A polypeptide composition with anti-obesity efficacy and application thereof

Abstract

The present application provides a polypeptide composition with anti-obesity effect, which is composed of capsaicin and polypeptides, and the amino acid sequence of the polypeptides comprises any one or more of SEQ ID NO. 1 to SEQ ID NO. 8. The polypeptides of sequences 1-8 are mixed uniformly at a mass ratio of 1-20:1-4:1-2:1-4:1:1:1:1, and the capsaicin is mixed with the mixed polypeptides at a ratio of 2-12:100 (w / w). The polypeptide composition proposed in the present application, in combination with capsaicin, not only promotes the weight loss effect of capsaicin, but also significantly reduces the fat index of the test mice after use, and can significantly reduce the total cholesterol concentration in the liver and the liver triglyceride concentration under a high-fat diet.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to a polypeptide composition that can enhance the anti-obesity effect of capsaicin and its application. Background Technology

[0002] Obesity is a chronic metabolic disease caused by excessive calorie intake or insufficient calorie expenditure, leading to fat accumulation. With economic development and improved living standards, the prevalence of obesity is increasing year by year, becoming a global health problem. Studies have shown a strong correlation between obesity and various diseases, including hypertension, coronary heart disease, type 2 diabetes, non-alcoholic fatty liver disease, and cancer. Therefore, the prevention and treatment of obesity are of paramount importance.

[0003] Chili peppers are a commonly consumed food, and capsaicin is the most abundant and biologically active small molecule compound in them. Capsaicin is a natural alkaloid derived from plants of the Capsicum genus, also known as trans-8-methyl-N-vanillyl-6-nonenylamide. It is a highly volatile, lipophilic, and hydrophobic white crystalline powder. Studies have shown that capsaicin exerts its anti-obesity effects by regulating lipid and glucose metabolism and influencing the gut microbiota. For example, capsaicin can inhibit adipocyte differentiation, induce browning of white adipocytes, increase thermogenesis, and reduce intracellular lipid content. It can also lower blood sugar by enhancing insulin secretion and inhibiting glucagon release. Furthermore, by directly or indirectly affecting the composition, abundance, and structure of the gut microbiota, capsaicin can also play a role in preventing obesity. Therefore, capsaicin has the potential to become a novel therapeutic ingredient for obesity.

[0004] However, as a strongly hydrophobic substance, capsaicin is almost insoluble in water at room temperature. This low solubility results in low bioavailability in the body, significantly limiting its anti-obesity effects. To enhance the weight-loss effect of capsaicin and reduce user discomfort, many researchers have combined capsaicin with other substances to create compositions that better exert its anti-obesity properties. Amphiphilic peptides are promising potential raw materials for combining with capsaicin to prepare anti-obesity compositions. Summary of the Invention

[0005] The purpose of this invention is to propose a polypeptide composition with anti-obesity effects, which is prepared by combining polypeptides and capsaicin, and can further enhance the anti-obesity effects of capsaicin.

[0006] The technical solution for achieving the above-mentioned objective of this invention is as follows:

[0007] A polypeptide composition with anti-obesity effects, comprising capsaicin and a polypeptide, wherein the amino acid sequence of the polypeptide includes the following sequence:

[0008] SEQ ID NO.1RGIIPL

[0009] SEQ ID NO.2FEGTVF

[0010] SEQ ID NO.3IDAAY

[0011] SEQ ID NO.4IILGPK

[0012] SEQ ID NO.5 KIADMF

[0013] SEQ ID NO.6TVFGSY

[0014] SEQ ID NO.7FRNTIF

[0015] SEQ ID NO.8DMFPF.

[0016] In this mixture, capsaicin and the polypeptide are mixed in a ratio of 2 to 12:100 (w / w).

[0017] PA2 protein consists of two polypeptide chains with a molecular weight of approximately 26 kDa. Each polypeptide chain contains three cysteine ​​residues, two of which form an intrachain disulfide bond and one forms an interchain disulfide bond. Because the conformation of PA2 protein is linked by only one covalent bond, it is flexible and can be easily altered. This application further screens for amphiphilic peptides of PA2 protein. It is generally believed that all proteins are amphiphilic, possessing both hydrophilic and hydrophobic amino acids, differing only in their affinity; however, strong hydrophilicity may prevent it from binding to hydrophobic molecules. This study selects peptides with higher hydrophobicity based on water-soluble peptides. This ensures that the peptides possess both a certain degree of hydrophilicity (soluble in water) and hydrophobicity (allowing them to bind to hydrophobic capsaicin). Experiments have confirmed that the binding of the aforementioned peptides to capsaicin can significantly enhance the weight-loss effect of capsaicin.

[0018] The present invention does not strictly limit the form of the polypeptide composition, which can be in the form of particles, liquid, emulsion, etc.

[0019] The polypeptide composition is ingested via the digestive tract, such as orally, chewing, lozenges, gavage, or tube feeding.

[0020] Preferably, the dosage form of the polypeptide composition is nanoparticles, powder, mixed solution, or capsule.

[0021] The following provides a method for preparing the polypeptide composition.

[0022] The polypeptide composition is prepared by dissolving the polypeptide in water to prepare a solution with a concentration of 0.1 to 2 mg / mL, dissolving capsaicin in ethanol at a concentration of 5 to 50 mg / mL, and mixing capsaicin and the polypeptide at a ratio of 2 to 12:100 (w / w).

[0023] The polypeptide is a mixed polypeptide obtained by mixing polypeptides of sequences 1-8 in a mass ratio of 1-22:1-5:1-2:1-4:1:1:1:1.

[0024] Further, the capsaicin and the polypeptide are vortexed at a ratio of 2–12:100 (w / w) for 10–30 s, centrifuged, and the supernatant is collected and freeze-dried to obtain the polypeptide composition; the centrifugation conditions are 5000–10000 × g for 5–30 min. The centrifugation temperature can be 4–25 °C.

[0025] This invention does not strictly limit the source of the polypeptide; it can be obtained by extraction from plants or by chemical synthesis. The plants can be peas, soybeans, etc.

[0026] The polypeptide is either artificially synthesized or derived from plant PA2 protein.

[0027] Preferably, artificial synthesis can be performed using the Fmoc method (9-fluorophthaloylmethoxycarbonyl protected herbinoamine-triphenylmethyl ester protected amino acid-solid phase synthesis) or other synthetic methods known in the art.

[0028] The PA2 protein is derived from pea protein.

[0029] Alternatively, the polypeptide may be synthesized using the Fmoc solid-phase synthesis method.

[0030] The beneficial effects of this invention are as follows:

[0031] The polypeptide composition proposed in this invention contains polypeptides with strong hydrophilicity, and selects highly hydrophobic peptide segments based on water-soluble polypeptides. The polypeptides proposed in this invention, when combined with capsaicin, can significantly enhance the weight-loss effect of capsaicin. This invention utilizes molecular biology techniques to combine polypeptides with capsaicin, not only promoting the weight-loss effect of capsaicin, but also significantly reducing the body fat index of test mice after use, and significantly reducing total cholesterol and triglyceride concentrations in the liver even under a high-fat diet. This polypeptide composition can better lower fasting blood glucose and regulate insulin levels. Attached Figure Description

[0032] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0033] Figures 1 to 5In this context, "NCD" represents the normal control group fed a low-fat diet, "HFD" represents the model group fed a high-fat diet, "HFD+CAP" represents the free capsaicin group fed a high-fat diet and administered free capsaicin by gavage, "HFD+PANP-CAP" represents the polypeptide composition group fed a high-fat diet and administered a polypeptide composition by gavage, and "HFD+PANP" represents the pure polypeptide group fed a high-fat diet and administered a mixed polypeptide by gavage.

[0034] Different letters in each graph indicate significant differences in the mean (P<0.05);

[0035] Figure 1 The weight of mice after 12 weeks of gavage in Example 3 of the present invention includes their weight at 12 weeks, weight gain from 0 to 12 weeks, weight changes from 0 to 12 weeks, and average daily food intake.

[0036] Figure 2 The fat index of mice at different sites after 12 weeks of gavage in Example 3 of this invention;

[0037] Figure 3 The liver fat status of mice after 12 weeks of gavage in Example 3 of the present invention includes liver weight, total cholesterol concentration in the liver, and triglyceride concentration in the liver.

[0038] Figure 4 The blood glucose concentration and serum insulin concentration of mice after 12 weeks of gavage in Example 3 of this invention;

[0039] Figure 5 This shows the relative mRNA expression of PPARγ and ACLY proteins in mice after 12 weeks of gavage in Example 3 of this invention. Detailed Implementation

[0040] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0041] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0042] Unless otherwise specified, all technical means used in this instruction manual are known in the field, and all raw materials used are commercially available.

[0043] It should be noted that all amino acid sequences in this article are "conservatively modified amino acid sequences". "Conservatively modified amino acid sequences" means that the modification does not significantly affect or change the biological characteristics of the polypeptide containing the amino acid sequence. The modification includes the addition and deletion of amino acids, and the number of conserved modifications does not exceed one or two.

[0044] Example 1

[0045] Amphiphilic peptides were synthesized using the Fmoc solid-phase synthesis method, with each peptide achieving a purity of over 90%. This was achieved through ExPasy (…). https: / / web.expasy.org / protparam / The hydrophilicity of the eight peptides was assessed (Table 1).

[0046] Table 1. Sequence listing of 8 polypeptides

[0047] Serial Number amino acid sequence Hydrophilicity score amino acid count SEQ ID NO.1 RGIIPL 1.05 6 SEQ ID NO.2 FEGTVF 0.867 6 SEQ ID NO.3 IDAAY 0.66 5 SEQ ID NO.4 IILGPK 1.15 6 SEQ ID NO.5 KIADMF 0.6 6 SEQ ID NO.6 TVFGSY 0.633 6 SEQ ID NO.7 FRNTIF 0.233 6 SEQ ID NO.8 DMFPF 0.48 5

[0048] Note: A positive hydrophilicity score indicates that the peptide is more hydrophobic, while a negative score indicates that the peptide is more hydrophilic.

[0049] The hydrophilicity scores of the eight peptides in Table 1 are all positive, indicating that the peptides are slightly hydrophobic, which is conducive to their interaction with capsaicin.

[0050] Example 2 Preparation of polypeptide composition

[0051] The peptides with sequences SEQ ID NO. 1-8 synthesized in Example 1 were mixed uniformly at a mass ratio of 20:4:1:4:1:1:1:1 (in previous studies, the mass ratio of the eight peptides in natural PA2 protein hydrolysate was determined to be 20:4:1:4:1:1:1:1 by mass spectrometry; therefore, after the artificial synthesis of the eight peptides, they were mixed according to the proportion of each peptide present in the natural PA2 protein hydrolysate) to obtain a mixed peptide; the mixed peptide was dissolved in water to prepare a solution with a concentration of 1 mg / mL. Capsaicin was dissolved in ethanol at a concentration of 25 mg / mL. Capsaicin and the mixed peptide were vortexed at a ratio of 8:100 (w / w) for 15 s, centrifuged (10000×g, 25℃, 10 min), and the supernatant was collected and lyophilized to obtain the peptide composition (in solid nanoparticle form).

[0052] Example 3 Evaluation of anti-obesity efficacy

[0053] Thirty-six 4-week-old male C57BL / 6J mice were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. After one week of acclimatization, the mice were divided into five groups: a normal control group (NCD) fed a low-fat diet, a model group (HFD), a free capsaicin group (HFD+CAP), a polypeptide combination group (HFD+PANP-CAP), and a pure polypeptide group (HFD+PANP) fed a high-fat diet. The free capsaicin group was administered 10 mg / kg of capsaicin daily by gavage, the polypeptide combination group was administered a polypeptide combination containing an equal amount of capsaicin by gavage, the pure polypeptide group was administered an equal amount of mixed polypeptides by gavage, and the control and model groups were administered an equal volume of physiological saline daily by gavage. Mouse body weight was measured weekly. After 12 weeks of continuous gavage, blood was collected from the orbital rim, the mice were euthanized by dislocation, and a series of indicators were measured.

[0054] like Figure 1 As shown, both the free capsaicin group and the peptide composition group reduced the weight gain induced by a high-fat diet in mice. After 12 weeks of feeding, compared with the body weight of the model group (45.69g), the mice in the free capsaicin group lost 5.10g, while the mice in the peptide composition group lost 9.27g, a weight loss 1.8 times that of the free capsaicin group. This indicates that the peptide composition group had a better weight-loss effect, and the peptide composition can promote the weight-loss effect of capsaicin. There was no significant difference in food intake among all groups, indicating that the changes in mouse body weight were not caused by changes in food intake.

[0055] like Figure 2 As shown, both free capsaicin and peptide composition intervention significantly reduced the fat index in the epididymis, groin, mesentery and perirenal region of mice. Compared with the free capsaicin group, the peptide composition group reduced each index by 35.93%, 32.24%, 23.47% and 28.48%, respectively, indicating that the peptide composition was more effective and significantly promoted the fat-reducing effect of capsaicin.

[0056] like Figure 3 As shown, a high-fat diet led to increased liver weight in mice and elevated total cholesterol and triglycerides in the liver. Intervention with free capsaicin and the polypeptide composition restored liver weight to normal levels and significantly reduced total cholesterol and triglyceride concentrations in the liver. Compared with free capsaicin, the total cholesterol and triglyceride concentrations in the liver were reduced by 6.82% and 11.38%, respectively, further demonstrating the anti-obesity efficacy of capsaicin promoted by the polypeptide composition.

[0057] like Figure 4 As shown, a high-fat diet induced increased fasting blood glucose and insulin secretion in mice. Compared with free capsaicin, the blood glucose concentration and serum insulin concentration of the polypeptide composition group decreased by 14.14% and 9.10%, respectively, indicating that the polypeptide composition can better reduce fasting blood glucose and regulate insulin levels, and promote the anti-obesity effect of capsaicin by regulating glucose metabolism.

[0058] like Figure 5 As shown, PPARγ can be activated by fatty acids and exogenous peroxisome proliferators, and regulates the expression of certain enzymes involved in lipid metabolism. ACLY acts as a bridge between glucose metabolism and fatty acid production, and is an enzyme that catalyzes an important step in fatty acid biosynthesis. The combination of free capsaicin and peptides reduced the expression of PPARγ and ACLY, thereby reducing lipid accumulation and inhibiting the de novo synthesis of fat. The peptide combination showed a better effect, indicating that capsaicin can exert an anti-obesity effect by regulating lipid metabolism, while the peptide combination can promote this process.

[0059] In short, Figures 1 to 5 Together, they demonstrate that the polypeptide composition proposed in this invention can enhance the anti-obesity effect of capsaicin.

[0060] Although the present invention has been described above through embodiments, those skilled in the art should understand that any improvements and modifications made to the present invention without departing from its spirit and essence should fall within the protection scope of the present invention.

Claims

1. A polypeptide composition with anti-obesity effects, characterized in that, Composed of capsaicin and a mixed polypeptide, said mixed polypeptide is obtained by mixing polypeptides with the following amino acid sequence in a mass ratio of 20:4:1:4:1:1:1:1:1: SEQ ID NO.1RGIIPL SEQ ID NO.2FEGTVF SEQ ID NO.3IDAAY SEQ ID NO.4IILGPK SEQ ID NO.5 KIADMF SEQ ID NO.6TVFGSY SEQ ID NO.7FRNTIF SEQ ID NO.8DMFPF; Capsaicin and the mixed polypeptide are mixed at a ratio of 2 to 12:100 (w / w).

2. The polypeptide composition with anti-obesity effect according to claim 1, characterized in that, The dosage form of the polypeptide composition is nanoparticles, powder, mixed solution or capsule.

3. The polypeptide composition with anti-obesity effects according to claim 1, characterized in that, The polypeptide composition is prepared by dissolving the mixed polypeptide in water to prepare a solution with a concentration of 0.1 to 2 mg / mL, dissolving capsaicin in ethanol at a concentration of 5 to 50 mg / mL, and mixing capsaicin and the mixed polypeptide at a ratio of 2 to 12:100 w / w.

4. The polypeptide composition with anti-obesity effects according to claim 3, characterized in that, The capsaicin and the mixed polypeptide were vortexed at a ratio of 2-12:100 (w / w) for 10-30 seconds, centrifuged, and the supernatant was collected and freeze-dried to obtain the polypeptide composition. The centrifugation conditions were 5000-10000×g for 5-30 minutes.

5. The polypeptide composition with anti-obesity effect according to any one of claims 1 to 4, characterized in that, The polypeptides with amino acid sequences 1-8 are artificially synthesized or derived from plant PA2 proteins.

6. The polypeptide composition with anti-obesity effects according to claim 5, characterized in that, The PA2 protein is derived from pea protein.

7. The polypeptide composition with anti-obesity effect according to claim 5, characterized in that, The polypeptide with amino acid sequences 1-8 was synthesized using the Fmoc solid-phase synthesis method.

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