Oligopeptide and application thereof in preparation of fat-reducing product

Abstract

The invention relates to oligopeptide and application thereof in preparation of fat-reducing products, and belongs to the technical field of biology. The invention provides an oligopeptide. The oligopeptide comprises at least one of the following amino acid sequences: FPLL, LPGFL, ITF and IVF. Four oligopeptides with different amino acid sequences can be obtained through solid-phase synthesis, and experiments prove that the obtained four oligopeptides all have the effect of reducing fat, have the advantages of being safe, capable of being used for a long time, free of side effects and the like, and can be applied to products for reducing fat 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

[0003] At present, fat-reducing products in the domestic market cannot meet the needs of consumers very well. There are relatively few fat-reducing drugs, and Orlistat is the only oral fat-reducing drug approved 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 insignificant weight-loss and fat-reducing effects. Oligopeptides are a type of peptide composed of 2 to 10 amino acids, which have various functions through different amino acid arrangements. There are currently no reports on the use of oligopeptides in fat reduction, so there is an urgent need to develop an oligopeptide with fat-reducing effects. Summary of the invention

[0004] 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 application in the preparation of fat-reducing products.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] 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.

[0007] 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.

[0008] As a preferred embodiment of the oligopeptide of the present invention, the oligopeptide is mainly synthesized by solid phase synthesis.

[0009] In a second aspect, the present invention provides an oligopeptide composition, the 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 are independently selected from at least one of FPLL, LPGFL, ITF and IVF. In addition to the fact that a single oligopeptide of the present invention can have the effect of reducing fat, a combination of two or more oligopeptides also has the effect of reducing fat.

[0010] In a third aspect, the present invention provides the use of the above oligopeptide in the preparation of fat-reducing products.

[0011] As a preferred embodiment of the application of the present invention, the products include medicines, foods, health products, cosmetics and biological products.

[0012] In a fourth aspect, the present invention provides a fat-reducing composition, comprising the above-mentioned oligopeptide or oligopeptide composition.

[0013] As a preferred embodiment of the fat-reducing composition of the present invention, the fat-reducing composition further comprises auxiliary materials.

[0014] As a preferred embodiment of the fat-reducing composition of the present invention, the auxiliary material includes at least one of a flavoring agent, a thickener, a disintegrant, and a filler.

[0015] 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.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention can obtain four oligopeptides with different amino acid sequences through solid phase synthesis. Experiments have confirmed that the obtained four oligopeptides 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

[0018] Figure 1 The liquid chromatograms of oligopeptides FPLL and LPGFL in Example 1 of the present invention, wherein Figure A is oligopeptide FPLL and Figure B is oligopeptide LPGFL;

[0019] Figure 2 The liquid chromatograms of oligopeptides ITF and IVF in Example 1 of the present invention, wherein Figure A is oligopeptide ITF, and Figure B is oligopeptide IVF;

[0020] Figure 3 The mass spectrum of the oligopeptide FPLL in Example 1 of the present invention;

[0021] Figure 4 This is the mass spectrum of the oligopeptide LPGFL in Example 1 of the present invention;

[0022] Figure 5 The mass spectrum of the oligopeptide ITF in Example 1 of the present invention;

[0023] Figure 6 The mass spectrum of oligopeptide IVF in Example 1 of the present invention;

[0024] Figure 7 This is the effect of different treatments on nematode fat in Effect Example 2 of the present invention. DETAILED DESCRIPTION

[0025] 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.

[0026] Unless otherwise specified, other materials, reagents, etc. used in the examples can be obtained from commercial sources.

[0027] Caenorhabditis elegans N2 was purchased from the Caenorhabditis Genetics Center at the University of Minnesota.

[0028] The techniques not described in detail in the following embodiments, 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).

[0029] In the following embodiments 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).

[0030] In the following embodiments and effect examples, FPLL refers to the oligopeptide with the amino acid sequence of SEQ ID NO.1, LPGFL refers to the oligopeptide with the amino acid sequence of SEQ ID NO.2, ITF refers to the oligopeptide with the amino acid sequence of SEQ ID NO.3, and IVF refers to the oligopeptide with the amino acid sequence of SEQ ID NO.4.

[0031] Example 1

[0032] This embodiment provides an oligopeptide, which is prepared by the following steps:

[0033] 1. Weigh 3 g of Rink Amide Resin (substitution degree 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 four times, and drain the resin for later use;

[0034] 2. Add a certain amount of 20% v / v piperidine (solvent is DMF) to the reactor, shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After removing the protecting group, 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 hydrated ninhydrin) method (two drops of test A and test B, react at 100°C for 1 minute). The resin has color, indicating that the deprotection group is successful;

[0035] 3. Weigh an appropriate amount of the first amino acid at the C-terminus and an appropriate amount of 1-hydroxy-benzotriazole (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, then place the reactor in a shaker at 30 ° C for 2 h, cap it with a certain amount of acetic anhydride (acetic anhydride: N, N-diisopropylethylamine: DCM = 1:1:2) for 0.5 h, then wash it four times with 3 times the volume of the resin in DMF, and drain it for use;

[0036] 4. Remove the Fmoc protecting group on the resin in the reactor according to the method of step 2;

[0037] 5. Weigh an appropriate amount of the second amino acid and an appropriate amount of HOBT into a 50mL centrifuge tube, add 25mL of DMF to dissolve it, then add 2.5mL of DIC and shake for 1min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30°C for 1h. Take a small amount of resin for detection and use the ninhydrin method to detect (two drops of test A and test B, 100°C for 1min). 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, 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 20min to remove the Fmoc protecting group on the resin. After deprotection, wash it four times with DMF, then drain it to detect whether the protection is removed;

[0038] 6. Connect the following amino acids in sequence according to steps 1-5. After the last amino acid is connected, remove the protection, wash four times with DMF, then drain the resin with methanol, and then cut the polypeptide from the resin with 95 cutting solution (trifluoroacetic acid: 1,2-dithiol: 3-isopropylsilane: water = 95:2:2:1) (add 10 mL of cutting solution per gram of resin), and centrifuge and precipitate four times with ice ether (cutting solution: ether = 1:9), and separate and purify by HPLC to obtain high-purity oligopeptides.

[0039] According to the above method, oligopeptides with amino acid sequences of FPLL, LPGFL, ITF and IVF can be synthesized respectively.

[0040] Effect Example 1

[0041] The oligopeptide obtained in Example 1 was characterized by HPLC-MS / MS, and the specific scheme is as follows:

[0042] A Waters 2695-ZQ2000 single quadrupole liquid spectrometer (Waters, USA) was used to separate and detect the samples, and an ODS-SP column (4.6×250 mm×5 μm) (Shimadzu, Japan) was used.

[0043] The mobile phase consisted of 0.1% (v / v) trifluoroacetic acid aqueous solution (A) and 0.1% (v / v) trifluoroacetic acid acetonitrile (B), the elution method was 0-25.00min 10%-70% B, 25.01-30.00min 100% B, flow rate 1mL / min, injection volume 30μL, column temperature: 40°C, the percentage of the above mobile phase means volume percentage, volume of mobile phase B / total volume of mobile phase A and mobile phase B.

[0044] 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 .

[0045] Table 1 Identification results of oligopeptides

[0046]

[0047] 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.

[0048] Effect Example 2

[0049] The fat-reducing effect of the oligopeptides (FPLL, LPGFL, ITF and IVF) obtained in Example 1 was evaluated, and the specific scheme was as follows:

[0050] This effect example uses Caenorhabditis elegans N2 as a model for experiment. When a large number of Caenorhabditis elegans N2 are in the early egg-laying stage, add alkaline lysis solution and shake manually for several minutes to break the nematodes into fragments. Wash and centrifuge to collect the eggs on a sterile NGM solid culture dish, place it in a 20°C biochemical incubator for 24 hours, and obtain a large number of uniform L1 stage nematodes.

[0051] The synchronized L1 stage nematodes were adjusted to a density of about 1 nematode / 10 μL using SM medium, and transferred to a 24-well plate (1000 μL / well). 100 μL of concentrated E. coli NA22 solution (hereinafter referred to as NA22) was added to each well, and the plate was placed in a 20°C shaker for culture until the D1 stage. D1 stage nematodes were collected, and except for the blank group, trehalose was not added. Trehalose was added to all other groups at a final concentration of 10 mg / mL. Orlistat and the oligopeptide to be tested were also added to the positive control sample and the oligopeptide group, 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 after precipitation, 600 μL of Oil Red O dye solution was added, and the mixture was shaken at room temperature for 30 min. The nematodes were washed 5 times with 0.5% v / v PBST. Slides were made and observed and photographed using a polarizing microscope. ImageJ was used for image analysis and numerical values ​​were recorded. The fat staining optical density value reflected the body fat content of Caenorhabditis elegans. The results are shown in Tables 2 and Figure 7 , the calculation formula is as follows:

[0052]

[0053] Table 2 Effects of different treatment groups on body fat content of nematodes

[0054]

[0055] As shown in Table 2 and Figure 7 As shown, there are significant differences in the fat staining optical density values ​​of the orlistat positive control group and the model group relative to the blank group, indicating that the model group was successfully modeled, and the fat staining optical density values ​​of the oligopeptides FPLL, LPGFL, ITF and IVF were significantly reduced relative to the model group, indicating that the oligopeptides have a fat-reducing effect. In particular, the FPLL and IVF oligopeptides have very strong fat-reducing effects, and the nematode fat staining optical density values ​​of the model group are reduced by 36.34% and 27.08%, respectively, which are lower than the body fat content of the blank group (no modeling), 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.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. An oligopeptide, characterized in that: The oligopeptide comprises at least one of the following amino acid sequences SEQ ID NO.1-4.

2. The oligopeptide according to claim 1, characterized in that The oligopeptide is mainly synthesized by solid phase synthesis method.

3. An oligopeptide composition, characterized in that: The oligopeptide composition comprises 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 are independently selected from at least one of SEQ ID NO.1-4.

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

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

6. A fat-reducing composition, characterized in that: The method comprises the oligopeptide according to any one of claims 1 to 2 or the oligopeptide composition according to claim 3.

7. The fat-reducing composition according to claim 6, characterized in that: The fat-reducing composition further comprises auxiliary materials.

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

9. The fat reducing composition according to claim 6, characterized in that: The dosage form of the fat-reducing composition is at least one of capsules, pills, oral liquids, tablets, granules, powders and injections.

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