MILK-DERIVED POLYPEPTIDE DERIVATIVE, COMPOSITION AND METHOD FOR PREVENTING AND TREATING OBESITY

Abstract

The present invention includes a milk-derived polypeptide derivative, composition and its method for using. The milk-derived polypeptide together with its cell penetrating peptide derivatives can slow down body weight gain of mice induced by high-fat diet, reduce blood glucose, serum triglycerides and insulin levels, and improve insulin sensitivity and glucose tolerance. In addition, it can function to reduce body weight and blood glucose of already established obese mice model. Therefore, it has potentials to prepare drugs, health care products and food additives for preventing and treating obesity and its complications and other related diseases.

Claims

1. A milk-derived polypeptide derivative comprising an active amino acid sequence of SEQ ID NO: 1.

2. The milk-derived polypeptide derivative according to claim 1, further comprising a cell penetrating amino acid sequence, wherein the cell penetrating amino acid sequence is located at N-terminal of the active amino acid sequence.

3. The milk-derived polypeptide derivative according to claim 2, wherein the polypeptide derivative is a modified or unmodified linear peptide chain consisting of 18-25 amino acids.

4. The milk-derived polypeptide derivative according to claim 2, wherein the cell penetrating amino acid sequence of the polypeptide derivative comprises at least one segment of oligo-arginine sequence.

5. The milk-derived polypeptide derivative according to claim 4, wherein the proportion of arginine in the cell penetrating amino acid sequence is no lower than 40%.

6. The milk-derived polypeptide derivative according to claim 5, wherein the polypeptide derivative is any one of SEQ ID NO: 2 to SEQ ID NO: 5.

7. A composition, comprising the milk-derived polypeptide derivative according to claim 1, and at least a pharmaceutically acceptable carrier or excipient.

8. A composition, comprising the milk-derived polypeptide derivative according to claim 2, and at least a pharmaceutically acceptable carrier or excipient.

9. A method for preventing and treating obesity or obesity complications with the milk-derived polypeptide derivative according to claim 2, comprising administrating the polypeptide derivative at a concentration to exerting biological activity of 5-10 mg/Kg.

10. The method for preventing and treating obesity or obesity complications with the milk-derived polypeptide derivative according to claim 9, wherein the polypeptide derivative is prepared as drugs, health care products or food additives.

11. The method for preventing and treating obesity and obesity complications with the milk-derived polypeptide derivative according to claim 10, wherein the obesity complications include hyperglycemia and hyperlipidemia.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 is a graph of different concentrations of milk-derived polypeptide derivatives in Embodiment 2 on body weight gain of mice with high-fat diet;

[0018] FIG. 2 is a bar chart illustrating different concentrations of milk-derived polypeptide derivatives in Embodiment 2 on food intake of mice with high-fat diet;

[0019] FIG. 3 illustrates different concentrations of milk-derived polypeptide derivatives in Embodiment 2 on adipose tissues of various parts of mice with high-fat diet;

[0020] FIG. 4 is a bar chart of different concentrations of milk-derived polypeptide derivatives in Embodiment 2 on wet weight of fat of mice with high-fat diet;

[0021] FIG. 5 is a bar chart of different concentrations of milk-derived polypeptide derivatives in Embodiment 2 on lean mass of mice with high-fat diet;

[0022] FIG. 6 is a graph of different concentrations of milk-derived polypeptide derivatives in Embodiment 3 on glucose tolerance (GTT) of mice with high-fat diet;

[0023] FIG. 7 is a graph of different concentrations of milk-derived polypeptide derivatives in Embodiment 3 on insulin sensitivity (ITT) of mice with high-fat diet;

[0024] FIG. 8 is a bar chart showing changes of fasting blood glucose, triglyceride and insulin levels of mice with high-fat diet after intervention of different concentrations of milk-derived polypeptide derivatives in Embodiment 4;

[0025] FIG. 9 is a diagram showing H&E staining and UCP1 protein immunohistochemistry of adipose tissues of mice with high-fat diet after intervention of different concentrations of milk-derived polypeptide derivatives in Embodiment 5;

[0026] FIG. 10 is a Western blot test chart of UCP1 protein in adipose tissues of mice with high-fat diet after intervention of different concentrations of milk-derived polypeptide derivatives in Embodiment 5;

[0027] FIG. 11 shows change of body weight and a fasting blood glucose level of obese model mice after intervention of milk-derived polypeptide derivatives in Embodiment 6 compared with a control group;

[0028] FIG. 12 is an anatomy of adipose tissues of various parts of obese model mice after intervention of milk-derived polypeptide derivatives in Embodiment 6 compared with a control group;

[0029] FIG. 13 a diagram showing H&E staining and UCP1 protein immunohistochemistry of adipose tissues from obese model mice after intervention of milk-derived polypeptide derivatives in Embodiment 6 compared with a control group; and

[0030] FIG. 14 is a Western blot test chart of UCP1 protein in adipose tissues of obese model mice after intervention of milk-derived polypeptide derivatives in Embodiment 6 compared with a control group.

DETAILED DESCRIPTION

[0031] The present disclosure will be further explained below with reference to the drawings and specific Embodiments.

Embodiment 1

[0032] Unless otherwise specified, peptide chains in the following embodiments of the disclosure were obtained from chemical synthesis by Shanghai Ketai Biological Co., Ltd., with purity of more than 98%.

[0033] An isolated linear milk-derived polypeptide is provided in this embodiment, which is a amino acid sequence shown in SEQ ID NO.1: Val-Lys-Glu-Ala-Met-Ala-Pro-Lys.

[0034] A derivative of this milk-derived polypeptide is further provided in this embodiment, with its N-terminal being connected to a cell penetrating sequence to form a linear nucleotide sequence of 18-25 amino acids in length: SEQ ID NO.2: GRKKRRQRRRVKEAMAPK, with a molecular formula of C.sub.93H.sub.175N.sub.41O.sub.22S, an average molecular weight of 2251.7 g/mol, and an average isoelectric pH of 12.471.

[0035] SEQ ID NO.3: CYGRKKRRQRRRVKEAMAPK, with a molecular formula of C.sub.108H.sub.197N.sub.45O.sub.26S, an average molecular weight of 2574.06 g/mol, and an average isoelectric pH of 12.471.

[0036] SEQ ID NO.4: GRKKRRQRRRPPQVKEAMAPK, with a molecular formula of C.sub.105H.sub.189N.sub.43O.sub.25S.sub.2, an average molecular weight of 2518.02 g/mol, and an average isoelectric pH of 11.911.

[0037] SEQ ID NO.5: GRKKRRQRRRPPQQVKEAMAPK, with a molecular formula of C.sub.113H.sub.205N.sub.47O.sub.28S, an average molecular weight of 2702.19 g/mol, and an average isoelectric pH of 12.471.

[0038] Combination of any one or more of the above amino acid sequences SEQ ID NO.2 to SEQ ID NO.5 as an active ingredient can be prepared into a composition for prevention and treatment of obesity and obesity complications by adding other pharmaceutically acceptable carriers or excipients.

[0039] Necessary modifications can be made by those skilled in the art without departing from protection scope of the present disclosure, including but not limited to protection/de-protection of specific groups, acylation, alkylation, amidation, esterification of a C-terminal and/or an N-terminal and/or a side chain, and other special coupling or chelating modifications.

Embodiment 2 Effect of Milk-Derived Polypeptide on Weight Increasing of Animal Model with High-Fat Diet

1. Experimental Method

[0040] C57BL/6J male mice at 6-8 weeks old were used as research subjects, and an experiment was carried out after adaptation to a SPF animal room for one week. The mice were fed with high-fat diet (with 60% of fat content) for 10 weeks to induce obesity phenotype in mice. At the same time, different drug concentrations (5 mg/kg and 10 mg/kg) were provided, and the milk-derived polypeptide derivatives corresponding to SEQ ID NO.5 were administered by intraperitoneal injection twice a week, with dissolved normal saline (Vehicle) as a control. The mice are weighed to obtain body weight and their food intake calculated every week. After the mice were killed, brown fat (BAT) at scapular, inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) were photographed, weighed to obtain wet weight and lean mass of the mice was calculated.

Experimental Results

[0041] Referring to FIG. 1 to FIG. 5, Compared with the control group (Vehicle), weight growth of the mice in a 5 mg/kg group started to slow down at a 8th week, while that of the mice in a 10 mg/kg group started to slow down at a 7th week. By a 10th week, weight of the mice in a milk-derived polypeptide derivative intervention group was significantly lower than that in the control group (Vehicle). After dissection, respective adipose tissues were photographed as shown in FIG. 3. Volumes of subcutaneous and abdominal white adipose tissues in the milk-derived polypeptide derivative intervention group decreased significantly. It is found in a wet weight test of the adipose tissues as shown in FIG. 4 that fat content of the mice in the milk-derived polypeptide derivative intervention group decreased significantly, while FIG. 5 showed that there was no significant change in the lean mass of the mice. The above indicates that the milk-derived polypeptide and its derivative can slow down obesity weight increasing induced by high-fat diet, which is caused by decrease of body fat content, and thus functions to prevent obesity.

Embodiment 3 Effect of Milk-Derived Polypeptide on Insulin Sensitivity and Glucose Tolerance of Animal Model with High-Fat Diet

1. Experimental Method

[0042] C57BL/6J male mice at 6-8 weeks old were used as research subjects, and an experiment was carried out after adaptation to a SPF animal room for one week. The mice were fed with high-fat diet (with 60% of fat content) for 10 weeks to induce obesity phenotype in mice. At the same time, different drug concentrations (5 mg/kg and 10 mg/kg) of the milk-derived polypeptide derivatives corresponding to SEQ ID NO.5 were provided, and were administered by intraperitoneal injection twice a week, with dissolved normal saline (Vehicle) as a control.

[0043] Insulin Sensitivity: The mice are fasted overnight for 12 hours, and at a next day, insulin was injected by intraperitoneal injection at a standard of 0.75 U per kg of weight. Blood samples were taken from tail vein at 0 min, 15 min, 30 min, 60 min, 90 min and 120 min after the injection, and changes of the blood glucose in the mice were measured by a blood glucose meter to evaluate the insulin sensitivity of the mice. Glucose tolerance test: after fasting for 6 hours, the mice were injected with glucose by intraperitoneal injection at a concentration of 1 g per kg of weight. Blood samples were taken from tail vein at 0 min, 15 min, 30 min, 60 min, 90 min and 120 min after the injection, and changes of the blood glucose in the mice were measured by a blood glucose meter to evaluate the glucose tolerance of the mice.

Experimental Results

[0044] FIG. 6 shows effects of different concentrations of milk-derived polypeptide derivatives on the glucose tolerance (GTT) in the obese mice. It can be seen that after continuous intervention for 10 weeks with the milk-derived polypeptide derivatives, fasting blood glucose levels of the mice were lower than those of the control group within 2 hours after glucose or insulin injection, and a curve of blood glucose rise was well controlled. After the intervention of 10 mg/kg concentration for 10 weeks, an area under the curve had significant statistical significance. It can be expected that intaking the milk-derived polypeptide derivative with a certain concentration for a long time helps to improve glucose tolerance of an individual, and provides corresponding drugs, health care products and food additives for prevention and treatment of obesity and its complications as active ingredients.

[0045] Similarly, FIG. 7 shows effects of different concentrations of milk-derived polypeptide derivatives on insulin sensitivity (ITT) in the obese mice. When an intake concentration is 5 mg/kg, it presents certain statistical significance, so it can be combined with prevention and treatment of hyperglycemia or diabetes to further improve the effects.

Embodiment 4 Effect of Milk-Derived Polypeptide on Blood Glucose, Blood Lipid and Insulin Levels of Animal Model with High-Fat Diet

1. Experimental Method

[0046] C57BL/6J male mice at 6-8 weeks old were used as research subjects, and an experiment was carried out after adaptation to a SPF animal room for one week. The mice were fed with high-fat diet (with 60% of fat content) for 10 weeks to induce obesity phenotype in mice. At the same time, different drug concentrations (5 mg/kg and 10 mg/kg) of the milk-derived polypeptide derivatives corresponding to SEQ ID NO.5 were provided, and were administered by intraperitoneal injection twice a week, with resolvent saline (Vehicle) as a control. The changes of the blood glucose in the mice were detected by a Roche blood glucose meter, and changes of blood lipid and insulin level in the mice were detected by Applygen and Millipore commercial kits respectively.

Experimental Results

[0047] FIG. 8 shows that after continuous intervention for 10 weeks, the fasting blood glucose, blood lipid and insulin levels of the mice were significantly reduced. Therefore, the milk-derived polypeptide derivative is used for preventing and treating obesity complications such as hyperglycemia and hyperlipidemia.

Embodiment 5 Effect of Milk-Derived Polypeptide on Morphology and Thermogenic Activity of Brown Fat and White Fat Droplets in Animal Model with High-Fat Diet

1. Experimental Method

[0048] C57BL/6J male mice at 6-8 weeks old were used as research subjects, and an experiment was carried out after adaptation to a SPF animal room for one week. The mice were fed with high-fat diet (with 60% of fat content) for 10 weeks to induce obesity phenotype in mice. At the same time, different drug concentrations (5 mg/kg and 10 mg/kg) were provided, and the milk-derived polypeptide derivatives corresponding to SEQ ID NO.5 were administered by intraperitoneal injection twice a week, with dissolved normal saline (Vehicle) as a control. After 10 weeks of intervention, the mice were killed, and brown fat (BAT) at scapula and inguinal subcutaneous white adipose tissues (iWAT) were taken to be fixed with a adipose tissue fixing solution (Solarbio, G2185) for 24 hours. H&E staining and a UCP1 protein immunohistochemistry study were carried out after dehydration, paraffin embedding and sectioning. The H&E staining is performed using a kit (ab245800) from Abcam, and specific steps can be found in kit instructions. Tissue sections were subjected to the immunohistochemistry study after dewaxing and antigen repairing. Specifically, the sections were incubated overnight with UCP1 antibody (1:200, Proteintech) at 4 degrees. The antibody was diluted with PBS containing 1% of BSA, and a color reaction was carried out next day with a DAB kit from Abcam. Specific operations can be referred to kit experimental steps. At the same time, total protein was extracted by a RIPA lysate, and expression of the UCP1 protein in the adipose tissues was detected by a Western blot method after quantifying, SDS-PAGE electrophoresis and transmembrane.

Experimental Results

[0049] As shown in FIG. 9, H&E staining results show that morphology sizes (white pore sizes) of lipid droplets in brown fat and white adipose tissues are significantly reduced, indicating that lipid accumulation in the adipose tissues is reduced and obesity performance is significantly improved. It is shown in UCP1 immunohistochemistry results that brown areas in both brown and white adipose tissues were significantly increased under action of the milk-derived polypeptide derivatives, suggesting that expression of the UCP1 protein was significantly increased and metabolic capacity of the adipose tissues was enhanced. Western blot results in FIG. 10 show that contents of the UCP1 protein in brown and white adipose tissues are also significantly increased, and it shows a certain dose-dependent relationship and a good effect can be provided at a low (5 mg/Kg) drug concentration. The above indicates that the milk-derived polypeptide and its derivative can serve to reduce lipid accumulation in the adipose tissues induced by high-fat diet, and this reduction is achieved by increasing thermogenic activity of the adipose tissues.

Embodiment 6 Effect of Milk-Derived Polypeptide on Body Weight and Blood Glucose of Obese Animal Model

1. Experimental Method

[0050] C57BL/6J male mice at 6-8 weeks old were used as research subjects, and an experiment was carried out after adaptation to a SPF animal room for one week. The mice were fed with high-fat diet (with 60% of fat content) for six months to induce obesity phenotype in mice. After six months, the milk-derived polypeptide derivative shown by SEQ ID NO.5 (10 mg/kg) was administered by intraperitoneal injection once a day and continuously for 8 weeks. The mice were weighed every week for their weight changes, and at the same time, blood glucose changes of the mice were detected by a blood glucose meter. After intervention, the mice were killed, and then brown fat (BAT) at scapular, inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) were photographed. As mentioned above, H&E staining was also performed to observe morphological changes of lipid droplets, and expression of the UCP1 protein in adipose tissues was detected by immunohistochemistry and the Western blot methods.

Experimental Results

[0051] As shown in FIG. 11, body weight of the obese mice induced by the high-fat diet can be significantly reduced after 8 weeks of administration of the milk-derived polypeptide derivatives, while weight of the control group has no significant change. In addition, fasting blood glucose of the mice in an administration group decreased, thus achieving effects of reducing the weight and reducing the blood glucose. After dissection, morphological observation shows that tissue volumes of white fat (subcutaneous and abdominal) and brown fat (scapular areas) in various parts are significantly reduced, suggesting that lipid accumulation is reduced. The results are shown in FIG. 12. The H&E staining also shows that sizes of the lipid droplets in the two tissues decreased significantly, which also indicated that lipid accumulation is decreased. The immunohistochemistry and the Western blot test in FIG. 13 and FIG. 14 indicate that the expression of UCP1 was increased in two kinds of adipose tissues. Details can be referred to brown areas in FIG. 13 and band thicknesses in FIG. 14. The above results show that this therapeutic effect is also achieved by activating the thermogenic activity of the adipose tissues and thus reducing lipid accumulation in the adipose tissues.