Hemp seed extract polypeptide and use thereof

Abstract

The present application relates to a kind of polypeptide extracted from hemp seed, its amino acid sequence is selected from any one of VADW, VAMP, YQLM, FPQS, FSPSSQQ, YGDQ, WLE, YTGD, VVDNNGRS, PSSQQ, YSYA, WNVN, WIAVK, LNAP, YNLP, FLQ, NYLP, LLY, FNPRG, YTPHW, FDGEL, WDSY, PQNHA, VFTPQ, PQNHA, IEQMPQRS, FNVDSE, PSSQQTR, FQL, LTTVNSY, YNL or YQL.The polypeptide can be applied in the preparation of related drugs such as hypoglycemic, lipid-lowering, liver protection, kidney protection, intestinal flora regulation and the like.

Description

Technical Field

[0001] This invention relates to the field of biology, and in particular to a polypeptide extracted from hemp seeds and its uses in lowering blood sugar, lowering blood lipids, protecting the liver, protecting the kidneys, and regulating intestinal flora. Background Technology

[0002] Hemp seeds are the dried, mature seeds of the cannabis plant (Cannabis sativa L.), a member of the Moraceae family. They are both food and medicine, cultivated throughout my country, and also found in semi-wild areas, widely distributed in Northeast, North, East, and Central South my country. Hemp seeds are neutral in nature and sweet in taste, possessing laxative properties. They are also rich in protein, vitamins, lecithin, volatile oils, and trace elements such as calcium and magnesium. While hemp seeds are a traditional Chinese medicine and food ingredient with various physiological functions, research on them is not in-depth, and their potential applications in medicine, health products, and food have not yet been fully explored.

[0003] Polypeptides are intermediate products and functionally active fragments of protein hydrolysis. With a molecular weight below 10,000 Daltons, they can permeate semipermeable membranes. Studies have found that after enzymatic hydrolysis of proteins in the digestive tract, they are mainly absorbed in the form of peptides. Furthermore, peptides derived from protein hydrolysis have better solubility, complete absorption, and biological activity than the original proteins. They not only meet the body's need for essential amino acid supplementation but also promote metabolism, growth and development, and regulate physiological and immune functions.

[0004] In recent years, naturally derived bioactive peptides have become a research hotspot in the development of pharmaceuticals and functional foods due to their advantages such as mild effects, well-defined functions, high safety, and few side effects. The preparation of hemp seed bioactive peptides from hemp seeds to develop bioactive peptide products with medicinal, health, and nutritional applications has broad application prospects. Summary of the Invention

[0005] Therefore, the purpose of this application is to provide a functional polypeptide derived from hemp seeds and its uses.

[0006] Specifically, this application provides a polypeptide whose amino acid sequence is selected from VADW (Seq ID No. 1), VAMP (Seq ID No. 2), YQLM (Seq ID No. 3), FPQS (Seq ID No. 4), FSPSSQQ (Seq ID No. 5), YGDQ (Seq ID No. 6), WLE, YTGD (Seq ID No. 7), VVDNNGRS (Seq ID No. 8), PSSQQ (Seq ID No. 9), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), VAMP (Seq ID No. 1), YGDQ (Seq ID No. 10), WLE, YTGD (Seq ID No. 11), VVDNNGRS (Seq ID No. 12), YGDQ (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), YGDQ (Seq ID No. 10), YGDQ (Seq ID No. 11), YGDQ (Seq ID No. 12), YGDQ (Seq ID No. 13), YGDQ (Seq ID No. 14), YGDQ (Seq ID No. 15), Y Any one of the following: No. 19), PQNH (Seq ID No. 20), VFTPQ (Seq ID No. 21), PQNHA (Seq ID No. 22), IEQMPQRS (Seq ID No. 23), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), FQL, LTTVNSY (Seq ID No. 26), YNL, or YQL.

[0007] This application provides the nucleic acid sequence encoding the polypeptide.

[0008] This application provides a composition comprising one or more amino acid sequences of the following sequences: VADW (Seq ID No. 1), VAMP (Seq ID No. 2), YQLM (Seq ID No. 3), FPQS (Seq ID No. 4), FSPSSQQ (Seq ID No. 5), YGDQ (Seq ID No. 6), WLE, YTGD (Seq ID No. 7), VVDNNGRS (Seq ID No. 8), PSSQQ (Seq ID No. 9), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19). Peptides of No. 19), PQNH (Seq ID No. 20), VFTPQ (Seq ID No. 21), PQNHA (Seq ID No. 22), IEQMPQRS (Seq ID No. 23), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), FQL, LTTVNSY (Seq ID No. 26), YNL, or YQL.

[0009] Preferably, the composition provided in this application further includes a pharmaceutically, food-grade, or health-grade acceptable carrier, excipient, or second active ingredient.

[0010] Preferably, the second active ingredient in the composition provided in this application is selected from one or more of the following: DPP-IV inhibitors, SGLT-2 inhibitors, GLP-1 receptor agonists, insulin secretagogues, α-glucosidase inhibitors, angiotensin-converting enzyme inhibitors, angiotensin receptor antagonists, calcium channel blockers, or β-receptor blockers.

[0011] This application provides amino acid sequences selected from VADW (Seq ID No. 1), VAMP (Seq ID No. 2), YQLM (Seq ID No. 3), FPQS (Seq ID No. 4), FSPSSQQ (Seq ID No. 5), YGDQ (Seq ID No. 6), WLE, YTGD (Seq ID No. 7), VVDNNGRS (Seq ID No. 8), PSSQQ (Seq ID No. 9), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 18), and WDSY (Seq ID No. 19). Use of any one of the following polypeptides, or a composition comprising one or more of the above polypeptides, in the preparation of a medicament for the prevention, treatment or improvement of hyperglycemia and related diseases in humans: No. 20), VFTPQ (Seq ID No. 21), PQNHA (Seq ID No. 22), IEQMPQRS (Seq ID No. 23), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), FQL, LTTVNSY (Seq ID No. 26), YNL or YQL.

[0012] This application provides amino acid sequences selected from VADW (Seq ID No. 1), VAMP (Seq ID No. 2), YQLM (Seq ID No. 3), FPQS (Seq ID No. 4), FSPSSQQ (Seq ID No. 5), YGDQ (Seq ID No. 6), WLE, YTGD (Seq ID No. 7), VVDNNGRS (Seq ID No. 8), PSSQQ (Seq ID No. 9), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 18), and WDSY (Seq ID No. 19). The use of any one of the following polypeptides, or a composition comprising one or more of the above polypeptides, in the preparation of a medicament having fat-reducing and metabolic-regulating functions: No. 20), VFTPQ (Seq ID No. 21), PQNHA (Seq ID No. 22), IEQMPQRS (Seq ID No. 23), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), FQL, LTTVNSY (Seq ID No. 26), YNL, or YQL.

[0013] This application provides amino acid sequences selected from VADW (Seq ID No. 1), VAMP (Seq ID No. 2), YQLM (Seq ID No. 3), FPQS (Seq ID No. 4), FSPSSQQ (Seq ID No. 5), YGDQ (Seq ID No. 6), WLE, YTGD (Seq ID No. 7), VVDNNGRS (Seq ID No. 8), PSSQQ (Seq ID No. 9), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 18), and WDSY (Seq ID No. 19). Use of any one of the following polypeptides (No. 20), VFTPQ (Seq ID No. 21), PQNHA (Seq ID No. 22), IEQMPQRS (Seq ID No. 23), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), FQL, LTTVNSY (Seq ID No. 26), YNL, or YQL, or a composition comprising one or more of the aforementioned polypeptides, in the preparation of a medicament that has hepatoprotective, liver-reducing, or liver-damaging effects.

[0014] This application provides amino acid sequences selected from VADW (Seq ID No. 1), VAMP (Seq ID No. 2), YQLM (Seq ID No. 3), FPQS (Seq ID No. 4), FSPSSQQ (Seq ID No. 5), YGDQ (Seq ID No. 6), WLE, YTGD (Seq ID No. 7), VVDNNGRS (Seq ID No. 8), PSSQQ (Seq ID No. 9), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 18), and WDSY (Seq ID No. 19). Use of any one of the following polypeptides (No. 20), VFTPQ (Seq ID No. 21), PQNHA (Seq ID No. 22), IEQMPQRS (Seq ID No. 23), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), FQL, LTTVNSY (Seq ID No. 26), YNL, or YQL, or a composition comprising one or more of the aforementioned polypeptides, in the preparation of a medicament for protecting the kidney and improving renal damage.

[0015] This application provides amino acid sequences selected from VADW (Seq ID No. 1), VAMP (Seq ID No. 2), YQLM (Seq ID No. 3), FPQS (Seq ID No. 4), FSPSSQQ (Seq ID No. 5), YGDQ (Seq ID No. 6), WLE, YTGD (Seq ID No. 7), VVDNNGRS (Seq ID No. 8), PSSQQ (Seq ID No. 9), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 18), and WDSY (Seq ID No. 19). Use of any one of the following polypeptides, or a composition comprising one or more of the above polypeptides, in the preparation of a medicament for improving intestinal flora imbalance: No. 20, VFTPQ (Seq ID No. 21), PQNHA (Seq ID No. 22), IEQMPQRS (Seq ID No. 23), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), FQL, LTTVNSY (Seq ID No. 26), YNL, or YQL.

[0016] This application provides a method for preventing, treating, or improving hyperglycemia and related diseases in humans. The method includes administering an effective amount of a polypeptide to a subject, the amino acid sequence of which is selected from: VADW (Seq ID No. 1), VAMP (Seq ID No. 2), YQLM (Seq ID No. 3), FPQS (Seq ID No. 4), FSPSSQQ (Seq ID No. 5), YGDQ (Seq ID No. 6), WLE, YTGD (Seq ID No. 7), VVDNNGRS (Seq ID No. 8), PSSQQ (Seq ID No. 9), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), and VADW (Seq ID 18). Any one of the following: No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 20), VFTPQ (Seq ID No. 21), PQNHA (Seq ID No. 22), IEQMPQRS (Seq ID No. 23), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), FQL, LTTVNSY (Seq ID No. 26), YNL, or YQL.

[0017] This application provides a method for fat reduction and metabolism regulation, comprising administering an effective amount of a polypeptide to a subject, wherein the amino acid sequence of the polypeptide is selected from: VADW (Seq ID No. 1), VAMP (Seq ID No. 2), YQLM (Seq ID No. 3), FPQS (Seq ID No. 4), FSPSSQQ (Seq ID No. 5), YGDQ (Seq ID No. 6), WLE, YTGD (Seq ID No. 7), VVDNNGRS (Seq ID No. 8), PSSQQ (Seq ID No. 9), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID ... Any one of the following: No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 20), VFTPQ (Seq ID No. 21), PQNHA (Seq ID No. 22), IEQMPQRS (Seq ID No. 23), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), FQL, LTTVNSY (Seq ID No. 26), YNL, or YQL.

[0018] This application provides a method for protecting the liver, reducing ALT and AST concentrations, or improving liver damage. The method includes administering an effective amount of a polypeptide to a subject, the amino acid sequence of which is selected from VADW (Seq ID No. 1), VAMP (Seq ID No. 2), YQLM (Seq ID No. 3), FPQS (Seq ID No. 4), FSPSSQQ (Seq ID No. 5), YGDQ (Seq ID No. 6), WLE, YTGD (Seq ID No. 7), VVDNNGRS (Seq ID No. 8), PSSQQ (Seq ID No. 9), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 16), and YTPHW (Seq ID No. 17). Any one of the following: No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 20), VFTPQ (Seq ID No. 21), PQNHA (Seq ID No. 22), IEQMPQRS (Seq ID No. 23), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), FQL, LTTVNSY (Seq ID No. 26), YNL, or YQL.

[0019] This application provides a method for protecting the kidney and improving kidney damage. The method includes administering an effective amount of a polypeptide to a subject, the amino acid sequence of which is selected from: VADW (Seq ID No. 1), VAMP (Seq ID No. 2), YQLM (Seq ID No. 3), FPQS (Seq ID No. 4), FSPSSQQ (Seq ID No. 5), YGDQ (Seq ID No. 6), WLE, YTGD (Seq ID No. 7), VVDNNGRS (Seq ID No. 8), PSSQQ (Seq ID No. 9), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID ... Any one of the following: No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 20), VFTPQ (Seq ID No. 21), PQNHA (Seq ID No. 22), IEQMPQRS (Seq ID No. 23), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), FQL, LTTVNSY (Seq ID No. 26), YNL, or YQL.

[0020] This application provides a method for improving gut microbiota dysbiosis, the method comprising administering to a subject an effective amount of a polypeptide, the amino acid sequence of which is selected from VADW (Seq ID No. 1), VAMP (Seq ID No. 2), YQLM (Seq ID No. 3), FPQS (Seq ID No. 4), FSPSSQQ (Seq ID No. 5), YGDQ (Seq ID No. 6), WLE, YTGD (Seq ID No. 7), VVDNNGRS (Seq ID No. 8), PSSQQ (Seq ID No. 9), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID 18), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID 18), FLQ, NYLP (Seq ID No. 18 ... FLQ, NYLP (Seq ID No. 19), LLY, FNPRG (Seq ID No. 10), Y Any one of the following: No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 20), VFTPQ (Seq ID No. 21), PQNHA (Seq ID No. 22), IEQMPQRS (Seq ID No. 23), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), FQL, LTTVNSY (Seq ID No. 26), YNL, or YQL.

[0021] The polypeptide provided in this application is extracted from hemp seeds, specifically prepared by a method including the following steps:

[0022] Oil was extracted from hemp seeds using organic solvents.

[0023] Extracting protein from hemp seeds,

[0024] Add protease for enzymatic hydrolysis.

[0025] Centrifugation was used to collect the supernatant, which was then freeze-dried to obtain the hemp seed polypeptide.

[0026] The organic solvent is selected from pure petroleum ether, n-hexane, n-butanol, ethanol, 5:2 petroleum ether / n-butanol, 5:2 n-hexane / n-butanol, 5:2 petroleum ether / ethanol, or 5:2 n-hexane / ethanol, preferably ethanol;

[0027] The method for extracting protein from hemp seeds is selected from ultrasound-assisted organic extraction, ultrasound-assisted alkaline extraction and acid precipitation extraction, alkaline extraction and acid precipitation extraction, or salt extraction, with salt extraction being preferred;

[0028] The protease is selected from pepsin, trypsin, α-chymotrypsin, papain, flavor protease, proteinase K, neutral protease, alkaline protease, thermophilic protease monoenzyme, gastric / pancreatic complex enzyme, or chymotrypsin / pancreatic complex enzyme, preferably thermophilic protease monoenzyme.

[0029] Invention Effects

[0030] 1. The hemp seed polypeptide provided in this application has an inhibitory effect on DPP-IV, can significantly increase the concentration of GLP-1 in plasma, reduce the concentration of GHb, improve glucose tolerance and improve insulin resistance, thereby achieving the effect of lowering blood sugar and promoting blood sugar metabolic balance.

[0031] 2. The hemp seed polypeptide can reduce the concentration of TC, TG and FFA in plasma and increase the concentration of PYY, thus having a certain fat-reducing effect.

[0032] 3. The hemp seed polypeptide can reduce the concentrations of ALT, AST, ALP and TP II in plasma, and has a certain liver protective function.

[0033] 4. The hemp seed polypeptide can reduce the concentration of creatinine and urea in plasma, and has a certain protective effect on the kidneys.

[0034] 5. The hemp seed polypeptide can effectively increase the abundance of beneficial bacteria genera including Lactobacillus, Akkermansia, and Bacteroides, while decreasing the abundance of harmful bacteria genus Prevotella, and increasing... Bacteroidetes / Firmicutes The proportion of [something] increases the α-diversity of the gut microbiota, which has a certain function of improving gut microbiota dysbiosis.

[0035] 6. The polypeptides and their compositions provided in this application are polypeptide sequences found in hemp seeds, a crop that is both food and medicine. They have virtually no side effects when consumed and are therefore safer for human use. Furthermore, they can achieve a hypoglycemic effect through oral administration, making them safer and more convenient to take. Attached Figure Description

[0036] Figure 1 The diagram shown is a schematic of the DPP IV inhibitory activity in the cell experiment of the synthesized polypeptide in Example 6.

[0037] Figure 2 The diagram shown is a schematic of the concentration of GLP-1 in the cell experiment of synthesized polypeptide in Example 6.

[0038] Figure 3 The diagram shown is a schematic of insulin concentration in the synthetic polypeptide cell experiment of Example 6.

[0039] Figure 4 The diagram shown is a schematic of the water drinking situation of mice in the animal experiment of Example 7.

[0040] Figure 5 The diagram shown is a schematic of the diet of mice in the animal experiment of Example 7.

[0041] Figure 6 The diagram shown is a schematic of the energy intake of mice in the animal experiment of Example 7.

[0042] Figure 7 The diagram shown is a schematic of the changes in mouse body weight during the animal experiment in Example 7.

[0043] Figure 8 The diagram shown is a schematic of the PYY concentration in mouse plasma during the animal experiment in Example 7.

[0044] Figure 9 The diagram shown is a schematic of the TG content in mouse plasma during the animal experiment in Example 7.

[0045] Figure 10 The diagram shown is a schematic of the TC content in mouse plasma during the animal experiment in Example 7.

[0046] Figure 11 The diagram shown is a schematic of the FFA content in mouse plasma during the animal experiment in Example 7.

[0047] Figure 12 The diagram shown is a schematic of the changes in blood glucose levels in mice during gavage in the animal experiment of Example 7.

[0048] Figure 13 The diagram shows the changes in glucose concentration (left) and the area under the curve (right) during the oral glucose tolerance test in the animal experiment of Example 7.

[0049] Figure 14 The diagram shown is a schematic of plasma GLP-1 concentration in the animal experiment of Example 7.

[0050] Figure 15 The diagram shown is a schematic of plasma insulin concentration and HOMA-IR in the animal experiment of Example 7.

[0051] Figure 16 The diagram shown is a schematic of plasma GHb concentration in the animal experiment of Example 7.

[0052] Figure 17 The diagram shown is a schematic of plasma ADPN concentration in the animal experiment of Example 7.

[0053] Figure 18 The diagram shown is a schematic diagram of gut microbiota α diversity in the animal experiment of Example 7.

[0054] Figure 19 The figure shows the abundance and distribution of Lactobacillus, Akkermansia, Bacteroides, and Prevotella in the animal experiment of Example 7. Bacteroidetes / Firmicutes A proportional diagram.

[0055] Figure 20 The diagram shown is a schematic diagram of the DPP IV inhibitory activity in the cell experiment of the enzymatic hydrolysis extract in Example 8.

[0056] Figure 21 The diagram shown is a schematic of the GLP-1 concentration in the cell experiment of the enzymatic hydrolysis extract in Example 8.

[0057] Figure 22 The diagram shown is a schematic of insulin concentration in the cell experiment of the enzymatic hydrolysis extract in Example 8.

[0058] Figure 23 The diagram shown is a schematic of the water drinking situation of mice in the animal experiment of Example 9.

[0059] Figure 24 The diagram shown is a schematic of the diet of mice in the animal experiment of Example 9.

[0060] Figure 25 The diagram shown is a schematic of the energy intake of mice in the animal experiment of Example 9.

[0061] Figure 26 The diagram shown is a schematic of the changes in mouse body weight during the animal experiment in Example 9.

[0062] Figure 27 The diagram shown is a schematic of the PYY concentration in mouse plasma during the animal experiment in Example 9.

[0063] Figure 28 The diagram shown is a schematic of the TG content in mouse plasma during the animal experiment in Example 9.

[0064] Figure 29 The diagram shown is a schematic of the TC content in mouse plasma during the animal experiment in Example 9.

[0065] Figure 30 The diagram shown is a schematic of the FFA content in mouse plasma during the animal experiment in Example 9.

[0066] Figure 31 The diagram shown is a schematic diagram of blood glucose changes in mice during gavage in the animal experiment of Example 9.

[0067] Figure 32The diagram shows the changes in glucose concentration (left) and the area under the curve (right) during the oral glucose tolerance test in the animal experiment of Example 9.

[0068] Figure 33 The diagram shown is a schematic of the concentration of GLP-1 in mouse plasma during the animal experiment in Example 9.

[0069] Figure 34 The diagram shown is a schematic of mouse plasma insulin concentration and HOMA-IR in the animal experiment of Example 9.

[0070] Figure 35 The diagram shown is a schematic of the GHb concentration in mouse plasma during the animal experiment in Example 9.

[0071] Figure 36 The diagram shown is a schematic of the ADPN concentration in mouse plasma during the animal experiment in Example 9.

[0072] Figure 37 The diagram shown is a schematic diagram of the α-diversity of gut microbiota in the animal experiment of Example 9.

[0073] Figure 38 The figure shows the abundance and distribution of Lactobacillus, Akkermansia, Bacteroides, and Prevotella in animal experiments in Example 9. Bacteroidetes / Firmicutes A proportional diagram. Detailed Implementation

[0074] The present application will now be described in further detail with reference to specific embodiments. The embodiments given are intended to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0075] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0076] This application provides a polypeptide extracted from hemp seeds and possessing multiple functional activities. Specifically, the polypeptide can inhibit the enzymatic activity of DPP-IV, increase the concentration of GLP-1, and has certain effects such as fat reduction, liver protection, kidney protection, and improvement of intestinal flora imbalance.

[0077] This application provides a polypeptide whose amino acid sequence is selected from VADW (Seq ID No. 1), VAMP (Seq ID No. 2), YQLM (Seq ID No. 3), FPQS (Seq ID No. 4), FSPSSQQ (Seq ID No. 5), YGDQ (Seq ID No. 6), WLE, YTGD (Seq ID No. 7), VVDNNGRS (Seq ID No. 8), PSSQQ (Seq ID No. 9), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19). Any one of the following: No. 19), PQNH (Seq ID No. 20), VFTPQ (Seq ID No. 21), PQNHA (Seq ID No. 22), IEQMPQRS (Seq ID No. 23), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), FQL, LTTVNSY (Seq ID No. 26), YNL, or YQL.

[0078] In the polypeptide sequence, the uppercase letters I, P, V, Q, E, S, T, and W each represent an amino acid or its amino acid residue. The correspondence between the uppercase letters and amino acids is shown in Table 1.

[0079] Table 1. Amino acids and their corresponding capital letters

[0080]

[0081] This application also provides the nucleic acid sequence encoding the said polypeptide.

[0082] This application provides a composition comprising one or more amino acid sequences of the following sequences: VADW (Seq ID No. 1), VAMP (Seq ID No. 2), YQLM (Seq ID No. 3), FPQS (Seq ID No. 4), FSPSSQQ (Seq ID No. 5), YGDQ (Seq ID No. 6), WLE, YTGD (Seq ID No. 7), VVDNNGRS (Seq ID No. 8), PSSQQ (Seq ID No. 9), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19). Peptides of No. 19), PQNH (Seq ID No. 20), VFTPQ (Seq ID No. 21), PQNHA (Seq ID No. 22), IEQMPQRS (Seq ID No. 23), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), FQL, LTTVNSY (Seq ID No. 26), YNL, or YQL.

[0083] Preferably, the composition provided in this application further includes a pharmaceutically, food-grade, or health-grade acceptable carrier, excipient, or second active ingredient.

[0084] Preferably, the second active ingredient in the composition provided in this application is selected from one or more of the following: DPP-IV inhibitors, SGLT-2 inhibitors, GLP-1 receptor agonists, insulin secretagogues, α-glucosidase inhibitors, angiotensin-converting enzyme inhibitors, angiotensin receptor antagonists, calcium channel blockers, or β-receptor blockers.

[0085] This application provides amino acid sequences selected from VADW (Seq ID No. 1), VAMP (Seq ID No. 2), YQLM (Seq ID No. 3), FPQS (Seq ID No. 4), FSPSSQQ (Seq ID No. 5), YGDQ (Seq ID No. 6), WLE, YTGD (Seq ID No. 7), VVDNNGRS (Seq ID No. 8), PSSQQ (Seq ID No. 9), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 18), and WDSY (Seq ID No. 19). Use of any one of the following polypeptides, or a composition comprising one or more of the above polypeptides, in the preparation of a medicament for the prevention, treatment or improvement of hyperglycemia and related diseases in humans: No. 20), VFTPQ (Seq ID No. 21), PQNHA (Seq ID No. 22), IEQMPQRS (Seq ID No. 23), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), FQL, LTTVNSY (Seq ID No. 26), YNL or YQL.

[0086] Hyperglycemia is a metabolic disorder caused by a significant increase in blood sugar. Related diseases caused by hyperglycemia include chronic diseases caused by long-term hyperglycemia, such as diabetes, retinopathy, nephropathy, diabetic nephropathy, diabetic retinopathy, arteriosclerosis, peripheral neuropathy, and autonomic neuropathy; it also includes acute and severe metabolic disorders caused by acute and significant increases in blood sugar, such as diabetic ketoacidosis and hyperosmolar hyperglycemia syndrome.

[0087] In some specific embodiments, the polypeptides provided in this application or compositions containing said polypeptides can inhibit DPP-IV activity, increase GLP-1 levels in vivo, and produce a blood glucose regulating effect, which can be applied to the development of drugs for hyperglycemia and related human diseases caused by hyperglycemia.

[0088] This application provides amino acid sequences selected from VADW (Seq ID No. 1), VAMP (Seq ID No. 2), YQLM (Seq ID No. 3), FPQS (Seq ID No. 4), FSPSSQQ (Seq ID No. 5), YGDQ (Seq ID No. 6), WLE, YTGD (Seq ID No. 7), VVDNNGRS (Seq ID No. 8), PSSQQ (Seq ID No. 9), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 18), and WDSY (Seq ID No. 19). The use of any one of the following polypeptides, or a composition comprising one or more of the above polypeptides, in the preparation of a medicament having fat-reducing and metabolic-regulating functions: No. 20), VFTPQ (Seq ID No. 21), PQNHA (Seq ID No. 22), IEQMPQRS (Seq ID No. 23), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), FQL, LTTVNSY (Seq ID No. 26), YNL, or YQL.

[0089] Obesity is a chronic metabolic disease characterized by excessive fat accumulation and overweight. Current medications primarily work by slowing down or reducing fat absorption or promoting the breakdown and metabolism of fat in the body to achieve fat reduction.

[0090] In some specific embodiments, the polypeptides provided in this application or compositions containing said polypeptides can reduce energy intake, slow down weight gain, and reduce the concentrations of TC, TG and FFA in plasma while increasing the concentration of PYY, and can be applied to the development of drugs related to fat reduction and metabolism regulation.

[0091] This application provides amino acid sequences selected from VADW (Seq ID No. 1), VAMP (Seq ID No. 2), YQLM (Seq ID No. 3), FPQS (Seq ID No. 4), FSPSSQQ (Seq ID No. 5), YGDQ (Seq ID No. 6), WLE, YTGD (Seq ID No. 7), VVDNNGRS (Seq ID No. 8), PSSQQ (Seq ID No. 9), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 18), and WDSY (Seq ID No. 19). Use of any one of the following polypeptides (No. 20), VFTPQ (Seq ID No. 21), PQNHA (Seq ID No. 22), IEQMPQRS (Seq ID No. 23), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), FQL, LTTVNSY (Seq ID No. 26), YNL, or YQL, or a composition comprising one or more of the aforementioned polypeptides, in the preparation of a medicament that has hepatoprotective, liver-reducing, or liver-damaging effects.

[0092] This application provides amino acid sequences selected from VADW (Seq ID No. 1), VAMP (Seq ID No. 2), YQLM (Seq ID No. 3), FPQS (Seq ID No. 4), FSPSSQQ (Seq ID No. 5), YGDQ (Seq ID No. 6), WLE, YTGD (Seq ID No. 7), VVDNNGRS (Seq ID No. 8), PSSQQ (Seq ID No. 9), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 18), and WDSY (Seq ID No. 19). Use of any one of the following polypeptides (No. 20), VFTPQ (Seq ID No. 21), PQNHA (Seq ID No. 22), IEQMPQRS (Seq ID No. 23), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), FQL, LTTVNSY (Seq ID No. 26), YNL, or YQL, or a composition comprising one or more of the aforementioned polypeptides, in the preparation of a medicament for protecting the kidney and improving renal damage.

[0093] This application provides amino acid sequences selected from VADW (Seq ID No. 1), VAMP (Seq ID No. 2), YQLM (Seq ID No. 3), FPQS (Seq ID No. 4), FSPSSQQ (Seq ID No. 5), YGDQ (Seq ID No. 6), WLE, YTGD (Seq ID No. 7), VVDNNGRS (Seq ID No. 8), PSSQQ (Seq ID No. 9), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 18), and WDSY (Seq ID No. 19). Use of any one of the following polypeptides, or a composition comprising one or more of the above polypeptides, in the preparation of a medicament having the function of improving intestinal flora imbalance: No. 20, VFTPQ (Seq ID No. 21), PQNHA (Seq ID No. 22), IEQMPQRS (Seq ID No. 23), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), FQL, LTTVNSY (Seq ID No. 26), YNL, or YQL.

[0094] The drugs, foods for special medical purposes, and health foods provided in this application may further include pharmaceutically acceptable carriers or excipients.

[0095] Specifically, examples of pharmaceutically acceptable carriers include excipients, binders, buffers, antioxidants, solubilizers, thickeners, lubricants, disintegrants, diluents, stabilizers, preservatives, colorants, flavorings, solubilizers, emulsifiers, isotonic agents, and the like.

[0096] The excipients may be selected from, but are not limited to, starch, lactose, sucrose, calcium carbonate, and calcium phosphate; the binders may be selected from, but are not limited to, starch, gum arabic, carboxymethyl cellulose, hydroxypropyl cellulose, crystalline cellulose, alginic acid, gels, and polyvinylpyrrolidone; the buffers may be selected from, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, borate buffer solutions, Tris salt buffer solutions, and the like; the antioxidants may be selected from, but are not limited to, butylated hydroxytoluene, butylated hydroxyanisole, sodium sulfite, sodium bisulfite, sodium metabisulfite, ascorbic acid, cysteine ​​hydrochloride, cystine, lipoic acid, thioglycerol, and the like; the lubricants may be selected from... However, the following are not limited to magnesium stearate, calcium stearate, and talc; the thickener may be selected from, but is not limited to, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, amorphous cellulose, polysaccharides (including starch derivatives), polyvinyl alcohol, and polyvinylpyrrolidone or mixtures thereof; the disintegrant may be selected from, but is not limited to, calcium carboxymethylcellulose and talc; the diluent may be selected from, but is not limited to, water for injection and saline; the preservative may be selected from, but is not limited to, sodium bisulfite, sodium bisulfite, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate, methylparaben, propylparaben, and phenylethanol; the isotonic agent may be selected from, but is not limited to, chlorides and sugars.

[0097] This application also provides that pharmaceutical products containing the said polypeptide can be prepared by mixing the polypeptide with a pharmaceutically acceptable carrier, for example, to obtain oral formulations such as tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets), capsules (including soft capsules, microcapsules), granules, powders, lozenges, syrups, emulsions, suspensions, films (e.g., orally disintegrating films), parenteral formulations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, drops), topical formulations (e.g., skin formulations, ointments), suppositories (e.g., rectal suppositories, vaginal suppositories), pills, nasal drops, respiratory formulations (inhalers), eye drops, etc. In addition, these formulations can be used as controlled-release formulations (e.g., sustained-release microcapsules), such as immediate-release formulations, sustained-release formulations, etc. Such formulations can be obtained by preparation methods conventionally used in this art.

[0098] This application provides a method for preventing, treating, or improving hyperglycemia and related diseases in humans. The method includes administering an effective amount of a polypeptide to a subject, the amino acid sequence of which is selected from: VADW (Seq ID No. 1), VAMP (Seq ID No. 2), YQLM (Seq ID No. 3), FPQS (Seq ID No. 4), FSPSSQQ (Seq ID No. 5), YGDQ (Seq ID No. 6), WLE, YTGD (Seq ID No. 7), VVDNNGRS (Seq ID No. 8), PSSQQ (Seq ID No. 9), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), and VADW (Seq ID 18). Any one of the following: No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 20), VFTPQ (Seq ID No. 21), PQNHA (Seq ID No. 22), IEQMPQRS (Seq ID No. 23), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), FQL, LTTVNSY (Seq ID No. 26), YNL, or YQL.

[0099] This application provides a method for fat reduction and metabolism regulation, comprising administering an effective amount of a polypeptide to a subject, wherein the amino acid sequence of the polypeptide is selected from: VADW (Seq ID No. 1), VAMP (Seq ID No. 2), YQLM (Seq ID No. 3), FPQS (Seq ID No. 4), FSPSSQQ (Seq ID No. 5), YGDQ (Seq ID No. 6), WLE, YTGD (Seq ID No. 7), VVDNNGRS (Seq ID No. 8), PSSQQ (Seq ID No. 9), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID ... Any one of the following: No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 20), VFTPQ (Seq ID No. 21), PQNHA (Seq ID No. 22), IEQMPQRS (Seq ID No. 23), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), FQL, LTTVNSY (Seq ID No. 26), YNL, or YQL.

[0100] This application provides a method for protecting the liver, reducing ALT and AST concentrations, or improving liver damage. The method includes administering an effective amount of a polypeptide to a subject, the amino acid sequence of which is selected from VADW (Seq ID No. 1), VAMP (Seq ID No. 2), YQLM (Seq ID No. 3), FPQS (Seq ID No. 4), FSPSSQQ (Seq ID No. 5), YGDQ (Seq ID No. 6), WLE, YTGD (Seq ID No. 7), VVDNNGRS (Seq ID No. 8), PSSQQ (Seq ID No. 9), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 16), and YTPHW (Seq ID No. 17). Any one of the following: No. 17), FDGEL (Seq ID No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 20), VFTPQ (Seq ID No. 21), PQNHA (Seq ID No. 22), IEQMPQRS (Seq ID No. 23), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), FQL, LTTVNSY (Seq ID No. 26), YNL, or YQL.

[0101] This application provides a method for protecting the kidney and improving kidney damage. The method includes administering an effective amount of a polypeptide to a subject, the amino acid sequence of which is selected from: VADW (Seq ID No. 1), VAMP (Seq ID No. 2), YQLM (Seq ID No. 3), FPQS (Seq ID No. 4), FSPSSQQ (Seq ID No. 5), YGDQ (Seq ID No. 6), WLE, YTGD (Seq ID No. 7), VVDNNGRS (Seq ID No. 8), PSSQQ (Seq ID No. 9), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID ... Any one of the following: No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 20), VFTPQ (Seq ID No. 21), PQNHA (Seq ID No. 22), IEQMPQRS (Seq ID No. 23), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), FQL, LTTVNSY (Seq ID No. 26), YNL, or YQL.

[0102] This application provides a method for improving gut microbiota dysbiosis, the method comprising administering to a subject an effective amount of a polypeptide, the amino acid sequence of which is selected from VADW (Seq ID No. 1), VAMP (Seq ID No. 2), YQLM (Seq ID No. 3), FPQS (Seq ID No. 4), FSPSSQQ (Seq ID No. 5), YGDQ (Seq ID No. 6), WLE, YTGD (Seq ID No. 7), VVDNNGRS (Seq ID No. 8), PSSQQ (Seq ID No. 9), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), WIAVK (Seq ID No. 12), LNAP (Seq ID No. 13), YNLP (Seq ID No. 14), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID No. 18), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID 18), FLQ, NYLP (Seq ID No. 15), LLY, FNPRG (Seq ID No. 16), YTPHW (Seq ID No. 17), FDGEL (Seq ID 18), FLQ, NYLP (Seq ID No. 18 ... FLQ, NYLP (Seq ID No. 19), LLY, FNPRG (Seq ID No. 10), Y Any one of the following: No. 18), WDSY (Seq ID No. 19), PQNH (Seq ID No. 20), VFTPQ (Seq ID No. 21), PQNHA (Seq ID No. 22), IEQMPQRS (Seq ID No. 23), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), FQL, LTTVNSY (Seq ID No. 26), YNL, or YQL.

[0103] Drugs containing the polypeptides described in this application can be administered to mammals (e.g., humans, mice, rats, rabbits, dogs, cats, cattle, horses, pigs, and monkeys). The administration route can be oral or parenteral (e.g., intravenous, intramuscular, subcutaneous, intra-organ, intranasal, intradermal, intravenous drip, intracerebral, rectal, vaginal, intraperitoneal, etc.).

[0104] The dosage of the peptides in this application administered to the subjects varies depending on the route of administration, symptoms, patient age, etc., and can be determined by the clinician in practice.

[0105] The drugs involved in this application can also be used in conjunction with other existing known drugs for treating hyperglycemia, type 2 diabetes, obesity, metabolic disorders, and drugs with hepatoprotective, renal, and intestinal flora regulating functions. When used together, there are no restrictions on the timing of administration of each drug; two or more different drugs can be administered simultaneously, or at different times. The dosage of the known drugs can be determined according to clinically used dosages and appropriately selected based on the patient, route of administration, etc.

[0106] In some embodiments, the polypeptides provided in this application may be produced by chemical synthesis. In some embodiments, the polypeptides may be produced by biosynthesis. In some embodiments, the polypeptides may be extracted from food or obtained through enzymatic hydrolysis.

[0107] In one specific embodiment, the polypeptide provided in this application is extracted from hemp seeds.

[0108] This application also provides a method for extracting the polypeptide from hemp seeds, comprising:

[0109] Oil was extracted from hemp seeds using organic solvents.

[0110] Extracting protein from hemp seeds,

[0111] Add protease for enzymatic hydrolysis.

[0112] Centrifugation was used to collect the supernatant, which was then freeze-dried to obtain the hemp seed polypeptide.

[0113] The organic solvent is selected from pure petroleum ether, n-hexane, n-butanol, ethanol, 5:2 petroleum ether / n-butanol, 5:2 n-hexane / n-butanol, 5:2 petroleum ether / ethanol, or 5:2 n-hexane / ethanol, preferably ethanol;

[0114] The method for extracting protein from hemp seeds is selected from ultrasound-assisted organic extraction, ultrasound-assisted alkaline extraction and acid precipitation extraction, alkaline extraction and acid precipitation extraction, or salt extraction, with salt extraction being preferred;

[0115] The protease is selected from pepsin, trypsin, α-chymotrypsin, papain, flavor protease, proteinase K, neutral protease, alkaline protease, thermophilic protease monoenzyme, gastric / pancreatic complex enzyme, or chymotrypsin / pancreatic complex enzyme, preferably thermophilic protease monoenzyme.

[0116] In this application, no restrictions are placed on the preparation method of hemp seed raw material powder. It can be prepared in accordance with conventional methods in the field, including but not limited to grinding.

[0117] In this application, hemp seed powder is added to an extraction reagent, stirred, and centrifuged to remove the oil from the hemp seed powder.

[0118] In one specific embodiment of this application, hemp seed powder is mixed with the organic solvent at a ratio of 1:10 to 1:20 (g / mL), and after stirring, centrifugation, and discarding the supernatant, defatted hemp seed powder is obtained.

[0119] For example, the ratio (g / mL) of hemp seed powder to organic solvent can be 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, etc.

[0120] In a preferred embodiment, hemp seed powder is mixed with anhydrous ethanol at a ratio of 1:20 (g / mL).

[0121] In this application, there are no restrictions on the method for extracting oil from hemp seed powder. It can be carried out in accordance with conventional methods in the art. In some specific embodiments, static stirring or ultrasonic extraction methods can be used.

[0122] In one specific embodiment, the oil extraction is carried out by static stirring: the mixture of hemp seed powder and extraction reagent is stirred at room temperature of 20-25 ℃ for 12 h, the supernatant is removed by centrifugation, the precipitate is added to the extraction reagent again and stirred at room temperature of 20-25 ℃ for 6 h, the supernatant is removed by centrifugation, the precipitate is first evaporated in a water bath at about 60 ℃ until no organic matter remains, and then dried and cooled to obtain defatted hemp seed powder.

[0123] In another specific embodiment, the oil is extracted using ultrasonic extraction: the mixture of hemp seed powder and extraction reagent is stirred at room temperature of 20-25 ℃ for 10 min, ultrasonicated (100 kHz) for 10 min, centrifuged to remove the supernatant, the precipitate is added to the extraction reagent again and stirred and ultrasonicated repeatedly, centrifuged to remove the supernatant, the precipitate is first evaporated in a water bath at about 60 ℃ until no organic matter remains, and then dried and cooled to obtain defatted hemp seed powder.

[0124] In a preferred embodiment of this application, the oil in hemp seed powder is extracted using a static stirring method.

[0125] In this application, no restrictions are placed on the extraction method of hemp seed protein. It can be carried out in accordance with conventional methods in the art. In some specific embodiments, the extraction method of hemp seed protein is selected from ultrasound-assisted organic extraction, ultrasound-assisted alkaline extraction and acid precipitation extraction, alkaline extraction and acid precipitation extraction, or salt extraction.

[0126] In one specific embodiment, hemp seed protein is extracted using an ultrasound-assisted organic extraction method: defatted hemp seed powder is mixed with n-hexane, centrifuged under ultrasound, and the supernatant is collected. The remaining substances can be repeated several times. The supernatants obtained are combined and evaporated until no organic matter remains, and then dried to obtain the final product.

[0127] Preferably, defatted hemp seed powder and n-hexane are mixed at a ratio of 1:30 to 1:50 (g / mL); preferably, the ultrasonic frequency is 2 kHz; preferably, the ultrasonic time is 20 to 40 min each time; preferably, the drying is carried out by water bath at 55 to 65 ℃; preferably, the drying is carried out by oven at 55 to 65 ℃.

[0128] In another specific embodiment, the protein in hemp seed is extracted by ultrasound-assisted alkaline extraction and acid precipitation: defatted hemp seed powder is mixed with ultrapure water (pH 8.5), ultrasonicated (200 W), and centrifuged to obtain the supernatant. The remaining substances can be repeated several times. The supernatants are combined and the pH is adjusted to 4.5 to precipitate the protein. After washing with water, the pH is adjusted to 7.0 and then dried to obtain the protein.

[0129] Preferably, defatted hemp seed powder is mixed with ultrapure water at a ratio of 1:10 to 1:30 (g / mL); preferably, the ultrasonic frequency is 100 to 300 W; preferably, the ultrasonic treatment temperature is 20 to 25 ℃; preferably, the ultrasonic treatment time is 20 to 40 min each time; preferably, the drying is carried out in an oven at 55 to 65 ℃.

[0130] In another specific embodiment, the protein in hemp seeds is extracted using an alkaline extraction and acid precipitation method: defatted hemp seed powder is mixed with ultrapure water (pH 10.0), stirred, centrifuged and the supernatant is collected. The remaining substances can be repeated several times. The supernatants are combined and the pH is adjusted to 5.0 to precipitate the protein. After washing with water, the pH is adjusted to 7.0 and then dried or freeze-dried to obtain the protein.

[0131] Preferably, defatted hemp seed powder is mixed with ultrapure water at a ratio of 1:5 to 1:20 (g / mL); preferably, the stirring temperature is 30 to 40°C; preferably, the stirring time is 1 to 3 hours each time; preferably, the product is dried in an oven at 55 to 65°C.

[0132] In another specific embodiment, hemp seed protein is extracted using salt extraction: defatted hemp seed powder is mixed with NaCl solution (pH 7.0), stirred, centrifuged and the supernatant is collected. The process can be repeated several times for the remaining material. Ultrapure water (pH 10.0) is added to the remaining material, stirred, centrifuged and the supernatant is collected. The supernatants are combined and the pH is adjusted to 4.5 to precipitate the protein. After washing with water, the pH is adjusted to 7.0, and the protein is dried or freeze-dried.

[0133] Preferably, the NaCl solution concentration is 0.5~1.0M; preferably, the defatted hemp seed powder is mixed with the NaCl solution at a ratio of 1:5~1:20 (g / mL); preferably, the stirring temperature is 30~40℃; preferably, the stirring time is 1~3h each time; preferably, the drying is carried out in an oven at 55~65℃.

[0134] In a preferred embodiment of this application, hemp seed protein is extracted using a salt extraction method.

[0135] In some embodiments, hemp seed protein is prepared into a protein solution of a certain mass concentration, and protease is added at a concentration of 8000~10000 U / g for enzymatic hydrolysis. Preferably, the protease hydrolysis is carried out at 35-60 °C; preferably, the protease hydrolysis is carried out under the condition of pH 7-11; preferably, the hydrolysis time is 3-6 h.

[0136] For example, enzymatic hydrolysis can be carried out at temperatures of 35℃, 40℃, 42℃, 45℃, 48℃, 50℃, 52℃, 55℃, 58℃, 60℃, 65℃, and 70℃.

[0137] Enzymatic hydrolysis with proteases can be carried out under conditions of pH 6, 7, 8, 9, 10, 11, etc.

[0138] The enzymatic hydrolysis time can be 3 h, 4 h, 5 h, 6 h, 7 h, etc.

[0139] In this application, the product after enzymatic hydrolysis is filtered, further centrifuged, and freeze-dried to obtain the hemp seed enzymatic hydrolysate.

[0140] Example

[0141] The hemp seeds used in the following examples of this application were obtained from Bama, Guangxi, and the thermophilic protease was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Other materials and reagents, unless otherwise specified, are commercially available.

[0142] Example 1: Optimization of Enzymatic Extraction Process and Method for Hemp Seeds

[0143] The enzymatic extraction method for hemp seeds disclosed in this embodiment includes the following specific steps: removing oil from hemp seeds using an organic solvent; extracting protein from defatted hemp seeds; adding protease to enzymatically hydrolyze the hemp seed protein; centrifuging the enzymatic hydrolysis product and collecting the supernatant to obtain the hemp seed enzymatic extract. The process conditions for each step of oil extraction, protein extraction, and enzymatic hydrolysis were screened through the following experiments to obtain a preferred preparation method for the hemp seed enzymatic extract.

[0144] (1) Screening of methods for extracting oil from hemp seeds

[0145] Pure petroleum ether, n-hexane, n-butanol, ethanol, 5:2 ratios of petroleum ether / n-butanol, 5:2 ratios of n-hexane / n-butanol, 5:2 ratios of petroleum ether / ethanol, and 5:2 ratios of n-hexane / ethanol were selected as extraction reagents, and the oil in hemp seeds was extracted at a solid-liquid ratio of 1:20. The extraction methods employed were static stirring and ultrasonic extraction. Detailed process parameters are as follows:

[0146] Static stirring method: Weigh hemp seeds (~2 g) → Add extraction reagent at a material-to-liquid ratio of 1:20 (w / v, g / mL) → Stir at room temperature (20-25 ℃) for 12 h → Centrifuge and discard the supernatant → Add extraction reagent at a material-to-liquid ratio of 1:20 (w / v, g / mL) to the precipitate again → Stir at room temperature (20-25 ℃) for 6 h → Centrifuge and discard the supernatant → Evaporate the precipitate in a water bath (60 ℃) until no organic matter remains → Dry in an oven (60 ℃) for 30 min → Cool to room temperature and weigh.

[0147] Ultrasonic extraction method: Weigh hemp seeds (~2g) → Add extraction reagent at a material-to-liquid ratio of 1:20 (w / v) → Stir at room temperature (20-25℃) for 10 min → Ultrasonicate at room temperature (20-25℃) (100 kHz) for 10 min → Centrifuge and discard supernatant → Add extraction reagent at a material-to-liquid ratio of 1:20 (w / v, g / mL) to the precipitate again → Stir at room temperature (20-25℃) for 10 min → Ultrasonicate at room temperature (20-25℃) (100 kHz) for 10 min → Centrifuge and discard supernatant → Evaporate the precipitate in a water bath (60℃) until no organic matter remains → Dry in an oven (60℃) for 30 min → Cool to room temperature and weigh.

[0148] The yields of the final raw materials using the above reagents and extraction methods are shown in Table 2. n-Butanol extraction with static stirring showed the lowest recovery rate, indicating its optimal oil and impurity removal effect. This was followed by ethanol extraction with static stirring and petroleum ether / n-butanol ultrasonic extraction. However, experiments revealed that the hemp seed residue after extraction with n-butanol or petroleum ether had an unpleasant, irritating odor. Furthermore, the residue obtained by ethanol extraction was found to be lighter in color compared to other extraction methods. Therefore, overall, the ethanol extraction with static stirring method was deemed the most effective for oil removal.

[0149] Table 2 Recovery rates of hemp seeds after oil removal obtained with different reagents and extraction methods (n=3)

[0150]

[0151] (2) Optimization of hemp seed protein extraction process

[0152] Hemp seed protein was extracted using ultrasound-assisted organic extraction, ultrasound-assisted alkaline extraction and acid precipitation, alkaline extraction and acid precipitation, and salt extraction, with the extraction process optimized. The specific operating procedures for each method are described below:

[0153] Ultrasonic-assisted organic extraction method: Weigh hemp seeds (~5 g) → Add n-hexane at a material-to-liquid ratio of 1:40 (w / v, g / mL) → Ultrasonicate at 30 ℃ and 2 kHz for 30 min → Centrifuge and collect the supernatant → Add n-hexane again to the remaining material at a material-to-liquid ratio of 1:40 (w / v, g / mL) → Ultrasonicate at 30 ℃ and 2 kHz for 30 min → Centrifuge and collect the supernatant → Combine the two supernatants and evaporate to dryness in a water bath (60 ℃) until no organic matter remains → Dry in an oven (60 ℃).

[0154] Ultrasonic-assisted alkaline extraction and acid precipitation extraction method: Weigh hemp seeds (~5g) → Add ultrapure water (pH 8.5) at a material-to-liquid ratio of 1:20 (w / v, g / mL) → Ultrasonicate (200 W) for 30 min at room temperature (20-25 ℃) → Centrifuge and collect the supernatant → Add ultrapure water (pH 8.5) again at a material-to-liquid ratio of 1:10 (w / v, g / mL) → Ultrasonicate (200 W) for 30 min at room temperature (20-25 ℃) → Centrifuge and collect the supernatant → Combine the supernatants from the two extractions and adjust the pH to 4.5 to precipitate the protein → Wash with water 3 times → Adjust the pH to 7.0 → Dry in an oven.

[0155] Alkaline extraction and acid precipitation extraction method: Weigh hemp seeds (~5g) → Add ultrapure water (pH10.0) at a material-to-liquid ratio of 1:10 (w / v, g / mL) → Extract at 35℃ with stirring for 2 h → Centrifuge and collect the supernatant → Add ultrapure water (pH10.0) to the remaining material again at a material-to-liquid ratio of 1:10 (w / v, g / mL) → Extract at 35℃ with stirring for 2 h → Centrifuge and collect the supernatant → Combine the supernatants removed in the two extractions and adjust the pH to 5.0 to precipitate the protein → Wash with water 3 times → Adjust the pH to 7.0 → Dry in an oven / freeze-dry for storage.

[0156] Salt extraction method: Weigh hemp seeds (~5g) → Add 0.8 M NaCl solution (pH 7.0) at a material-to-liquid ratio of 1:10 (w / v, g / mL) → Extract at 35℃ with stirring for 2 h → Centrifuge and collect the supernatant → Add ultrapure water (pH 10.0) to the remaining material again at a material-to-liquid ratio of 1:10 (w / v, g / mL) → Extract at 35℃ with stirring for 2 h → Centrifuge and collect the supernatant → Combine the supernatants removed in the two extractions and adjust the pH to 4.5 to precipitate the protein → Wash with water 3 times → Adjust the pH to 7.0 → Dry in an oven / freeze-dry for storage.

[0157] The final protein yields using the four methods described above are shown in Table 3. Table 3 shows that the salt extraction freeze-drying method yielded the highest protein yield, reaching 24.15%. Furthermore, comparison revealed that the protein obtained using the salt extraction method was significantly lighter in color than that obtained using the alkaline extraction method. Therefore, in summary, the salt extraction method is the most effective for protein extraction.

[0158] Table 3 Protein extraction yield of different extraction methods (n=3)

[0159]

[0160] (3) Optimization of enzymatic hydrolysis process of hemp seed protein

[0161] a. Enzymatic hydrolysis of hemp seed protein

[0162] Hemp seed protein was enzymatically hydrolyzed using pepsin, trypsin, chymotrypsin, papain, flavorzyme, proteinase K, neutral protease, alkaline protease, thermolysin, and gastric / pancreatic (trypsin / pepsin) and chymotrypsin / pancreatic (trypsin / chymotrypsin) complex enzymes, respectively. The specific method was as follows: a 0.1% (w / w) protein solution was prepared, and proteases were added at a protein sample concentration of 10000 U / g (8000 U / g for thermolysin). Enzymatic hydrolysis was performed for 5 h according to the optimal conditions reported in the literature for each enzyme. After hydrolysis, the mixture was centrifuged at 5000 rpm for 20 min, the supernatant was collected, and the product was freeze-dried to obtain the hemp seed enzymatic hydrolysate. The optimal conditions for each enzyme hydrolysis are shown in Table 4.

[0163] Table 4. Types of enzymes and their enzymatic hydrolysis conditions

[0164]

[0165] b. Ultrafiltration and fractionation of enzymatic hydrolysate

[0166] The enzymatic hydrolysate was fractionated using a 3 kDa ultrafiltration tube. The centrifugation conditions were 4200 rpm / min, with each centrifugation lasting 25 min. The centrifugation was repeated until no filtrate was transferred to the collection tube. The filtrates from each centrifugation were then combined for later use.

[0167] c. Determination of protein / peptide concentration in enzymatic hydrolysate

[0168] The protein / peptide concentration in the fractionation solution was determined using the biuret method. The specific steps were as follows: Take 200 μL of each enzymatic digest / standard solution (5 mg / mL bovine serum albumin). Add 1 mL of chromogenic reagent, mix well, and let stand for 15 min. Then, transfer 200 μL of the reaction solution to a 96-well plate and measure the absorbance at 540 nm. The formula for calculating the protein / peptide concentration in the test solution is as follows:

[0169]

[0170] Where A0 is the absorbance of the blank (i.e., the sample solvent well), A1 is the absorbance of the sample to be tested, and A2 is the absorbance of the standard. The protein / peptide concentrations of each separated component are calculated according to this formula, as shown in Table 5.

[0171] Table 5. Concentration and percentage of each fraction after enzyme digestion

[0172]

[0173] Table 5 shows that thermophilic protease, flavor protease and proteinase K have better enzymatic hydrolysis effects, especially thermophilic protease, in which the content of enzymatic hydrolysis components <3 kDa is greater than 86.31%.

[0174] Example 2 Preparation of enzymatic hydrolysis extract of hemp seed

[0175] Based on the optimized process obtained from the above experiments, the enzymatic hydrolysis extract of hemp seeds was prepared using the following steps and conditions:

[0176] The raw hemp seeds were crushed using a grinder. A certain amount of hemp seed powder was weighed and mixed with anhydrous ethanol at a material-to-liquid ratio of 1:20 (w / v, g / mL). The mixture was stirred at 20-25 ℃ for 12 h. After centrifugation at 5000 rpm for 10 min, the supernatant was discarded and the precipitate was collected. The above mixing, stirring and centrifugation steps were repeated 3 times. All the precipitates were then combined and dried in a 60 ℃ water bath until the ethanol evaporated, resulting in oil-free hemp seed powder.

[0177] Weigh a certain amount of the above hemp seed powder, add 0.8 M NaCl solution (pH 7.0) at a material-to-liquid ratio of 1:10 (w / v, g / mL), stir at 35 ℃ for 2 h, centrifuge at 8000 rpm for 10 min and collect the supernatant. Add ultrapure water (pH 10.0) to the remaining material again at a material-to-liquid ratio of 1:10 (w / v, g / mL), stir at 35 ℃ for 2 h, centrifuge and collect the supernatant. Combine the supernatants removed in the two processes and adjust the pH to 4.5 to precipitate the protein. Wash with water 3 times, adjust the pH to 7.0, and freeze dry to obtain hemp seed protein powder.

[0178] The above-mentioned hemp seed protein powder was mixed with ultrapure water to prepare a protein solution with a mass concentration of 0.1%. Thermophilic protease was added at a protein sample concentration of 8000 U / g. The pH was adjusted to 7.0, and enzymatic hydrolysis was carried out at 55 °C for 5 h. After enzymatic hydrolysis, the solution was centrifuged at 5000 rpm for 20 min, the supernatant was collected, and the extract was freeze-dried to obtain the hemp seed enzymatic hydrolysate.

[0179] Example 3: Identification of polypeptides in enzymatic hydrolysis extract of hemp seed

[0180] The hemp seed enzymatic hydrolysis extract obtained in Example 2 was prepared into a 5 mg / mL solution with ultrapure water, then desalted using a desalting column, freeze-dried, and then reconstituted with acetonitrile aqueous solution. The peptide concentration was quantified using NannoDrop and uniformly prepared to 2 mg / mL. Finally, the solution was filtered through a 0.22 μM membrane and detected using a Thermo Fisher QExactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer. The data were analyzed using Maxquant to obtain the peptide composition of the hemp seed enzymatic hydrolysis fraction. Table 6 shows the top ten peptides with the highest relative abundance: LLY, VFTPQ (Seq ID No. 21), VADW (Seq ID No. 1), YNLP (Seq ID No. 14), LNAP (Seq ID No. 13), VAMP (Seq ID No. 2), NYLP (Seq ID No. 15), FNPRG (Seq ID No. 16), and IEQMPQRS (Seq ID No. 14). No.23), PQNH (Seq ID No.20).

[0181] Table 6. Top 10 peptides with the highest relative abundance identified by mass spectrometry

[0182]

[0183] These 10 peptides were artificially synthesized for subsequent in vitro DPP-IV inhibitory activity verification.

[0184] Example 4: Screening of DPP-IV inhibitory peptides based on molecular docking technology

[0185] Using the A chain of DPP-IV after pretreatment of the 5YP3 crystal structure as the receptor, and the identified hemp seed polypeptide library as the receptor, molecular docking was performed using MOE software. Then, the hemp seed polypeptide sequence with the strongest potential DPP-IV inhibitory activity was screened based on the docking score. Finally, 22 peptides were selected for subsequent in vitro DPP-IV inhibitory activity verification: YGDQ (Seq ID No. 6), LTTVASY (Seq ID No. 26), WIAVK (Seq ID No. 12), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), PQNHA (Seq ID No. 22), WDSY (Seq ID No. 19), YTGD (Seq ID No. 7), YQLM (Seq ID No. 3), FSPSSQQ (Seq ID No. 5), FDGEL (Seq ID No. 18), YTPHW (Seq ID No. 17), PSSQQ (Seq ID No. 9), YQL, FPQS (Seq ID No. 4), FLQ, YNL, FQL, WLE, and VVDNNGRS (Seq ID No. 8). The specific screening results are shown in Table 7.

[0186] Table 7 Molecular docking results

[0187]

[0188] Example 5: Determination of the in vitro DPP IV inhibitory activity of the peptide

[0189] Thirty-two peptides obtained in Examples 3 and 4 were artificially synthesized, and the IC50 of the peptides on DPP-IV inhibition was determined. 50 The value was used to evaluate its inhibitory effect on DPP IV.

[0190] Experimental Methods: Five sample concentration gradients (1.0, 0.4, 0.08, 0.016, and 0.0032 mg / mL) were prepared for each peptide. A 100 μL reaction system was constructed in a 96-well microplate as follows: ① First, 60 μL of the peptide solutions at different concentrations was added; ② 20 μL of 2.0 mM Gly-Pro-pNA solution (final concentration 0.40 mM) was added, and the mixture was stirred for 1 minute using a plate shaker; ③ 20 μL of rhDPP-IV solution was added to achieve a final rhDPP-IV activity of 0.025 Unit / mL. After adding rhDPP-IV, the plate was immediately incubated at 37°C for 60 min, with absorbance measured at 405 nm every 10 min. For any sample, select two points where the absorbance value changes within a linear range to calculate the rate of change of absorbance with respect to time, S = (Abs2-Abs1) / (t2-t1), and calculate the DPP-IV inhibition rate using the following formula (2-1):

[0191]

[0192] In equation (2-1), The representative sample's inhibition rate against rhDPP-IV, The rate (slope) of change in absorbance of the negative control, i.e., the solvent group, in the detection system. The rate of change in absorbance represents the sample group. All samples and control groups underwent three replicates, and the standard deviation was calculated. The experimental results are shown in Table 8. The results show that the IC50 values ​​of VAMP, YQLM, FPQS, FSPSSQQ, YGDQ, and WLE peptides are... 50 Less than 1.0 mmol / L, especially the IC50 of peptide VAMP 50 The concentration was 1.0 μmol / L; the IC50 values ​​for peptides YTGD, VVDNNGRS, PSSQQ, VADW, YSYA, WNVN, WIAVK, and LNAP were... 50 Less than 10 mmol / L; IC50 of YNLP, FLQ, NYLP, and LLY peptides 50 Less than 100 mmol / L; among which the IC50 of peptides FNVDSE, PSSQQTR, FQL, LTTVNSY, YNL, and YQL is less than 100 mmol / L. 50 Relatively high.

[0193] Table 8 IC50 values ​​of each synthetic polypeptide 50

[0194]

[0195] Example 6: Evaluation of the hypoglycemic activity of the peptide in cellular experiments

[0196] The 32 polypeptides obtained in Examples 3 and 4 were artificially synthesized, and their DPP-IV inhibitory activity, GLP-1 concentration, and insulin concentration in Caco2 cells were measured to evaluate their hypoglycemic activity.

[0197] Experimental methods:

[0198] (1) The concentration is 1×10 5 Caco2 cells were seeded at a density of 10 cells / mL in 24-well plates and cultured for 24 h. The culture medium was then replaced with medium containing either 2 mg / mL of each synthetic peptide or 5 mg / mL of protein hydrolysate and cultured for another 24 h. The supernatant was then collected, and the activity of DPP-IV and the concentration of GLP-1 were measured. The inhibitory activity of DPP-IV was determined using the DPP-IV-Globe assay. TM The GLP-1 concentration was determined using the Protease Assay (Promega) and Solarbio GLP-1 ELSA reagent. The results are as follows: Figure 1 and Figure 2 As shown.

[0199] (2) 1000 μL of a concentration of 1×10 5 INS-1 cells at a density of 10 cells / mL were seeded in 24-well plates and cultured for 24 h. The culture medium was then replaced with medium containing 2 mg / mL of each synthetic peptide and cultured for another 24 h. The cells were then washed once with KRBH buffer (Krebs-Ringer Bicarbonate HEPES Buffer), and 1000 μL of KRBH buffer was added to each well, followed by incubation for 1 h. The KRBH buffer was then removed, and KRBH buffer containing 16.7 mM glucose solution was added, followed by incubation for 2 h. The supernatant was collected, and the insulin concentration was measured using a Solarbio Insulin kit. The results are shown below. Figure 3 As shown.

[0200] The analysis results show that all synthetic peptides can inhibit DPP-IV activity and promote insulin secretion to some extent. Among them, peptides VADW, WDSY, VAMP, WIAVK, and FNPRG are the most effective in inhibiting DPP-IV activity; peptides FPQS, VFTPQ, FNVDSE, YTGD, and YQLM are the most effective in promoting GLP-1 secretion; and peptides YSYA, PSSQQTR, VFTPQ, PQNHA, and VADW are the most effective in promoting insulin secretion.

[0201] Example 7: Evaluation of the efficacy of polypeptides in lowering blood sugar, reducing fat, protecting the liver and kidneys, and regulating intestinal flora - animal experiments

[0202] The relative abundance and IC50 of the comprehensive mass spectrometry identification 50 Based on the results of value and cellular activity evaluation, three peptides, VADW, VAMP, and YQLM, were selected for in vivo activity verification. The specific experimental protocol is as follows:

[0203] Sixty male C57BL / 6J mice (6 weeks old, weighing 18-22 g) were selected (purchased from Zhejiang Vitonlife Laboratory Animal Technology Co., Ltd.). After one week of acclimatization, the mice were used to establish the model and were administered the mice by gavage.

[0204] The experiment was divided into 6 groups, with 10 mice in each group. The animals were distinguished by ear tagging. The groups were: normal group (WideType, WT), model group (High-fat-diet, HFD), positive drug group (Sitagliptin, PC), and 3 peptide sample groups (VADW, VAMP, and YQLM). The mice were administered the drugs by gavage.

[0205] During the experiment, all mice except the normal group were fed a high-fat diet (20 kcal of protein, 20 kcal of carbohydrates, and 60 kcal of fat) to induce the model. No other special treatments were administered. All groups were gavaged once daily at a dose of 0.2 mL / 20 g mouse body weight. The normal group (Wide Type, WT) and the model group (High-fat-diet, HFD) were gavaged daily with water (sample solvent). The positive control group was gavaged daily with 1.25 mg / kg of sitagliptin phosphate (purchased from Sigma). The VADW, VAMP, and YQLM peptide sample groups were gavaged with 50 mg / kg of their respective samples. The experiment was terminated when the body weight and fasting blood glucose levels of the model group mice were more than 20% higher than those of the normal group.

[0206] During the gavage period, the mice's diet, water intake, blood glucose, and body weight were measured and statistically analyzed weekly. The results showed that after 8 weeks of gavage, the body weight and fasting blood glucose levels of the model group mice were 20% higher than those of the normal group. Therefore, the entire animal experiment was terminated after 8 weeks of gavage.

[0207] (1) Evaluation of the fat-reducing efficacy of peptides

[0208] Eight weeks after gavage, mice were treated and their blood was collected. Plasma was obtained after centrifugation. Total cholesterol (TC) and total triglycerides (TG) in the plasma were analyzed using an automated animal chemistry analyzer. Simultaneously, non-esterified fatty acids (FFA) and poly(PPY) in the plasma were determined using an Elasa kit (Elabscience). Statistical results are shown below. Figure 4-11 As shown.

[0209] Analysis of the results revealed that gavage administration of the three peptide samples significantly reduced the intake of food, water, and energy in mice, and slowed the increase in mouse weight. Simultaneously, gavage administration of the three peptides also significantly reduced the concentrations of TC, TG, and FFA in mouse plasma, and increased the concentration of PYY, indicating that all the peptides had a fat-reducing effect.

[0210] (2) Evaluation of the hypoglycemic efficacy of polypeptides

[0211] Fasting blood glucose levels in mice were measured weekly during the experiment, and oral glucose tolerance was evaluated in week 8. The procedure was as follows: fasting blood glucose was measured at 0 min, followed by gavage administration of 2 g / kg glucose. Blood glucose levels were then measured at 15 min, 30 min, 60 min, 90 min, and 120 min, and the area under the curve (AUC) was calculated. Simultaneously, the concentrations of insulin, glucagon-like peptide-1 (GLP-1), glycated hemoglobin (GHb), and adiponectin in mouse plasma were measured using an Elasa kit (Elabscience). The insulin resistance index (HOMA-IR) was calculated using the following formula to evaluate insulin resistance in mice. Experimental results are as follows: Figure 12-17 As shown.

[0212]

[0213] Where C(Insulin): plasma insulin concentration; C(FBS): fasting glucose concentration.

[0214] Analysis of the results revealed that gavage administration of VADW, VAMP, and YQLM significantly increased plasma GLP-1 concentration, decreased fasting blood glucose in mice, increased oral glucose tolerance, and increased plasma ADPN concentration, thereby increasing insulin sensitivity. A decrease in plasma GHb concentration was also observed, indicating that gavage administration of VADW, VAMP, and YQLM peptides can promote glucose homeostasis in mice.

[0215] (3) Evaluation of the protective effects of polypeptides on the liver and kidneys

[0216] After an 8-week gavage experiment, the plasma levels of T-bil-D-II (direct bilirubin) in mice were measured; elevated levels indicated hepatocyte damage. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP II), and albumin (ALB II) were measured to evaluate the hepatoprotective effects of VADW, VAMP, and YQLM peptides. Simultaneously, plasma creatinine and urea concentrations were measured to evaluate the renal protective effects of the peptides. The specific results are shown in Table 9.

[0217] Table 9. Changes in blood biochemical parameters after peptide intervention (n=6)

[0218]

[0219] Note: There are significant differences in the meanings represented by different letters. p <0.05. T-bil-D-II: direct bilirubin; ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP: alkaline phosphatase; TP II: total protein; ALB II: albumin; Creas: creatinine; Urea: urea.

[0220] Analysis revealed that oral administration of VADW, VAMP, and YQLM significantly reduced the concentrations of ALT, AST, ALP, and TP II in plasma, indicating that these three peptides have a certain hepatoprotective effect. Simultaneously, analysis also showed that the concentrations of creatinine and urea in plasma were significantly lower in the three peptide oral administration groups than in the model group, indicating that these three peptides have a certain renal protective effect.

[0221] (4) Evaluation of the efficacy of peptides in regulating intestinal flora

[0222] After an 8-week gavage experiment, colonic contents were collected from mice, and the intestinal microbiota composition was analyzed using 16S rRNA sequencing. Specific results are as follows: Figure 18 , 19 As shown. By Figure 18The results showed that gavage administration of several peptides significantly increased the α-diversity of the colonic gut microbiota, including increasing the Chao1 and Observed_species indices and decreasing the Goods_coverage index. Simultaneously, gavage administration of hemp seed peptides effectively increased the abundance of beneficial bacteria genera including Lactobacillus, Akkermansia, and Bacteroides, while decreasing the abundance of harmful bacteria genus Prevotella. Bacteroidetes / Firmicutes proportion ( Figure 19 This indicates that oral administration of hemp seed polypeptides has a certain function in improving intestinal flora imbalance.

[0223] Example 8 Evaluation of the hypoglycemic activity of hemp seed enzymatic hydrolysis extract (cell experiment)

[0224] Experimental method: The concentration was 1×10 5 Caco2 cells were seeded at a density of 10 cells / mL in 24-well plates and cultured for 24 h. The culture medium was then replaced with medium containing 5 mg / mL of each hemp seed protease hydrolysate and cultured for another 24 h. The supernatant was then collected, and the activity of DPP IV and the concentration of GLP-1 were measured. The concentration of DPP IV was determined using the Elabscience Elasa kit, and the concentration of GLP-1 was determined using the Solarbio GLP-1 ELSA kit. The results are shown below. Figure 20 , 21 As shown.

[0225] 1000 μL of a concentration of 1×10 5 INS-1 cells at a density of 10 cells / mL were seeded in 24-well plates and cultured for 24 h. The culture medium was then replaced with medium containing 5 mg / mL of each hemp seed protein hydrolysate and cultured for another 24 h. The cells were then washed once with KRBH buffer (Krebs-Ringer Bicarbonate HEPES Buffer), and 1000 μL of KRBH buffer was added to each well, followed by incubation for 1 h. The KRBH buffer was then removed, and KRBH buffer containing 16.7 mM glucose solution was added, followed by incubation for 2 h. The supernatant was collected, and the insulin concentration was measured using a Solarbio Insulin kit. The results are shown below. Figure 22 As shown.

[0226] In the above experiments, the hemp seed proteases were the 11 proteases listed in Table 4 of Example 1. A control group was also set up with sitagliptin (…). Sitagliptin (10 μM).

[0227] A comprehensive evaluation of the cell experiments showed that each protease hydrolysis component could reduce the concentration of DPPIV to some extent and increase the concentrations of GLP-1 and Insulin, thus possessing potential hypoglycemic effects. Among them, the hydrolysis product obtained by thermophilic bacteria protease hydrolyzing hemp seed protein had the best potential hypoglycemic effect, and was more effective than sitagliptin, a commonly used DPP IV inhibitor hypoglycemic drug in the existing technology.

[0228] Experiment Example 9: Evaluation of the effects of hemp seed enzymatic hydrolysis extract on fat reduction, blood sugar reduction, liver protection, kidney protection, and intestinal flora regulation (animal experiment)

[0229] Using the hemp seed protein and enzymatic extract obtained in Example 2 as research subjects, animal experiments were conducted to evaluate their efficacy in fat reduction, blood sugar reduction, liver protection, kidney protection, and intestinal flora regulation. The experimental methods are as follows:

[0230] Six-week-old (18-22 g) male C57BL / 6J mice (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) were selected and modeled after one week of acclimatization.

[0231] The experiment was divided into 7 groups, with 10 mice in each group. The animals were distinguished by ear tagging. The groups were: normal group (WideType, WT), model group (High-fat-diet, HFD), positive drug group (Sitagliptin, PC), 3 hemp seed enzymatic hydrolysis extract sample groups (low-dose group, TPH-L; medium-dose group, TPH-M; high-dose group, TPH-H) and hemp seed protein group (Pro). The mice were administered the drugs by gavage.

[0232] During the experiment, except for the normal group, all mice were fed a high-fat diet (20 kcal of protein, 20 kcal of carbohydrates, and 60 kcal of fat) to induce the model. No other special treatments were given. All groups were administered 0.2 mL / 20 g of mouse body weight via gavage once daily. The normal group (Wide Type, WT) and the model group (High-fat-diet, HFD) were administered water (sample solvent) daily. The positive control group was administered 1.25 mg / kg of sitagliptin phosphate (purchased from Sigma) daily. The low-dose (TPH-L), medium-dose (TPH-M), and high-dose (TPH-H) groups of hemp seed enzymatic hydrolysis extract were administered 80, 160, and 320 mg / kg of hemp seed enzymatic hydrolysis extract, respectively. The hemp seed protein group (Pro) was administered 320 mg / kg of unhydrolyzed hemp seed protein via gavage. The gavage treatment lasted for 8 weeks, during which the mice's diet, water intake, and body weight were measured and recorded weekly. The experiment was terminated when the body weight and fasting blood glucose level of the model group mice were more than 20% higher than those of the normal group.

[0233] During the gavage period, the mice's diet, water intake, blood glucose, and body weight were measured and statistically analyzed weekly. The results showed that after 8 weeks of gavage, the body weight and fasting blood glucose levels of the model group mice were 20% higher than those of the normal group. Therefore, the entire animal experiment was terminated after 8 weeks of gavage.

[0234] (1) Evaluation of the fat-reducing effect of hemp seed enzymatic hydrolysis extract

[0235] Eight weeks after gavage, mice were treated and their blood was collected. Plasma was obtained after centrifugation. Total cholesterol (TC) and total triglycerides (TG) in the plasma were analyzed using an automated animal chemistry analyzer. Simultaneously, non-esterified fatty acids (FFA) and peptide YY (PYY) in the plasma were determined using an Elasa kit (Elabscience). Statistical results are shown below. Figure 23-30 As shown.

[0236] Analysis of the results revealed that all three doses of hemp seed enzymatic hydrolysate administered by gavage reduced the intake of food, water, and energy in mice, slowed the increase in mouse weight, and significantly reduced the concentrations of TC, TG, and FFA in mouse plasma while increasing the concentration of PYY, indicating that the hemp seed enzymatic hydrolysate has a fat-reducing effect.

[0237] (2) Evaluation of the hypoglycemic effect of hemp seed enzymatic hydrolysis extract

[0238] Fasting blood glucose levels in mice were measured weekly during the experiment, and oral glucose tolerance was evaluated in week 8. The procedure was as follows: fasting blood glucose was measured at 0 min, followed by gavage administration of 2 g / kg glucose. Blood glucose levels were then measured at 15 min, 30 min, 60 min, 90 min, and 120 min, and the area under the curve (AUC) was calculated. Simultaneously, the concentrations of insulin, glucagon-like peptide-1 (GLP-1), glycated hemoglobin (GHb), and adiponectin in mouse plasma were measured using an Elasa kit (Elabscience). The insulin resistance index (HOMA-IR) was calculated using the following formula to evaluate insulin resistance in mice. Experimental results are as follows: Figures 31-36 As shown.

[0239] HOMA-IR=C(Insulin)*C(FBS) / 22.5

[0240] Where C(Insulin): plasma insulin concentration; C(FBS): fasting glucose concentration.

[0241] Analysis of the results revealed that gavage administration of hemp seed enzymatic hydrolysate significantly increased the concentration of GLP-1 in plasma, reduced fasting blood glucose in mice, increased oral glucose tolerance, increased the concentration of ADPN in plasma, and thus increased insulin sensitivity in mice. It was also found that the concentration of GHb in the plasma of mice decreased, indicating that gavage administration of hemp seed enzymatic hydrolysate can promote glucose metabolism balance in mice.

[0242] (3) Evaluation of the protective effects of hepatitis B and kidneys on hemp seed enzymatic hydrolysis extract

[0243] After an 8-week gavage experiment, the levels of T-bil-D-II (direct bilirubin) in mouse plasma were measured. Elevated levels indicated hepatocellular damage. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP II), and albumin (ALB II) were also measured to evaluate the hepatoprotective effect of the hemp seed enzymatic hydrolysate. Simultaneously, plasma creatinine and urea concentrations were measured to evaluate the renal protective effect of the hemp seed enzymatic hydrolysate. The specific results are shown in Table 10.

[0244] Table 10 Changes in blood biochemical parameters after intervention with protein and protease hydrolysate extracts (n=6)

[0245]

[0246] Note: There are significant differences in the meanings represented by different letters. p <0.05. T-bil-D-II: direct bilirubin; ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP: alkaline phosphatase; TP II: total protein; ALB II: albumin; Creas: creatinine; Urea: urea.

[0247] Analysis revealed that oral administration of hemp seed enzymatic hydrolysate significantly reduced the concentrations of ALT, AST, ALP, and TP II in plasma, indicating that the hepatoprotective effect of oral administration of hemp seed enzymatic hydrolysate. Simultaneously, the concentrations of creatinine and urea in plasma were also significantly lower in the hemp seed enzymatic hydrolysate administration group than in the model group, indicating that the hemp seed enzymatic hydrolysate administration of hemp seed enzymatic hydrolysate had a certain renal protective effect.

[0248] (4) Evaluation of the efficacy of hemp seed enzymatic hydrolysis extract in regulating intestinal flora

[0249] After an 8-week gavage experiment, colonic contents were collected from mice, and the intestinal microbiota composition was analyzed using 16S rRNA sequencing. Specific results are as follows: Figure 37, 38 As shown. By Figure 37 The results showed that gavage administration of hemp seed enzymatic hydrolysate significantly increased the α-diversity of the colonic intestinal flora, including increasing the Chao1 and Observed_species indices (higher values ​​indicate higher flora richness) and decreasing the Goods_coverage index (indicating higher sample richness). Simultaneously, gavage administration of hemp seed enzymatic hydrolysate effectively increased the abundance of beneficial bacteria genera including Lactobacillus, Akkermansia, and Bacteroides, while decreasing the abundance of harmful bacteria genus Prevotella. Bacteroidetes / Firmicutes (The ratio of Bacteroides / Firmwallis, according to literature, is inversely correlated with obesity) Figure 38 This indicates that oral administration of hemp seed enzymatic hydrolysate has a certain function in improving intestinal flora imbalance.

[0250] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application, without departing from the scope of the technical solution of this application, shall still fall within the protection scope of this application.

Claims

1. A polypeptide, characterized in that, Its amino acid sequence is selected from VADW (Seq ID No. 1) or VAMP (Seq ID No. 2).

2. A nucleic acid encoding the polypeptide of claim 1.

3. A composition comprising one or two polypeptides having an amino acid sequence of VADW (Seq ID No. 1) or VAMP (Seq ID No. 2).

4. The composition according to claim 3, characterized in that, The composition also includes a pharmaceutically, food-, or health-product-acceptable carrier, excipient, or secondary active ingredient.

5. The composition according to claim 4, characterized in that, The second active ingredient is selected from one or more of the following: DPP-IV inhibitors, SGLT-2 inhibitors, GLP-1 receptor agonists, insulin secretagogues, α-glucosidase inhibitors, angiotensin-converting enzyme inhibitors, angiotensin receptor antagonists, calcium channel blockers, or β-receptor blockers.

6. Use of the polypeptide of claim 1 or the composition of any one of claims 3 to 5 in the preparation of a medicament for the prevention or treatment of hyperglycemia.

7. Use of the polypeptide of claim 1 or the composition of any one of claims 3 to 5 in the preparation of a medicament with fat-reducing function.

8. Use of the polypeptide of claim 1 or the composition of any one of claims 3 to 5 in the preparation of a medicament that improves liver damage caused by a high-fat diet.

9. Use of the polypeptide of claim 1 or the composition of any one of claims 3 to 5 in the preparation of a medicament that improves kidney damage caused by a high-fat diet.

10. Use of the polypeptide of claim 1 or the composition of any one of claims 3 to 5 in the preparation of a medicament that improves intestinal flora imbalance caused by a high-fat diet.

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