CN114805483B

CN114805483B - Five pancreatic lipase inhibitors derived from small red bean protein and application thereof

Info

Publication number: CN114805483B
Application number: CN202210345491.8A
Authority: CN
Inventors: 沈群; 赵卿宇; 付永霞; 赵亮星
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2022-04-02
Filing date: 2022-04-02
Publication date: 2022-12-13
Anticipated expiration: 2042-04-02
Also published as: CN114805483A

Abstract

The present application provides peptides having pancrelipase inhibitor effect, the sequence of which is selected from SEQ ID No.1-5; the application also provides application of the peptide in preparing medicines, foods and health-care products for treating hyperlipemia. The peptide is a natural substance which is plant-derived, non-toxic and harmless, and experiments prove that the peptide has obvious pancreatic lipase inhibitory activity and lipid-lowering efficacy; the medicine has clear action mechanism and clear target, is expected to become a substitute medicine for preventing and treating hyperlipemia, and has good market prospect in the medicine and functional food industry.

Description

Five pancreatic lipase inhibitors derived from small red bean protein and application thereof

Technical Field

The application belongs to the field of protein, and particularly provides five pancreatic lipase inhibitors derived from small red bean protein and application thereof.

Background

Dietary fat exists primarily in the form of triglycerides, but such molecules cannot be absorbed in the gut unless hydrolyzed to smaller molecules such as monoglycerides and free fatty acids. Pancreatic lipase is a water-soluble lipolytic enzyme secreted by the pancreas, plays an important role in the digestion of triglycerides, can digest 50-70% of the dietary fat in food, and is the principal lipolytic enzyme of the human body. The lipid ingested by diet is firstly hydrolyzed by pancreatic lipase, decomposed into chylomicron, then forms micelle with cholesterol, bile salt and the like, and is finally absorbed by cells in intestinal tracts, but the excessive hydrolysis can cause the development of diseases such as hyperlipidaemia and the like.

Hyperlipidemia is mainly manifested by elevated levels of triglycerides or cholesterol in the body. In recent years, with the improvement of living standard and the change of eating habits of people, the proportion of patients with hyperlipidemia is increased rapidly. According to the guidelines for preventing and treating dyslipidemia of adults in China (revised 2016), the overall prevalence rate of dyslipidemia of adults in China is as high as 40.40%, and the hyperlipidemia is one of the diseases with high morbidity in modern society. At present, the research and development of drugs and auxiliary functional foods for treating hyperlipidemia have become a research hotspot in the pharmaceutical, food biotechnology and nutrition communities. Pancreatic lipase plays an extremely important role in maintaining the balance of lipid metabolism in the body as a key enzyme for digestion and absorption of lipids, and the activity thereof largely determines the occurrence of hyperlipidemia. The pancrelipase inhibitor can effectively inhibit the absorption of fat in diet by inhibiting the activity of pancrelipase, thereby achieving the purpose of treating hyperlipidemia. Orlistat is a pancreatic lipase inhibitor widely accepted and purchased in the market, but has side effects of insomnia, hypodynamia, nausea, vomiting, flatulence, even arrhythmia, heart failure, sudden death and the like. Therefore, there is a need to find more effective and safer pancreatic lipase inhibitors from natural products, and especially, polypeptides having lipid-lowering activity obtained from food proteins have a promising prospect as alternative drugs for preventing and treating hyperlipidemia. Previous researches find that the small red bean protein hydrolysate can obviously inhibit the activity of pancreatic lipase. Based on the method, lipid-lowering peptides in the small red bean protein hydrolysate are screened out by technologies such as ultrafiltration and mass spectrometry sequencing, and functional verification is carried out, so that technical support is provided for developing safe and effective pancreatic lipase inhibitors.

Disclosure of Invention

In one aspect, the present application provides a peptide having pancrelipase inhibitor effect, characterized in that the sequence of said peptide is selected from SEQ ID No.1 to 5.

Further, the sequence of the peptide is SEQ ID NO.4 or SEQ ID NO.5.

In another aspect, the present application provides a composition, wherein the composition comprises the above peptide and pharmaceutically, food or nutraceutical acceptable excipients.

In another aspect, the present application provides the use of the above peptide or composition for the preparation of pancreatic lipase inhibitors.

In another aspect, the present application provides the use of the above peptide or composition for the preparation of a medicament for the treatment of hyperlipidemia.

In another aspect, the application provides the use of the above peptide or composition in the preparation of a food or health product suitable for hyperlipidemic people.

In another aspect, the present application provides a method for screening the above peptide, comprising:

(1) In vitro simulated digestion: hydrolyzing small red bean protein by using an enzyme method to obtain a protein hydrolysate;

(2) Screening of lipid-lowering peptides: and (3) screening the parts with the optimal lipid-lowering activity from different ultrafiltration fractions of the small red bean protein hydrolysate by taking the pancreatic lipase activity inhibition rate as an evaluation index. Then, a peptide segment with good docking effect with pancreatic lipase is found out through mass spectrum sequencing and virtual screening technology;

(3) Analysis of inhibition effect and mechanism: the inhibition effect of the lipid-lowering peptide prepared by adopting the Fmoc solid-phase synthesis method is evaluated by taking the pancreatic lipase activity inhibition rate as an evaluation index, and the inhibition mechanism of the lipid-lowering peptide is further clarified by molecular docking. In order to degrade more properties of lipopeptides, toxicity, isoelectric point, steric hindrance, overall average hydrophilicity and human intestinal absorbability were finally evaluated based on computer software.

Further, the hydrolysis uses pepsin and pancreatin.

Further, the ultrafiltration fraction is divided into <3kDa, 3-10kDa and >10kDa fractions.

Has the advantages that:

the five lipid-lowering peptides (YLQGFGKNIL, LKQAHGGELLPN, FPFKLPAWQGHGHGHGHGHGHGHPDH, LLGGLDSSLLPH and IFNNDPNNHP) which are firstly discovered from the small red bean protein are natural substances which are plant-derived, nontoxic and harmless, and have obvious lipid-lowering efficacy. Pancreatic lipase plays an extremely important role in maintaining the balance of lipid metabolism in the body, and its activity largely determines the occurrence of hyperlipidemia. YLQGFGKNIL, LKQAKGQEFLPFLPNP, FPFKLPAWEQGHD, LLGGLDSSLLPH and IFNNDPNNHP achieve the effect of reducing blood fat by inhibiting the activity of pancreatic lipase, and the effect is also verified by in vitro enzyme activity inhibition experiments and molecular docking. Therefore, the YLQGFGKNIL, LKQAHGQEFLPNPN, FPFKLPAWEQGHD, LLGGLDSSLLPH and IFNNDPNNHP as the lipid-lowering active ingredients have good lipid-lowering effect, are nontoxic, have clear action mechanism and definite target spots, are expected to become alternative medicines for preventing and treating the hyperlipidaemia, and have good market prospects in the industries of medicines and functional foods.

Drawings

FIG. 1 shows the results of the inhibition of pancreatic lipase activity by different ultrafiltration fractions;

FIG. 2 shows the results of screening peptide fragments for inhibition of pancreatic lipase activity;

FIG. 3 is an overview and details of the optimal positions of screened peptide fragments after docking with pancreatic lipase at the active site (A: YLQGFGKNIL; B: LKQAKGQEFFLPNP; C: FPFKLPAWEQGHD; D: LLGGLDSSLLPH; E: IFNNDPNNHP); wherein pancreatic lipase residues are represented by the yellow bar model. The blue, gray, yellow and green dotted lines represent hydrogen bonds, hydrophobic interactions, salt bridges and pi-pi stacking, respectively.

Detailed Description

Example 1 extraction of Small Red Bean protein

The small red bean protein is extracted by adopting an alkali-dissolving and acid-precipitating method. Specifically, defatted red soybean powder and distilled water were mixed at a feed-to-liquid ratio of 1 (w/v). After shaking the water bath at 40 ℃ for 1h, the supernatant was collected and after adjusting the pH of the supernatant to 4.5, the supernatant was allowed to stand at room temperature for 1h to precipitate the protein. The precipitate was collected, washed 3 times with distilled water, and the pH of the protein was adjusted to 7.0. Finally, it was freeze-dried and stored at-20 ℃.

Example 2 in vitro simulated digestion

The small red bean protein is hydrolyzed by a double enzyme (pepsin and pancreatin) method to obtain a protein hydrolysate. Specifically, firstly, adding 4% (w/w) pepsin into a 5% small red bean protein solution, carrying out enzymolysis at the pH of 2.0 and the temperature of 37 ℃ for 2 hours; subsequently, the pH value of the enzymolysis liquid is adjusted to 5.3, the pH value is maintained at 7.5, 4 percent of pancreatin (w/w) is added, the enzymolysis temperature is 37 ℃, and the enzymolysis time is2 hours. After enzymolysis, inactivating enzyme in boiling water bath for 10min, centrifuging enzymolysis liquid, and collecting supernatant.

Example 3 screening for lipopeptides

The supernatant collected in step (2) was subjected to ultrafiltration by using 10kDa and 3kDa centrifugal filters to obtain different fractions (> 10kDa, 3-10kDa and <3 kDa). The effect of each fraction on pancreatic lipase activity was observed at a concentration of 4mg/mL using the pancreatic lipase activity inhibition rate as an evaluation index, and the results showed that the <3kDa fraction had the best inhibitory effect on pancreatic lipase activity (see FIG. 1). The specific process of the pancreatic lipase activity inhibition experiment is as follows:

in a 96-well plate, 50. Mu.L of the sample, 40. Mu.L of a 2.5mg/mL pancreatic lipase solution and 50. Mu.L of 10mM p-nitrophenyl butyrate were incubated at 37 ℃ for 30min in a phosphate buffer at pH 7.3. The microplate reader recorded the absorbance at 405 nm. Orlistat was used as a positive control and calculated according to equation (1).

In formula (1): a: absorbance of the control; b: absorbance of control blank; c: absorbance of the sample; d: absorbance of sample blank.

Performing mass spectrometry on the <3kDa fraction by adopting liquid chromatography-tandem mass spectrometry, virtually screening the obtained peptide sequence by using Dock 6.9, and screening according to the butt-joint score of the peptide segments (< -110 kcal/mol) to obtain peptide segments YLQGFGKNIL, LKQAHFLPNP, FPFKLPAWQGHGH, LLGGLDSSLLPH and IFNNDPNNHP (shown in table 1) with good butt-joint effect with pancreatic lipase, wherein the peptide segments with good butt-joint effect with the pancreatic lipase are virtually screened

Example 4 inhibitory Effect and mechanism analysis

The Fmoc solid phase synthesis method is adopted to prepare the lipid-lowering peptides, and the purity of each peptide is determined to be more than 95% through high performance liquid chromatography and mass spectrometry. Pancreatic lipase inhibition experiments found that at a concentration of 4mg/mL, the pancreatic lipase inhibition rates of YLQGFGKNIL, LKQAHGGEFLPN, FPFKLPAWEQGHD, LLGGLDSSLLPH, and IFNNDPNNHP were 40.33%, 46.01%, 46.49%, 60.26%, and 62.60%, respectively (see FIG. 2).

The crystal structure of pancreatic lipase (PDB number: 1 ETH) is obtained from RCSB Protein Data Bank database (http:// www. RCSB. Org /), YLQGFGKNIL, LKQHGQEFFLPNP, FPFKLPAWEQGHD, LLGGLDSSLLPH, IFNNDPNNHP and pancreatic lipase are respectively subjected to semi-flexible docking by adopting DOCK 6.9, and the key amino acid residues and interaction force of the pancreatic lipase and the pancreatic lipase are determined (see figure 3). As can be seen from fig. 3, the ylqgfgknll peptide can interact with 9 amino acid residues, and the interaction includes hydrogen bonding and hydrophobic interaction. The YLQGFGKNIL peptide is capable of forming hydrophobic interactions with substrate binding residues (Phe 78, ile79, trp253, phe 216) (see FIG. 3A). The lkqahgqefpnp peptide can interact with 7 amino acid residues, and the interaction includes only hydrophobic interactions (see fig. 3B). The fpfklpawefghd peptide can interact with 8 amino acid residues, and interactions include hydrophobic interactions and salt bridges. The FPFKLPAWEQGHD peptide is able to form hydrophobic interactions with substrate binding residues (Phe 216, ile79, trp 253) (see fig. 3C). The LLGGLDSSLLPH peptide can interact with 11 amino acid residues, and interactions include hydrogen bonding, hydrophobic interactions, and salt bridges. The LLGGLDSSLLPH peptide is capable of forming hydrogen and salt bridges with catalytic residues (Ser 153 and His 264) and hydrophobic interactions and salt bridges with substrate binding residues (Phe 78, phe216, his 152) (see figure 3D). IFNNDPNNHP peptides can interact with 9 amino acid residues and interactions include hydrogen bonding, hydrophobic interactions, salt bridges and pi-pi stacking. IFNNDPNNHP peptides were able to form hydrogen and salt bridges with catalytic residues (Ser 153 and His 264) and hydrophobic interactions, salt bridges and pi-pi stacking with substrate binding residues (Phe 216, arg257, his 152) (see figure 3E). In summary, lipid lowering peptides inhibit pancreatic lipase activity primarily by occupying catalytic or substrate binding sites.

Computer software was used to predict the properties of YLQGFGKNIL, LKQAHGQEFFLPNP, FPFKLPAWEQGHD, LLGGLDSSLLPH, IFNNDPNNHP, where toxicity and steric hindrance were assessed by ToxinPred (https:// webbs. Iiit. Edu. In/raghava/ToxinPred/index. Html), total average hydrophilicity by ExPasy (https:// web. Expass. Org/protparam /), human intestinal absorption by admAR (http:// lmd. Environment. Edu. Cn/admeastar 1/home), and isoelectric point by Pepdraw (http:// w. Tune. Edge. Edu. Biochem/WW/Petrad /). As shown in table 2, all five lipid lowering peptides were non-toxic. YLQGFGKNIL and LKQAHGGEFLPN have isoelectric points greater than 7, indicating that it is basic, while FPFKLPAWEQGHD, LLGGLDSSLLPH and IFNNDPNNHP are acidic. The overall average hydrophilicity can be used to characterize the hydrophilicity and hydrophobicity of the protein, where a more positive value indicates more hydrophobic and a more negative value indicates more hydrophilic, so LKQAKQEFLPFLPNP, FPFKLPAWAWQGHGHGH and IFNNDPNNHP have more hydrophilic, and YLQGFGKNIL and LLGGLDSSLLPH have more hydrophobic. The steric hindrance values of the 5 lipopeptides are between 0.52 and 0.66, with LLGGLDSSLLPH having the lowest steric hindrance value, which is beneficial for enhancing pancreatic lipase inhibition.

TABLE 2 prediction of properties of the screened peptide fragments

SEQUENCE LISTING

<110> university of agriculture in China

<120> five pancreatic lipase inhibitors derived from small red bean protein and application thereof

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 10

<212> PRT

<213> artificial

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Tyr Leu Gln Gly Phe Gly Lys Asn Ile Leu

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Leu Lys Gln Ala His Gly Gln Glu Phe Leu Pro Asn Pro

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<212> PRT

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Phe Pro Phe Lys Leu Pro Ala Trp Glu Gln Gly His Asp

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Leu Leu Gly Gly Leu Asp Ser Ser Leu Leu Pro His

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Ile Phe Asn Asn Asp Pro Asn Asn His Pro

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Claims

1. A peptide having a pancreatic lipase inhibitor effect, characterized in that the sequence of said peptide is SEQ ID No.4 or SEQ ID No.5.

2. A composition comprising the peptide of claim 1 and a pharmaceutically acceptable excipient.

3. Use of a peptide according to claim 1 or a composition according to claim 2 for the preparation of a pancrelipase inhibitor.

4. Use of the peptide according to claim 1 or the composition according to claim 2 for the preparation of a medicament for the treatment of hyperlipidemia.