Α-amylase inhibitory peptide and use thereof

Abstract

An α-amylase inhibitory peptide. The peptide comprises amino acid sequences of eight adjacent amino acids X1, X2, X3, X4, X5, X6, X7, and X8, wherein each amino acid of the peptide is independently selected from a D-amino acid or an L-amino acid, and at least one of X2, X4, and X6 is W. The peptide has α-amylase inhibitory activity, has the effects of losing weight and lowering blood sugar, and can be used for preparing α-amylase inhibitors, weight-losing drugs, hypoglycemic drugs, or foods, health-care products or drugs for preventing or treating obesity-related diseases.

Description

α-amylase inhibitory peptide and its application

[0001] This application claims priority to the Chinese patent application filed on February 5, 2024 (application number: CN202410162962.0, invention name: α-amylase inhibitory peptide and its application), and the entire contents of the priority patent application are incorporated by reference into this patent application. Technical Field

[0002] The present invention belongs to the field of biomedicine, and in particular relates to an α-amylase inhibitory peptide and application thereof. Background Art

[0003] Obesity is both an independent disease and a significant contributing factor to numerous chronic conditions, including cardiovascular and cerebrovascular disease, various cancers, type 2 diabetes, and hypertension. With rapid socioeconomic development and improved living standards, coupled with significant changes in dietary patterns and the emergence of numerous unhealthy habits, obesity has become a serious health hazard, becoming a focal point of public health concern.

[0004] Peptides are a major category of biopharmaceuticals, and medicinal peptides have significant advantages. First, peptides are derived directly or indirectly from nature, which aligns with the trend of screening new drugs from natural products. Second, peptides have much stronger specificity than small molecules and much lower antigenicity than proteins, eliminating the shortcomings of small molecule and protein drugs. Third, peptide molecules, especially oligopeptides, can often circumvent gastrointestinal digestion, overcoming the drawback that protein molecules are broken down by digestive enzymes and cannot be taken orally. Finally, peptide molecules may neither be metabolized in the liver nor accumulated in adipose tissue, thus avoiding liver toxicity and cumulative toxicity.

[0005] Therefore, it is necessary to develop peptide drugs with hypoglycemic effects. Summary of the Invention

[0006] Based on the crystal structure of α-amylase, the present invention provides a series of novel α-amylase inhibitory peptides. These peptides possess α-amylase inhibitory activity, simultaneously exhibit weight loss and blood sugar lowering effects, are safe, have no toxic side effects, and have the potential for oral administration. In some embodiments, these peptides also have lipid-lowering effects.

[0007] One aspect of the present invention provides a peptide comprising an amino acid sequence of 8 adjacent amino acids X1X2X3X4X5X6X7X8, wherein at least one of X2, X4 and X6 is W.

[0008] In some embodiments, each amino acid of the peptide is independently selected from D-amino acids or L-amino acids, and satisfies any one of the following conditions (1) to (3):

[0009] (1) X2, X4 and X6 are all W, and X1 is selected from D, R, L, N, S, E or V, X3 is selected from A, P, V, G, Y, K or R, X5 is selected from E, F, G, R, H or P, X7 is selected from D, S, R, V, W, N or Y, and X8 is A;

[0010] (2) X1 is selected from D, X2 is selected from W or F, X3 is selected from A, X4 is selected from W or Y, X5 is selected from E or D, X6 is selected from W, X7 is selected from D, X8 is selected from A, and only one of X2 and X4 is W;

[0011] (3) X1 is selected from D, E or A, X2 is F, X3 is selected from A, G or L, X4 is selected from W or Y, X5 is selected from D or A, X6 is selected from F or W, X7 is selected from D or E, X8 is selected from P, W or A, and only one of X4 and X6 is W.

[0012] In some embodiments, the peptide comprises an amino acid sequence of 8 adjacent amino acids X1WX3WX5WX7A, wherein X1 is selected from D, R, L, N, S, E or V, X3 is selected from A, P, V, G, Y, K or R, X5 is selected from E, F, G, R, H or P, and X7 is selected from D, S, R, V, W, N or Y.

[0013] In some embodiments, in the peptide, X2, X4 and X6 are all W, and X1 is selected from D, R, L, N, S, E or V, X3 is selected from A, P, V, G, Y, K or R, X5 is selected from E, F, G, R, H or P, X7 is selected from D, S, R, V, W, N or Y, and X8 is A.

[0014] In some embodiments, in the peptide, X2, X4 and X6 are all W, and X1 is D, X3 is A, X5 is E, X7 is D, and X8 is A, or X1 is R, X3 is P, X5 is F, X7 is S, and X8 is A, or X1 is L, X3 is V, X5 is G, X7 is R, and X8 is A, or X1 is N, X3 is G, X5 is F, X7 is R, and X8 is A, or X1 is S, X3 is Y, X5 is R, X7 is V, and X8 is A, or X1 is E, X3 is K, X5 is H, X7 is W, and X8 is A, or X1 is L, X3 is G, X5 is R, X7 is N, and X8 is A, or X1 is V, X3 is R, X5 is P, X7 is Y, and X8 is A.

[0015] In some embodiments, the peptide comprises any of the following amino acid sequences:

[0016] DWAWEWDA (SEQ ID NO: 1), RWPWFWSA (SEQ ID NO: 2), LWVWGWRA (SEQ ID NO: 3), NWGWFWRA (SEQ ID NO: 4), SWYWRWVA (SEQ ID NO: 5), EWKWHWWA (SEQ ID NO: 6), LWGWRWNA (SEQ ID NO: 7), VWRWPWYA (SEQ ID NO: 8).

[0017] In some embodiments, the peptide comprises the amino acid sequence set forth in any one or more of SEQ ID NO: 14, SEQ ID NO: 18, and SEQ ID NO: 22-30.

[0018] In some embodiments, the peptide comprises an amino acid sequence of 8 adjacent amino acids X1FX3X4X5X6X7X8, X1 is selected from D, E or A, X3 is selected from A, G or L, X4 is selected from W or Y, X5 is selected from D or A, X6 is selected from F or W, X7 is selected from D or E, X8 is selected from P, W or A, and only one of X4 and X6 is W.

[0019] In some embodiments, in the peptide, X1 is selected from D, E or A, X2 is F, X3 is selected from A, G or L, X4 is selected from W or Y, X5 is selected from D or A, X6 is selected from F or W, X7 is selected from D or E, X8 is selected from P, W or A, and only one of X4 and X6 is W.

[0020] In some embodiments, in the peptide, X1 is D, X2 is F, X3 is A, X4 is W, X5 is D, X6 is F, X7 is D, X8 is P, or X1 is selected from E or A, X2 is F, X3 is selected from G or L, X4 is selected from Y, X5 is selected from D or A, X6 is W, X7 is selected from D or E, and X8 is selected from W or A.

[0021] In some embodiments, the peptide comprises the amino acid sequence of 1) or 2) below:

[0022] 1) DFAWDFDP (SEQ ID NO: 9), or

[0023] 2) X1FX3YX5WX7X8, wherein X1 is selected from E or A, X3 is selected from G or L, X5 is selected from D or A, X7 is selected from D or E, and X8 is selected from W or A.

[0024] In some embodiments, in the peptide, X1 is D, X2 is F, X3 is A, X4 is W, X5 is D, X6 is F, X7 is D, and X8 is P.

[0025] In some embodiments, in the peptide, X1 is selected from E or A, X2 is F, X3 is selected from G or L, X4 is selected from Y, X5 is selected from D or A, X6 is W, X7 is selected from D or E, and X8 is selected from W or A.

[0026] In some embodiments, the peptide comprises an amino acid sequence of 8 adjacent amino acids X1FX3YX5WX7X8, wherein X1 is selected from E or A, X3 is selected from G or L, X5 is selected from D or A, X7 is selected from D or E, and X8 is selected from W or A.

[0027] In some embodiments, the peptide comprises any of the following amino acid sequences:

[0028] DFAWDFDP (SEQ ID NO:9), EFGYAWEW (SEQ ID NO:10), AFLYDWDA (SEQ ID NO:11).

[0029] In some embodiments, in the peptide, X1 is E, X2 is F, X3 is G, X4 is Y, X5 is A, X6 is W, X7 is E, and X8 is W.

[0030] In some embodiments, in the peptide, X1 is A, X2 is F, X3 is L, X4 is Y, X5 is D, X6 is W, X7 is D, and X8 is A.

[0031] In some embodiments, the peptide comprises the amino acid sequence set forth in any one or more of SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 31, and SEQ ID NO: 32.

[0032] In some embodiments, in the peptide, X1 is selected from D, X2 is selected from W or F, X3 is selected from A, X4 is selected from W or Y, X5 is selected from E or D, X6 is selected from W, X7 is selected from D, X8 is selected from A, and only one of X2 and X4 is W.

[0033] In some embodiments, the peptide comprises an amino acid sequence of 8 consecutive amino acids DX2AX4X5WDA, wherein X2 is selected from W or F, X4 is selected from W or Y, X5 is selected from E or D, and only one of X2 and X4 is W.

[0034] In some embodiments, in the peptide, X1 is selected from D, X2 is selected from W or F, X3 is selected from A, X4 is selected from W or Y, X5 is selected from E or D, X6 is selected from W, X7 is selected from D, X8 is selected from A, and only one of X2 and X4 is W.

[0035] In some embodiments, the peptide comprises any of the following amino acid sequences:

[0036] DWAYEWDA (SEQ ID NO: 12), DFAWDWDA (SEQ ID NO: 13).

[0037] In some embodiments, in the peptide, X2 and X6 are W, X1 is D, X3 is A, X4 is Y, X5 is E, X7 is D, and X8 is A.

[0038] In some embodiments, in the peptide, X4 and X6 are W, X1 is D, X2 is F, X3 is A, X5 is D, X7 is D, and X8 is A.

[0039] In some embodiments, the peptide comprises the amino acid sequence shown in any one or more of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 20.

[0040] In some embodiments, each amino acid of the peptide is independently selected from a D-amino acid or an L-amino acid.

[0041] In some embodiments, in the peptide, X4 is a D-amino acid or an L-amino acid.

[0042] In some embodiments, in the peptide, X4 is a D-type amino acid or an L-type amino acid, and X1, X2, X3, X5, X6, X7, and X8 are L-type amino acids.

[0043] In some embodiments, in the peptide, X4 is a D-amino acid, and X1, X2, X3, X5, X6, X7, and X8 are L-amino acids.

[0044] In other embodiments, in the peptide, X1 to X8 are all L-amino acids.

[0045] In some embodiments, the peptide is a polypeptide consisting of 8 amino acids.

[0046] In some embodiments, the peptide is a linear peptide or a cyclic peptide.

[0047] In some embodiments, the peptide has amylase inhibitory activity.

[0048] In some embodiments, the peptide has an in vitro amylase inhibition rate of greater than 10% at a concentration of 0.5 mg / mL.

[0049] In some embodiments, the peptide has lipase inhibitory activity.

[0050] In some embodiments, the peptide has colipase inhibitory activity.

[0051] In some embodiments, the peptide has an amino acid sequence as shown in any one or more of SEQ ID NOs: 14-32 below.

[0052] Another aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the peptide.

[0053] Another aspect of the present invention provides an expression vector comprising the nucleic acid molecule of the present invention.

[0054] Another aspect of the present invention provides a host cell comprising the nucleic acid molecule of the present invention or the expression vector of the present invention.

[0055] In some embodiments of the invention, the host cell is a bacterium.

[0056] Another aspect of the present invention provides a pharmaceutical composition comprising the peptide, nucleic acid molecule, expression vector or host cell of the present invention; and a pharmaceutically acceptable carrier.

[0057] In some embodiments, the pharmaceutical composition can be formulated for administration via a specific route of administration. For example, the pharmaceutical composition can be formulated for oral, intravenous, intratumoral, intraperitoneal, intradermal, subcutaneous, intranasal or other routes of administration.

[0058] In some embodiments, the dosage form of the pharmaceutical composition includes (but is not limited to): injection, aerosol, pellet, external ointment, controlled release or sustained release or nanoformulation.

[0059] One aspect of the present invention provides the use of the peptide, pharmaceutical composition, nucleic acid molecule, expression vector or host cell in the preparation of α-amylase inhibitors, lipase inhibitors, colipase inhibitors, weight loss drugs, hypoglycemic drugs, hypolipidemic drugs or foods, health products or drugs for preventing or treating obesity-related diseases.

[0060] In some embodiments, the present invention provides the use of the peptide, pharmaceutical composition, nucleic acid molecule, expression vector, or host cell in the preparation of an α-amylase inhibitor, a weight-loss drug, a hypoglycemic drug, or a food, health product, or drug for preventing or treating obesity-related diseases. Another aspect of the present invention provides any of the above peptides, pharmaceutical compositions, nucleic acid molecules, expression vectors, or host cells for use as a food, health product, or drug; in some embodiments, the food, health product, or drug is used to prepare an α-amylase inhibitor, a lipase inhibitor, a colipase inhibitor, a weight-loss drug, a hypoglycemic drug, a hypolipidemic drug, or for preventing or treating obesity-related diseases.

[0061] Another aspect of the present invention provides a method for preventing or treating a disease, comprising administering to a subject in need thereof a therapeutically effective amount of any of the foregoing peptides, pharmaceutical compositions, nucleic acid molecules, expression vectors or host cells; in some embodiments, the disease is an obesity-related disease.

[0062] Another aspect of the present invention provides a food or health product comprising any of the peptides, pharmaceutical compositions, nucleic acid molecules, expression vectors or host cells described above.

[0063] In some embodiments, the obesity-related diseases include one or more of obesity, cardiovascular and cerebrovascular diseases, cancer, type 2 diabetes, hyperglycemia, hypertension, hyperlipidemia, fatty liver, coronary heart disease, atherosclerosis, stroke and acute pancreatitis.

[0064] In some embodiments, the obesity-related diseases include one or more of obesity, cardiovascular and cerebrovascular diseases, cancer, type 2 diabetes, hyperglycemia, and hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] FIG1 shows the results of an in vitro enzyme activity inhibition experiment of TS-1.

[0066] Figure 2 shows the experimental flow of the animal experiments.

[0067] Figure 3 shows the postprandial blood glucose changes of mice in the animal experiment, where white kidney bean extract was the positive control, Vehicle was the negative control, TSC-high was 150 mg / Kg TS-1, TSC-med was 75 mg / Kg TS-1, and TSC-low was 37.5 mg / Kg TS-1.

[0068] Figure 4 shows the weight changes of mice in the animal experiment, where the white kidney bean extract was used as a positive control, Vehicle was used as a negative control, TSC-high was 150 mg / Kg TS-1, TSC-med was 75 mg / Kg TS-1, and TSC-low was 37.5 mg / Kg TS-1.

[0069] Figure 5 shows the changes in red blood cell (RBC) and platelet counts in the blood of mice in the animal experiment, where the white kidney bean extract was used as a positive control, Vehicle was used as a negative control, TSC-high was 150 mg / Kg TS-1, TSC-med was 75 mg / Kg TS-1, and TSC-low was 37.5 mg / Kg TS-1.

[0070] Figure 6 shows the changes in white blood cell count and body weight of mice in the animal experiment, wherein the white kidney bean extract was used as a positive control, Vehicle was used as a negative control, TSC-high was 150 mg / Kg TS-1, TSC-med was 75 mg / Kg TS-1, and TSC-low was 37.5 mg / Kg TS-1.

[0071] Figure 7 shows the changes in alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels in the liver blood of mice in the animal experiment, wherein the white kidney bean extract was used as a positive control, Vehicle was used as a negative control, TSC-high was 150 mg / Kg TS-1, TSC-med was 75 mg / Kg TS-1, and TSC-low was 37.5 mg / Kg TS-1.

[0072] Figure 8 shows the changes in urea nitrogen (BUN), creatinine-S (CRE-S), albumin (ALB), and total protein (TP) levels in the renal blood of mice in the animal experiment, where white kidney bean extract was used as a positive control, Vehicle was used as a negative control, TSC-high was 150 mg / Kg TS-1, TSC-med was 75 mg / Kg TS-1, and TSC-low was 37.5 mg / Kg TS-1.

[0073] Figure 9 shows the changes in chloride ion (Cl) and sodium ion (Na) levels in the blood of mice in the animal experiment, where the white kidney bean extract was used as a positive control, Vehicle was used as a negative control, TSC-high was 150 mg / Kg TS-1, TSC-med was 75 mg / Kg TS-1, and TSC-low was 37.5 mg / Kg TS-1.

[0074] Figure 10 shows the changes in chloride ion (Cl) and sodium ion (Na) levels in the blood of mice in the animal experiment, where the white kidney bean extract was the positive control, Vehicle was the negative control, TSC-high was 150 mg / Kg TS-1, TSC-med was 75 mg / Kg TS-1, and TSC-low was 37.5 mg / Kg TS-1.

[0075] Figure 11 shows the changes in relative liver weight of mice in the animal experiment, where white kidney bean extract was used as a positive control, Vehicle was used as a negative control, TSC-high was 150 mg / Kg TS-1, TSC-med was 75 mg / Kg TS-1, and TSC-low was 37.5 mg / Kg TS-1.

[0076] Figure 12 shows the changes in the relative weights of other organs of mice in the animal experiment, where the white kidney bean extract was used as the positive control, Vehicle was used as the negative control, TSC-high was 150 mg / Kg TS-1, TSC-med was 75 mg / Kg TS-1, and TSC-low was 37.5 mg / Kg TS-1.

[0077] FIG13 shows the results of the stability test of TS-1 and commercially available α-amylase inhibitors in pepsin; in each time group, from left to right are: TS peptide, Xiuzheng, Ruilin, Lucuimei, Yolanda, and Swanson.

[0078] FIG14 shows the serum TG concentration on day 1 of the mouse OLTT experiment.

[0079] FIG15 shows the area under the curve of serum TG on day 1 of the mouse OLTT experiment.

[0080] FIG16 shows the serum TG concentration on day 8 of the mouse OLTT experiment.

[0081] FIG17 shows the area under the curve of serum TG on day 8 of the mouse OLTT experiment.

[0082] FIG18 shows the serum TG concentration on day 14 of the mouse OLTT experiment.

[0083] FIG19 shows the area under the curve of serum TG on day 14 of the mouse OLTT experiment. DETAILED DESCRIPTION

[0084] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and examples. The specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention in any way. In addition, descriptions of known structures and techniques are omitted in the following description to avoid unnecessary confusion about the concepts of the present disclosure. Such structures and techniques are also described in many publications.

[0085] the term

[0086] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0087] Unless the context clearly dictates otherwise, as used herein, the expressions "a" and "an" include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0088] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0089] As used herein, numerical ranges are to be understood as including all numbers within the range. For example, a range of 1 to 20 is to be understood as including any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0090] Herein, in the amino acid sequence of the peptide, A represents alanine (Ala), R represents arginine (Arg), N represents aspartic acid (Asn), D represents aspartic acid (Asp), C represents cysteine ​​(Cysteine, Cys), Q represents glutamine (Gln), and E represents glutamic acid (Gln). In the embodiment of the present invention, the amino acid residues (residues) are d-terminal or l-terminal. The amino acid residues (residues) mentioned herein may be D-type or L-type. The amino acid sequences mentioned herein may be in the order of N-terminus to C-terminus from left to right unless otherwise stated.

[0091] The peptides of the present invention may comprise peptides obtained by replacing one or more conservative amino acids in the peptide. In some embodiments, conservative amino acid replacement can mean that an amino acid residue is replaced with a biologically similar residue. Particularly preferred substitutions are generally conservative in nature, that is, those that occur within an amino acid family. For example, amino acids are generally divided into four families: (1) acidic - aspartic acid (N) and glutamic acid (E); (2) basic - lysine (K), arginine (R), histidine (H); (3) non-polar - alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W); (4) uncharged polar - glycine (G), asparagine (D), glutamine (Q), cysteine ​​(C), serine (S), threonine (T), tyrosine (Y). Phenylalanine, tryptophan and tyrosine are sometimes classified as aromatic amino acids. Examples of conservative changes include substitution of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, for another hydrophobic residue, or substitution of one polar residue for another, such as arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, or similar conservative substitutions of amino acids with structurally related amino acids that do not significantly affect biological activity. Thus, proteins having an amino acid sequence substantially identical to a reference molecule but with minor amino acid substitutions that do not substantially affect protein activity are within the definition of a reference polypeptide.

[0092] The term "pharmaceutically acceptable carrier" as used herein refers to a component of a pharmaceutical composition other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In some embodiments, the pharmaceutically acceptable carrier includes: a polymer of acrylic acid or methacrylic acid, maleic anhydride, and an alkenyl derivative polymer; an immunostimulatory sequence (ISS), such as an oligodeoxyribonucleotide sequence (CpG ODN) having one or more unmethylated CpG units; an oil-in-water (W / O) adjuvant, an oil-in-water (O / W) adjuvant, or an oil-in-water-in-oil (W / O / W) adjuvant, such as Freund's adjuvant, SPT emulsion, MF59, ISA 206, ISA72, adjuvant-65, SAF, etc.; a cationic lipid containing a quaternary ammonium salt such as DDA; a cytokine; aluminum hydroxide or aluminum phosphate; a saponin (e.g., Quil A, QS-21, GPI-0100); or any combination or mixture thereof.

[0093] Materials and reagents

[0094] 2-Cl-CTC-polymer resin was purchased from Xi'an Lanxiao Technology New Materials Co., Ltd.;

[0095] Piperidine was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0096] Fmoc-L-alanine (Fmoc-Ala-OH), Fmoc-Pbf-L-arginine (Fmoc-Arg(pbf)-OH), Fmoc-N-trityl-L-asparagine (Fmoc-Asp(OtBu)-OH), Fmoc-S-trityl-L-cysteine ​​(Fmoc-Cys(Trt)-OH), Fmoc-N-trityl-L-glutamine (Fmoc-Gl n(Trt)-OH), Fmoc-O-tert-butyl-L-glutamate (Fmoc-Glu(OtBu)-OH), Fmoc-glycine (Fmoc-Gly-OH), N-Fmoc-N'-trityl-L-histidine (Fmoc-His(Trt)-OH), Fmoc-L-isoleucine (Fmoc-Ile-OH), Fmoc-L-leucine (Fmoc-Leu-OH), N- alpha-Fmoc-N-epsilon-tert-butyloxycarbonyl-L-lysine (Fmoc-Lys(Boc)-OH), Fmoc-L-methionine (Fmoc-Met-OH), Fmoc-L-phenylalanine (Fmoc-Phe-OH), Fmoc-L-proline (Fmoc-Pro-OH), Fmoc-O-tert-butyl-L-serine (Fmoc-Ser(tBu)-OH) , Fmoc-O-tert-butyl-L-threonine (Fmoc-Thr(tBu)-OH), N-alpha-fluorenylmethoxycarbonyl-N-in-tert-butyloxycarbonyl-L-tryptophan (Fmoc-Trp(Boc)-OH), Fmoc-O-tert-butyl-L-tyrosine (Fmoc-Tyr(tBu)-OH), and Fmoc-L-valine (Fmoc-Val-OH) were purchased from Jier Biochemical (Shanghai) Co., Ltd.;

[0097] Chemical reagents such as trifluoroacetic acid (TFA), HATU, N,N-diisopropylethylamine (DIEA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1,2-ethanedithiol (EDT), triisopropylsilane (TIPS), N,N-dimethylformamide (DMF), and dichloromethane (DCM) were purchased from Anaiji Chemical Reagent Co., Ltd.

[0098] PBS (pH 7.2-7.4) was purchased from Guangzhou Jiete Biofiltration Co., Ltd.

[0099] Pepsin 1:3000, purchased from Solebao Biotechnology Co., Ltd.;

[0100] Trypsin 1:250 was purchased from Solebao Biotechnology Co., Ltd.;

[0101] Hydrochloric acid 12N was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0102] Porcine pancreatic α-amylase (11 u / mg) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0103] α-Amylase inhibitor (kidney bean) 75u / mg was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (A17HS188175).

[0104] Example 1 Synthesis method

[0105] The linear peptide compound and the cyclic peptide compound with the sequence shown in Table 2 were synthesized according to the following method, and the purity of the synthesized experimental peptides was tested according to the following method.

[0106] Purity testing method: Instrument: Agilent 6545XT Ultra-High-Pressure Liquid-Mass Spectrometer; Chromatographic column: ZORBAX RRHD SB-C18, 2.1×50mm, 1.8µm. Testing conditions: Mobile phase A: 0.5% FA in water, mobile phase B: 0.5% FA in acetonitrile, injection volume: 5µL, flow rate: 0.4mL / min. Gradient elution was performed according to the program in Table 1.

[0107] Table 1

[0108] (1) Chemical synthesis of peptide compounds

[0109] Taking TS-1 as an example, the specific linear peptide compound was chemically synthesized according to the following synthesis method.

[0110] Take TS-1 as an example:

[0111] 2-Cl-CTC-polymer resin was selected. According to the characteristics of the amino acid sequence Asp-Trp-Ala-Trp-Glu-Trp-Asp-Ala (DWAWEWDA), the carboxyl group of Fmoc-Ala-OH at the C-terminus was first connected to the resin in the form of a covalent bond. Then, the amino group of Fmoc-Ala-OH and the carboxyl group of Fmoc-Asp(OtBu)-OH were connected by a shrinkage reaction. After removing the unreacted raw materials, Fmoc-Trp(Boc)-O H, Fmoc-Glu(OtBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Trp(Boc)-OH, and Fmoc-Asp(OtBu)-OH, a shrinkage reaction occurs, so that the polypeptide fragments on the resin are sequentially extended in the order from the C-terminus to the N-terminus of the polypeptide TS-1 to obtain the target polypeptide connected to the resin. Finally, the resin is removed using a lysis buffer (TFA:EDT:TIPS:H2O=95:2:1:2) to obtain the target polypeptide.

[0112] The product obtained after excision was purified by high-performance liquid chromatography using a Phenomenex C18 column, 4.6 × 150 mm in size. Mobile phase A: water containing 0.1% (v / v) trifluoroacetic acid (TFA); mobile phase B: acetonitrile containing 0.01% (v / v) TFA. The concentration of phase B was increased from 5.0% to 50.0% over 50 minutes at a flow rate of 3.0 mL / min and detection at 214 nm. The purified product was collected, quickly frozen in liquid nitrogen, and freeze-dried to obtain TS-1.

[0113] MS identification showed that the purity of TS-1 obtained by freeze-drying was 96.3%, and the molecular weight was 1077.42, which was consistent with the theoretical molecular weight (1077.42), proving that the prepared TS-1 was consistent with the theoretical design.

[0114] (2) Chemical synthesis of cyclic peptide compounds

[0115] Taking TS-9 as an example, the specific cyclic peptide compound was chemically synthesized according to the following synthesis method.

[0116] 2-Cl-CTC-polymer resin was selected, and according to the characteristics of the amino acid sequence Asp-Trp-Ala-Trp-Glu-Trp-Asp-Ala (DWAWEWDA), the carboxyl group of Ala at the C-terminus was first connected to the resin in the form of a covalent bond, and then the amino group of Ala and the carboxyl group of Asp were connected through a shrinkage reaction. After removing the unreacted raw materials, Trp, Glu, Trp, Ala, Trp, and Asp were added in sequence with reference to the above steps, and a shrinkage reaction occurred, so that the polypeptide fragments on the resin were sequentially extended in the order from the C-terminus to the N-terminus of the polypeptide TS-1 to obtain the target polypeptide connected to the resin. The resin was cut with a cleavage solution (TFA:DCM=0.5:99.5) to obtain a target polypeptide intermediate. After purification, the intermediate was cyclized with EDCI to obtain a cyclic peptide intermediate. The protecting group on the cyclic peptide intermediate was removed with a cleavage solution (TFA:EDT:TIPS:H2O=95:2:1:2) to obtain the target cyclic peptide.

[0117] The target cyclic peptide was purified by high-performance liquid chromatography using a Phenomenex C18 column, 4.6 × 150 mm. Mobile phase A was water containing 0.1% (v / v) trifluoroacetic acid (TFA); mobile phase B was acetonitrile containing 0.01% (v / v) TFA. The concentration of phase B was increased from 5.0% to 50.0% over 50 minutes at a flow rate of 3.0 mL / min. Detection was performed at 214 nm. The purified product was collected, quickly frozen in liquid nitrogen, and freeze-dried to obtain TS-9.

[0118] MS identification showed that the purity of TS-9 obtained by freeze-drying was 97.3%, and the molecular weight was 1059.39, which was consistent with the theoretical molecular weight (1059.41), proving that the prepared TS-9 was consistent with the theoretical design.

[0119] The remaining peptides can be prepared by referring to the method of TS-1 or TS-9, and the results of MS identification are shown in Table 2 below.

[0120] Table 2

[0121] Note: In the amino acid sequences in the table above, lowercase letters represent D-type amino acids, and uppercase letters represent L-type amino acids.

[0122] Example 2 In vitro stability experiment

[0123] The stability test of the experimental peptide of the present application was carried out according to the following method:

[0124] Preparation of standard solution: Weigh 20 mg of experimental peptide and dissolve it in 20 mL of PBS to make the standard solution; dilute the standard solution 4 times with PBS to make the control solution;

[0125] Preparation of simulated artificial gastric fluid: Weigh 10 mg of pepsin and dissolve it in 4 mL of PBS. Adjust the pH to 4-5 with 1N hydrochloric acid and make up to 10 mL. Dilute the simulated artificial gastric fluid 4-fold with PBS to serve as the simulated gastric fluid background. Preparation of simulated artificial intestinal fluid: Weigh 10 mg of trypsin and dissolve it in 10 mL of PBS.

[0126] Simulated artificial intestinal fluid was diluted 4-fold with PBS and used as the simulated intestinal fluid background.

[0127] (1) Stability test of experimental peptides in simulated gastric fluid

[0128] 1 mL of the standard solution was aspirated and added to 1 mL of simulated artificial gastric fluid to react at 37°C for 1 hour. Samples were taken and the purity and abundance of the experimental peptides were detected by liquid chromatography-mass spectrometry (LCMS). The LCMS test results after subtracting the simulated gastric fluid background were used to obtain the stability test results of the experimental peptides in the simulated gastric fluid reaction solution.

[0129] (2) Stability test of experimental peptides in simulated intestinal fluid

[0130] 1 mL of the standard solution was aspirated and added to 1 mL of simulated artificial intestinal fluid for reaction at 37°C for 1 hour. Samples were taken and the purity and abundance of the experimental peptides were detected by liquid chromatography-mass spectrometry (LCMS). The LCMS test results after subtracting the simulated intestinal fluid background were used to obtain the stability test results of the experimental peptides in the simulated intestinal fluid reaction solution.

[0131] The experimental peptide was subjected to a stability test and the results of LCMS detection showed that in a 1 mg / mL pepsin solution or trypsin solution, the experimental peptide of the present application (e.g., TS-1) can stably exist for 1 hour without degradation. This means that the experimental peptide will not be decomposed under the action of pepsin or trypsin, and can maintain its chemical structure and active state. Since LCMS is a highly sensitive analytical method, the results indicate that the experimental peptide has high stability in pepsin solution and may have potential for oral administration.

[0132] Example 3 In vitro long-term stability experiment

[0133] The long-term stability test of the experimental peptide of the present application was carried out according to the following method:

[0134] The experimental peptide solid and its PBS solution were placed at 4°C, room temperature, and 37°C, respectively, and tested for deterioration.

[0135] (1) Synthetic peptide solid stability test:

[0136] (1-1) 210 mg of the test peptide was placed in a sealed PE tube and placed in a 4°C refrigerator, room temperature, and a 37°C incubator.

[0137] (1-2) Use a temperature control device (refrigerator, incubator) to adjust the temperature inside the container to the target temperature (4°C for refrigerator, 37°C for incubator) and maintain a constant temperature.

[0138] (1-3) During the experiment, the temperature of the temperature-controlled equipment (refrigerator, incubator) was observed daily and purity was tested every 7 days. Purity Testing Method: Purity was tested using LCMS, using the same method as described in Example 1.

[0139] (2) Stability test of synthetic peptide in PBS solution:

[0140] (2-1) 210 mg of the test peptide was dissolved in 210 mL of PBS solution, divided equally into three portions, sealed in PE tubes, and placed in a 4°C refrigerator, room temperature, and a 37°C incubator, respectively.

[0141] (2-2) Use a temperature control device (refrigerator, incubator) to adjust the temperature inside the container to the target temperature (4°C for refrigerator, 37°C for incubator) and maintain a constant temperature.

[0142] (2-3) During the experiment, the temperature of the temperature control equipment (refrigerator, incubator) was observed every day and purity testing was performed every 7 days using the same method as the "Purity Testing Method" in Example 1.

[0143] The results of LCMS analysis of long-term stability experiments on the experimental peptides showed that the experimental peptides (e.g., TS-1) of the present application were stable for 30 days in both solid form and PBS solution at 4°C, room temperature, and 37°C. This indicates that the experimental peptides have high stability under different temperature conditions and are suitable for storage and application within these temperature ranges.

[0144] Example 4 In vitro amylase inhibition activity test experiment

[0145] The in vitro amylase inhibitory activity test of the experimental peptide of the present application was performed according to the following method:

[0146] (1) Reagent preparation:

[0147] Preparation of DNS reagent: Solution A: Dissolve 6.9 g of crystalline phenol in 15.2 mL of 10% NaOH solution, dilute to 69 mL with distilled water, and add 6.9 g of sodium bisulfite to this solution; Solution B: Dissolve 255 g of potassium sodium tartrate in 300 mL of 10% NaOH solution, and then add 880 mL of 1% 3,5-dinitrosalicylic acid solution; Mix solutions A and B to obtain the yellow DNS reagent, which is stored in a brown bottle for 7-10 days before use.

[0148] Porcine pancreatic α-amylase solution: Dissolve 50 mg of porcine pancreatic α-amylase in 100 mL of PBS and store the solution in a refrigerator at 4°C. This condition ensures that the α-amylase remains active for 14 days.

[0149] Starch solution: Weigh 2.0g of starch and add 100mL of water. Heat to 80°C and dissolve. Stir for 3 minutes and then allow to stand at room temperature. The starch solution remains active at room temperature for 24 hours.

[0150] Yangshen solution: Weigh 10 mg of α-amylase inhibitor (kidney bean) and dissolve it in 10 mL of PBS. Store the solution in a refrigerator at 4°C. This condition ensures that the α-amylase inhibitor (kidney bean) remains active for 3 days.

[0151] (2) The experimental groups are as follows:

[0152] Sample group: 200 μL of porcine pancreatic α-amylase solution (concentration of 5 mg / mL) was mixed with 200 μL of α-amylase inhibitor (experimental peptide or α-amylase inhibitor (kidney bean) ( Yangshen ));

[0153] Blank group: no porcine pancreatic α-amylase or α-amylase inhibitor (experimental peptide) was added;

[0154] Blank control group: no α-amylase inhibitor (experimental peptide or Yangshen);

[0155] Inhibition control group: no porcine pancreatic α-amylase was added.

[0156] After mixing the solutions from each experimental group, use a turbo shaker to mix thoroughly. Incubate in a 37°C waterbath for 30 minutes. Add 150 μL of 2% soluble starch and incubate in a 37°C waterbath for 30 minutes. Add 350 μL of DNS reagent and incubate in a boiling water bath for 10 minutes. Cool to room temperature, then dilute a certain volume of the solution 4-fold and spot-dip into a 96-well plate. Measure the absorbance at 540 nm.

[0157] The inhibition rate of α-amylase inhibitors was calculated by the following formula:

[0158] Inhibition rate = {1-(A4-A3) / (A2-A1)}×100%;

[0159] A1, A2, A3, and A4 are the absorbance values ​​(Abs) of the blank group, blank control group, inhibition control group, and sample group at 540 nm, respectively.

[0160] The results of the in vitro enzyme activity inhibition experiment (such as Table 3 and Figure 1) prove that the experimental peptide of the present application has good amylase inhibition function. For example, when the concentration of TS-1 reaches 1 mg / mL, the inhibition rate can reach 44% (see Figure 1).

[0161] Reference peptides TS-D1 (GHWYYRCW, SEQ IN DO: 33), TS-D2 (FQSPRYSQ, SEQ IN DO: 34) and TS-D3 (PLPLHMLP, SEQ IN DO: 35) were synthesized as controls according to the chemical synthesis method of the linear peptide compound in Example 1.

[0162] The inhibition rates marked in Table 3 below are the inhibition rates of each experimental peptide molecule at a concentration of 0.5 mg / mL. It can be seen that each experimental peptide of the present application can meet the inhibitory function of amylase, and its inhibition rate reaches more than 10%, which is significantly higher than the inhibition rate of the control peptides TS-D1, TS-D2 and TS-D3.

[0163] Table 3

[0164] Example 5: Blood sugar reduction experiment in mice using α-amylase inhibitory peptide

[0165] Referring to the experimental protocol for the obesity model method in the "Weight Loss Function Evaluation Method" for health products, we commissioned Nanjing Bosi Biopharmaceutical Technology Co., Ltd. to conduct a third-party sample weight loss and blood sugar lowering function mouse experiment. The experimental process is shown in Figure 2.

[0166] Adaptation period: Mice (C57b1 / 6J mice, purchased from Shanghai Bikeyi Biotechnology Co., Ltd.) were fed with maintenance diet under the barrier system and observed for 7 days.

[0167] Modeling screening period:

[0168] Mice were intubated to establish a model of obesity. After the acclimation period, they were randomly divided into two groups based on body weight. Five mice were fed a maintenance diet (purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd., Catalog No. AIN-93M) as a blank control group, and 40 mice were fed a high-calorie diet (45% fat-based energy diet, purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd., Catalog No. XTFH45) as a model group. Food intake, food spillage, and food leftovers were recorded weekly, and body weight was measured once. After one week of feeding, mice fed the high-calorie diet were ranked by weight gain, and the one-third of obesity-resistant mice with the lowest weight gain were eliminated.

[0169] After the modeling period, male mice weighing 50-100 g were selected and housed in single cages for one week. All animals were weighed, and those with uniform weight were selected. They were randomly divided into groups of 5 animals per group according to Table 4. The animals were fed a high-calorie diet. Food intake was recorded. The experiment lasted for 2 weeks.

[0170] Table 4

[0171] The day of grouping was designated D0. During the experiment, animals were fasted but had free access to water. The test substance was administered via enema three times daily. Food was distributed between 07:00 and 09:00, 13:00 and 15:00, and 19:00 and 21:00, and food intake was recorded. The enema volume was 1 mL. Blood glucose was measured once daily after the animals had consumed food and administered the drug. Blood glucose was measured at 0, 90, and 180 minutes after the animals had consumed the drug.

[0172] The animals' general appearance, behavior, poisoning symptoms, and deaths were observed and recorded daily.

[0173] At the end of the 15-day experiment, the animals were killed and blood was collected for the following examinations: routine blood test: determination of red blood cell count, hemoglobin concentration, hematocrit, white blood cell count and classification, platelet count, reticulocyte count if necessary depending on the test substance, bone marrow smear cytology, etc.

[0174] Blood biochemical indexes were measured: alanine aminotransferase, aspartate aminotransferase, glutamyl transpeptidase, alkaline phosphatase, urea nitrogen, creatinine, blood glucose, serum albumin, total protein, total cholesterol and triglyceride, chloride, potassium and sodium.

[0175] The absolute weights of the heart, thymus, adrenal glands, liver, kidneys, spleen, and testes were weighed to calculate the relative weights (organ / body weight ratio). Perirenal fat, peritesticular fat, and perihepatic fat were removed and weighed to calculate the fat / body weight ratio.

[0176] The results show that the experimental peptide of the present application has a significant effect on reducing blood glucose levels after a meal (i.e., starch load). The changes in blood glucose levels in mice after a meal of the exemplary experimental peptide TS-1 are shown in Figure 3. The medium-dose group of the TS-1 experimental peptide achieved an effect basically equivalent to that of Yang Shen when the dosage was half that of Yang Shen.

[0177] The experimental peptides of the present application have a significant effect on reducing the weight of mice. The weight changes of mice during the experimental process of the exemplary experimental peptide TS-1 are shown in Figure 4. The medium-dose group of the TS-1 experimental peptide achieved an effect basically equivalent to that of Yang Shen when the dosage was half that of Yang Shen.

[0178] The blood of mice in the three dose groups of the experimental peptide of the present application at the end of the experiment was tested. The results showed that routine blood tests such as red blood cells, platelets, lymphocytes, monocytes, and white blood cells (neutrophils, eosinophils, and basophils) did not show any abnormalities; enzymes or protein markers related to liver and kidney function did not show any abnormal expression; other blood indicators did not show any abnormal expression. Some experimental results of the exemplary experimental peptide TS-1 are shown in Figures 5 to 10.

[0179] The three dose groups of the experimental peptide of the present application, high, medium and low, were tested on mouse organs at the end of the experiment. The results showed that the relative weights of organs such as the liver, kidney, heart, spleen, thymus, adrenal gland, and testicles of the mice did not show any abnormal changes or effects. The experimental results of the exemplary experimental peptide TS-1 are shown in Figures 11 and 12.

[0180] That is, in the above-mentioned animal experiments, by monitoring the blood sugar changes of mice loaded with high-calorie food, it was found that the experimental peptides (such as TS-1) in the high, medium and low dose groups had a significant control effect on the postprandial blood sugar of mice, and had the effect of controlling the weight of mice. The mice were in good condition during the entire administration period, and no abnormal toxicological reactions were observed. The routine blood and biochemical indicators of mice in the experimental peptide groups with different doses were normal, and there were no significant changes in the organs, and no obvious lesions or lesions were observed, indicating that the experimental peptides (such as TS-1) of the present application have no toxic side effects on mice.

[0181] Example 6 α-amylase inhibitory peptide pepsin activity detection experiment

[0182] 1. Experimental reagents:

[0183] Starch solution: 0.3% (0.3g + 100mL) starch mother liquor;

[0184] Iodine test phosphate buffer (pH = 6): weigh 11.3075 g of disodium hydrogen phosphate (NaHPO4·12H2O) and 2.0175 g of citric acid (C6H8O7·H2O), dissolve in water and dilute to 250 mL, and use after pH correction;

[0185] Pepsin (purchased from Solebao Biotechnology Co., Ltd.): 1 mg / mL concentration (0.1 g dissolved in 100 mL water) was adjusted to pH 3 with dilute hydrochloric acid;

[0186] α-amylase inhibitor: The experimental peptide of the present invention (referred to as TS peptide) and the commercially available white kidney bean extract of Xi'an Ruilin Biotechnology Co., Ltd. (referred to as Ruilin), the white kidney bean extract of Xi'an Youlanda Biotechnology Co., Ltd. (referred to as Youlanda), the white kidney bean concentrated powder of Green Cuimei (referred to as Green Cuimei), the revised white kidney bean chewable tablet candy (referred to as Xiuzheng), and the white kidney bean blocker capsule of Swanson (referred to as Swanson) were respectively mixed with an acidic aqueous solution with a pH of 3 to prepare 1 mg / mL (10 mg + 10 mL water) of α-amylase inhibitor.

[0187] 2. Time gradient: α-amylase inhibitors were digested in pepsin solution for 30, 60, 90, and 120 min.

[0188] 3. Experimental steps:

[0189] (1) Pepsin digestion:

[0190] (1.1) Mix 1 mL of 1 mg / mL α-amylase inhibitor solution (sample) with 1 mL of pepsin in a 1:1 volume ratio in a test tube and incubate the mixture in a shaking water bath at 37°C for digestion.

[0191] (1.2) After 0, 15, 30, 60, and 90 min of digestion, add 6.5 μL of 0.5 M NaOH to neutralize the reaction solution. Do not perform any other inactivation procedures on pepsin, and immediately determine the α-amylase activity by the iodine assay.

[0192] (2) Determination of α-amylase activity inhibition by iodine test

[0193] (2.1) Mix α-amylase (1 mg / mL aqueous solution, porcine pancreatic α-amylase, purchased from Shanghai Yuanye Biotechnology Co., Ltd., the same below) and α-amylase inhibitor (the supernatant of the digestion reaction in (1.2) above) in a volume ratio of 1:1 and preheat in a water bath at 37°C ± 0.2°C for 8 min.

[0194] (2.2) Add the reagent solutions to each test tube in sequence according to Table 5 below, mix thoroughly, and incubate the test tubes in a 37°C water bath for 5 min. Then, add 5 mL of 0.1 mol / L dilute hydrochloric acid to terminate the reaction. Take 1 mL of the solution from each test tube and add it to 10 mL of dilute iodine solution. Mix thoroughly, measure the absorbance (A) at 660 nm, and record the data. During the experiment, set up a blank tube, an enzyme control tube, and a control blank tube (see Table 5 for the reagent solution addition).

[0195] Table 5. Amount of each solution added (unit: mL)

[0196] The inhibitory activity value (U / g) of α-amylase inhibitory peptide was determined with reference to the group standard “T.CCCMHPIE+1.26—2018” “Plant Extract White Kidney Bean Extract”.

[0197] U / g: The amount of α-amylase inhibitor that inhibits α-amylase from cleaving starch chains into 1 μmol of maltose within 1 minute at 37°C and pH 6.0 is 1 U / g of amylase inhibitor activity.

[0198] U / g is calculated using the following formula:

[0199] Where:

[0200] M1--the amount of starch remaining in the test tube with inhibitor (g);

[0201] M2--the amount of starch remaining in the test tube without inhibition (g);

[0202] U / g--α-amylase inhibitor activity of the sample (U / g);

[0203] M 取样量 - Sample size (g);

[0204] B--the slope of the starch standard curve regression equation;

[0205] A1--absorbance of amylase reference;

[0206] A2--absorbance of the sample;

[0207] 342.3--the molar mass of maltose (g / mol);

[0208] T--reaction time (min);

[0209] F--conversion factor of the sample, 1000;

[0210] 10 6 --Unit conversion coefficient between moles and micromoles.

[0211] 4. Experimental results:

[0212] The experimental results show that even under pepsin hydrolysis conditions, the experimental peptide of the present invention still has good α-amylase inhibitory activity and is significantly better than various commercially available white kidney bean extracts. The experimental results of an exemplary experimental peptide TS-1 of the present invention are shown in Figure 13.

[0213] Example 7 In vitro inhibition of lipase and colipase activity by α-amylase inhibitory peptides

[0214] 1. In vitro Lipase Inhibitory Activity Assay

[0215] (1) Reagents:

[0216] Substrate p-NPP: isopropanol was prepared to 10 mM (38 mg + 10 mL isopropanol);

[0217] Lipase (purchased from Beijing Yinuokai Technology Co., Ltd.): 1 mg / mL, dissolved in D-PBS, prepared in 50 mL;

[0218] TS peptide (experimental peptide of the present invention): 1 mg / mL, dissolved in Tris-HCl;

[0219] Tris-HCl: 50 mM, pH = 8;

[0220] The reaction concentration of lipase was approximately 13 μM; the reaction concentration of TS peptide (0.1 mg / mL) was 4 μM and 36 μM.

[0221] (2) Experimental steps:

[0222] (2.1) According to Table 6, the peptide, Tris-HCl, and lipase were mixed in a constant temperature shaker at 37°C and incubated for 10 minutes.

[0223] (2.2) Add the substrate p-NPP, shake and mix in a constant temperature shaker at 37°C, and incubate for 20 min;

[0224] (2.3) Termination of the reaction: 98°C for 5 min, cooling with cold water, and centrifugation at 12,000 rpm for 5 min;

[0225] (2.4) OD value measurement: aspirate 200 μL of supernatant and measure the absorbance at 405 nm in a microplate reader;

[0226] (2.5) Calculate the Lipase inhibition rate according to the following formula:

[0227] Wherein, “OD control” refers to the OD value of the control experimental group, “OD blank” refers to the OD value of the blank control group, and “OD sample” refers to the OD value of the sample group to be tested.

[0228] Table 6

[0229] Note: Negative peptide (amino acid sequence: LNPDSQPK (SEQ ID NO: 36), a polypeptide composed of 8 L-amino acids that does not have α-amylase inhibitory function)

[0230] The experimental results show that the experimental peptides of the present invention have good lipase inhibitory activity. The experimental results of TS-1 of the present invention are shown in Table 7.

[0231] Table 7. Results of the α-amylase inhibitory peptides' lipase activity inhibition experiment

[0232] 2. In vitro Colipase Inhibitory Activity Assay

[0233] (1) Reagents:

[0234] Substrate p-NPP: isopropanol was prepared to 10 mM (38 mg + 10 mL isopropanol);

[0235] Lipase (purchased from Beijing Yinuokai Technology Co., Ltd.): 45 kDa, 1 mg / mL, PBS (containing Ca 2+ Mg 2+ ) dissolve, prepare 50 mL, store at 4℃, and place on ice;

[0236] Colipase (colipase, purchased from Shanghai Titan Technology Co., Ltd.): 11 kDa, dissolved in pure water to a stock solution of 2 mg / mL, sealed in aliquots and stored at -20°C, diluted with pure water to 0.2 mg / mL before use;

[0237] Bile salts: 100 mM, water-soluble, 521 mg + 10 mL water;

[0238] TS peptide (experimental peptide of the present invention): 1 mg / mL, dissolved in Tris-HCl;

[0239] Tris-HCl: 50 mM, pH = 8;

[0240] The reaction concentration of lipase is about 13 μM; the reaction concentration of colipase is about 1.1 μM; the reaction concentration of TS peptide is 4 μM and 24 μM;

[0241] (2) Experimental steps:

[0242] (2.1) According to Table 8, add (colipase + TS peptide) to a 1.5 mL centrifuge tube, mix well, and place in a shaker at 37°C. Incubate for 15 minutes, then cool to room temperature.

[0243] (2.2) Pour (20 μL substrate p-NPP + 30 μL bile salt system) into a new 2 mL centrifuge tube and shake gently to mix. Transfer 150 μL of system ①, which has been cooled to room temperature, to system ② as soon as possible, shake thoroughly to mix, and incubate at 37°C in a shaker for 15 min.

[0244] (2.3) After removing the reaction solution from the shaker, immediately add 300 μL of Lipase, mix thoroughly by inversion, and incubate at 37°C on a shaker for 30 min.

[0245] (2.4) Termination of the reaction: The reaction system was incubated at 98°C for 5 min, cooled with cold water, and centrifuged at 12,000 rpm for 5 min.

[0246] (2.5) OD value measurement: aspirate 200 μL of supernatant and measure the absorbance at 405 nm in a microplate reader;

[0247] (2.6) Calculate the colipase inhibition rate according to the following formula:

[0248] (where “OD Colipase " refers to the OD value of the Colipase group, "OD bile salt" refers to the OD value of the bile salt control group, and "OD sample" refers to the OD value of the test sample experimental group)

[0249] Table 8

[0250] Note: System ① in the table refers to the sample (TS peptide) and colipase (colipase) incubation system; System ② refers to the bile salt and fat incubation system, i.e., the substrate system; ①+② refers to the mixture of system ① and system ②; negative peptide (amino acid sequence: LNPDSQPK (SEQ ID NO: 36), a polypeptide composed of 8 L-amino acids that does not have α-amylase inhibitory function).

[0251] The experimental results show that the experimental peptides of the present invention have good colipase inhibitory function. The experimental results of the exemplary experimental peptide TS-1 are shown in Table 9.

[0252] Table 9: Results of the experiment on the inhibition of colipase activity by α-amylase inhibitory peptides

[0253] Example 8: Lipid-lowering Experiment in Mice Using α-Amylase Inhibitory Peptides

[0254] 1. Experimental reagents are shown in Table 10.

[0255] Table 10

[0256] 2. Experimental Methods

[0257] C57BL / 6J mice (male, weighing 20±2g, 8-9 weeks old, purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) were housed in an SPF-grade clean environment with free access to food and a light-dark cycle (12h:12h). After 7 days of adaptive feeding, each mouse underwent duodenal intubation surgery. Anti-inflammatory and postoperative care were performed every day after surgery, and the mice recovered for one week after surgery. After one week of recovery, the dead mice and those weighing less than 20g were eliminated, and the animals that underwent successful surgery were randomly divided into 3 groups with 4 mice in each group, namely Group-1 (Vehicle group, also known as negative control, ddH2O (double distilled water)), Group-7 (ALST group, ALST), and Group-8 (TS group, experimental peptide of the present invention). The day of grouping was recorded as Day 0, and the mice were fasted for 12 hours in preparation for the OLTT experiment the next day.

[0258] Starting on Day 1, intraduodenal administration was administered three times daily at a volume of 0.1 mL / 10 g body weight for 14 consecutive days. Specific dosing methods are shown in Table 11. Animal weight, general appearance, behavior, signs of poisoning, and mortality were observed daily. The presence of diarrhea and oily stools were also observed. Daily food intake was recorded during the dosing period.

[0259] Table 11. Dosage Methods

[0260] OLTT experiments were performed after the first administration on Day 1, the first administration on Day 8, and the last administration on Day 14: Before the OLTT experiment, mice in each group were fasted for 12 hours, and blood was collected before gavage with soybean oil as the 0-hour sample. Subsequently, mice in each group were gavage with 5 mL / kg soybean oil, and blood was collected 1 hour, 2 hours, 3 hours, and 4 hours after gavage with soybean oil to detect serum triglycerides (TG), and the area under the serum TG curve (AUC) was calculated. After blood collection on Day 1, mice were fed with a high-fat diet.

[0261] 3. Clinical Observation of Mice in Experiments

[0262] Inspection time: Day 1-Day 14;

[0263] Examination subjects: mice in each group;

[0264] Inspection Method: Observe and record the general appearance, behavior, signs of poisoning, and mortality of the mice. Observe whether the mice have diarrhea and whether their feces are oily. The observation results are coded as shown in Table 12 below.

[0265] Table 12. Clinical observations in mice

[0266] 4. Mouse OLTT Experimental Detection

[0267] Inspection time: after the first dose on Day 1, after the first dose on Day 8, and after the last dose on Day 14;

[0268] Examination subjects: mice in each group;

[0269] Examination method: Each group of mice was fasted for 12 hours. Blood was collected before oral gavage with soybean oil as the 0-min sample. Then, each group of mice was gavage with 5 mL / kg soybean oil. Blood was collected 1 hour, 2 hours, 3 hours, and 4 hours after oral gavage with soybean oil to detect serum TG, and the area under the serum TG curve (AUC) was calculated.

[0270] Blood was collected from the inner canthus of mice before (0 h) and at 1, 2, 3, and 4 h after soybean oil administration. 100 μL of blood was collected each time and centrifuged at 4000 r / min for 10 min. Serum was separated and analyzed for serum triglycerides according to the kit instructions. Serum and working solution were incubated in a 96-well plate according to Table 13. OD values ​​were measured using a microplate reader, and serum triglyceride levels were calculated using the formula.

[0271] Table 13.

[0272] Wherein, C standard = 2.68mmol / L

[0273] Statistical Analysis: Data were analyzed and graphed using Graphpad Prism 9 (Version 9.4.0). All data are expressed as mean ± SD. Statistical differences between groups were analyzed using one-way ANOVA and two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs Vehicle. P values ​​less than 0.05 were considered significant.

[0274] 5. Experimental Results

[0275] Clinical observations of mice showed that the mice were generally in good condition during the administration period, and no abnormal toxicological reactions were observed;

[0276] The OLTT test results showed that compared with the Vehicle group, the serum triglyceride concentration and serum triglyceride curve area (AUC) of the TS-administered group were significantly reduced, and the tolerance to lipids was enhanced. Among them, the experimental results of the exemplary experimental peptide TS-1 are shown in Figures 14-19 and Tables 14-19 below.

[0277] Table 14. Serum TG concentrations (mmol / L) in Day 1 OLTT experiment

[0278] Table 15 Day 1 OLTT test serum TG area under the curve, AUC (mmol / L)

[0279] Table 16 Serum TG concentration (mmol / L) in the OLTT experiment on Day 8

[0280] Table 17 Area under the curve of serum TG in the OLTT experiment on Day 8, AUC (mmol / L)

[0281] Table 18 Serum TG concentration (mmol / L) in the OLTT experiment on Day 14

[0282] Table 19 Area under the curve of serum TG in the OLTT experiment on Day 14

[0283] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A peptide, characterized in that An amino acid sequence comprising eight adjacent amino acids X1X2X3X4X5X6X7X8, wherein each amino acid of the peptide is independently selected from a D-type amino acid or an L-type amino acid, wherein at least one of X2, X4, and X6 is W, and any one of the following conditions (1) to (3) is satisfied: (1) X2, X4 and X6 are all W, and X1 is selected from D, R, L, N, S, E or V, X3 is selected from A, P, V, G, Y, K or R, X5 is selected from E, F, G, R, H or P, X7 is selected from D, S, R, V, W, N or Y, and X8 is A; (2) X1 is selected from D, X2 is selected from W or F, X3 is selected from A, X4 is selected from W or Y, X5 is selected from E or D, X6 is selected from W, X7 is selected from D, X8 is selected from A, and only one of X2 and X4 is W; (3) X1 is selected from D, E or A, X2 is F, X3 is selected from A, G or L, X4 is selected from W or Y, X5 is selected from D or A, X6 is selected from F or W, X7 is selected from D or E, X8 is selected from P, W or A, and only one of X4 and X6 is W.

2. The peptide according to claim 1, characterized in that X1 is D, X3 is A, X5 is E, X7 is D, or X1 is R, X3 is P, X5 is F, X7 is S, or X1 is L, X3 is V, X5 is G, X7 is R, or X1 is N, X3 is G, X5 is F, X7 is R, or X1 is S, X3 is Y, X5 is R, X7 is V, or X1 is E, X3 is K, X5 is H, X7 is W, or X1 is L, X3 is G, X5 is R, X7 is N, or X1 is V, X3 is R, X5 is P, and X7 is Y.

3. The peptide according to claim 1 or 2, characterized in that The peptide comprises any of the following amino acid sequences: DWAWEWDA (SEQ ID NO: 1), RWPWFWSA (SEQ ID NO: 2), LWVWGWRA (SEQ ID NO: 3), NWGWFWRA (SEQ ID NO: 4), SWYWRWVA (SEQ ID NO: 5), EWKWHWWA (SEQ ID NO: 6), LWGWRWNA (SEQ ID NO: 7), VWRWPWYA (SEQ ID NO: 8).

4. The peptide according to claim 1 or 2, characterized in that The peptide comprises the amino acid sequence shown in any one or more of SEQ ID NO: 14, SEQ ID NO: 18, and SEQ ID NO: 22-30.

5. The peptide according to claim 1, characterized in that In the peptide, X1 is D, X2 is F, X3 is A, X4 is W, X5 is D, X6 is F, X7 is D, and X8 is P; or X1 is selected from E or A, X2 is F, X3 is selected from G or L, X4 is selected from Y, X5 is selected from D or A, X6 is W, X7 is selected from D or E, and X8 is selected from W or A.

6. The peptide according to claim 1 or 5, characterized in that The peptide comprises any of the following amino acid sequences: DFAWDFDP (SEQ ID NO:9), EFGYAWEW (SEQ ID NO:10), AFLYDWDA (SEQ ID NO:11).

7. The peptide according to claim 1 or 5, characterized in that The peptide comprises the amino acid sequence shown in any one or more of SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 31, and SEQ ID NO:

32.

8. The peptide according to claim 1, characterized in that The peptide comprises any of the following amino acid sequences: DWAYEWDA (SEQ ID NO: 12), DFAWDWDA (SEQ ID NO: 13).

9. The peptide according to claim 1 or 8, characterized in that The peptide comprises the amino acid sequence shown in any one or more of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO:

20.

10. The peptide according to any one of claims 1 to 9, characterized in that The peptide is a polypeptide consisting of 8 amino acids; and / or The peptide is a linear peptide or a cyclic peptide; and / or The peptide has an in vitro amylase inhibition rate of more than 10% at a concentration of 0.5 mg / mL; and / or The peptides have amylase inhibitory activity, lipase inhibitory activity and / or colipase inhibitory activity.

11. The peptide according to any one of claims 1 to 10, characterized in that In the peptide, X4 is a D-amino acid, and X1, X2, X3, X5, X6, X7 and X8 are L-amino acids; or In the peptide, X1 to X8 are all L-type amino acids.

12. The peptide according to any one of claims 1 to 11, characterized in that The peptide has an amino acid sequence shown in any one or more of SEQ ID NOs: 14-32.

13. A nucleic acid molecule comprising a nucleotide sequence encoding the peptide according to any one of claims 1 to 12. An expression vector comprising the nucleic acid molecule of claim 13 . A host cell comprising the nucleic acid molecule of claim 13 or the expression vector of claim 14. 16 . A pharmaceutical composition comprising: the peptide according to claim 1 , the nucleic acid molecule according to claim 13 , the expression vector according to claim 14 , or the host cell according to claim 15 ; and a pharmaceutically acceptable carrier.

17. Use of the peptide according to any one of claims 1 to 12, the nucleic acid molecule according to claim 13, the expression vector according to claim 14, the host cell according to claim 15, or the pharmaceutical composition according to claim 16 in the preparation of an α-amylase inhibitor, a lipase inhibitor, a colipase inhibitor, a weight-loss drug, a blood sugar-lowering drug, a blood lipid-lowering drug, or a food, health product, or drug for preventing or treating obesity-related diseases.

18. The use according to claim 17, characterized in that The obesity-related diseases include one or more of obesity, cardiovascular and cerebrovascular diseases, cancer, type 2 diabetes, hyperglycemia, hypertension, hyperlipidemia, fatty liver, coronary heart disease, atherosclerosis, stroke and acute pancreatitis.

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