hydrolysate at 2000 g for 10 min, taking the supernatant; and filtering the obtained supernatant with an ultrafiltration membrane to retain polypeptide molecules with a molecular weight between 4000 and 6000 Da;

[0026] (6) The retained polypeptide molecules are sequenced by Obitrap Fusion Lumos mass spectrometer and analyzed by high performance liquid chromatography to identify the purity.

[0027] Preferably, the method for extracting crocodile bone peptide comprises the following steps:

[0028] (1) taking crocodile bones, removing the remaining meat and remaining fat, and using a grinder to obtain crocodile bone mince;

[0029] (2) adding 6 times of water to the obtained crocodile bone powder and adjusting the pH thereof to 7.4;

[0030] (3) adding 70% (v / v) composite protease, stirring continuously at 70° C. for 20 h, and centrifuging the supernatant to obtain crocodile bone protein solution; wherein the composite protease is composed of papain and alkaline protease, and the usage ratio of the two is 5:9;

[0031] (4) After enzymatic hydrolysis, the enzyme was inactivated at 100°C and then cooled to room temperature;

[0032] (5) centrifuging the cooled enzymatic hydrolysate at 2200 g for 15 min, taking the supernatant; and filtering the obtained supernatant with an ultrafiltration membrane to retain polypeptide molecules with a molecular weight between 4000 and 6000 Da;

[0033] (6) The retained peptide molecules are sequenced by Obitrap Fusion Lumos mass spectrometer and analyzed by high performance liquid chromatography to determine the purity.

[0034] Another aspect of the present invention provides the use of crocodile bone peptide in the preparation of anti-tumor drugs, characterized in that the crocodile bone peptide is a main active short peptide prepared by the method.

[0035] Preferably, the sequence of the crocodile bone peptide can be selected from one or more of those shown in SEQ ID NO.1-2.

[0036] Preferably, the sequence of the crocodile bone peptide can be selected from the combination of SEQ ID NO.1-2.

[0037] Further preferably, the mass ratio of SEQ ID NO.1 to SEQ ID NO.2 in the crocodile bone peptide is 1-5:1-8.

[0038] Preferably, the mass ratio of SEQ ID NO.1 to SEQ ID NO.2 in the crocodile bone peptide is 1:1.

[0039] Preferably, the tumor includes, but is not limited to, hepatocellular carcinoma, breast cancer, pancreatic cancer, lung cancer, gastric cancer, esophageal cancer, colon cancer, bladder cancer, kidney cancer, prostate cancer, ovarian cancer or uterine cancer.

[0040] Preferably, the tumor is selected from hepatocellular carcinoma.

[0041] Preferably, the dosage of the crocodile bone peptide is preferably 45-100 μg / kg.

[0042] The advantages of the invention are as follows: the present invention extracts two kinds of active short peptides from crocodile bones for the first time, the active short peptides prepared by the separation method have high purity, and activity analysis shows that the active short peptides have good anti-tumor effects, which expands the economic value of crocodile bones and provides a positive reference for the treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Analysis of the activity of the crocodile bone peptide on tumor cell proliferation;

[0044] Figure 2 This is an analysis of the tumor killing effect of the crocodile bone peptide described in the present invention on model mice. DETAILED DESCRIPTION

[0045] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0046] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0047] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0048] Example 1

[0049] A method for extracting crocodile bone peptide comprises the following steps:

[0050] (1) taking crocodile bones, removing the remaining meat and remaining fat, and using a grinder to obtain crocodile bone mince;

[0051] (2) adding 4 times the amount of water to the obtained crocodile bone powder and adjusting the pH thereof to 7.0;

[0052] (3) adding 50% (v / v) composite protease, stirring continuously for enzymolysis at 60° C. for 12 h, and centrifuging the supernatant to obtain crocodile bone protein solution; wherein the composite protease is composed of papain and alkaline protease, and the dosage ratio of papain to alkaline protease is 1:2;

[0053] (4) After enzymatic hydrolysis, the enzyme was inactivated at 100°C and then cooled to room temperature;

[0054] (5) centrifuging the cooled enzymatic hydrolysate at 1500 g for 5 min, taking the supernatant; and filtering the obtained supernatant with an ultrafiltration membrane to retain polypeptide molecules with a molecular weight between 4000 and 6000 Da;

[0055] (7) The retained polypeptide molecules were sequenced by Obitrap Fusion Lumos mass spectrometer, and two major active peptides were obtained after identification and analysis, and the sequences were shown in SEQ ID NO.1-2, respectively, and spray-dried or vacuum-freeze-dried for use. After high performance liquid chromatography analysis and identification, the purity of the two major active peptides prepared by the method of the present invention reached 91.6% and 93.2%, respectively.

[0056] Example 2

[0057] A method for extracting crocodile bone peptide comprises the following steps:

[0058] (1) taking crocodile bones, removing the remaining meat and remaining fat, and using a grinder to obtain crocodile bone mince;

[0059] (2) adding 5 times the amount of water to the obtained crocodile bone powder and adjusting the pH thereof to 7.2;

[0060] (3) adding 60% (v / v) composite protease, stirring continuously at 65° C. for 18 h, and centrifuging the supernatant to obtain crocodile bone protein solution; wherein the composite protease is composed of papain and alkaline protease, and the usage ratio of the two is 3:4;

[0061] (4) After enzymatic hydrolysis, the enzyme was inactivated at 100°C and then cooled to room temperature;

[0062] (5) centrifuging the cooled enzymatic hydrolysate at 2000 g for 10 min, taking the supernatant; and filtering the obtained supernatant using an ultrafiltration membrane with a molecular weight cutoff of 4000-6000 Da polypeptide molecules;

[0063] (7) The retained polypeptide molecules were sequenced by Obitrap Fusion Lumos mass spectrometer, and two major active peptides were obtained after identification and analysis, and the sequences were shown in SEQ ID NO.1-2, respectively, and spray-dried or vacuum-freeze-dried for use. After high performance liquid chromatography analysis and identification, the purity of the two major active peptides prepared by the method of the present invention reached 94.2% and 96.5%, respectively.

[0064] Example 3

[0065] A method for extracting crocodile bone peptide comprises the following steps:

[0066] (1) taking crocodile bones, removing the remaining meat and remaining fat, and using a grinder to obtain crocodile bone mince;

[0067] (2) adding 6 times of water to the obtained crocodile bone powder and adjusting the pH thereof to 7.4;

[0068] (3) adding 70% (v / v) composite protease, stirring continuously at 70° C. for 20 h, and centrifuging the supernatant to obtain crocodile bone protein solution; wherein the composite protease is composed of papain and alkaline protease, and the usage ratio of the two is 5:9;

[0069] (4) After enzymatic hydrolysis, the enzyme was inactivated at 100°C and then cooled to room temperature;

[0070] (5) centrifuging the cooled enzymatic hydrolysate at 2200 g for 15 min, taking the supernatant; and filtering the obtained supernatant using an ultrafiltration membrane with a molecular weight cutoff of 4000-6000 Da polypeptide molecules;

[0071] (7) The retained polypeptide molecules are sequenced by Obitrap Fusion Lumos mass spectrometer, and two major active peptides are obtained through identification and analysis, and the sequences are shown in SEQ ID NO.1-2, respectively, and then spray-dried or vacuum-freeze-dried for later use.

[0072] According to high performance liquid chromatography analysis and identification, the purity of the two main active peptides prepared by the method of the present invention reached 95.6% and 98.2% respectively.

[0073] Example 4

[0074] Analysis of the activity of crocodile bone peptide on tumor cell proliferation

[0075] 1) Culture hepatocellular carcinoma HEPG2 cells at 1×10 5 Cells / well were inoculated into a 96-well cell culture plate, 150 μL per well, and placed in an incubator for 12 h for adhesion treatment;

[0076] 2) using cell culture medium to prepare a crocodile bone peptide solution represented by SEQ ID NO.1-2 with a final concentration of 25 μg / mL;

[0077] 3) Add 50 μL of the crocodile bone peptide solution described in step 2) to the cell culture plate shown in step 1) and incubate for 12 hours.

[0078] The specific groups include: treatment group 1 is the crocodile bone peptide solution shown in SEQ ID NO.1, treatment group 2 is the crocodile bone peptide solution shown in SEQ ID NO.2, treatment group 3 is the crocodile bone peptide solution mixed with SEQ ID NO.1 and 2 (the mass ratio of SEQ ID NO.1 and 2 is 1:1), and treatment group 4 is the control group, which is a simple culture medium. The proliferation activity of tumor cells was analyzed using the MTT cell proliferation and cytotoxicity detection kit.

[0079] The results are as follows Figure 1 As shown, the two crocodile bone peptides prepared by the present invention have good activity in inhibiting tumor cell proliferation, especially when the two crocodile bone peptides are used in combination, the inhibitory effect on tumor cell proliferation is most obvious.

[0080] 2. In vivo antitumor activity of crocodile bone peptide

[0081] Immunodeficient NSG mice were injected with 1 × 10 8 HEPG2 cells were used to construct a mouse liver cancer tumor model. When the tumor volume was about 90 mm 3 The treated group was given 45 μg / kg mixed crocodile bone peptide solution (the mass ratio of SEQ ID NO.1 and 2 was 1:1) via the tail vein; the blank group was not treated during the period. After continuous monitoring for 3 weeks, the abdominal tumor volume of the mice was counted. Figure 2 It can be seen that compared with the blank group, the mixed crocodile bone peptide solution can significantly inhibit tumor growth and reduce tumor volume.

[0082] It is necessary to point out that the above embodiments are limited to further elaboration and explanation of the technical solution of the present invention, and are not further limitations of the technical solution of the present invention. The method of the present invention is only a preferred implementation scheme, and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A crocodile bone peptide, characterized in that: The peptide sequence is selected from one or more of those shown in SEQ ID NO.1-2.

2. The method for extracting crocodile bone peptide according to claim 1, characterized in that: The following steps are involved: (1) taking crocodile bones, removing the remaining meat and remaining fat, and using a grinder to obtain crocodile bone mince; (2) adding 4-6 times the mass of water to the obtained crocodile bone powder and adjusting the pH thereof to 7.0-7.4; (3) adding 50-70% (v / v) composite protease, stirring continuously for enzymolysis at 60-70° C. for 12-20 hours, centrifuging, and taking the supernatant to obtain crocodile bone protein solution; wherein the composite protease is composed of papain and alkaline protease in a dosage ratio of (1-5): (2-9); (4) After enzymolysis, the product is inactivated and cooled; (5) centrifuging the cooled enzymatic hydrolysate at 1500-2200 g for 5-15 min, taking the supernatant; and filtering the obtained supernatant with an ultrafiltration membrane to retain polypeptide molecules with a molecular weight between 4000-6000 Da; (6) The retained polypeptide molecules are sequenced by Obitrap Fusion Lumos mass spectrometer and analyzed by high performance liquid chromatography to identify the purity.

3. The method for extracting crocodile bone peptide as claimed in claim 2, characterized in that, The following steps are involved: (1) taking crocodile bones, removing the remaining meat and remaining fat, and using a grinder to obtain crocodile bone powder; (2) adding 4 times the amount of water to the obtained crocodile bone powder and adjusting the pH thereof to 7.0; (3) adding 50% (v / v) composite protease, stirring continuously for enzymolysis at 60° C. for 12 h, and centrifuging the supernatant to obtain crocodile bone protein solution; wherein the composite protease is composed of papain and alkaline protease, and the dosage ratio of the two is 1:2; (4) After enzymatic hydrolysis, the enzyme was inactivated at 100°C and then cooled to room temperature; (5) centrifuging the cooled enzymatic hydrolysate at 1500 g for 5 min, taking the supernatant; and filtering the obtained supernatant with an ultrafiltration membrane to retain polypeptide molecules with a molecular weight between 4000 and 6000 Da; (6) The retained polypeptide molecules are sequenced by Obitrap Fusion Lumos mass spectrometer and analyzed by high performance liquid chromatography to identify the purity.

4. The method for extracting crocodile bone peptide as claimed in claim 2, characterized in that: The following steps are involved: (1) taking crocodile bones, removing the remaining meat and remaining fat, and using a grinder to obtain crocodile bone mince; (2) adding 5 times the amount of water to the obtained crocodile bone powder and adjusting the pH thereof to 7.2; (3) adding 60% (v / v) composite protease, stirring continuously at 65° C. for 16 h, and centrifuging the supernatant to obtain crocodile bone protein solution; wherein the composite protease is composed of papain and alkaline protease, and the usage ratio of the two is 3:4; (4) After enzymatic hydrolysis, the enzyme was inactivated at 100°C and then cooled to room temperature; (5) centrifuging the cooled enzymatic hydrolysate at 2000 g for 10 min, taking the supernatant; and filtering the obtained supernatant with an ultrafiltration membrane to retain polypeptide molecules with a molecular weight between 4000 and 6000 Da; (6) The retained polypeptide molecules are sequenced by Obitrap Fusion Lumos mass spectrometer and analyzed by high performance liquid chromatography to identify the purity.

5. The method for extracting crocodile bone peptide as claimed in claim 2, characterized in that, The following steps are involved: (1) taking crocodile bones, removing the remaining meat and remaining fat, and using a grinder to obtain crocodile bone mince; (2) adding 6 times of water to the obtained crocodile bone powder and adjusting the pH thereof to 7.4; (3) adding 70% (v / v) composite protease, stirring continuously for enzymolysis at 70° C. for 18 h, and centrifuging the supernatant to obtain crocodile bone protein solution; wherein the composite protease is composed of papain and alkaline protease composite enzyme, and the usage ratio of the two is 5:9; (4) After enzymatic hydrolysis, the enzyme was inactivated at 100°C and then cooled to room temperature; (5) centrifuging the cooled enzymatic hydrolysate at 2200 g for 15 min, taking the supernatant; and filtering the obtained supernatant with an ultrafiltration membrane to retain polypeptide molecules with a molecular weight between 4000 and 6000 Da; (6) The retained polypeptide molecules are sequenced by Obitrap Fusion Lumos mass spectrometer and analyzed by high performance liquid chromatography to identify the purity.

6. Use of crocodile bone peptide in the preparation of anti-tumor drugs, characterized in that: The crocodile bone peptide is prepared by the method according to any one of claims 2 to 5, wherein the sequence of the peptide is selected from one or more of SEQ ID NO.1-2, and the tumor is hepatocellular carcinoma.

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