An oligopeptide and its application in preparing fat-reducing products

Abstract

The present invention relates to an oligopeptide and its use in the preparation of fat-reducing products, belonging to the field of biotechnology. The present invention provides an oligopeptide comprising at least one of the following amino acid sequences: FPLL, LPGFL, ITF, and IVF. Four oligopeptides with different amino acid sequences are synthesized through solid-phase synthesis. Experimental results confirm that all four oligopeptides exhibit fat-reducing efficacy and possess advantages such as safety, long-term suitability, and lack of side effects. These oligopeptides can be used in products for fat-reducing and related symptoms.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an oligopeptide and application thereof in the preparation of fat-reducing products. Background Art

[0002] Currently, fat-reducing products on the domestic market cannot fully meet consumer demand. There are relatively few fat-reducing drugs. Orlistat is the only approved oral fat-reducing drug in China, but it also has certain side effects, such as gastrointestinal discomfort, liver damage, and kidney damage. Although there are many common meal replacements and dietary fiber weight-loss functional foods, they often have problems such as ineffective weight loss and fat reduction. Oligopeptides are a type of peptide composed of 2 to 10 amino acids. Different amino acid arrangements have various functions. There are currently no reports on the use of oligopeptides in fat reduction, so there is an urgent need to develop oligopeptides with fat-reducing effects. Summary of the Invention

[0003] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an oligopeptide with fat-reducing effect and its use in the preparation of fat-reducing products.

[0004] To achieve the above object, the technical solution adopted by the present invention is:

[0005] In a first aspect, the present invention provides an oligopeptide comprising at least one of the following amino acid sequences: FPLL, LPGFL, ITF and IVF.

[0006] The present invention can obtain four oligopeptides with different amino acid sequences through solid phase synthesis, and experiments have confirmed that the obtained four oligopeptides all have the effect of reducing fat.

[0007] As a preferred embodiment of the oligopeptide of the present invention, the oligopeptide is mainly synthesized by solid phase synthesis.

[0008] In a second aspect, the present invention provides an oligopeptide composition comprising at least two of oligopeptide A, oligopeptide B, oligopeptide C, and oligopeptide D; the amino acid sequences of oligopeptide A, oligopeptide B, oligopeptide C, and oligopeptide D being independently selected from at least one of FPLL, LPGFL, ITF, and IVF. In addition to the ability of a single oligopeptide to reduce fat, a combination of two or more oligopeptides of the present invention also has fat-reducing effects.

[0009] In a third aspect, the present invention provides the use of the above oligopeptide in the preparation of fat-reducing products.

[0010] As a preferred embodiment of the application of the present invention, the products include medicines, foods, health products, cosmetics and biological products.

[0011] In a fourth aspect, the present invention provides a fat-reducing composition comprising the above-mentioned oligopeptide or oligopeptide composition.

[0012] As a preferred embodiment of the fat-reducing composition of the present invention, the fat-reducing composition further comprises auxiliary materials.

[0013] As a preferred embodiment of the fat-reducing composition of the present invention, the auxiliary materials include at least one of a flavoring agent, a thickener, a disintegrant, and a filler.

[0014] As a preferred embodiment of the fat-reducing composition of the present invention, the dosage form of the fat-reducing composition is at least one of capsules, pills, oral liquids, tablets, granules, powders and injections.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention can obtain four oligopeptides with different amino acid sequences through solid-phase synthesis. Experiments have confirmed that the four oligopeptides obtained all have the effect of reducing fat, and have the advantages of being safe, usable for a long time, and having no side effects. They can be applied to products for fat reduction and its related symptoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The liquid chromatograms of oligopeptides FPLL and LPGFL in Example 1 of the present invention are shown, wherein Figure A is oligopeptide FPLL and Figure B is oligopeptide LPGFL;

[0018] Figure 2 The liquid chromatograms of oligopeptides ITF and IVF in Example 1 of the present invention are shown, wherein Figure A is oligopeptide ITF and Figure B is oligopeptide IVF;

[0019] Figure 3 This is the mass spectrum of the oligopeptide FPLL in Example 1 of the present invention;

[0020] Figure 4 This is the mass spectrum of the oligopeptide LPGFL in Example 1 of the present invention;

[0021] Figure 5 This is the mass spectrum of the oligopeptide ITF in Example 1 of the present invention;

[0022] Figure 6 This is the mass spectrum of the oligopeptide IVF in Example 1 of the present invention;

[0023] Figure 7 This is the effect of different treatments on nematode fat in Effect Example 2 of the present invention. DETAILED DESCRIPTION

[0024] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0025] Unless otherwise specified, other materials and reagents used in the examples can be obtained from commercial sources.

[0026] Caenorhabditis elegans N2 was purchased from the Caenorhabditis Genetics Center at the University of Minnesota.

[0027] The techniques not described in detail in the following examples, comparative examples and effect examples are all commonly used techniques in the art. Please refer to "Molecular Biology Experiment Manual" (Ma Wenli, People's Military Medical Publishing House), "Molecular Biology Experiment (Second Edition)" (Zhejiang University Press), and "Cell Biology Experiment" (Yang Hongbing, Hou Lixia, Zhang Yuxi, Higher Education Press).

[0028] In the following examples and effect examples, "V" refers to valine (Val), "P" refers to proline (Pro), "G" refers to glycine (Gly), "H" refers to histidine (His), "R" refers to arginine (Arg), "K" refers to lysine (Lys), "I" refers to isoleucine (Ile), "F" refers to phenylalanine (Phe), "L" refers to leucine (Leu), "W" refers to tryptophan (Trp), "A" refers to alanine (Ala), "M" refers to methionine (Met), "C" refers to cysteine ​​(Cys), "N" refers to asparagine (Asn), "S" refers to serine (Ser), "Q" refers to glutamine (Gln), "Y" refers to tyrosine (Tyr), "D" refers to aspartic acid (Asp), "E" refers to glutamic acid (Glu), and "T" refers to threonine (Thr).

[0029] In the following examples and effect examples, FPLL refers to the oligopeptide with an amino acid sequence of SEQ ID NO.1, LPGFL refers to the oligopeptide with an amino acid sequence of SEQ ID NO.2, ITF refers to the oligopeptide with an amino acid sequence of SEQ ID NO.3, and IVF refers to the oligopeptide with an amino acid sequence of SEQ ID NO.4.

[0030] Example 1

[0031] This embodiment provides an oligopeptide, which is prepared by the following steps:

[0032] 1. Weigh 3 g of Rink Amide Resin (degree of substitution 0.3 mmol / g) into a 150 mL reactor, soak it in 50 mL of dichloromethane (DCM) for 2 h, wash the resin with 3 times the volume of the resin in nitrogen-dimethylformamide (DMF), and then drain it. Repeat this process four times. Drain the resin and set it aside for later use.

[0033] 2. Add a certain amount of 20% v / v piperidine (solvent is DMF) to the reactor and shake on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After the protecting group is removed, wash it four times with DMF 3 times the volume of the resin and then drain it. Take a small amount of resin and test it with the ninhydrin (nine-well ninhydrin hydrate) method (two drops each of test A and test B, react at 100°C for 1 minute). If the resin is colored, it means that the protecting group is successfully removed.

[0034] 3. Weigh an appropriate amount of the first amino acid at the C-terminus and an appropriate amount of 1-hydroxybenzotriazole (HOBT) into a 50 mL centrifuge tube, add 20 mL of DMF to dissolve them, then add 3 mL of N, N-diisopropylcarbodiimide (DIC) and shake for 1 min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30°C for 2 h. Seal the head with a certain amount of acetic anhydride (acetic anhydride: N, N-diisopropylethylamine: DCM = 1:1:2) for 0.5 h, then wash four times with 3 times the volume of the resin with DMF, and drain for use;

[0035] 4. Remove the Fmoc protecting group from the resin in the reactor according to the method of step 2;

[0036] 5. Weigh an appropriate amount of the second amino acid and an appropriate amount of HOBT into a 50 mL centrifuge tube, add 25 mL of DMF to dissolve it, then add 2.5 mL of DIC and shake for 1 min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30 ° C for 1 h. Take a small amount of resin for detection and detect it with the ninhydrin method (two drops of test A and test B, 100 ° C for 1 min). If the resin is colorless, it means that the reaction is complete; if the resin has color, it means that the condensation is incomplete and the reaction is continued; after the reaction is complete, wash the resin four times with DMF and then drain it. Add a certain amount of 20% v / v piperidine (solvent is DMF) to the reactor and shake it on a decolorization shaker for 20 min to remove the Fmoc protecting group on the resin. After deprotection, wash it four times with DMF and then drain it to detect whether the protection is removed.

[0037] 6. Connect the subsequent amino acids in sequence according to steps 1-5. After the last amino acid is connected, remove the protection, wash four times with DMF, and then drain the resin with methanol. Then use 95 cutting solution (trifluoroacetic acid: 1,2-dithiol: 3-isopropylsilane: water = 95:2:2:1) to cut the polypeptide from the resin (add 10 mL of cutting solution per gram of resin), and centrifuge four times with ice ether (cutting solution: ether = 1:9). Separate and purify by HPLC to obtain a high-purity oligopeptide.

[0038] According to the above method, oligopeptides with amino acid sequences of FPLL, LPGFL, ITF and IVF can be synthesized respectively.

[0039] Effect Example 1

[0040] The oligopeptide obtained in Example 1 was characterized by HPLC-MS / MS. The specific scheme is as follows:

[0041] The samples were separated and detected using a Waters 2695-ZQ2000 single quadrupole liquid spectrometer (Waters, USA) using an ODS-SP column (4.6×250 mm×5 μm) (Shimadzu, Japan).

[0042] The mobile phase consisted of 0.1% (v / v) trifluoroacetic acid aqueous solution (A) and 0.1% (v / v) trifluoroacetic acid in acetonitrile (B). The elution method was 10% to 70% B from 0 to 25.00 min and 100% B from 25.01 to 30.00 min. The flow rate was 1 mL / min, the injection volume was 30 μL, and the column temperature was 40°C. The percentages of the mobile phases are by volume, i.e., the volume of mobile phase B / the total volume of mobile phase A and mobile phase B.

[0043] Mass spectrometry detection method: scanning cycle 0.1S, ESI ion source temperature 100℃, positive ion mode, spray voltage 3000V, scanning range 300-1999Da. The identification results are shown in Table 1 and Figure 1-6 .

[0044] Table 1 Identification results of oligopeptides

[0045]

[0046] As shown in Table 1 and Figure 1-6 As shown, the amino acid sequences of the oligopeptides prepared in Example 1 identified by HPLC-MS / MS technology are FPLL, LPGFL, ITF and IVF, which are the same as the expected oligopeptide amino acid sequences.

[0047] Effect Example 2

[0048] The fat-reducing effect of the oligopeptides (FPLL, LPGFL, ITF, and IVF) obtained in Example 1 was evaluated. The specific scheme is as follows:

[0049] This experiment used C. elegans N2 as a model. When a large number of C. elegans N2 were in the early egg-laying stage, alkaline lysis buffer was added and the nematodes were manually shaken for several minutes to break into fragments. The eggs were then washed and centrifuged onto sterile NGM solid culture dishes and incubated in a 20°C biochemical incubator for 24 hours to obtain a large number of uniform L1-stage nematodes.

[0050] Synchronized L1-stage nematodes were adjusted to a density of approximately 1 nematode / 10 μL using SM medium and transferred to a 24-well plate (1000 μL / well). 100 μL of concentrated Escherichia coli NA22 (hereinafter referred to as NA22) was added to each well and cultured in a shaking incubator at 20°C until the D1 stage. D1-stage nematodes were collected and, except for the blank group, trehalose was added to all other groups to a final concentration of 10 mg / mL. Orlistat and the oligopeptide to be tested were also added to the positive control and oligopeptide groups, respectively. The loading concentrations are shown in Table 2. After loading, the well plate was placed on a shaker and continued to be cultured until the D4 stage. The nematodes were collected into EP tubes, 40% isopropanol was added, the supernatant was discarded, 600 μL of Oil Red O staining solution was added, and the cells were shaken at room temperature for 30 minutes. The nematodes were washed repeatedly with 0.5% v / v PBST five times; slides were made, and polarizing microscope was used to observe and photograph the images. ImageJ was used for image analysis and numerical values ​​were recorded. The optical density value of fat staining reflected the body fat content of Caenorhabditis elegans. The results are shown in Tables 2 and Figure 7 , the calculation formula is as follows:

[0051]

[0052] Table 2 Effects of different treatment groups on body fat content of nematodes

[0053]

[0054] As shown in Table 2 and Figure 7 As shown, the fat staining optical density values ​​of the orlistat positive control group and the model group were significantly different from those of the blank group, indicating that the model group was successfully established. The fat staining optical density values ​​of the oligopeptides FPLL, LPGFL, ITF, and IVF were significantly reduced compared to the model group, indicating that the oligopeptides have fat-reducing effects. In particular, the FPLL and IVF oligopeptides had very strong fat-reducing effects, with the nematode fat staining optical density values ​​of the model group reduced by 36.34% and 27.08%, respectively. These values ​​were lower than the body fat content of the blank group (no model established), indicating that the oligopeptides of the present invention have a good fat-reducing effect and can be used in products for fat-reducing and related symptoms.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An oligopeptide, characterized in that The amino acid sequence of the oligopeptide is shown in SEQ ID NO.

2.

2. An oligopeptide composition, characterized in that The oligopeptide composition comprises the oligopeptide according to claim 1 and at least one oligopeptide selected from SEQ ID NO. 1, 3 and 4.

3. Use of the oligopeptide according to claim 1 or the oligopeptide composition according to claim 2 in the preparation of fat-reducing products.

4. The use according to claim 3, characterized in that The fat-reducing products include medicines, foods and health products.

5. A fat-reducing composition, characterized in that: The method comprises the oligopeptide according to claim 1 or the oligopeptide composition according to claim 2.

6. The fat-reducing composition according to claim 5, wherein: The fat-reducing composition further comprises auxiliary materials.

7. The fat-reducing composition according to claim 6, wherein: The auxiliary materials include at least one of flavoring agents, thickeners, disintegrants, and fillers.

8. The fat-reducing composition according to claim 5, wherein: The dosage form of the fat-reducing composition is at least one of capsules, pills, oral liquid, tablets, granules, powders and injections.

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