A low molecular weight peptide and its application in the preparation of products for delaying muscle atrophy

Abstract

The present invention relates to an oligopeptide with the efficacy of delaying muscle atrophy and its application in the preparation of products for delaying muscle atrophy, belonging to the field of biotechnology. The present invention provides an oligopeptide, which comprises tetrapeptides and / or tripeptides; the amino acid sequence of the tetrapeptide is LWPF and / or LPGF; the amino acid sequence of the tripeptide is FEP. Through solid-phase synthesis, the present invention can obtain oligopeptides with 3 different amino acid sequences. Experiments confirm that the 3 obtained oligopeptides all have the efficacy of improving muscle atrophy, and have the advantages of being safe, can be used for a long time, and have no side effects, and can be applied to products for improving muscle atrophy and its related symptoms.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an oligopeptide and its application in the preparation of products for delaying muscle atrophy. Background Art

[0002] Muscle atrophy is a degenerative syndrome in which the total muscle mass decreases, muscle strength declines, and the physiological function of muscles deteriorates progressively with age. Muscle atrophy can trigger a series of serious health problems, such as falls, fractures, disability, and incapacity; it can also significantly increase the infection rate, hospitalization rate, and mortality rate, forming a causal chain of high mortality in the elderly population. In addition, muscle atrophy increases the risk of various chronic diseases, such as osteoporosis, chronic enteritis, diabetes, chronic obstructive pulmonary disease, tumors, etc. At present, the prevalence of sarcopenia is very high. According to statistics, the incidence rate in the elderly aged 60 - 70 is 5% - 13%, and the incidence rate in the elderly over 80 is as high as 11% - 50%. The older the age, the higher the incidence rate. With the aggravation of social aging, the prevalence of sarcopenia will show an obvious increasing trend, becoming a major public health problem and social and economic problem threatening the health of the whole people, reducing the quality of life, and consuming medical resources.

[0003] However, the current technical means for delaying muscle atrophy are still relatively limited. It mainly focuses on supplementing high-quality protein and exercise. An oligopeptide is a peptide composed of 2 - 10 amino acids, and different amino acid compositions have different biological activities. Therefore, through a large number of experiments, the present invention has developed an oligopeptide with the effect of delaying muscle atrophy. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an oligopeptide and its application in the preparation of products for delaying muscle atrophy.

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

[0006] In the first aspect, the present invention provides an oligopeptide, which includes a tetrapeptide and / or a tripeptide; the amino acid sequence of the tetrapeptide is LWPF and / or LPGF; the amino acid sequence of the tripeptide is FEP.

[0007] The present invention can obtain oligopeptides with 3 different amino acid sequences through solid-phase synthesis, and it is experimentally confirmed that all 3 obtained oligopeptides have the effect of improving muscle atrophy.

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

[0009] Second aspect, the present invention provides an oligopeptide composition having the effect of delaying muscle atrophy. The oligopeptide composition includes at least two of oligopeptide I, oligopeptide II, and oligopeptide III; the amino acid sequences of oligopeptide I, oligopeptide II, and oligopeptide III are independently selected from at least one of LWPF, LPGF, and FEP. In addition to the fact that a single oligopeptide of the present invention can play the role of delaying muscle atrophy, a composition of two or more oligopeptides also has the effect of delaying muscle atrophy.

[0010] As a preferred embodiment of the oligopeptide composition of the present invention, the oligopeptide I, oligopeptide II, and oligopeptide III are mainly synthesized by solid-phase synthesis.

[0011] Third aspect, the present invention provides the use of the above-mentioned oligopeptide or oligopeptide composition in the preparation of products for delaying muscle atrophy.

[0012] Fourth aspect, the present invention provides the use of the above-mentioned oligopeptide or oligopeptide composition in the preparation of products for improving muscle motor ability.

[0013] As a preferred embodiment of the use of the present invention, the products include drugs and cosmetics.

[0014] Fifth aspect, the present invention provides a composition for delaying muscle atrophy, including the above-mentioned oligopeptide or oligopeptide composition.

[0015] As a preferred embodiment of the composition for delaying muscle atrophy of the present invention, the composition for delaying muscle atrophy further includes excipients.

[0016] As a preferred embodiment of the composition for delaying muscle atrophy of the present invention, the excipients include at least one of flavoring agents, binders, lubricants, disintegrants, antioxidants, stabilizers, thickeners, and fillers.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] The present invention can obtain three oligopeptides with different amino acid sequences through solid-phase synthesis. Experiments have confirmed that the three obtained oligopeptides all have the effect of improving muscle atrophy, and have the advantages of safety, long-term use, and no side effects, and can be applied to products for improving muscle atrophy and its related symptoms. Description of the Drawings

[0019] Figure 1 It is the liquid chromatogram of different oligopeptides in Effect Example 1 of the present invention. Among them, Figure A is oligopeptide LWPF, Figure B is oligopeptide LPGF, and Figure C is oligopeptide FEP;

[0020] Figure 2 It is the mass spectrum of oligopeptide LWPF in Effect Example 1 of the present invention;

[0021] Figure 3 It is the mass spectrum of the oligopeptide LPGF in the effect example 1 of the present invention;

[0022] Figure 4 It is the mass spectrum of the oligopeptide FEP in the effect example 1 of the present invention;

[0023] Figure 5 It is the influence of different treatments on the nematode muscle in the effect example 2 of the present invention;

[0024] Figure 6 It is the influence of different samples on the pharyngeal pumping times of nematodes in the effect example 3 of the present invention;

[0025] Figure 7 It is the influence of different samples on the body swing times of nematodes in the effect example 3 of the present invention. Detailed implementation manners

[0026] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0027] Other materials, reagents, etc. used in the embodiments can be obtained from commercial channels without special instructions.

[0028] Escherichia coli NA22 and Caenorhabditis elegans N2 are both purchased from the Caenorhabditis Genetics Center of the University of Minnesota, USA.

[0029] The technologies not described in detail in the following embodiments, comparative examples and effect examples are all common technologies in the art, and reference can be made to "Molecular Biology Experiment Manual" (Ma Wenli, People's Military Medical Press), "Molecular Biology Experiments (Second Edition)" (Zhejiang University Press), "Cell Biology Experiments" (Yang Hongbing, Hou Lixia, Zhang Yuxi, Higher Education Press).

[0030] 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), "T" refers to threonine (Thr).

[0031] In the following examples and effect examples, LWPF refers to the oligopeptide Leu-Trp-Pro-Phe, LPGF refers to the oligopeptide Leu-Pro-Gly-Phe, and FEP refers to the oligopeptide Phe-Glu-Pro.

[0032] Example 1

[0033] This embodiment provides an oligopeptide, which is prepared by the following steps:

[0034] 1. Weigh 3g of Rink Amide Resin (substitution degree 0.3mmol / g) in a 150mL reactor, soak it in 50mL of dichloromethane (DCM) for 2h, 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;

[0035] 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℃ for 1 minute). The resin has color, indicating that the deprotection group is successful;

[0036] 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;

[0037] 4. Remove the Fmoc protecting group on the resin in the reactor according to the method of step B2;

[0038] 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℃ 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℃ 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 continue the reaction; 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;

[0039] 6. Connect the following amino acids in sequence according to steps B1-B5. 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 for each 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.

[0040] According to the above method, polypeptides with amino acid sequences of LWPF, LPGF and FEP can be synthesized respectively.

[0041] Effect Example 1

[0042] The oligopeptide obtained in Example 1 was characterized by HPLC-MS / MS, and the specific scheme is as follows:

[0043] 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.

[0044] 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℃, 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.

[0045] Mass spectrometry detection method: Scanning period is 0.1S, ESI ion source temperature is 100°C, positive ion mode, spray voltage is 3000V, scanning range is 300 - 1999Da, and the identification results are shown in Table 1 and Figures 1-4 。

[0046] Table 1 Identification results of oligopeptides

[0047]

[0048] As shown in Table 1 and Figures 1-4 shown, the amino acid sequences of the oligopeptides identified by the present invention using HPLC-MS / MS technology are LWPF, LPGF, and FEP respectively.

[0049] Effect Example 2

[0050] The efficacy of delaying muscle loss and protecting muscle tissue of the oligopeptides (LWPF, LPGF, and FEP) obtained in Example 1 was evaluated through Caenorhabditis elegans N2. The specific scheme is as follows:

[0051] The experiment was carried out with reference to the following literature: Zhang Huijie, The Role and Mechanism of Chrysanthelide in Anti-aging and the Treatment of Sarcopenia [D] Nanchang University 2022-5-23.

[0052] RW1596 nematodes were used. The density of nematodes was adjusted to about 1 nematode / 10 μL with S.M medium and transferred to a 24-well plate (1000 μL / well). 100 μL of NA22 was added to each well of the 24-well plate and the drug was administered. After intervention at 20°C until the 15th day of adulthood, the Caenorhabditis elegans were washed into a 5 mL centrifuge tube. After sedimentation, the supernatant was discarded, and the washing was repeated 2 - 3 times. 20 μL was pipetted onto a glass slide, and a drop of vegetable oil was added to cover the nematode body to prevent dehydration of the nematodes. Subsequently, it was inverted and photographed under a fluorescence microscope at a magnification to observe the changes in the sarcomere structure of Caenorhabditis elegans. The experimental groups were set as a blank group, a positive drug group, and a sample group. The positive drug group was 500 μg / mL of Ginkgo biloba extract, and the sample groups were 10 mg / mL of the oligopeptides (LWPF, LPGF, and FEP) obtained in Example 1. The results are shown in Figure 5 。

[0053] As Figure 5 shown, in the blank control group, severe fractures occurred in the sarcomere lines in the body wall muscles of Caenorhabditis elegans, and the green lines were no longer parallel. In contrast to the blank group, in the oligopeptide sample groups, the muscle lines of the nematodes showed a complete and neatly arranged network, and multiple green parallel lines were shown in each body wall muscle. It shows that the oligopeptides LWPF, LPGF, and FEP have a good effect of delaying muscle loss and can maintain the good structure of muscle fibers.

[0054] Effect Example 3

[0055] The effects of the oligopeptides (LWPF, LPGF, and FEP) obtained in Example 1 on improving muscle motor function were evaluated using Caenorhabditis elegans N2. The specific protocol is as follows:

[0056] The following literature was referred to for the experiment on improving muscle motor function:

[0057] Dhondt I, Verschuuren C, Zečić A. Prediction of biological age by morphological staging of sarcopenia in Caenorhabditis elegans. Dis Model Mech. 2021 Nov 1;14(11):dmm049169.

[0058] Chow DK, Glenn CF, Johnston JL, Goldberg IG, Wolkow CA. Sarcopenia in the Caenorhabditis elegans pharynx correlates with muscle contraction rate over lifespan. Exp Gerontol. 2006 Mar;41(3):252-260.

[0059] Caenorhabditis elegans N2 was used. First, the worm density was adjusted to about 1 worm / 10 μL with S.M medium and transferred to a 24-well plate (1000 μL / well). Then, 100 μL of NA22 was added to each well of the 24-well plate and the drug was administered. After intervention at 20 °C until the 15th day of adulthood, the detection was carried out.

[0060] Detection of body swing index: 100 μL of the worm solution was pipetted onto a glass slide and spread evenly with a pipette tip so that it was distributed evenly and did not overlap. It was placed under a 5X objective lens of a microscope. After standing for 5 min, a 30-s movement video was recorded. The video was opened and played at a speed of 0.5 times. The nematodes in the video were tracked and the number of head swings was counted.

[0061] Detection of pharyngeal pumping index: 10 μL of the worm solution was pipetted onto a glass slide and placed under a microscope. After standing for 1 min, a 30-s pharyngeal pumping video was recorded under a 10X objective lens. The video was opened and played at a speed of 0.5 times. The nematodes in the video were tracked and the number of head pharyngeal pumpings was counted.

[0062] The above results are shown in Figures 6-7 and Table 2.

[0063] The experimental groups were set as the blank group, the positive drug group, and the oligopeptide group. Among them, the positive drug group was the ginkgo biloba extract at 500 μg / mL, and the oligopeptide group was 1 mg / mL.

[0064] Table 2 Statistics of the number of pharyngeal pumps and body swings of nematodes in different treatment groups

[0065]

[0066] As shown in Table 2 and Figures 6-7 as indicated, the number of pharyngeal pumps and body swings of nematodes in the oligopeptide group were significantly increased compared with those in the blank control group, indicating that the oligopeptides LWPF, LPGF, and FEP of the present invention can improve the motor ability of muscles.

[0067] In summary, the oligopeptides and oligopeptide compositions of the present invention can delay muscle loss, maintain the good structure of muscle fibers, improve the motor ability of muscles, and thus improve sarcopenia.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A oligopeptide, characterized in that, The oligopeptide is a tetrapeptide; the amino acid sequence of the tetrapeptide is LWPF.

2. The oligopeptide according to claim 1, wherein The oligopeptide is mainly synthesized by solid-phase synthesis.

3. An oligopeptide composition with the efficacy of delaying muscle atrophy, characterized in that, The oligopeptide composition includes at least two of oligopeptide I, oligopeptide II, and oligopeptide III; the amino acid sequences of oligopeptide I, oligopeptide II, and oligopeptide III are LWPF, LPGF, and FEP, respectively.

4. The oligopeptide composition according to claim 3, wherein The oligopeptide I, oligopeptide II, and oligopeptide III are mainly synthesized by solid-phase synthesis.

5. Use of the oligopeptide according to any one of claims 1-2 or the oligopeptide composition according to any one of claims 3-4 in the preparation of a product for delaying muscle atrophy.

6. The application according to claim 5, characterized in that, The product includes drugs and cosmetics.

7. A muscle atrophy delaying composition, characterized in that, It includes the oligopeptide according to any one of claims 1-2 or the oligopeptide composition according to any one of claims 3-4.

8. The muscle atrophy delaying composition according to claim 7, wherein The composition for delaying muscle atrophy further includes excipients.

9. The composition for delaying muscle atrophy according to claim 8, wherein, The excipients include at least one of flavoring agents, binders, lubricants, disintegrants, antioxidants, stabilizers, thickeners, and fillers.

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