A hexapeptide with sleep-promoting activity and its application

Abstract

The present invention belongs to the field of food health technology, and specifically relates to a hexapeptide with sleep-promoting activity and its application. The hexapeptide is YAKPAA, that is, the amino acid sequence is Tyr-Ala-Lys-Pro-Ala-Ala. The hexapeptide YAKPAA is derived from food-derived casein hydrolysate, is safe and nutritious, and has no toxic side effects. YAKPAA can be combined with GABA A Receptor binding, binding sites are: SER205, ASN60, ASP192, TYR210, TYR58, THR207, THR142, GLU189, activate GABA A receptors, thus playing a role in helping sleep. 5 mg / kg of YAKPAA can significantly shorten the latency of mice to fall asleep and prolong their sleep time. This provides a reference for the development of new food-derived bioactive peptide functional peptides with sleep-promoting activity, as well as functional products with casein hydrolysate as a sleep-promoting active ingredient.

Description

Technical Field

[0001] The invention belongs to the technical field of food health, and specifically relates to a hexapeptide with sleep-promoting activity and application thereof. Background Art

[0002] As the number of people suffering from sleep disorders increases and their age becomes younger, consumers' demand for sleep-aid products has also increased dramatically, among which melatonin and γ-aminobutyric acid are the most popular.

[0003] Although sedatives and hypnotic drugs represented by benzodiazepines on the market are effective, long-term use will bring many side effects, such as drug resistance and addiction. Therefore, it is necessary to find and develop safer food-derived bioactive substances as a natural therapy. Food-derived active peptides can not only provide rich nutrients for humans, but also have many biological functions, such as lowering blood pressure, anti-inflammatory, anti-oxidation, anti-type 2 diabetes and anti-cancer. Cow's milk itself is regarded as a healthy food that can calm the nerves and soothe the nerves. Casein accounts for more than 80% of the total protein in cow's milk. At present, many studies have shown that bioactive peptides derived from cow's milk casein can have the effect of calming the nerves and helping sleep.

[0004] Chinese patent CN 112409470 A "A casein peptide with sleep-improving effect, preparation method and application thereof" discloses casein octapeptide KLPPVGPY (Lys-Leu-Pro-Pro-Val-Gly-Pro-Tyr) and decapeptide APIASLLPPY (Ala-Pro-Ile-Ala-Ser-Leu-Leu-Pro-Pro-Tyr) with sleep-improving effect.

[0005] U.S. Patent US005846939A "USE OF A DECAPEPTIDE WITH BENZODIAZEPINE-TYPEACTIVITY FOR PREPARING MEDICNES AND FOOD SUPPLEMENTS" discloses an αS1-casein-derived α-CZP homologous sequence anti-anxiety decapeptide YLGYLEQLLR (Tyr-Leu-Gly-Tyr -Leu-Glu-Gln-Leu-Leu-Arg) with Tyr at the N-terminus and a molecular weight of 1267 Da.

[0006] Chinese patent CN 102083453 A discloses an anti-anxiety composition comprising αS1-casein peptides, and proposes a biosynthetic or chemical synthesis method to prepare two peptides: the pentapeptide YLGYL (Tyr-Leu-Gly-Tyr-Leu) with a molecular weight of 627 Da and the hexapeptide YLGYLE (Tyr-Leu-Gly-Tyr-Leu-Glu) having antianxiety activity.

[0007] Chinese patent CN 114106096 A "A hexapeptide with sleep-improving activity and its application" discloses a hexapeptide with sleep-improving activity and its application. The primary structure amino acid sequence of the hexapeptide is YPVEPF (Tyr-Pro-Val-Glu-Pro-Phe) and the molecular weight is 750.85 Da.

[0008] Chinese patent CN 113754751 A "A novel sleep-promoting milk-derived active peptide CCL-2S and its preparation method and application" discloses a sleep-promoting milk-derived active octapeptide FALPQYLK (Phe-Ala-Leu-Pro-Gln-Tyr-Leu-Lys) with a molecular weight of 979.17 Da.

[0009] Chinese patent CN 111632130 A "Application of milk-derived active peptide CCL-3S in the preparation of sleep-promoting products" discloses a sleep-promoting milk-derived active 15-peptide with an amino acid sequence of HQGLPQEVLNENLLR (His-Gln-Gly-Leu-Pro-Gln-Glu-Val-Leu-Asn-Glu-Asn-Leu-Leu-Arg) and a molecular weight of 1759.998 Da.

[0010] Chinese patent CN 113735957 A "A novel sleep-promoting milk-derived active peptide CCL-4S and its preparation method and application" discloses a novel sleep-promoting milk-derived active undecapeptide with an amino acid sequence of VAPFPEVFGKE (Val-Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys-Glu) and a molecular weight of 1219.38 Da.

[0011] In the prior art, casein hydrolysates containing the anti-anxiety decapeptide YLGYLEQLLR (α-CZP) have been shown to significantly prolong the sleep time of rats. In the prior art, casein hydrolysates containing the hexapeptide YPVEPF have been shown to significantly prolong the sleep time of mice. However, it is still unknown whether casein hydrolysates contain other peptides with sleep-improving activity. In addition, published Chinese patents disclose that octapeptides, decapeptides, 15-peptides, etc. derived from bovine milk casein have the effect of improving sleep, but research on food-derived small molecule peptides with sleep-improving activity is still insufficient, and new active peptides with sleep-promoting effects need to be further developed. Summary of the invention

[0012] In order to develop new food-borne bioactive functional peptides with sleep-promoting activity and provide a reference for functional products with casein hydrolysate as a sleep-promoting active ingredient, the present invention uses casein as raw material, hydrolyzes it, and separates and identifies a new peptide with sleep-promoting activity.

[0013] One of the technical solutions provided by the present invention is a hexapeptide, the amino acid sequence of the hexapeptide is YAKPAA (Tyr-Ala-Lys-Pro-Ala-Ala), the molecular weight is 619.7097 Da, and the active peptide segment is derived from κ-casein (f61-66).

[0014] Furthermore, the hexapeptide YAKPAA can be prepared by chemical solid phase synthesis;

[0015] Furthermore, the hexapeptide YAKPAA can also be separated and obtained from casein hydrolysate; the casein is bovine casein or other mammalian casein;

[0016] Furthermore, the method for obtaining the hexapeptide YAKPAA from casein hydrolysate is as follows:

[0017] Take 250-300 kg of casein and add it to 1000 kg of water, stir and soak it at room temperature for 30-60 min, stir and heat it to 40-45°C, adjust the pH of the solution to 7.0±0.2 with sodium hydroxide aqueous solution, and stir and dissolve it fully for 30-90 min at 40-45°C; after there are no obvious white particles in the solution, add 0.1-10.0‰ of composite protease ("‰" is calculated by the mass of casein substrate), after enzymolysis for 30-60 min, adjust the pH of the solution to 7.0±0.2 with sodium hydroxide aqueous solution, continue enzymolysis for 2-2.5 hours, and then add 0.1-10.0‰ of papain ("‰" is calculated by the mass of casein substrate), and continue enzymolysis for 1-2 hours; after the enzymolysis is completed, centrifuge the enzymatic hydrolyzate, take the supernatant, concentrate and sterilize it, and spray dry it to obtain the final casein hydrolyzate. The casein hydrolysate was subjected to Q Exactive™ Hybrid Quadrupole-Orbitrap™ LC-MS / MS analysis. The raw files of the original mass spectrometry results were subjected to De novo analysis, and multiple peptides including YAKPAA were obtained.

[0018] Furthermore, the composite protease is composed of neutral protease, papain and trypsin in a mass ratio of 1-5:1-9:1-3.

[0019] The second technical solution provided by the present invention is the application of the hexapeptide YAKPAA, especially in improving sleep, more particularly in preparing medicines for preventing, improving or treating sleep disorders, or in preparing foods for improving sleep disorders, or in preparing health products for improving sleep disorders;

[0020] The sleep disorders include, but are not limited to: reduced sleep time, difficulty falling asleep, insomnia, poor sleep quality, etc.;

[0021] Furthermore, the hexapeptide YAKPAA can be used alone in the preparation of the food, health care product or medicine; or it can be used in combination with other components having sleep-aiding activity;

[0022] The components with sleep-promoting activity include, but are not limited to, Chinese medicinal materials that are both medicinal and edible (such as spiny jujube seeds, longan, lily, jujube, gardenia, tuckahoe, lotus seeds, honey, licorice, nutmeg, bergamot and mame, etc.), tryptophan, melatonin, γ-aminobutyric acid (GABA), etc. and other protein peptides;

[0023] Furthermore, the hexapeptide YAKPAA is prepared into beverages, oral liquids, capsules, pills, sustained-release preparations, injections, microcapsule powders, tablets, granules or emulsion tablets;

[0024] Preferably, the hexapeptide YAKPAA is prepared into microcapsules to improve the gastrointestinal digestion stability, bioavailability and shelf life of the hexapeptide, so that it can be better applied in the food industry and health care fields.

[0025] The third technical solution provided in this scheme is a composition comprising the hexapeptide YAKPAA, wherein the composition has YAKPAA as the only active ingredient and may also contain other components having sleep-aiding activity and / or auxiliary materials.

[0026] Beneficial Effects

[0027] The hexapeptide YAKPAA with sleep-promoting activity provided by the present invention can be chemically synthesized or directionally prepared from bovine casein enzymatic hydrolysates or other mammalian casein enzymatic hydrolysates, can be used alone in health products and medicines for improving sleep, can be used in combination with other components with sleep-promoting activity, and can also be prepared into microcapsules to improve the gastrointestinal digestion stability, bioavailability and shelf life of the hexapeptide, so that it can be better used in the food industry and health care fields.

[0028] The peptide YAKPAA provided by the present invention is derived from food-derived casein hydrolysate, which is safe, nutritious, and has no toxic side effects. A Receptor binding, binding sites are: SER205, ASN60, ASP192, TYR210, TYR58, THR207, THR142, GLU189, activate GABA A 5 mg / kg of YAKPAA can significantly shorten the latency to fall asleep and prolong the sleep time of mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the total ion diagram of casein hydrolysis products.

[0030] Figure 2 This is the secondary mass spectrum of YAKPAA.

[0031] Figure 3 is the peptide sequence of YAKPAA.

[0032] Figure 4 GABA A Schematic diagram of molecular docking of receptor 6HUO and hexapeptide YAKPAA.

[0033] Figure 5 The effects of YAKPAA on sleep latency and sleep duration in mice;

[0034] Where A is the sleep latency and B is the sleep time.

[0035] Figure 6 The effect of YAKPAA on sleep-related neurotransmitters in mouse serum;

[0036] Among them, A is 5-HT, B is GABA, C is NE, D is Glu, E is 5-HIAA, F is β-EP, G is cortisol, and H is GABA / Glu;

[0037] Note: “*”: compared with the control group, p<0.05; “**”: compared with the control group, p<0.01; “***”: compared with the control group, p<0.001.

[0038] Figure 7 The effect of YAKPAA on sleep-related neurotransmitters in mouse brain tissue;

[0039] Among them, A is 5-HT, B is GABA, C is NE, D is Glu, E is β-EP, F is cortisol, and G is GABA / Glu;

[0040] Note: “*”: compared with the control group, p<0.05; “**”: compared with the control group, p<0.01; “***”: compared with the control group, p<0.001; “****”: compared with the control group, p<0.0001. DETAILED DESCRIPTION

[0041] The present invention is described below by specific embodiments. The technical means not particularly described in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0042] The present invention is further explained below through specific implementation modes.

[0043] Example 1 Analysis and Identification of YAKPAA

[0044] (1) Casein pretreatment

[0045] Take 250 kg of casein (cow milk casein) and add it to 1000 kg of water. Stir and soak it at room temperature for 60 min. After stirring and heating to 40-45°C, adjust the pH of the liquid to 7.0±0.2 with 7.5% sodium hydroxide aqueous solution, and stir and fully dissolve it at 40-45°C for 60 min.

[0046] (2) Casein enzymatic hydrolysis

[0047] After no obvious white particles are present in the feed liquid, 0.4‰ (w / w, "‰" is calculated based on the mass of the casein substrate) of a composite protease is added, including neutral protease, papain, and trypsin in a mass ratio of 1:3:1 (the neutral protease is derived from Aspergillus oryzae fermentation, with a preservation number of CGMCC No.7586, and a specific preparation method is described in Example 1 of Chinese invention patent ZL201510176420.X). After half an hour of enzymolysis, the pH of the feed liquid is adjusted to 7.0±0.2 with a 7.5% sodium hydroxide aqueous solution, and the enzymolysis is continued for 2.5 hours, and then 0.5‰ (w / w, "‰" is calculated based on the mass of the casein substrate) of papain is added, and the enzymolysis is continued for 1.5 hours; after the enzymolysis is completed, the enzymatic hydrolyzate is separated by a butterfly separator, and the supernatant is taken, concentrated and sterilized, and then spray-dried to obtain the final casein hydrolyzate.

[0048] (3) Peptide analysis

[0049] The hydrolysate was subjected to LC-MS / MS peptide spectrum analysis to obtain the peptides contained in the casein hydrolysate, and the peptides were sorted and classified. Finally, the bioinformatics method was used to further perform molecular docking on the initially screened peptides, and the final theoretical effective peptides were determined based on the scores. The specific analysis process is as follows:

[0050] ① Pretreatment of peptide samples:

[0051] The spray-dried casein hydrolysate sample was re-dissolved with 100 μL 50 mM NH4HCO3, and DTT solution was added to make the final concentration 10 mmol / L, and reduced in a 56°C water bath for 1 h. Then IAA solution was added to make the final concentration 50 mmol / L, and the reaction was protected from light for 40 min. Finally, the sample was desalted using a self-filled desalting column, and the solvent was evaporated in a 45°C vacuum centrifugal concentrator.

[0052] ② Analysis of casein hydrolysate samples by LC-MS / MS

[0053] Capillary liquid chromatography conditions:

[0054] 1) Analytical column: 150 μm id × 150 mm, packed with Acclaim PepMap RPLC C18, 1.9 μm, 100Å;

[0055] 2) Mobile phase A: 0.1% formic acid; Mobile phase B: 0.1% formic acid, 80% ACN;

[0056] 3) Flow rate: 600 mL / min;

[0057] 4) Analysis time for each component: 66 min. Mobile phase method: 0 min, 4%B; 2 min, 8%B; 45 min, 28%B; 55 min, 40%B; 56 min, 95%B; 66 min, 95%B.

[0058] Mass spectrometry parameters:

[0059] 1) Primary mass spectrometry parameters: Resolution: 70,000; AGCtarget: 3e6; MaximumIT: 100 ms;

[0060] Scanrange: 300 to 1800 m / z.

[0061] 2) Secondary mass spectrometry parameters: Resolution: 17,500; AGCtarget: 1e5; MaximumIT: 50 ms; TopN: 20; NCE / steppedNCE: 28.

[0062] The raw file generated by LC-MS / MS data acquisition of casein hydrolysate sample is opened with Xcalibur, and you can see Figure 1 Total ion current chromatogram shown.

[0063] ③Molecular docking

[0064] The mass spectrometry raw files were analyzed for peptide sequences using the De novo method, and the peptides identified by mass spectrometry were combined with GABA using Schrodinger software. A The receptor 6HUO was docked in batches, and the docking module selected the ligant docking module high-throughput screening option. A The identified peptides were classified and screened based on the docking site of the receptor protein 6HUO, the number of hydrogen bonds between the ligand and the receptor, the number of repetitions of the binding site with benzodiazepines (BZDs), and the protein source. Finally, two pentapeptides (TPTLN, VPYPQ) and two hexapeptides (YVPLGT, YAKPAA) were determined for in vitro chemical synthesis and in vivo animal experimental verification (as shown in Table 1).

[0065] Among them, the secondary mass spectrum of the hexapeptide YAKPAA is as follows Figure 2 The peptide sequence is shown in Figure 3As shown in the figure, the fragment ions of peptides include: N-terminal fragment ions (a, b, c types) and C-terminal fragment ions (x, y, z types). The side chains of a, y, and z type ions break to form d, v, and w type ions respectively. In addition, there are internal ions formed by the breakage of both ends. The b and y series ions are the most common. The primary structure of the peptide can be analyzed and inferred based on the b or y series fragment ions of the peptide. Figure 2 m / z 310.67285 is 1 / 2 of the hexapeptide [M+H + ] ion signal, which is consistent with the molecular weight of the hexapeptide of 619.7097. The hexapeptide was further analyzed by secondary mass spectrometry using in-source collision-induced dissociation technology, and the primary structure of the hexapeptide was determined to be Tyr-Ala-Lys-Pro-Ala-Ala.

[0066] The docking results of the ligant docking module (accuracy option selected) of the hexapeptide YAKPAA and the animal experiments are shown in Examples 2 and 3.

[0067] Table 1: Molecular docking results of four casein hydrolyzed peptides using Schrodinger

[0068]

[0069] Note: The lower the docking score, the better the binding between the small molecule peptide ligand and the protein receptor.

[0070] Example 2: GABA A Molecular docking of receptor 6HUO and hexapeptide YAKPAA

[0071] The YAKPAA hexapeptide in casein hydrolysate has the potential functional activity of helping sleep. In Example 1, the peptides obtained by mass spectrometry identification were subjected to batch molecular docking using the Schrodinger software ligant docking module (high throughput screening option). On this basis, in order to further analyze the structure of YAKPAA and the binding effect of the receptor protein 6HUO, the hexapeptide YAKPAA and GABA were also docked. A The receptor was docked using the ligant docking module (accurate docking option), and the specific results are as follows:

[0072] YAKPAA and GABA A The receptor binding sites are: SER205, ASN60, ASP192, TYR210, TYR58, THR207, THR142, GLU189 ( Figure 4 as shown).

[0073] GABA and GABA A Receptor binding, activation of GABA AReceptor, GABA A The acceptor is a Cl - Channel, GABA A Activation of the receptor leads to channel opening, Cl - The influx of YAKPAA into nerve cells causes the hyperpolarization of the cell membrane, inhibits the excitation of the nerve cells, and thus plays a sedative and hypnotic role. A Receptor binding, directly activating GABA A receptors, increasing GABA A The sensitivity of the receptor to sodium pentobarbital is increased, thereby improving sleep. Based on the molecular docking technology analysis (ligant docking module is selected for molecular docking, and the accuracy docking option is selected), the casein hydrolysate hexapeptide YAKPAA and GABA A The binding energy of receptor binding is relatively high, and the binding energy and the number of hydrogen bonds are -10.316 and 8, respectively (this result is somewhat different from Table 1, because both of them are ligant docking modules selected, but the high-throughput screening option selected for batch docking in Table 1 has a low docking accuracy. After determining the peptide segment according to the screening results in Table 1, the accuracy docking option is reselected to obtain the binding energy and number of hydrogen bonds for accurate docking in this embodiment).

[0074] Example 3: Determination of sleep-promoting activity of YAKPAA

[0075] 1. Experimental Materials

[0076] Experimental animals: ICR mice, male, 8 weeks old (20 ± 2 g), were purchased from Shanghai Slake Experimental Animal Co., Ltd.;

[0077] Normal saline;

[0078] Diazepam tablets (diazepam, Tianjin Lisheng Pharmaceutical Co., Ltd.);

[0079] Sodium pentobarbital (Beijing Chemical Reagent Company);

[0080] GABA (Tianjin Century Weikang Biotechnology Co., Ltd.);

[0081] Hexapeptide YAKPAA (commissioned to Shanghai Jier Biochemical Co., Ltd. for chemical synthesis, SEQ ID NO.1).

[0082] 2. Experimental methods

[0083] ICR mice were allowed to eat and drink freely. All mice were allowed to adapt to the environment for one week (7 days) before the experiment. Then the experimental mice were divided into four groups for drug administration, with 10 mice in each group, namely the control group (Control), diazepam group, GABA group and YAKPAA group. The details are as follows:

[0084] Blank control group: daily gavage with purified water;

[0085] Diazepam group: daily dose was 1 mg / kg bodyweight, twice a day;

[0086] GABA group: daily dose was 83.3 mg / kg bodyweight;

[0087] YAKPAA group: daily dose was 5 mg / kg bodyweight.

[0088] The drug was administered continuously for 7 days, and each group had free access to food and water during the administration period. The experiment started 30 minutes after the last administration. At the beginning of the last administration of the mice, a stopwatch was used to count down for 30 minutes, and then 42 mg / kg of sodium pentobarbital was intraperitoneally injected into the four groups of mice. The injection time T1 was recorded at the same time. When the righting reflex of the mice disappeared, the righting reflex disappearance time T2 was recorded, which was the time when the mice began to fall asleep. When the righting reflex of the mice was restored, it was the time when the mice woke up T3. The sleep latency and sleep duration of the mice were calculated respectively. The shorter the sleep latency and the longer the sleep duration, the better the sleep-aiding effect of the peptide. The calculation formula is as follows:

[0089] Sleep latency = T2-T1; sleep time = T3-T2.

[0090] After the sleep recording was completed, the drug was administered at the above dose for 4 days. One hour after the last administration, the mice were euthanized, blood was collected from the eyeballs, plasma was collected, centrifuged at 3000 rpm, 4°C for 15 min, the supernatant was collected, and frozen in a -80°C refrigerator for subsequent analysis of changes in sleep-related neurotransmitters in serum, including A: 5-HT, B: GABA, C: NE, D: Glu, E: 5-HIAA, F: β-EP, G: cortisol, H: GABA / Glu. At the same time, the mouse brain was collected and placed at -80°C for subsequent analysis of changes in sleep-related neurotransmitters in mouse brain tissue, including A: 5-HT, B: GABA, C: NE, D: Glu, E: β-EP, F: cortisol, and G: GABA / Glu.

[0091] 3. Experimental results

[0092] (1) Sleep time results and analysis

[0093] Figure 5 Middle A and Figure 5 B in the middle represents the sleep latency and sleep time of YAKPAA (the sleep latency and sleep time are also shown in Table 2). The experiment used diazepam as a positive control. The results showed that diazepam can significantly shorten the sleep latency of mice and prolong the sleep time of mice. GABA (83.3 mg / kg) was used as a comparative product in the experiment. The dosage of GABA was selected based on the maximum dose of GABA for humans of 500 mg / 60 kg body weight, which was converted to the corresponding dose of 83.3 mg / kg body weight for mice. The results showed that 5 mg / kg of hexapeptide can significantly shorten the sleep latency of mice (p<0.05), and its effect is better than GABA; at the same time, GABA and YAKPAA were shown to significantly prolong the sleep time of mice (p<0.01), and there was no significant difference between the two effects.

[0094] Table 2 Sleep latency and sleep time in different groups

[0095]

[0096] Note: “*”: compared with the control group, p<0.05; “**”: compared with the control group, p<0.01; “***”: compared with the control group, p<0.001.

[0097] (2) Analysis of the sleep-inducing, sedative and stress-relieving effects of YAKPAA

[0098] The occurrence of sleep is closely related to neurotransmitters in the brain. For example, 5-HT in the brain can participate in the regulation of physiological functions such as appetite, pain, sleep and body temperature. This experiment also analyzed the changes in sleep-related neurotransmitters in mouse serum and brain ( Figure 6 and Figure 7 ) Further confirm the sleep-aiding effect of YAKPAA from multiple angles.

[0099] 5-HT, also known as serotonin, is an important inhibitory neurotransmitter widely present in mammals. Its level is very high in the cerebral cortex and synapses, mainly distributed in the pineal gland and hypothalamus. The increase in the level of 5-HT can play a certain sedative and sleep-promoting role. The experimental results show that compared with the control group, diazepam, GABA and YAKPAA can significantly increase the content of 5-HT in mouse serum ( Figure 6 A), and YAKPAA is better than diazepam in increasing 5-HT levels ( Figure 6 A, p<0.01 and p<0.05), and YAKPAA can also significantly increase the 5-HT content in the mouse brain ( Figure 7 In Figure A, it increased the level of 5-HT better than GABA (p<0.05). This further verifies the sleep-promoting effect of YAKPAA.

[0100] 5-Hydroxyindoleacetic acid (5-HIAA) is one of the metabolites of 5-HT. YAKPAA can also significantly reduce the content of 5-HIAA in mouse serum ( Figure 6 E) (p<0.05), although diazepam and GABA can also partially reduce the content of 5-HIAA in mouse serum, but neither has a significant effect (p>0.05). These results suggest that YAKPAA can partially inhibit the decomposition of 5-HT and play a role in promoting sleep.

[0101] On the other hand, compared with the control group, diazepam, GABA and YAKPAA could significantly reduce the levels of Glu in the serum and brain of mice ( Figure 6 Medium D and Figure 7 D), and YAKPAA is significantly better than diazepam in reducing the level of Glu in the brain ( Figure 7 D, p<0.0001 and p<0.01). Glu is the most abundant excitatory neurotransmitter in the brain. In the Glu system, Glu can activate Glu receptors on the postsynaptic membrane of Glu neurons, thereby triggering excitatory impulses. This suggests that YAKPAA can play a good role in helping sleep by reducing the level of Glu in the brain.

[0102] In addition, diazepam, GABA and YAKPAA can significantly increase the ratio of GABA / Glu ( Figure 6 Medium H and Figure 7 Its effect of upregulating the ratio of GABA / Glu in the brain is similar to that of GABA ( Figure 6 Medium B and Figure 7 Many substances that improve sleep work by improving the imbalance of GABA / Glu inhibition / excitation metabolism and regulating the GABA / Glu ratio.

[0103] On the other hand, diazepam, GABA, and YAKPAA had no significant effect on the NE content in mouse serum ( Figure 6 C), but both can significantly reduce the NE content in the mouse brain ( Figure 7 C), and the reduction effect of YAKPAA is stronger than that of diazepam and GABA (p<0.0001 and p<0.01). Norepinephrine (NE) is also a neurotransmitter that regulates the sleep-wake process. Its release peaks in the wakeful state, and the reduction of NE helps promote sleep. This further shows that YAKPAA can promote sleep by inhibiting the secretion of NE in the mouse brain.

[0104] In addition, YAKPAA can also promote the pleasure and relaxation of mice to a certain extent. The experimental results show that compared with the control group, diazepam, GABA and YAKPAA can significantly increase the content of β-endorphin (β-EP) in the serum of mice ( Figure 6 In contrast, the level of β-endorphin in the serum of mice in the YAKPAA group increased significantly (p<0.01 and p<0.05). However, there was no significant change in β-EP in the brain ( Figure 7 E). β-endorphin is an endogenous morphine-like substance belonging to the opioid peptide family. β-endorphin in serum can cross the blood-brain barrier and produce a sedative effect similar to opium in mice, which has analgesic effects and promotes a sense of pleasure. The increase in β-endorphin content can play a certain role in helping sleep.

[0105] At the same time, YAKPAA can relieve stress to a certain extent. Cortisol is a glucocorticoid. Increased cortisol can lead to anxiety. The experimental results show that compared with the control group, YAKPAA can reduce the content of cortisol in the serum and brain of mice ( Figure 6 Medium G and Figure 7 F), and the reduction reached a significant level in the brain ( Figure 7 (middle F, p<0.05).

[0106] In summary, YAKPAA can promote sleep in mice by increasing 5-HT in the serum and brain of mice, inhibiting the levels of NE and Glu in the brain, and regulating the ratio of GABA / Glu, and has a calming and stress-relieving effect by regulating the secretion of β-endorphin and cortisol.

[0107] Although the present invention has been disclosed as above in terms of preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations in form and details to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. Application of hexapeptide YAKPAA in the preparation of medicines for preventing, improving or treating sleep disorders.

2. Application of hexapeptide YAKPAA in the preparation of food for improving sleep disorders.

3. Application of hexapeptide YAKPAA in the preparation of health products for improving sleep disorders.

4. The use according to any one of claims 1 to 3, characterized in that: The sleep disorders include: reduced sleep time, difficulty falling asleep, insomnia or poor sleep quality.

5. The use according to any one of claims 1 to 3, characterized in that: The hexapeptide YAKPAA is used alone in the preparation of the food, health care product or medicine, or is used in combination with other components having sleep-aiding activity.

6. The use according to any one of claims 1 to 3, characterized in that: The hexapeptide YAKPAA is prepared in the form of beverage, oral liquid, capsule, microcapsule powder, tablet, granule or emulsion.

7. The use according to any one of claims 1 to 3, characterized in that: The hexapeptide YAKPAA is prepared by chemical solid phase synthesis.

8. The use according to any one of claims 1 to 3, characterized in that: The hexapeptide YAKPAA is separated and obtained from casein hydrolysate; the casein is bovine milk casein.

Images