Polypeptide lllle derived from sturgeon cartilage and application thereof

Abstract

The present application relates to a polypeptide LLLE derived from sturgeon cartilage and its application, in particular to a polypeptide LLLE derived from sturgeon cartilage and its application in improving obesity caused by food pesticide residues. The sequence of the polypeptide LLLE is: Leu-Leu-Leu-Glu; based on it, various pharmaceutically acceptable dosage forms can be prepared, including but not limited to injection, tablet, capsule, pill, granule, syrup and other oral liquid, suppository; it can effectively alleviate fat accumulation and blood lipid abnormality caused by chlorpyrifos chronic exposure and high-fat diet, that is, control obesity caused by chlorpyrifos chronic exposure, significantly reduce the content of serum total glyceride, total cholesterol and low-density lipoprotein cholesterol, improve the content of serum high-density lipoprotein cholesterol and regulate blood lipid. Moreover, the food-derived oligopeptide is stable and easy to absorb, safe and environmentally friendly, and has no toxic and harmful effects on human body.

Description

TECHNICAL FIELD

[0001] The present application relates to a polypeptide LLLE derived from sturgeon cartilage and its application, in particular to a polypeptide LLLE derived from sturgeon cartilage and its application in improving obesity caused by food pesticide residues. BACKGROUND

[0002] Obesity has developed rapidly in the past few decades and has become a worldwide concern. It is known that lifestyle and genetic factors are the key to inducing obesity. Pesticides are one of the main environmental pollutants, and long-term exposure can induce human chronic diseases such as obesity and diabetes, which has attracted widespread attention. After the new rules limiting the use of organophosphorus, chlorpyrifos and diazinon pesticides were introduced, pyrethroid pesticides gradually replaced organophosphorus to control pests and were listed as the fourth type of pesticides by the World Health Organization. Pyrethroid pesticides are divided into type I and type II pyrethroids, and chlorpyrifos is a type I pyrethroid, which accounts for about 60% of the market share of pyrethroid pesticides. Studies have shown that long-term exposure to trace amounts of pyrethroid pesticides can disrupt the glucose and lipid metabolism of animals, promote the production of triglycerides by adipocytes, and long-term exposure to low doses of chlorpyrifos can exacerbate obesity and insulin resistance induced by high-fat diet in mice. Under the induction of high-fat diet, long-term exposure to low doses of chlorpyrifos can increase the content of triglycerides (TG) and total cholesterol (TC) in the serum and liver of mice, exacerbate fat accumulation in the body and cause obesity.

[0003] Many research reports have shown that polypeptides from food proteins have ideal biological activities, such as lipid-lowering, antioxidant, anti-hypertensive and anti-inflammatory activities. Fish is a good source of bioactive peptides, and in recent years, domestic and foreign research on sturgeon polypeptides has mainly focused on the preparation, process optimization and biological activity of sturgeon cartilage peptides and sturgeon skin collagen peptides. Studies have shown that fish collagen peptides can inhibit lipid accumulation and regulate lipid metabolism to achieve weight loss, but these studies are induced by high-fat diet and do not take into account the influence of food pesticide residues on modern people in addition to high-fat diet, and there are relatively few reports on this aspect. Therefore, it is of good application prospect and significance in the food and medical fields to research and develop safe and effective bioactive peptide products to alleviate or eliminate the chronic toxicity caused by pesticide residue exposure. SUMMARY

[0004] In view of the deficiencies of the prior art, the present application aims to solve one of the problems; the present application provides a polypeptide LLLE derived from sturgeon cartilage and its application in improving obesity caused by the chronic toxicity of food pesticide residues.

[0005] A polypeptide LLLE derived from sturgeon cartilage, the sequence of which is: Leu-Leu-Leu-Glu.

[0006] Use of a polypeptide LLLE derived from sturgeon cartilage in the preparation of a medicament for preventing or treating obesity caused by pesticide residues. The pesticide residue is a chlorpyrifos.

[0007] A polypeptide preparation based on the polypeptide LLLE;

[0008] The polypeptide preparation can be prepared in various dosage forms acceptable in pharmacy, including but not limited to injections, tablets, capsules, pills, granules, syrups and other oral liquids, suppositories.

[0009] Further, the LLLE powder of the present application is low in solubility, and is added to a 5% sodium carboxymethyl cellulose solution to prepare a suspension (within an effective amount range), which is administered orally to mice in different treatment groups daily, and evaluated based on physiological indicators of the mice. Here, the "effective amount" refers to the amount of an effective component that can improve obesity, which can generally be determined by experiments by those skilled in the art. Generally, when the effective amount is based on the weight of the composition as a whole, the content is within the range of 0.001% to 99.990% by weight.

[0010] Further, the effect of improving obesity can be evaluated by measuring the weight gain, organ weight, and fat weight (accumulation amount) of the test mice, and the serum total triglyceride, total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol contents.

[0011] The inventors of the present application found that, after a series of experiments, LLLE can reduce further weight gain caused by a high-fat diet under chlorpyrifos pesticide exposure without affecting energy intake.

[0012] At the same time, LLLE alleviates abnormal weight gain and abnormal fat accumulation in mice on a high-fat diet induced by chlorpyrifos, as well as the increase in serum total triglyceride, total cholesterol, and low-density lipoprotein cholesterol contents and the decrease in high-density lipoprotein cholesterol.

[0013] Therefore, based on these experimental results, it can be considered that the LLLE of the present application has an effect of improving obesity caused by a high-fat diet under pesticide exposure.

[0014] At the same time, based on these experimental results, the contents of the present application are completed.

[0015] The series of detailed processing methods will be shown in the later embodiments.

[0016] The "improvement" described in the specification includes prevention, improvement, treatment of obesity caused by fat accumulation caused by chlorpyrifos, prevention of obesity-related syndromes, and delay of the manifestation of these symptoms.

[0017] It should be noted that the administration dose of the medicine is not particularly limited, and in practical application, it can be flexibly selected according to the physical condition of the administration subject. According to a specific embodiment of the present application, the preparation is a polypeptide suspension obtained by dissolving granules, and the administration dose is 10 mg / kgBW / day;

[0018] According to this dose, animal experiments (mice) were carried out, and the intragastric volume was 60-90 μL. The body weight of the intervened mice was significantly reduced, the fat content was significantly reduced, and the serum indicators were significantly improved.

[0019] The present application has the following beneficial effects:

[0020] The polypeptide segment LLLE according to the present application can effectively alleviate the fat accumulation and blood lipid abnormality caused by chronic exposure to chlorpyrifos and high-fat diet, that is, control obesity caused by chronic exposure to chlorpyrifos, has the effects of significantly reducing the content of serum total triglyceride, total cholesterol and low-density lipoprotein cholesterol, improving the content of serum high-density lipoprotein cholesterol, and regulating blood lipid. Moreover, the food-derived oligopeptide is stable and easy to absorb, safe and environmentally friendly, and has no toxic and harmful effects on the human body.

[0021] Other terms not specifically defined in the specification have the meanings given in a Chinese dictionary or are understood by those of ordinary skill in the art. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The food intake of each group of experimental mice in Example 2 after intervention is shown in the figure, the abscissa represents the group, and the ordinate represents the average food intake per mouse per day; ns represents no significant difference.

[0023] Figure 2 The body weight change and fat content of each group of experimental mice in Example 2 after intervention are shown in the figure; Figure A shows the weekly body weight change of each group of mice, the abscissa represents the number of weeks, and the ordinate represents the average body weight; Figure B shows the final body weight (body weight at the 14th week) of each group of mice, the abscissa represents the grouping, and the ordinate represents the average body weight; Figure C shows the epididymal white adipose tissue content of different groups of mice, the abscissa represents the grouping, and the ordinate represents the content; Figure D shows the ratio of epididymal white adipose tissue to body weight of different groups of mice, the abscissa represents the grouping, and the ordinate represents the ratio; * represents P<0.05.

[0024] Figure 3 The serum index content of each group of experimental mice in Example 3 after intervention is shown in the figure; Figure A shows the serum total triglyceride content of each group of mice; Figure B shows the serum total cholesterol content of each group of mice; Figure C shows the serum low-density lipoprotein cholesterol content of each group of mice; and Figure D shows the serum high-density lipoprotein cholesterol content of each group of mice. The abscissa of each figure represents the grouping, and the ordinate represents the content. * represents P<0.05, and ** represents P<0.01.

[0025] Note, attached Figures 1-3 In this context, CC represents the control group; NC represents the high-fat diet group exposed to permethrin; and ND represents the high-fat diet group exposed to permethrin supplemented with LLLE. Detailed Implementation

[0026] The following description of embodiments is for illustrative purposes only and is not intended to limit the scope of the invention. Furthermore, the provided embodiments enable the invention to be displayed more clearly and completely, and allow those skilled in the art to fully understand the invention.

[0027] The synthesis of the polypeptide LLLE was provided by Shanghai Chutai Biotechnology Co., Ltd.; the permethrin pesticide was provided by Shanghai Bailingwei Technology Co., Ltd.

[0028] Meanwhile, the evaluation of the improvement effect on obesity caused by chronic toxicity of pesticide residues was conducted in a live organism, specifically in high-fat diet (HFD) mice exposed to permethrin pesticide. All mice were 3-4 week old male C57 / BL6J mice purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (Nanjing, China).

[0029] Example 1: Extraction and synthesis of polypeptide LLLE

[0030] Preparation of sturgeon cartilage peptides

[0031] (1) Pre-processing:

[0032] The meat, fat, and connective tissue on the sturgeon cartilage are removed, and then the spinal cord inside the cartilage is removed by heating and boiling to obtain the cleaned cartilage. The cartilage is then chopped at 4°C using a chopper to obtain cartilage particles.

[0033] (2) Enzymatic hydrolysis process:

[0034] Hot-Pressure Extraction (Liquefaction): Cartilage particles were mixed with distilled water at a ratio of 1:2.5 (w / v). The cartilage was immersed in the distilled water to obtain a mixture. After hot-pressing (120℃, 90 min), the mixture was homogenized at 1500 r / min for 30 s using a homogenizer. This homogenization was repeated 3 times to obtain the hot-pressed extract (denoted as HPE).

[0035] Double-enzyme stepwise enzymatic hydrolysis: HPE was taken and the substrate concentration was adjusted with distilled water to make the protein content 5% (w / w) to obtain HPE mixture; then enzymatic hydrolysis was carried out under the following conditions: 37℃ / 60℃, pH 7.0; enzymatic hydrolysis time: 2h:2h; enzyme amount 0.65% (trypsin / HPE mixture, w(g) / v(ml)): 0.5% (papain / HPE mixture, w(g) / v(ml)). After the end of enzymatic hydrolysis, the enzyme was inactivated by heating at 90℃ for 15 min; after inactivation, the upper clear liquid was quickly cooled to room temperature, and then centrifuged at 10000 rpm for 30 min to remove oil and insoluble substances; the upper clear liquid was filtered with a 0.2 μm membrane to obtain the filtrate, which was freeze-dried for 48 h to obtain sturgeon cartilage hydrolysate (denoted as SCH).

[0036] Alcohol extraction: SCH was mixed with distilled water, wherein SCH:distilled water = 1:20 (w(g) / v(ml)), and anhydrous ethanol was added to the mixed solution to a final ethanol concentration of 85%, and the mixture was stirred with a magnetic stirrer during the process; after mixing, the mixture was placed at 4℃ for 24 h. Then the mixture was centrifuged at 4℃ and 10000 rpm for 30 min, and the supernatant was filtered and subjected to low-pressure rotary evaporation (45℃, 60 r / min) to remove ethanol; the rotary evaporated liquid was freeze-dried for 48 h to obtain sturgeon cartilage alcohol-soluble hydrolysate (denoted as ESCH).

[0037] (3) Gel filtration chromatography column:

[0038] According to the molecular weight distribution of the obtained ESCH, gel filtration chromatography column G-15 (1.2×60 cm, GE Healthcare) was used to separate the sturgeon cartilage alcohol-soluble hydrolysate, deionized water was used for elution, and the ultraviolet detection wavelength was 280 nm, and three components were mainly obtained, which were named F1, F2 and F3. Among them, F3 had the highest NO inhibition rate and showed good antioxidant activity.

[0039] (4) SP-120-50-ODS column chromatography:

[0040] SP-120-50-ODS column chromatography (Φ1.2×50 cm; Daiso gel) was used to further purify and separate the F3 component with anti-inflammatory activity. Linear gradient elution was carried out using a gradient mixer, and monitoring was carried out at a wavelength of 280 nm; after ODS hydrophobic chromatography of F3, four components were separated, which were named ODS-1, ODS-2, ODS-3 and ODS-4, and ODS-3 had the highest inhibition rate.

[0041] (5) Peptide sequence identification:

[0042] The ODS-3 was desalted and analyzed by liquid chromatography-mass spectrometry (LC-MS / MS) to screen five peptide sequences (amino acids) that matched the protein database, including LTGP (Leu-Thr-Gly-Pro), LLLE (Leu-Leu-Leu-Glu), LLEL (Leu-Leu-Glu-Leu), VGPAGPAGP (Val-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro), and FSGLDGAKGD (Phe-Ser-Gly-Leu-Asp-Gly-Ala-Lys-Gly-Asp).

[0043] Synthesis of polypeptide LLLE

[0044] The identified polypeptide sequence LLLE was synthesized and used in animal experiment research to analyze its improvement in obesity caused by chronic toxicity of food and pesticide residues. The polypeptide was synthesized by Shanghai Chupai Biotechnology Co., Ltd. with a purity of more than 98%.

[0045] The following is the identification of LLLE:

[0046] Certificate of Analysis

[0047]

[0048] From the above information, it can be seen that the purity of LLLE reaches more than 98%, and the molecular weight of LLLE is 486.6.

[0049] Example 2: Animal experiment with test mice for tissue experiment analysis

[0050] The experiment was conducted in a SPF (specific pathogen free) barrier environment with a temperature of (25±3) °C, a relative humidity of (50±10) %, and a 12h light / dark cycle. After acclimation for 1 week, 30 mice that met the above conditions were randomly divided into 3 groups (n=10), all of which were fed with 45% high-fat feed. Permethrin and polypeptide solution were administered according to different groups in the morning and evening, respectively. Permethrin was dissolved in corn oil, and the gavage dose was 0.5ug / g BW / day. The polypeptide solution was prepared weekly and suspended in CMC-Na (carboxymethyl cellulose sodium) solution, with a gavage volume of 60-90μL per mouse and a gavage dose of 10mg / kg BW / day.

[0051] The total duration of the experiment was 14 weeks, and the specific group names and treatment methods are shown below:

[0052] CC: control group, gavage with corn oil and CMC-Na (carboxymethyl cellulose sodium) solution, fed with 45% high-fat feed.

[0053] NC: experimental group, gavage with chlorpyrifos and CMC-Na (carboxymethyl cellulose sodium) solution, fed with 45% high-fat feed.

[0054] ND: polypeptide group, gavage with chlorpyrifos and polypeptide solution, fed with 45% high-fat feed.

[0055] HFD (45% fat, XTHF45 purchased from Jiangsu Cooperation Bioengineering Co., Ltd., 45% refers to the proportion of energy), the specific formula includes: 23.7% protein, 40.9% carbohydrate, 23.6% fat, 5.8% cellulose, 5.8% mineral, 0.1% vitamin.

[0056] During the entire experiment, the mice were given sufficient food and water, and the body weight and food intake of the test mice were recorded every week. At the end of the treatment, the mice were fasted for 12 h and sacrificed by CO2 asphyxiation. Blood was drawn from the heart of the test mice and serum was separated by centrifugation at 3000g at 4°C for 20 min. The epididymal white adipose tissue of the mice was weighed and quickly frozen in liquid nitrogen and stored at -80°C for use.

[0057] It was found that after 8 weeks of administration to the test mice, the body weight of the NC group under chlorpyrifos pesticide exposure was significantly higher than that of the control group CC, while the body weight of the pesticide-exposed mice in the ND group supplemented with the sample decreased and approached the level of the control group CC Figure 2 A, B), and the weight of epididymal fat (E-WAT) and the ratio of E-WAT / BW were also significantly higher in the NC group than in the CC group, and the ND group was significantly lower than the NC group, approaching the level of the control group Figure 2 C, D).

[0058] At the same time, during the 14-week treatment, there was no significant change in the average daily food intake per mouse among the groups Figure 1 , which indicates that neither chlorpyrifos treatment nor LLLE intervention changed the energy intake of the test mice.

[0059] In summary, these results all indicate that LLLE reduced the body weight gain and fat accumulation of high-fat diet mice under pesticide exposure without affecting energy intake.

[0060] Example 3: Evaluation of LLLE supplementation on blood lipid levels of test mice

[0061] According to the experimental protocol and treatment method described in Example 2, the levels of total triglycerides (TG), total cholesterol (T-CHO), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in the collected serum were detected using the kits. Specific detection methods are detailed in the kit instructions. The TG, T-CHO, HDL-C, and LDL-C kits were all purchased from Nanjing Jiancheng Biological Research Institute.

[0062] The experimental results are shown in Figure 3 Compared with the control group, permethrin exposure significantly increased the levels of total triglycerides (TG), total cholesterol (T-CHO), and low-density lipoprotein cholesterol (LDL-C) in mouse serum. Figure 3 AC), supplemental intervention for LLLE significantly reduced these three indicators, bringing them close to the control group levels. Figure 3 AC); while exposure to permethrin significantly reduced the level of high-density lipoprotein cholesterol (HDL-C). Figure 3 D), supplemental intervention with LLLE reversed this trend, and HDL-C levels increased. Figure 3 D); This indicates that the polypeptide LLLE does indeed have the effect of improving dyslipidemia in permethrin-induced hyperlipidemic mice.

[0063] Based on the above description, the main component of this invention, LLLE, is a polypeptide segment derived from sturgeon cartilage. Without affecting energy intake and organ weight changes, it reduces weight gain and adipose tissue mass increase caused by a high-fat diet with permethrin exposure, while inhibiting the increase of serum TG, TC and LDL-C levels and improving the trend of HDL-C reduction.

[0064] The subjects described above were all eligible mice. This invention can also be used in human clinical or health experiments and has certain reference value.

[0065] In addition, as mentioned above, the scope of protection of this invention also includes the utilization form of the composition (such as beverages or dry powder), and this invention can be widely applied in the food and pharmaceutical fields.

[0066] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. Use of a polypeptide LLLE derived from sturgeon cartilage in the preparation of a medicament for preventing or treating obesity caused by pesticide residues, characterized in that, The pesticide residue is permethrin; the polypeptide LLLE sequence derived from sturgeon cartilage is Leu-Leu-Leu-Glu. ​

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