Compositions containing probiotics and use thereof in treating insomnia

Abstract

The present invention provides a probiotic composition and its use in treating insomnia. More specifically, the composition is further added with a sleep-inducing peptide CSM-3 obtained by specific screening, which can improve the activity pattern of insomnia rats in an open field and promote the synthesis of 5-HIAA to improve sleep. The combination of the polypeptide and probiotics can significantly promote the increase of sleep in mice, and has good application prospects.

Description

Technical Field

[0001] The present application relates to the biological field, and in particular to a composition comprising probiotics and use thereof in treating insomnia. Background Art

[0002] Physiological insomnia, as a common disease, has serious adverse effects on people's normal work and life, and may even induce or aggravate a variety of physical diseases. Surveys and studies have shown that about 27% of the world's population suffers from insomnia. And with the increase of life pressure, the incidence of insomnia has shown a clear upward trend. Now research has shown that the body's sleep state is not a simple end of the wakefulness state, but a process of sleep-wakefulness regulated by the central nervous system and actively produced with the participation of related neurotransmitters, cytokines and other substances. The sleep-wakefulness process is a rhythmic physiological behavior that involves active regulation of multiple centers and systems. Therefore, the causes of physiological insomnia also involve a wide range, but it is basically the result of dysfunction of the body's central nervous system, especially the disorder of various neurotransmitters related to sleep.

[0003] Treatments for insomnia include cognitive behavioral therapy, drug therapy, and physical therapy. Early drug therapy was mainly based on benzodiazepines, but due to cognitive decline, falls, mental and motor impairment, changes in sleep structure, drug dependence, and rebound insomnia, it was gradually replaced by non-benzodiazepines. Over time, peptides have been shown to be useful for the treatment of insomnia. For example, studies have shown that bovine milk contains peptides that promote sleep. The results of the study showed that bovine milk-derived sleep-promoting peptides also promote human sleep. Long-term administration of bovine milk-derived phospho-casein peptides had a significant effect on various aspects of sleep over a 4-week period. Subjects treated with bovine milk-derived casein peptides showed a significant decrease in the total score of PSQI-J (Pittsburgh Sleep Quality Index) after 14 days of treatment. This improvement was greater after 28 days of treatment and was still detectable one week after the treatment was stopped. In addition, studies have shown that bovine milk phospho-casein peptides have sleep-promoting properties and enhance pentobarbital sodium-induced sleep in mice. It also promotes slow-wave (δ) EEG activity in rats; indicating a pattern of sleep or relaxation. Experiments have shown that its sleep-promoting effect may be mediated by GABAA (γ-aminobutyric acid type A) receptor-chloride ion channel. Studies have shown that α-casein can reduce anxiety symptoms in cats. DL-4-chlorophenylalanine (PCPA) is an inhibitor of tryptophan hydroxylase (TDH), which is the rate-limiting enzyme for the synthesis of 5-HT (serotonin). After the PCPA-induced insomnia rat model was successfully replicated, the neurotransmitter 5-HT in the brain of the insomnia rat decreased significantly, resulting in the disappearance of the rat's circadian rhythm and insomnia in the rat. Ziziphus jujuba seed and lily are two medicinal and edible ingredients with sedative and hypnotic effects, and are often used in combination with other plants that help sleep. Studies have shown that Ziziphus jujuba seed extract can reduce the number of spontaneous activities in mice, have an inhibitory effect on the central nervous system, and can increase the content of neurotransmitter 5-HT in the central nervous system of mice. Some studies have shown that Ziziphus jujuba seed extract acts on the GABA system; some studies have shown that lily extract acts on the 5-HT pathway and prolongs the sleep time induced by sodium pentobarbital.

[0004] In recent years, scientific research has also shown that some probiotics have good therapeutic effects on body diseases and functions, and have potential beneficial effects on human health, such as regulating the number of bacterial flora; improving host immune defense and barrier function; reducing oxidative stress, etc. Studies have shown that probiotics can be used to treat insomnia. However, the effect of using probiotics alone needs to be further improved. Summary of the invention

[0005] The invention provides a sleep-inducing peptide screened and identified from spinach seeds. The sleep-inducing peptide is named CSM-3, and its amino acid sequence is shown in SEQ ID NO:1.

[0006] Specifically, the sleep-inducing peptide of the present invention comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 1.

[0007] More specifically, the polypeptide may also have conservative substitutions.

[0008] Conservative substitution tables that provide functionally similar amino acids are known to those skilled in the art. For example, the characteristics of amino acid side chains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains with the following common functional groups or characteristics: aliphatic side chains (G, A, V, L, I, P); hydroxyl containing side chains (S, T, Y); sulfur atoms containing side chains (C, M); carboxylic acids and amino compounds containing side chains (D, N, E, Q); bases containing side chains (R, K, H); and aromatic hydrocarbons containing side chains (H, F, Y, W).

[0009] Furthermore, the present invention also provides the use of the sleep-inducing peptide CSM-3 in preparing a pharmaceutical composition for promoting sleep.

[0010] The medicine may further comprise a second medicine for promoting sleep.

[0011] Specifically, the second drug can be a probiotic.

[0012] More specifically, the probiotic is Bacillus coagulans CGMCC9951.

[0013] Specifically, the medicine of the present invention further contains a pharmaceutically acceptable carrier.

[0014] For oral administration, the pharmaceutical composition can be in the form of, for example, tablets, capsules, liquid capsules, suspensions or liquids. The pharmaceutical composition is preferably manufactured in the form of a dosage unit containing a specific amount of active ingredient. For example, the pharmaceutical composition can be provided in tablets or capsules, and the tablets or capsules are included in the active ingredient of the amount in the range of from about 0.1 to 1000 mg, preferably from about 0.25 to 250 mg, more preferably from about 0.5 to 100 mg. Depending on the patient's condition and other factors, the suitable daily dose for people or other mammals may vary greatly, but can be determined using conventional methods.

[0015] Any pharmaceutical composition considered herein can be, for example, delivered orally via any acceptable and suitable oral formulation. Exemplary oral formulations include, but are not limited to, for example, tablets, lozenges, lozenges, aqueous and oily suspensions, dispersible powders or granules, emulsions, hard and soft capsules, liquid capsules, syrups and elixirs. The pharmaceutical composition intended for oral administration can be prepared according to any method known in the art for making the pharmaceutical composition intended for oral administration. In order to provide pharmaceutically palatable preparations, the pharmaceutical composition according to the present invention can contain at least one agent selected from sweeteners, flavoring agents, coloring agents, demulcents, antioxidants and preservatives.

[0016] Pharmaceutically acceptable carriers, adjuvants and vehicles that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-α-tocopheryl polyethylene glycol 1000 succinate, surfactants for pharmaceutical dosage forms such as Tweens, polyethoxylated castor oils such as CREMOPHOR surfactant (BASF), or other similar polymer delivery matrices, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin. Cyclodextrins such as α-, β- and γ-cyclodextrin or chemically modified derivatives such as hydroxyalkylcyclodextrins (including 2- and 3-hydroxypropyl-cyclodextrin) or other solubilized derivatives may also be advantageously used to enhance the delivery of the polypeptides and probiotics described herein.

[0017] In some embodiments, compositions or preparations may include excipients. In some cases, excipients may be pharmaceutically acceptable excipients. Excipients may include, but are not limited to, water, fluidizing agents, lubricants, adhesives, surfactants, acidulants, alkalizers, agents for adjusting pH, antimicrobial preservatives, antioxidants, antistatic agents, buffers, chelating agents, wetting agents or wetting agents. Excipients may also include colorants, coating agents, sweeteners, flavoring agents and aromatics or masking agents. Compositions and preparations may include therapeutic agents with a single excipient or with a variety of excipients in any suitable combination, with or without carriers. In some cases, excipients may include glycerol.

[0018] In some cases, pharmaceutically acceptable excipients can include gum arabic, acesulfame potassium, acetic acid, glacial acetic acid, acetone, acetyl tributyl citrate, acetyl triethyl citrate, agar, albumin, alcohol, alginic acid, fatty polyester, alitame, almond oil, alpha tocopherol, aluminum hydroxide adjuvant, aluminum oxide, aluminum phosphate adjuvant, aluminum stearate, ammonia solution, ammonium alginate, ascorbic acid, ascorbyl palmitate, aspartame, attapulgite, bentonite, benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, benzyl benzoate, boric acid, bronopol, butylated hydroxyanisole, butylated hydroxytoluene, butyl paraben, calcium alginate, calcium carbonate, anhydrous calcium hydrogen phosphate, dihydrated calcium hydrogen phosphate, tribasic calcium phosphate, Calcium stearate, calcium sulfate, rapeseed oil, carbomer, carbon dioxide, carboxymethylcellulose calcium, carboxymethylcellulose sodium, carrageenan, castor oil, hydrogenated castor oil, cellulose (e.g., microcrystalline cellulose, powdered cellulose, silicified microcrystalline cellulose, cellulose acetate, cellulose acetate phthalate), carob bean gum, cetearyl alcohol, cetyl trimethylamine bromide, cetyl alcohol, cetylpyridinium chloride, chitosan, chlorhexidine, chlorobutanol, chlorocresol, chlorodifluoroethane, chlorofluorocarbon, chloroxylenol, cholesterol, citric acid monohydrate, colloidal silicon dioxide, colorant, copovidone, corn oil, cottonseed oil, cresol, croscarmellose sodium, crospovidone, cyclodextrin, cyclomethicone, denatonium benzoate, dextran, dextrin, dextrose, dibutyl phthalate , dibutyl sebacate, diethanolamine, diethyl phthalate, difluoroethane, dimethicone, dimethyl ether, dimethyl phthalate, dimethyl sulfoxide, dimethylacetamide, disodium edetate, sodium docusate, edetate, isoascorbic acid, erythritol, ethyl acetate, ethyl lactate, ethyl maltol, ethyl oleate, ethyl vanillin, ethyl cellulose, ethylene glycol palmitostearate, ethylene vinyl acetate, ethyl paraben, fructose, fumaric acid, gelatin, glucose, glycerin, glyceryl behenate, glyceryl monooleate, glyceryl monostearate, glyceryl palmitostearate, tetraethylene glycol ether (glycofurol), guar gum, hectorite, heptafluoropropane, hexetidine, hydrocarbons, hydrochloric acid, hydroxyethyl cellulose, hydroxyethyl Methylcellulose, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, hydroxypropyl starch, hypromellose, hypromellose acetate succinate, hypromellose phthalate, honey, imidazolidinyl urea, inulin, iron oxide, isomalt, isopropyl alcohol, isopropyl myristate, isopropyl palmitate, kaolin, lactic acid, lactitol, anhydrous lactose, lactose monohydrate, spray-dried lactose, lanolin, lanolin alcohol, hydrous lanolin, lauric acid, lecithin, leucine, linoleic acid, polyethylene glycol hydroxystearate, aluminum magnesium silicate, magnesium carbonate, magnesium oxide, magnesium silicate, magnesium stearate, magnesium trisilicate, malic acid, maltitol, maltitol solution, maltodextrin, maltol, maltose, mannitol, medium chain triglycerides, meglumine,Menthol, methylcellulose, methylparaben, mineral oil, light mineral oil, monoethanolamine, monosodium glutamate, monothioglycerol, myristic acid, neohesperidin dihydrochalcone, nitrogen, nitrous oxide, octyldodecanol, oleic acid, oleyl alcohol, olive oil, palmitic acid, paraffin, peanut oil, pectin, petrolatum, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, phosphoric acid, polacrilin potassium, poloxamer, polycarbophil, polydextrose, polyethylene glycol, polyethylene oxide, polymethacrylate, poly(methyl vinyl ether) / Maleic anhydride), polyoxyethylene alkyl ether, polyoxyethylene castor oil derivative, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene stearate, polyvinyl acetate phthalate, polyvinyl alcohol, potassium alginate, potassium benzoate, potassium bicarbonate, potassium chloride, potassium citrate, potassium hydroxide, potassium metabisulfite, potassium sorbate, povidone, propionic acid, propyl gallate, propylene carbonate, propylene glycol, propylene glycol alginate, propylparaben, 2-pyrrolidone, raffinose, saccharin, saccharin sodium, saponite, sesame oil, shellac, simethicone, ethyl sodium alginate, sodium ascorbate, sodium benzoate, sodium bicarbonate, sodium borate, sodium chloride, sodium citrate dihydrate, sodium cyclamate, sodium hyaluronate, sodium hydroxide, sodium lactate, sodium lauryl sulfate, sodium metabisulfite, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium propionate, sodium starch glycolate, sodium stearyl fumarate, sodium sulfite, sorbic acid, sorbitan esters (sorbitan fatty acid esters), sorbitol, soybean oil, starch, starch (e.g., pregelatinized, sterilized corn), stearic acid, stearyl alcohol, sucralose, sucrose, sugar, Compressed sugar, candy, sugar spheres, sulfobutyl ether beta-cyclodextrin, sulfuric acid, sunflower oil, suppository base, hard fat, talc, tartaric acid, tetrafluoroethane, sweet protein, thimerosal, thymol, titanium dioxide, tragacanth gum, trehalose, triacetin, tributyl citrate, triethanolamine, triethyl citrate, vanillin, hydrogenated vegetable oil, water, wax, anionic emulsifier, wax (such as carnauba wax, cetyl esters, microcrystalline, non-ionic emulsifier, white wax, yellow wax), xanthan gum, xylitol, zein, zinc acetate, zinc stearate or any combination thereof.

[0019] In some embodiments, the composition or formulation may include a buffer. In some cases, the buffer may include potassium phosphate, sodium acetate, sodium citrate, sodium phosphate, trisodium citrate, or a combination thereof. In some cases, the composition or formulation may include a pH adjusting agent, such as acetic acid, citric acid, hydrochloric acid, sodium hydroxide, sulfuric acid, or a combination thereof.

[0020] In some embodiments, compositions described herein or preparation can include penetration enhancers. In some cases, penetration enhancers can be materials that can promote drug penetration into skin, mucous membrane, nerve sheath or through another barrier (e.g., mucous membrane tissue). In some cases, penetration enhancers can be at least partially: inert, nontoxic, nonirritating, non-allergic, compatible with medicine and excipient, odorless, tasteless, colorless or any combination thereof. In some cases, penetration enhancers can be fatty acids, alcohols, surfactants, solvents, hydrogen bond receptors or any combination thereof. In some cases, the penetration enhancer can include azone (1-dodecylazacycloheptane-2-one), dimethyl sulfoxide, dimethylacetamide, dimethylformamide, ethanol, propylene glycol, N-methylpyrrolidone, oleic acid, lauryl alcohol, ketone terpenes, terpenes, sulfoxides, alkanols, organic acids, alcohols, polyols, pyrrolidone, glycols, urea and urea derivatives, enzymes, iminosulfurane, cyclodextrin, fatty acid esters, surfactants, polymers, glyceryl monooleate, oxazolone or any combination thereof. In some cases, the penetration enhancer can include cyclodextrin, sodium hyaluronate, cremophor RH40, chitosan, cyclopentyl adenosine, dextran or any combination thereof. In some cases, the preparation herein can include an enzyme regulator.

[0021] Furthermore, the probiotics are used at a concentration of 1-9×10 8 CFU / mL, 1-50mL / kg. More preferably, the dosage is 1×10 8 CFU / mL, 20mL / kg.

[0022] The dosage of the polypeptide is 1-10 mg / kg·d, more preferably 50 mg / kg·d.

[0023] The polypeptide of the present invention was administered intragastrically to mice at a high dose of 100 mg / kg / d for 40 consecutive days, and the mice had no obvious toxic side effects.

[0024] Beneficial Effects

[0025] The present invention provides a probiotic composition and its use in treating insomnia. More specifically, the composition is further added with a sleep-inducing peptide CSM-3 obtained by specific screening, which can improve the activity pattern of insomnia rats in an open field, promote the synthesis of 5-HIAA, and thus improve sleep. The combination of the polypeptide and probiotics can significantly promote the effect of increasing sleep in mice, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The effect of peptides on 5-HIAA, a metabolite of 5-HT, in the hypothalamus of rats with insomnia induced by PCPA DETAILED DESCRIPTION

[0027] The specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although the specific embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to enable the scope of the present invention to be fully communicated to those skilled in the art.

[0028] Example 1 Screening and identification of sleep-promoting peptides

[0029] Take the Chinese jujube kernel, crush it and pass it through a No. 4 sieve, soak it in petroleum ether [solid-liquid ratio 1:5 (g / mL)] for 24 hours, centrifuge it at 3000r / min for 10 minutes at room temperature 25°C, dry the precipitate in a fume hood, pass it through a No. 4 sieve, and obtain defatted Chinese jujube kernel powder. Take the defatted Chinese jujube kernel powder, add distilled water to dissolve it at a solid-liquid ratio of 1:25 (g / mL), adjust the solution pH to 11 with 1mol / L NaOH solution, extract it at a constant temperature of 57°C for 50 minutes, centrifuge it at 3000r / min for 20 minutes, filter it to obtain the supernatant, adjust the solution pH to 4 with 1mol / L hydrochloric acid solution, let it stand at 4°C for 2 hours, centrifuge it at 3000r / min for 10 minutes, redissolve the precipitate with water, adjust the solution pH to 7 with 1mol / L NaOH, dialyze it for 48 hours, and freeze-dry it to obtain Chinese jujube kernel protein. Take the jujube kernel protein, add distilled water to dissolve it into a 5% protein solution, and adjust the alkaline protease and papain to the optimal temperature and optimal pH value according to the mass fraction of the enzyme to the substrate of 3%, and perform enzymolysis for 4 hours respectively. After the enzymolysis is completed, place the solution in a 90°C water bath to inactivate the enzyme for 10 minutes, centrifuge at 6000r / min for 20 minutes, take the supernatant and freeze-dry.

[0030] The supernatant was prepared into a 300 mg / ml solution with ultrapure water, extracted with a syringe and filtered through a 0.22 μm filter membrane, and then the hypnotic peptide was initially separated by preparative high performance liquid chromatography (LC-8A, Shimadzu). Chromatographic conditions: The chromatographic column was a self-assembled glass column (20 mm × 450 mm, C18 filler (10 μm, MachereyNagel, France); mobile phase: A pump is ultrapure water (containing 0.1% TFA), B pump is acetonitrile (containing 0.1% TFA); flow rate: 10mL / min; injection volume: 5mL; detection wavelength: 214nm, 280nm; elution conditions: 10%-20% (0.01-30min), 20%-35% (30-90min), 35%-52.5% (90-110min), 52.5%-90% (110-125min). Seven components were obtained by preliminary separation, and the samples of the seven components were subjected to preliminary activity evaluation using the patch clamp model. It was found that component 4 had a relatively good sleep-promoting effect, and then component 4 was separated and purified. Chromatographic conditions: Welch C18 column (4.6 mm × 250 mm, 5 μm, Ultimate); mobile phase: ultrapure water (containing 0.1% TFA) for pump A, acetonitrile (containing 0.1% TFA) for pump B; injection volume: 30 ul, flow rate: 1.5 mL / min; detection wavelength: 214 nm, 280 nm; elution conditions: 10%-20% (0.01-15 min), 20%-30% (15-35 min), 30%-90% (35-50 min). According to the activity peak, the No. 4 component sample was divided into four sections. The activity of the four segments was evaluated by the patch clamp model, and the second segment product had the best activity. The second segment product was further separated by Welch C18 column (4.6 mm × 250 mm, 5 μm, Ultimate); mobile phase: A pump is ultrapure water (containing 0.1% TFA), B pump is acetonitrile (containing 0.1% TFA); injection volume: 20 μL; flow rate: 1.5 mL / min; detection wavelength: 214 nm, 280 nm; elution conditions: 10%-20% (0.01-8 min), 20%-30% (8-30 min), 30%-90% (30-480 min). Three active fragments were further obtained, among which the first segment had the highest activity identified by the patch clamp model. The first segment was subjected to mass spectrometry sequence identification and then artificial peptide synthesis, named CSM-3, and its amino acid sequence is shown in SEQ ID NO: 1.

[0031] Example 2 Identification of CSM-3 sleep-promoting peptide activity

[0032] After 7 days of adaptation to the basic diet, SPF SD rats were randomly divided into 5 groups (n=10) according to their body weight. During the experiment, each group of animals was housed in a single cage, with an indoor temperature of 22±2℃ and a humidity of 50%±10%. The breeding environment was automatically controlled with 12h light and 12h dark, and the animals were free to eat and drink. After the rats adapted for 7 days, except for the blank group, the rats in the other groups were intraperitoneally injected with PCPA 300mg / kg at 8:00-8:30 am every day, with a gavage volume of 10mL / kg, once a day, for 2 consecutive days. The blank group was intraperitoneally injected with the same volume of weakly alkaline 0.9% sodium chloride injection as PCPA.

[0033] After the rat insomnia model was successfully replicated, the drug-administered groups were given samples by gavage at 8:00 am every day. The peptide dosages were 10 mg / kg·d (low-dose group) and 50 mg / kg·d (high-dose group), respectively; the diazepam control group was given a dosage of 3 mg / kg·d. The drugs were administered for 7 consecutive days, and the blank group and the model group were given an equal amount of normal saline, and the gavage volume of each group was 20 mL / kg. Fasting for 12 hours before the last administration (dose on the 7th day). The mine field experiment was carried out after the last administration. The open field reaction box was 50 cm high and 50 cm long on the bottom side. The internal area of ​​the open field was set as a 4×4 grid, with the middle 4 grids combined as the central area and the outer grids combined as the peripheral area. The open field was placed directly under the incandescent lamp, and the camera position was adjusted to be directly above the open field. The open field experiment was carried out in a quiet environment. The experimenter placed the animals at a uniform angle in the center of the bottom surface of the box and then took pictures and timed them. The activities of the rats were spontaneously recorded by the camera. Each rat was tested for 10 minutes. After the test, the open field was cleaned and the inner wall and bottom of the box were cleaned to prevent odor and excrement from being left behind. The test indicators included: the total distance of the rat's open field movement, the number of times the rat stayed in the center of the open field, the central stay time, etc. The results are shown in Table 1.

[0034] Table 1 Effects of peptides on open field exercise in rats with insomnia induced by PCPA

[0035] Group Central stop times Time spent in Central Area Total movement distance / cm Low dose group 12.69±0.32# 15.87±0.80# 3546.47±84.13# High dose group 10.99±0.17# 13.78±0.62# 3484.71±56.47# Model Group 19.15±0.34 39.41±0.77 5079..34±93.54 Blank group 10.53±0.28# 13.56±0.56# 3897.52±72.11# Positive control group 12.48±0.45# 18.59±0.43# 3598.18±45.73#

[0036] Compared with the insomnia model group, #p<0.05.

[0037] From Table 1, the number of times the rats in the insomnia model group stayed in the center, the time they stayed in the central area, and the total distance they moved were significantly different from those in the blank group (p<0.05). The above indicators reflect to a certain extent that the rats in the insomnia model are more nervous and more active in the open field. The number of times the rats in the low-dose group and the high-dose group stayed in the center, the time they stayed in the central area, and the total distance they moved were significantly different from those in the insomnia model group (p<0.05), and there was no significant difference compared with the blank group (p>0.05). This result suggests that the polypeptide of the present invention can improve the number of times the rats in the insomnia model group stayed in the center, the time they stayed in the central area, and the total distance they moved.

[0038] Furthermore, each group was intraperitoneally injected with 10% chloral hydrate at a dose of 0.3 g / kg 60 minutes after gavage, and then the rat brain was taken out for ultra-performance liquid chromatography-tandem mass spectrometry to determine the effect of the peptide on the metabolite 5-HIAA of 5-HT in the hypothalamus of PCPA-induced insomnia rats. The results are as follows: Figure 1 shown.

[0039] Studies have shown that sleep is positively correlated with cerebrospinal fluid pentylhydroxyindoleacetic acid (5-HIAA), which is Figure 1 The results showed that the 5-HIAA content in the hypothalamus of rats in the insomnia model group was significantly lower than that in the blank group (p<0.05); after treatment, the 5-HIAA content in the low-dose and high-dose peptide groups was significantly increased compared with the model group, indicating that the peptide promoted the synthesis of 5-HIAA and thus improved sleep.

[0040] Example 3 Experiment on the combined application of CSM-3 sleep-inducing peptide and probiotics

[0041] Bacillus coagulans CGMCC9951 was purchased from the Microbial Breeding and Metabolic Regulation Laboratory of the School of Food and Bioengineering, Henan University of Science and Technology. The bacteria were activated to prepare seed liquid, which was fermented in a 5L fermenter at 37°C for 96 hours. The fermentation liquid was mixed with 5% corn starch by mass and spray-dried at 160°C to obtain bacterial powder. Accurately weigh the Bacillus coagulans CGMCC9951 powder in 0.85% sterile saline to obtain 1×10 8 CFU / mL. After preparation, store at 4℃ for later use.

[0042] Clean-grade female ICR mice, weighing 20 ± 2 g, were purchased from Fujian Anbuli Biotechnology Co., Ltd. The animals used in each experiment were randomly divided into 5 groups, with 10 mice in each group.

[0043] Peptide group: intragastric administration, intragastric dose 50 mg / kg·d, 20 mL / kg;

[0044] Probiotic group: intragastric administration of Bacillus coagulans 1×10 8 CFU / mL, 20mL / kg;

[0045] Peptide combined with probiotics group: Oral administration, peptide oral administration, 50 mg / kg·d, 20 mL / kg; after an interval of 2 hours, oral administration of 1×10 Bacillus coagulans 8 CFU / mL, 20mL / kg;

[0046] Positive control group: intragastric administration, the intragastric dose of diazepam was 3 mg / kg·d, 20 mL / kg;

[0047] The above groups were given drugs continuously for 30 days, and the blank control group was gavaged with the same volume of distilled water.

[0048] 15 minutes after the last administration, each group of mice was intraperitoneally injected with sodium pentobarbital 50 mg / kg, with an injection volume of 0.2 mL / 20 g. The disappearance of the righting reflex of mice was used as an indicator to observe whether the test sample could prolong the sleeping time of sodium pentobarbital. If the sleeping time of mice in the drug administration group increased and was significantly different from that of the control group, the result was judged to be positive. The results are shown in Table 2.

[0049] Table 2 Effects of each group on prolonging sleep time with sodium pentobarbital

[0050] Group Sleep time (min) Peptide Group 56.6±1.9# Probiotics group 52.7±2.0# Peptide combined with probiotics group 64.5±3.7# Positive control group 55.8±3.6# Blank control group 48.7±2.5

[0051] As shown in Table 3, under the hypnotic effect of sodium pentobarbital (50 mg / kg), the sleep time of mice in the polypeptide group, probiotic group, polypeptide combined with probiotic group and positive control group was significantly prolonged compared with the blank control group (P < 0.05), especially after the polypeptide combined with probiotics, it can synergistically increase the sleep time of mice, with better effect. The above results show that polypeptides and / or probiotics can effectively prolong the sleep time of mice induced by sodium pentobarbital.

[0052] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A sleep-inducing peptide screened and identified from Ziziphus jujuba seeds, wherein the sleep-inducing peptide is CSM-3, and its amino acid sequence is shown in SEQ ID NO:

1.

2. Use of the sleep-promoting peptide CSM-3 as claimed in claim 1 in the preparation of a pharmaceutical composition for promoting sleep.

3. A medicine box for treating insomnia, characterized in that The invention is composed of the sleep-inducing peptide CSM-3 described in claim 1 and the probiotic Bacillus coagulans CGMCC9951.

4. Use of the sleep-promoting peptide CSM-3 and the probiotic Bacillus coagulans CGMCC9951 according to claim 1 in preparing a medicine kit for treating insomnia.

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