A hexapeptide CK6 with sleep-improving effect and its preparation method and application

Abstract

The present invention discloses a hexapeptide CK6 with a sleep-improving effect, a preparation method and an application thereof, and belongs to the field of biotechnology. The amino acid sequence of the hexapeptide CK6 is CDAKVK, as shown in the sequence table SEQ ID NO: 3, and has a sleep-improving effect. The hexapeptide CK6 is extracted from the enzymatic hydrolysate of the sea cucumber body wall and is combined with GABA A R has potential interactions, which can increase the sleep rate of mice and significantly prolong the sleep time of mice. At the same time, it can also significantly increase the content of 5-hydroxytryptamine and γ-aminobutyric acid in the mouse brain. It can be used to develop functional foods that assist in improving sleep. It has a good effect on improving sleep, few toxic side effects, and high safety.

Description

Technical Field

[0001] The present invention relates to a small molecule peptide and a preparation method and application thereof, and in particular to a hexapeptide CK6 with a sleep-improving effect and a preparation method thereof and application thereof in functional foods that assist in improving sleep, and belongs to the field of biotechnology. Background Art

[0002] Sleep can restore human functions, consolidate memory and maintain normal brain function. It is an important physiological function of the human body. Sleep problems caused by staying up late and going to bed late have become one of the hot issues of global concern. Neurotransmitters 5-hydroxytryptamine (5-HT), γ-aminobutyric acid (GABA) and dopamine (DA) have been shown to have an important impact on sleep and insomnia. Drug therapy is one of the ways to improve sleep problems. For example, sedative hypnotic drugs can make people fall asleep, but after waking up, they will have side effects such as dizziness, fatigue, drowsiness, and lack of energy. In addition, some drugs will produce adverse reactions such as drug resistance, addiction, and withdrawal. In order to overcome the potential side effects of drugs, more attention should be paid to natural substances that have sleep-improving effects.

[0003] The idea that milk can calm nerves and help people sleep is deeply rooted in people's minds. Liu Xingyu and others evaluated the role of sleep-promoting peptides from bovine milk in the article "Evaluation of the in vitro antioxidant activity of sleep-promoting peptides from bovine milk and their in vivo antioxidant effects on Caenorhabditis elegans". At present, a series of sleep-promoting peptides such as HQGLPQEVLNENLLR, YKVPQLEIVPNSAEER, HPIKHQGLPQEVLN ENLLR, VPQLEIVPNSAEER, and YLGYLEQLLR have been isolated from milk protein hydrolysate, but there are few reports on sleep-promoting peptides from other protein sources, especially aquatic products.

[0004] Sea cucumber is a traditional tonic food, and the preparation and biological activity of sea cucumber peptides have attracted much attention. The biological activities of sea cucumber peptides that have been discovered and evaluated include antioxidant, anti-diabetic, ACE inhibition, immunomodulatory, anti-cancer, anti-fatigue, anti-aging, neuroprotection and trace metal chelation, but there are few reports on the improvement of sleep by sea cucumber peptides. Summary of the invention

[0005] The purpose of the present invention is to provide a small molecule peptide screened from sea cucumber body wall enzymatic hydrolysate, which has a sleep-improving effect and can be applied to functional foods that assist in improving sleep.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A hexapeptide CK6 with a sleep-improving effect. The amino acid sequence of the hexapeptide CK6 is CDAKVK, as shown in SEQ ID NO: 3 in the sequence table, and has a sleep-improving effect.

[0008] The application of the aforementioned hexapeptide CK6 with sleep-improving effect in functional foods that assist in improving sleep.

[0009] A method for preparing the aforementioned hexapeptide CK6 having a sleep-improving effect adopts a solid phase synthesis method, specifically:

[0010] The solid phase synthesis was carried out by using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid phase carrier and adopting the Fmoc solid phase synthesis strategy.

[0011] Another method for preparing the aforementioned hexapeptide CK6 having a sleep-improving effect is to use an enzymatic method, specifically:

[0012] (1) taking a sea cucumber, adding 9 times the volume of water as the sea cucumber, adding a composite protease at a ratio of 0.5% of enzyme to substrate for enzymolysis, the enzymolysis temperature is 55° C., the pH value is 8.0, the enzymolysis time is 6 hours, and an enzymolysis product is obtained, wherein the composite protease is a mixture of alkaline protease and neutral protease at a mass ratio of 1:1;

[0013] (2) After the enzymatic hydrolysis is completed, the enzymatic hydrolysis product is filtered through eight layers of gauze to remove the residue and obtain a clarified polypeptide enzymatic hydrolysate;

[0014] (3) freeze-drying the clarified polypeptide hydrolysate to obtain a sea cucumber peptide sample, wherein the sea cucumber peptide sample contains a large amount of hexapeptide CK6.

[0015] The benefits of the present invention are as follows: the hexapeptide CK6 obtained by screening from the sea cucumber body wall enzymatic hydrolysate of the present invention can improve the sleep rate of mice and significantly prolong the sleep time of mice. In addition, it can also significantly increase the content of 5-hydroxytryptamine and γ-aminobutyric acid in the mouse brain. Compared with the reported decapeptide YLGYLEQLLR with hypnotic and anti-anxiety effects, the sleep time of mice treated with the hexapeptide CK6 is significantly increased, and the content of γ-aminobutyric acid in the brain tissue is significantly increased. The hexapeptide CK6 can be used to develop functional foods that assist in improving sleep, and has a good sleep improvement effect, few toxic and side effects, and high safety in use. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Hexapeptide CDAKVK and GABA A Schematic diagram of the binding mode of R;

[0017] Figure 2 for Figure 1 A partial enlarged schematic diagram of

[0018] Figure 3 This is the effect of hexapeptide CDAKVK treatment on the sleep rate of mice, * indicates P < 0.05;

[0019] Figure 4This is a graph showing the effect of hexapeptide CDAKVK treatment on the sleep latency of mice;

[0020] Figure 5 This is a graph showing the effect of hexapeptide CDAKVK treatment on the sleep duration of mice, ** indicates P < 0.01;

[0021] Figure 6 The figure shows the effect of hexapeptide CDAKVK treatment on the content of neurotransmitters in the mouse brain, where A shows the effect on the content of 5-hydroxytryptamine, B shows the effect on the content of γ-aminobutyric acid, and C shows the effect on the content of dopamine. * indicates P<0.05, and *** indicates P<0.001.

[0022] Figure 7 The graphs show the effects of hexapeptide CDAKVK treatment on gene transcription levels in mice, where A shows the effects on GABRA1 gene transcription levels, and B shows the effects on GABRB2 gene transcription levels. * indicates P<0.05, and ** indicates P<0.01. DETAILED DESCRIPTION

[0023] The present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0024] 1. Preparation of sea cucumber peptide samples

[0025] Take a sea cucumber, add 9 times the volume of water as the sea cucumber (the volume concentration of the sea cucumber is 10%), add a composite protease (mixed by alkaline protease and neutral protease in a mass ratio of 1:1) at an enzyme-substrate ratio of 0.5% for enzymatic hydrolysis, the enzymatic hydrolysis temperature is 55°C, the pH value is 8.0, and the enzymatic hydrolysis time is 6 hours.

[0026] After the enzymatic hydrolysis is completed, the enzymatic hydrolysate is filtered through eight layers of gauze to remove the residue and obtain a clarified polypeptide enzymatic hydrolysate.

[0027] The clarified polypeptide hydrolysate is freeze-dried to obtain a sea cucumber peptide sample.

[0028] 2. Obtaining the polypeptide sequence in the sea cucumber peptide sample

[0029] The sea cucumber peptide samples obtained above were determined by LC-MS / MS, and the determination results were analyzed by mass spectrometry analysis software to obtain several polypeptide sequences.

[0030] The LC-MS / MS assay conditions were:

[0031] (1) In the liquid phase method: the chromatographic column is C18, 3μm, 250mm×75μm (Eksigent), phase A is water, 0.1% formic acid; phase B is acetonitrile, 0.1% formic acid, the flow rate is 300nL / min, the injection volume is 4μL, and the chromatographic gradient is 60min. The specific elution gradient is: 0-48min, phase A is uniformly reduced from 95% to 60%; 48-55min, phase A is uniformly reduced from 60% to 30%; 55-56min, phase A is uniformly reduced from 30% to 0; 56-60min, maintain 0% phase A;

[0032] (2) Mass spectrometry method: Orbitrap Exploris 480 (Thermofisher), positive ion detection mode, primary resolution of 120,000, AGC setting of 300, scanning range of 200-1600 m / z. MIPS mode of peptide, valence states of 1-5, secondary resolution of 15,000, separation window of 1.6 m / z.

[0033] 3. Screening peak area ≥10 7 Active oligopeptides with amino acid number ≤ 6

[0034] From the several peptide sequences obtained above, 18 peptide sequences with peak areas ≥10 7 The screening results of active oligopeptides with amino acid number ≤ 6 are shown in Table 1.

[0035] Table 1 Peak area ≥10 in sea cucumber peptide samples 7 Active oligopeptide sequence with amino acid number ≤ 6

[0036]

[0037] 4. Screening and GABA A Active oligopeptides with strong R protein binding ability

[0038] The 18 peptide sequences in Table 1 were respectively linked to the γ-aminobutyric acid receptor (GABA A Before docking, the 2D structure of the peptide was converted into a 3D structure by energy minimization, and the GABA A A polypeptide sequence with strong R protein binding ability. Protein GABA A The 3D structure of R can be downloaded from the RCSB protein database (PDB ID: 6HUO). The docking results are expressed as docking scores (-CiE). The larger the -CiE value, the closer the peptide is to GABA. A The stronger the interaction, the stronger the R. The molecular docking results of 18 peptide sequences are shown in Table 2.

[0039] Table 2 Sea cucumber peptide components and GABA A R interaction prediction results

[0040]

[0041] 5. Molecular docking analysis

[0042] Among the 18 active peptides tested, the hexapeptide CDAKVK (denoted as CK6, SEQ ID NO: 3) was highly expressed in the presence of GABA. A The peak of -CiE (122.259 kcal / mol) docked by R was the largest, and the peak area (3.67×10 7 ) ranked third. Therefore, the hexapeptide CDAKVK was selected for further prediction analysis.

[0043] After analysis, the hexapeptide CDAKVK and GABA A The binding mode of R is Figure 1 and Figure 2 The molecular docking situation is as follows:

[0044] Hexapeptide CDAKVK and GABA A There are 6 HH bond interactions, 1 CH bond interaction, 1 salt bridge interaction and 3 electrostatic interactions between R, and 11 amino acid residues are involved in the binding of the hexapeptide CDAKVK with GABA A The interaction between R.

[0045] VI. Evaluation of the sleep-improving effect of hexapeptide CDAKVK

[0046] 1. Solid phase synthesis of hexapeptide CDAKVK and decapeptide YLGYLEQLLR

[0047] The decapeptide YLGYLEQLLR has been shown to have hypnotic and antianxiety effects and has been commercialized. It was selected as a control for the hexapeptide CDAKVK.

[0048] The hexapeptide CDAKVK and the decapeptide YLGYLEQLLR were synthesized in the solid phase by using the Fmoc solid phase synthesis strategy, Fmoc protected amino acids as raw materials and polystyrene resin as solid phase carrier.

[0049] 2. Animal grouping and active peptide treatment

[0050] Male Kunming mice aged 6-8 weeks were selected, and the temperature was 20℃-26℃, the air humidity was 50%-60%, the light and dark were alternating for 12h, and food and water were freely available. After 7 days of adaptive feeding, they were randomly divided into groups (control group, YLGYLEQLLR group, CDAKVK group), with 10 mice in each group. Among them, the control group was gavaged with normal saline once a day, the YLGYLEQLLR group was gavaged with solid-phase synthesized ten-peptide YLGYLEQLLR (dissolved in normal saline, concentration of 1 mg / mL) once a day, and the CDAKVK group was gavaged with solid-phase synthesized hexapeptide CDAKVK (dissolved in normal saline, concentration of 1 mg / mL) once a day, with a gavage volume of 0.1mL / 10g, and the gavage was continued for 30 days.

[0051] 3. Hypnosis experiment induced by sodium pentobarbital

[0052] The experiment was conducted in a quiet environment at 24-25℃. 15 minutes after the mice received the last oral administration (day 30), the maximum subthreshold hypnotic dose of sodium pentobarbital (35 mg / kg) was intraperitoneally injected, and the number of animals that fell asleep within 30 minutes (those with loss of righting reflex for more than 1 minute) was recorded.

[0053] Effects of hexapeptide CDAKVK treatment on the sleep rate of mice Figure 3 .

[0054] Depend on Figure 3 It can be seen that under the induction of sodium pentobarbital, the sleep rate of mice in the control group was 30.5%, the sleep rate of mice in the YLGYLEQLLR group was 36.1%, and the sleep rate of mice in the CDAKVK group was 44.47.8%; compared with the control group, the CDAKVK group significantly increased the sleep rate of mice (p<0.05), while there was no significant difference between the YLGYLEQLLR group and the control group (p>0.05); compared with the YLGYLEQLLR group, there was no significant difference in the sleep rate of the CDAKVK group (p>0.05).

[0055] 4. Sleep latency experiment induced by sodium pentobarbital

[0056] The experiment was conducted in a quiet environment at 24℃-25℃. 15 minutes after the mice received the last oral administration (day 30), a suprathreshold hypnotic dose of sodium pentobarbital (50 mg / kg) was intraperitoneally injected, and the effect of the test sample on the sleep latency of mice was observed using the disappearance of the righting reflex as an indicator.

[0057] Effects of hexapeptide CDAKVK on sleep latency in mice Figure 4 .

[0058] Depend on Figure 4It can be seen that under the induction of sodium pentobarbital, the latency of the control group mice to fall asleep was 5.12±0.89min, the latency of the YLGYLEQLLR group mice to fall asleep was 4.31±0.46min, and the latency of the CDAKVK group mice to fall asleep was 4.43±0.60min; there was no significant difference between the YLGYLEQLLR group and the CDAKVK group and the control group (p>0.05), and there was no significant difference between the YLGYLEQLLR group and the CDAKVK group (p>0.05).

[0059] 5. Sleep duration experiment induced by sodium pentobarbital

[0060] The experiment was conducted in a quiet environment at 24℃-25℃. 15 minutes after the mice received the last oral administration (day 30), a suprathreshold hypnotic dose of sodium pentobarbital (50 mg / kg) was intraperitoneally injected, and the disappearance of the righting reflex was used as an indicator to observe whether the test sample could prolong the sleep time of the mice.

[0061] Effects of hexapeptide CDAKVK treatment on sleep duration in mice Figure 5 .

[0062] Depend on Figure 5 It can be seen that under the induction of sodium pentobarbital, the sleep time of mice in the control group was 19.42±4.86min, the sleep time of mice in the YLGYLEQLLR group was 27.70±3.51min, and the sleep time of mice in the CDAKVK group was 35.24±6.21min; compared with the control group, both the YLGYLEQLLR group and the CDAKVK group significantly prolonged the sleep time of mice (p<0.01); compared with the YLGYLEQLLR group, the CDAKVK group had no significant effect on the sleep time of mice (p>0.05).

[0063] 6. Determination of neurotransmitter content

[0064] After the 30-day experiment, the mice were killed and their brain tissues were isolated. An appropriate amount of brain tissue was weighed and homogenized with PBS buffer at a volume ratio of 1:9, centrifuged at 3000 rpm and 4°C for 20 min, and the supernatant was taken to measure the contents of 5-hydroxytryptamine (5-HT), γ-aminobutyric acid (GABA), and dopamine (DA) using a kit.

[0065] Effects of hexapeptide CDAKVK treatment on 5-HT, GABA and DA levels in mouse brain Figure 6 .

[0066] Depend on Figure 6The results showed that the contents of 5-HT, GABA and DA in the brain of mice in the control group were 117.67±11.86 ng / mL, 3.34±0.17 μmol / mL and 1.27±0.16 pg / mL, respectively; the contents of 5-HT, GABA and DA in the brain of mice in the YLGYLEQLLR group were 135.3±15.21 ng / mL, 3.61±0.29 μmol / mL and 1.22±0.08 pg / mL, respectively; the contents of 5-HT, GABA and DA in the brain of mice in the CDAKVK group were 148.17±3.43 ng / mL, 3.90±0.06 μM / mL and 1.175±0.12 pg / mL, respectively; compared with the control group, the content of 5-HT in the brain of mice in the YLGYLEQLLR group was significantly increased ( p <0.05), the content of 5-HT in the brain of mice in the CDAKVK group was significantly increased (p<0.001), and the content of GABA in the brain of mice in the YLGYLEQLLR group was not significantly different from that in the control group ( p >0.05), the CDAKVK group significantly increased the GABA content in the brain of mice (p<0.001), and the DA content in the brain of mice in the YLGYLEQLLR group and CDAKVK group had no significant difference compared with the control group (p>0.05). Compared with the YLGYLEQLLR group, the CDAKVK group had no significant effect on the 5-HT, GABA and DA contents in the brain of mice (p>0.05).

[0067] 7. Real-time fluorescence quantitative PCR detection

[0068] (1) Real-time fluorescence quantitative PCR primer design

[0069] GABAA (γ-aminobutyric acid receptor) subunits GABRA1 (γ-aminobutyric acid receptor A1) and GABRB2 (γ-aminobutyric acid receptor B2) were selected, and real-time fluorescence quantitative PCR (qRT-PCR) primers were designed using the NCBI website (https: / / www.ncbi.nlm.nih.gov / ) and Primer 5 software from Premier Company of Canada. The specific information of the primers is shown in Table 3.

[0070] Table 3 Primer sequences for real-time fluorescence quantitative PCR

[0071]

[0072] (2) Total RNA extraction from tissues

[0073] 100 mg of mouse brain tissue was placed in a sterile mortar precooled with liquid nitrogen, and the mixture was quickly and thoroughly ground with liquid nitrogen. Then 1 mL of TrasZol Up reagent was added, and the mixture was transferred to a 1.5 mL centrifuge tube without DNase, and 0.2 mL of chloroform was added. The mixture was fully vortexed for 30 seconds, allowed to stand for 10 minutes, and then centrifuged at 4°C and 12000×g for 15 minutes. The supernatant was taken to another 1.5 mL centrifuge tube without RNase, and 0.5 mL of isopropanol was added. The liquid in the tube was gently mixed, allowed to stand at room temperature for 10 minutes, and then centrifuged again at 4°C and 12000×g for 10 minutes. The supernatant was discarded, and 1 mL of 75% ethanol prepared with RNase-free water was added to the precipitate. The precipitate was gently washed, and centrifuged at 4°C and 7500×g for 5 minutes. The supernatant was discarded, and the centrifuge tube cap was opened to evaporate the ethanol. Then 100 μL of RNase-free water was added, and the total RNA concentration and quality of the sample were quickly determined using Nano Drop 2000c. The RNA was aliquoted into RNase-free centrifuge tubes and stored at -80°C.

[0074] (3) cDNA template synthesis

[0075] Take 1 µg of total RNA and synthesize cDNA using the TransScript All-in-One First-Strand cDNA SynthesisSuperMix for qPCR (One-Step gDNA Removal) Kit.

[0076] (4) Gene expression detection

[0077] Preparation of qRT-PCR system: After appropriately diluting the cDNA template, take 5 μL and add it to the total system. Take 0.4 μL of forward primer and reverse primer respectively and add them to the total system. Then take 10 μL of 2×TransStartTM Green qPCR SuperMix (containing SYBR Green dye) and 4.2 μL of enzyme-free water and add them to the total system. The total system is 20 μL.

[0078] The qRT-PCR three-step method was as follows: denaturation at 94°C for 10 min; amplification reaction was performed for 40 cycles, including denaturation at 94°C for 15 s, annealing at 50°C for 15 s, and extension at 72°C for 45 s; and extension at 72°C for 10 min.

[0079] The effect of hexapeptide CDAKVK treatment on the transcriptional levels of GABRA1 and GABRB2 genes in mice is shown in Figure 7 .

[0080] Depend on Figure 7It can be seen that compared with the control group, the YLGYLEQLLR group and the CDAKVK group can significantly upregulate the transcription levels of the GABRA1 gene and the GABRB2 gene; compared with the YLGYLEQLLR group, the CDAKVK group had no significant effect on the transcription levels of the GABRA1 gene and the GABRB2 gene (p>0.05).

[0081] In summary, the hexapeptide CDAKVK (CK6) obtained by screening from the sea cucumber body wall enzymatic hydrolysate in the present invention has a sleep-improving effect and can be applied to functional foods that assist in improving sleep.

[0082] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A hexapeptide CK6 having a sleep-improving effect, characterized in that: The amino acid sequence of the hexapeptide CK6 is CDAKVK, as shown in SEQ ID NO: 3 in the sequence table, and has a sleep-improving effect.

2. Use of the hexapeptide CK6 with sleep-improving effect as claimed in claim 1 in the preparation of functional foods that assist in improving sleep.

3. The method for preparing the hexapeptide CK6 having sleep-improving effect according to claim 1, characterized in that: The solid phase synthesis method is adopted, specifically: The solid phase synthesis was carried out by using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid phase carrier and adopting the Fmoc solid phase synthesis strategy.

4. The method for preparing the hexapeptide CK6 having sleep-improving effect according to claim 1, characterized in that: Enzymatic hydrolysis is used, specifically: (1) taking a sea cucumber, adding 9 times the volume of water as the sea cucumber, adding a composite protease at a ratio of 0.5% of enzyme to substrate for enzymolysis, the enzymolysis temperature is 55° C., the pH value is 8.0, the enzymolysis time is 6 hours, and an enzymolysis product is obtained, wherein the composite protease is a mixture of alkaline protease and neutral protease at a mass ratio of 1:1; (2) After the enzymatic hydrolysis is completed, the enzymatic hydrolysis product is filtered through eight layers of gauze to remove the residue and obtain a clarified polypeptide enzymatic hydrolysate; (3) freeze-drying the clarified polypeptide hydrolysate to obtain a sea cucumber peptide sample, wherein the sea cucumber peptide sample contains a large amount of hexapeptide CK6.

Images