Use of bovine spleen peptide powder in improving sleep, preventing or treating depression, or improving intestinal function

Abstract

The present invention provides the use of bovine spleen peptide powder in the manufacture of a food product for improving sleep in a subject, alleviating depression in a subject, or improving bowel function in a subject.The present invention also provides the use of bovine spleen peptide powder in the manufacture of a medicament for use in preventing or treating sleep disorders or depression in a subject, or for use in improving bowel function in a subject.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to Chinese Patent Applications Nos. 202211570537.2, 202211570333.9, and 202211570539.1, filed on December 8, 2022, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates generally to the fields of biomedicine and food, and in particular, the present invention relates to the use of bovine spleen peptide powder in the manufacture of a food product for use in improving sleep in a subject, alleviating depression in a subject, or improving bowel function in a subject, or in the manufacture of a medicament for use in preventing or treating a sleep disorder or depression in a subject, or in the manufacture of a medicament for use in improving bowel function in a subject. [Background technology]

[0003] Sleep disorders refer to various functional disorders that occur during the sleep-wake process, such as insomnia, hypersomnia, sleep-disordered breathing, and sleep behavior disorders due to various causes. Clinical symptoms mainly include abnormal sleep quantity (excess or insufficient) and narcoleptic disorders during sleep (sleepwalking, sleep talking, nightmares, night terrors, teeth grinding, involuntary movements of muscles and limbs). Many sleep disorders are caused by long-term ideological conflict, excessive mental strain, mental labor, improper management of the balance between work and rest over a long period of time, and weakness following illness.

[0004] According to the 2017 Guidelines for the Diagnosis and Treatment of Insomnia in China, insomnia is the most common clinical sleep disorder and is characterized by frequent and persistent difficulty falling asleep and / or staying asleep, resulting in sleep dissatisfaction. Among adults, 10% to 15% meet the diagnostic criteria for insomnia, and insomnia often has a chronic course, with nearly half of severe cases lasting for 10 years or more. Insomnia can cause fatigue, anxiety, general discomfort, lethargy, sluggish reactions, headaches, and attention deficits. In severe cases, it can lead to schizophrenia, depression, anxiety disorders, and functional disorders such as neurological dysfunction, as well as disorders of various systems (e.g., circulatory system, digestive system, etc.).

[0005] Currently, Western medicine primarily uses pharmaceutical treatments, which can be divided into six categories: (1) benzodiazepines, (2) antidepressants, (3) antihistamines, (4) barbiturates and non-barbiturates, (5) antipsychotics and other sedatives, and (6) melatonin. Benzodiazepines are the most commonly used, and their main pharmacological effects are anxiolytic, sedative, hypnotic, anticonvulsant, and skeletal muscle relaxant. Their main mechanism of action is to stimulate the release of inhibitory neurotransmitters such as gamma-aminobutyric acid (GABA) and glycine from inhibitory neurons in the brain, thereby suppressing the production and release of 5-HT, thereby producing anxiolytic effects. However, commonly used benzodiazepines and non-benzodiazepine sedatives and hypnotics can cause serious side effects when used in large doses over a long period of time, making patients susceptible to tolerance and dependence.

[0006] In traditional Chinese medicine conditioning, treatments such as herbal prescriptions, acupuncture, and acupressure massage are advocated, but these have the drawback of requiring a long treatment period and slow effectiveness.

[0007] Depression is a common mental disorder that manifests as a unique combination of symptoms, including persistently low mood, lack of interest, lack of sexual desire, difficulty concentrating, sleep disturbances, fatigue, suicidal thoughts, and physical impairment.

[0008] Currently, depression is the leading cause of disability worldwide, its annual economic burden is among the highest among clinical diseases, and it has become a serious public health problem. Regarding the use of antidepressants, the Expert Opinion on the Treatment and Management of Depression (2022) states the following:

[0009] (1) According to guidelines in China and other countries, the first-line drugs recommended for monotherapy of depression are (1) serotonin (5-hydroxytryptamine) reuptake inhibitors (SSRIs), (2) serotonin-norepinephrine reuptake inhibitors (SNRIs), (3) norepinephrine-dopamine reuptake inhibitors (NDRIs), and (4) other antidepressants. Among these, melatonin receptor MT1 and MT2 agonists and serotonin receptor 5-HT2C antagonists are represented by agomelatine.

[0010] (2) Combination of antidepressants: In the clinical treatment of depression, the use of a single drug is often ineffective, especially in improving anxiety symptoms, so the use of a combination of antidepressants is very common. Although the combination of venlafaxine and mirtazapine is more widely used clinically, this regimen has always lacked advanced clinical evidence. Furthermore, the combination of drugs inevitably requires high doses, has problems with drug interactions, and often leads to more side effects. Its actual therapeutic efficacy is controversial, so it is not currently recommended.

[0011] Bowel dysfunction refers to gastrointestinal dysfunction, and patients with gastrointestinal dysfunction often experience constipation. The American Gastroenterological Association (AGA) and the American College of Gastroenterology (ACG) have published evidence-based practical recommendations for the pharmacological treatment of CIC in adults, including the use of various over-the-counter (OTC) and prescription medications. Among these, (1) fiber, (2) osmotic laxatives, (3) stimulant laxatives, (4) secretagogues, and (5) 5-HT4 receptor agonists are commonly used. However, abdominal distension and flatulence, nausea, and diarrhea are common dose-dependent side effects.

[0012] There is a great need to develop natural foods (special medical formulated foods, health foods, etc.) that are safe, mild, and have no side effects, and that are effective in improving sleep, alleviating depression, or improving intestinal function in subjects, as well as pharmaceuticals that are effective in preventing and treating sleep disorders and depression in subjects, or improving intestinal function, in order to alleviate and resolve patients' suffering.

[0013] Bovine spleen peptides are typically made from pure, natural bovine spleen, and are processed through a series of techniques, including pulverization, sterilization, biological enzyme hydrolysis, enzyme inactivation, purification, concentration, centrifugal spraying, and drying, to produce a low-molecular-weight bovine spleen peptide powder. This peptide powder is highly active and easily absorbed by the human body. It helps relieve fatigue, boosts immunity, relieves the effects of alcohol, protects the liver, and lowers blood lipids and blood pressure. Summary of the Invention

[0014] According to a first aspect, the present invention provides the use of bovine spleen peptide powder in the manufacture of a food product for improving sleep in a subject, or in the manufacture of a medicament for preventing or treating a sleep disorder in a subject.

[0015] In some embodiments of the first aspect, the subject suffers from a sleep disorder. In some embodiments of the first aspect, the sleep disorder is one or more selected from the group consisting of insomnia, sleep-disordered breathing, central hypersomnia, circadian rhythm sleep-wake disorder (CRSWD), parasomnia, and sleep movement disorder.

[0016] In some embodiments of the first aspect, improving sleep in a subject or treating a sleep disorder in a subject comprises one or more of increasing the subject's sleep time, increasing the subject's sleep efficiency, shortening the subject's sleep onset latency, decreasing the subject's amount of wakefulness activity after sleep onset, and shortening the subject's wakefulness activity time after sleep onset.

[0017] According to a second aspect, the present invention provides the use of bovine spleen peptide powder in the manufacture of a food product for alleviating depression in a subject, or in the manufacture of a medicament for preventing or treating depression in a subject.

[0018] According to a third aspect, the present invention provides the use of bovine spleen peptide powder in the manufacture of a food product for improving bowel function in a subject, or in the manufacture of a medicament for improving bowel function in a subject. In some embodiments of the third aspect, improving bowel function in the subject comprises lubricating the intestinal tract and / or preventing, ameliorating, or treating constipation.

[0019] In some embodiments of any of the first to third aspects, the bovine spleen peptide powder is used at a concentration of 250 μg / mL or less. In some embodiments of any of the first to third aspects, the bovine spleen peptide powder, or the bovine spleen peptide powder together with a food supplement, is manufactured as a food product. In some embodiments of any of the first to third aspects, the food product is a special medical formulated food (a food product formulated for a special medical purpose). In some embodiments of any of the first to third aspects, the food is a health food.

[0020] In some embodiments of any of the first to third aspects, the bovine spleen peptide powder is formulated into a pharmaceutically acceptable dosage form together with a pharmaceutically acceptable carrier. In some embodiments of any of the first to third aspects, the medicament is administered orally, subcutaneously, intramuscularly, or intraperitoneally. In some embodiments of any of the first through third aspects, the subject is a vertebrate. In some embodiments of any of the first through third aspects, the subject is a fish, a mammal, a cyclostome, an amphibian, a reptile, or a bird. In some embodiments of any of the first through third aspects, the subject is a fish or a mammal.

[0021] In some embodiments of any of the first through third aspects, the bovine spleen peptide powder is prepared by the following steps: crushing and homogenizing bovine spleens to obtain a spleen slurry; a step of freezing and thawing the spleen slurry; subjecting the frozen-thawed spleen slurry to solid-liquid separation and recovering the supernatant; filtering the supernatant to obtain a bovine spleen extract; sterilizing the bovine spleen extract; drying the sterilized bovine spleen extract to obtain the bovine spleen peptide powder; The method is produced by a method including at least one of the following steps.

[0022] In some embodiments of any of the first to third aspects, the content of the polypeptide in the bovine spleen peptide powder is 150 to 200 mg / g. In some embodiments of any of the first to third aspects, the molecular weight of the polypeptide molecules in the bovine spleen peptide powder is less than 17 KD. In some embodiments of any of the first to third aspects, the content of amino acids in the bovine spleen peptide powder is 400 to 450 mg / g. According to a fourth aspect, the present invention provides a method for improving sleep in a subject, comprising administering to said subject an effective amount of bovine spleen peptide powder. According to a fifth aspect, the present invention provides a method for preventing or treating a sleep disorder in a subject, comprising administering to the subject an effective amount of bovine spleen peptide powder.

[0023] According to a sixth aspect, the present invention provides a method for alleviating depression in a subject, comprising administering to the subject an effective amount of bovine spleen peptide powder. According to a seventh aspect, the present invention provides a method for preventing or treating depression in a subject, comprising administering to the subject an effective amount of bovine spleen peptide powder. According to an eighth aspect, the present invention provides a method of improving bowel function in a subject, comprising administering to the subject an effective amount of bovine spleen peptide powder. According to a ninth aspect, the present invention provides a method for preventing, ameliorating or treating constipation in a subject, comprising administering to the subject an effective amount of bovine spleen peptide powder. [Brief explanation of the drawings]

[0024] [Figure 1] FIG. 1 shows a flow chart for the production of bovine spleen peptide powder. [Figure 2] FIG. 1 shows a standard curve graph for detecting polypeptide content in bovine spleen peptide powder. [Figure 3] FIG. 1 shows the results of SDS-PAGE electrophoresis of bovine spleen peptide powder. [Figure 4] FIG. 1 shows the results of HPLC detection of amino acid standards. [Figure 5] FIG. 1 shows the results of HPLC detection of the amino acid content of bovine spleen peptide powder. [Figure 6A-B] Representative figures showing the migration trajectories of zebrafish treated with bovine spleen peptide powder, where the black line in A corresponds to the black line in B and represents the distance traveled at slow speeds, the green line in A corresponds to the light gray line in B and represents the distance traveled at intermediate speeds, and the red line in A corresponds to the dark gray line in B and represents the distance traveled at fast speeds. [Figure 7]FIG. 1 shows the level of waking activity in zebrafish after administration of bovine spleen peptide powder, where * indicates p<0.05 and *** indicates p<0.001 when compared with the model control. [Figure 8] FIG. 1 shows the waking activity time of zebrafish after treatment with bovine spleen peptide powder, where * indicates p<0.05 and *** indicates p<0.001 when compared to the model control. [Figure 9] FIG. 1 shows the percentage of bright field movement time in zebrafish after treatment with bovine spleen peptide powder, where * represents p<0.05 and ** represents p<0.01 when compared with model control. [Figure 10] FIG. 1 shows the bright field movement distance of zebrafish after treatment with bovine spleen peptide powder, where *** indicates p<0.001 when compared with the model control. [Figure 11] FIG. 1 is a typical diagram showing the number of intestinal goblet cells in zebrafish after treatment with bovine spleen peptide powder. The dashed line represents the intestinal tract in the analyzed area, and the particles represent goblet cells. [Figure 12] FIG. 1 shows the number of intestinal goblet cells after treatment with bovine spleen peptide powder, where *** indicates p<0.001 when compared with model control. [Figure 13] FIG. 1 is a typical diagram showing the fluorescence intensity of the gastrointestinal tract of zebrafish after treatment with bovine spleen peptide powder. The dashed box in the exemplary graph is the analyzed region of the gastrointestinal tract of zebrafish. [Figure 14] FIG. 1 shows the gastrointestinal fluorescence intensity of zebrafish after treatment with bovine spleen peptide powder, where *** indicates p<0.001 when compared with the model control. DETAILED DESCRIPTION OF THE INVENTION

[0025] Detailed Description of the Invention The present inventors have experimentally found that bovine spleen peptide powder contributes to improving sleep quality, antidepressant effects, and improving intestinal function (e.g., intestinal lubrication, prevention, amelioration, or treatment of constipation) in subjects. Therefore, the bovine spleen peptide powder of the present invention can be used to improve a subject's sleep, alleviate a subject's depression (e.g., effectively alleviate depressed mood and the anxiety and tension associated therewith), or to manufacture foods (e.g., special medical formula foods or health foods) for improving a subject's intestinal function, or to manufacture pharmaceuticals for preventing or treating sleep disorders or depression in a subject, or for improving a subject's intestinal function, thereby opening up new uses for bovine spleen peptide powder.

[0026] The practice of the present invention will employ, unless otherwise indicated, techniques of molecular biology, microbiology, cell biology, biochemistry and immunology, which are conventional in the art. Unless otherwise specified, terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0027] [Definition] In this specification, a state in which the zebrafish's movement speed exceeds 20 mm / s is considered to be an awake state, a state in which the movement speed is less than 4 mm / s is considered to be a sleep state, and a state in which the movement speed is between 4 and 20 mm / s is considered to be a state corresponding to sleep onset latency.

[0028] In this specification, the term "special medical formula food" refers to special foods that provide nutritional supplements to people with special health conditions, and the term "health food" refers to foods with health functions. The definitions of these two types of foods vary depending on the regulations and requirements of each country.

[0029] In China, foods for special medical purposes (FSMP) refer to a special category of foods developed to provide nutritional supplementation to people with specific diseases or special health conditions. They have beneficial effects on improving disease treatment efficacy and postoperative rehabilitation efficacy, improving patients' nutritional status, enhancing the body's own resistance, and improving the patient's overall health level. Special medical foods are specially processed and formulated to meet the special nutritional and dietary needs of people with dietary restrictions, digestive and absorption disorders, metabolic disorders, or specific disease conditions. Such products should be taken alone or in combination with other foods under the guidance of a doctor or clinical nutritionist.

[0030] In China, health foods refer to foods that have health functions or are intended to supplement nutrients such as vitamins and minerals. Health foods are foods that are suitable for consumption by specific people (groups), have the function of regulating the body, are not intended to treat diseases, and do not cause acute, subacute, or chronic harm to the human body.

[0031] In the United States, the categories closest to the classification of health foods are dietary supplements and medical foods, which are classified by function. Dietary supplements are orally ingested products containing "dietary ingredients" used to supplement the diet, which include substances such as vitamins, minerals, herbal medicines, amino acids, enzymes, organ tissues, glands, and metabolites. Medical foods are defined in Section 5(b)(3) of the Orphan Drug Act (21 USC 360ee(b)(3)) as foods taken orally under the supervision of a physician for the primary purpose of providing a specific dietary management for a disease or condition.

[0032] In Australia, complementary medicines are the Chinese equivalent of health foods and refer to therapeutic products that contain, in whole or in part, one or more specified ingredients, each of which has clearly defined properties and traditional uses, particularly those listed in complementary medicines.

[0033] In Japan, foods are classified into three categories: Food for Specified Health Uses (FOSHU), functional foods, and functionally labeled foods. Of these, FOSHU is defined as a food that contains functional ingredients that affect the physiological functions of the human body and contribute to the promotion of health and the alleviation of disease. Functional foods are foods that are intended to supplement specific nutrients. Functional foods are foods that are produced based on scientific evidence and are not intended for patients, but are intended to help maintain and improve health through the intake of the "active ingredients" contained in the food.

[0034] As used herein, the term "amount of awake activity" refers to a distance traveled with a velocity of more than 20 mm / s.

[0035] As used herein, the term "wakeful activity time" refers to movement time with a movement velocity greater than 20 mm / s. According to a first aspect, the present invention provides the use of bovine spleen peptide powder in the manufacture of a food product for improving sleep in a subject.

[0036] In some embodiments of the first aspect, improving the subject's sleep comprises one or more of increasing the subject's sleep time, increasing the subject's sleep efficiency, shortening the subject's sleep onset latency, shortening the subject's amount of wakefulness after sleep onset, and shortening the subject's wakefulness after sleep onset.

[0037] According to a second aspect, the present invention provides the use of bovine spleen peptide powder in the manufacture of a food product for alleviating depression in a subject. In some embodiments of the second aspect, the food product can effectively alleviate depressed mood and the anxiety and tension associated therewith.

[0038] According to a third aspect, the present invention provides the use of bovine spleen peptide powder in the manufacture of a food product for improving bowel function in a subject. In some embodiments of any of the first to third aspects, the bovine spleen peptide powder, or the bovine spleen peptide powder together with a food supplement, is manufactured as a food product. In some embodiments of any of the first to third aspects, the food supplement comprises a sweetener, an acidulant, and / or a preservative. In some embodiments of any of the first to third aspects, the sweetener is one or more selected from the group consisting of sorbitol, fructose, glucose, lactose, mannitol, maltitol, and xylitol. In some embodiments of any of the first through third aspects, the acidulant is one or more selected from the group consisting of citric acid, lemon concentrate, tartaric acid, malic acid, lactic acid, and acetic acid. In some embodiments of any of the first to third aspects, the preservative is one or more selected from the group consisting of benzoic acid, sodium benzoate, potassium sorbate, and sodium lactate. In some embodiments of any of the first to third aspects, the food product is a formulated food for special medical use.

[0039] In some embodiments of any of the first to third aspects, the food is a health food.

[0040] In some embodiments of any of the first to third aspects, the food product is a tablet, powder, granules, tea, hard capsule, soft capsule, oral liquid, pill, tincture, ointment, beverage, confectionery, liquid milk, biscuit, confectionery, raw extract, or nutritional premix.

[0041] According to a fourth aspect, the present invention provides the use of bovine spleen peptide powder in the manufacture of a medicament for preventing or treating a sleep disorder in a subject. In some embodiments of the fourth aspect, the sleep disorder is one or more selected from the group consisting of insomnia, sleep-disordered breathing, central hypersomnia, circadian rhythm sleep-wake disorder, parasomnia, and sleep movement disorder. In some embodiments of the fourth aspect, the sleep disorder is insomnia.

[0042] In some embodiments of the fourth aspect, treating the sleep disorder in the subject comprises one or more of increasing the subject's sleep time, increasing the subject's sleep efficiency, decreasing the subject's sleep onset latency, decreasing the subject's amount of wakefulness activity after sleep onset, and decreasing the subject's wakefulness time after sleep onset.

[0043] According to a fifth aspect, the present invention provides the use of bovine spleen peptide powder in the manufacture of a medicament for preventing or treating depression in a subject. According to a sixth aspect, the present invention provides the use of bovine spleen peptide powder in the manufacture of a medicament for improving bowel function in a subject. In some embodiments of any of the fourth to sixth aspects, the bovine spleen peptide powder is formulated into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable carrier.

[0044] In some embodiments of any of the fourth to sixth aspects, the pharmaceutically acceptable carrier refers to a carrier that does not inhibit the biological activity of the active ingredient, including those conventionally used in the pharmaceutical field. The pharmaceutically acceptable carrier of the present invention may be solid or liquid, containing pharmaceutically acceptable excipients, buffers, emulsifiers, stabilizers, preservatives, diluents, encapsulating agents, fillers, etc. For example, pharmaceutically acceptable buffers further include phosphates, acetates, citrates, borates, and carbonates. In some embodiments, a medicament for preventing or treating a sleep disorder in a subject can be prepared by any method known in the pharmaceutical field. All methods include combining the active ingredient of the present invention with one or more pharmaceutically acceptable carriers. Typically, the composition is prepared by combining the active ingredient with a liquid carrier, a solid carrier, or both, and then shaping the resulting product as needed.

[0045] In some embodiments of any of the fourth to sixth aspects, the bovine spleen peptide powder is formulated together with a pharmaceutically acceptable carrier into an oral liquid, capsule, powder, tablet, granule, pill, syrup, suppository, or injection. In some embodiments of any of the fourth through sixth aspects, the pharmaceutical agent is administered orally, subcutaneously, intramuscularly, or intraperitoneally. In some specific embodiments of any of the fourth through sixth aspects, the pharmaceutical agent is administered orally.

[0046] In some embodiments of any of the above aspects, the bovine spleen peptide powder is used at a concentration of 250 μg / mL or less, e.g., the bovine spleen peptide powder is used at a concentration of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 60.5, 61, 61.5, 62, 62.5, 63, 63.5, 64, 64.5, 65, 65.5, 66, 66.5, 67, 67.5, 68, 68.5, 69, 69.5, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 1 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250 μg / mL, or a value or range between any two of these values.

[0047] In some embodiments of any of the above aspects, the bovine spleen peptide powder is used at a concentration of 62.5 to 250 μg / mL. For example, the bovine spleen peptide powder is used at a concentration of 62.5, 63, 63.5, 64, 64.5, 65, 65.5, 66, 66.5, 67, 67.5, 68, 68.5, 69, 69.5, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 85, 90, 95, 100, 105, 110, 115, 116, 117, 118, 120, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 20, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250 μg / mL, or a value or range between any two of these values.

[0048] In some embodiments of any of the above aspects, the bovine spleen peptide powder is used at a concentration of 62.5, 125, or 250 μg / mL. In some embodiments of any of the above aspects, the subject is a vertebrate. In some embodiments of any of the above aspects, the subject is a fish, a mammal, a cyclostome, an amphibian, a reptile, or a bird. In some embodiments of any of the above aspects, the subject is a fish (e.g., a zebrafish) or a mammal (e.g., a human).

[0049] In some embodiments of the first or fourth aspect, the subject suffers from a sleep disorder. In some embodiments, the sleep disorder is one or more selected from the group consisting of insomnia, sleep-disordered breathing, central hypersomnia disorder, circadian rhythm sleep-wake disorder, parasomnia, and sleep movement disorder. In some embodiments of the second or fifth aspect, the subject exercises for longer periods after ingesting the food product or administering the pharmaceutical agent.

[0050] In some embodiments of the second or fifth aspect, the subject moves an increased distance after ingesting the food product or administering the pharmaceutical agent. In some embodiments of the second or fifth aspect, after ingesting the food product or after administering the pharmaceutical agent, the subject's percentage of light field movement time increases. In some embodiments of the second or fifth aspect, after ingesting the food product or after administering the pharmaceutical agent, the subject exhibits an increased photopic movement distance.

[0051] In some embodiments of the second or fifth aspect, the depression comprises one or more of the following clinical symptoms: 1. Emotional symptoms: These are the core symptoms of depressive disorders and manifest as persistently low mood, loss of interest, and lack of energy. 2. Physical symptoms: loss of appetite, insomnia, weight loss, decreased libido, 3. Cognitive symptoms: inattention, decreased memory, decreased thinking ability, and in severe cases, negative thoughts and even suicidal behavior.

[0052] In some embodiments of the third or sixth aspect, the bovine spleen peptide powder can increase the number of goblet cells in the intestinal tract, and goblet cells can synthesize and secrete mucin, thereby lubricating the intestinal tract. In some embodiments of the third or sixth aspect, improving bowel function in the subject comprises lubricating the intestinal tract. In some embodiments of the third or sixth aspect, improving bowel function in the subject comprises preventing, ameliorating, or treating constipation.

[0053] According to a seventh aspect, the present invention provides a method for improving sleep in a subject, comprising administering to said subject an effective amount of bovine spleen peptide powder. According to an eighth aspect, the present invention provides a method for preventing or treating a sleep disorder in a subject, comprising administering to said subject an effective amount of bovine spleen peptide powder.

[0054] According to a ninth aspect, the present invention provides a method for alleviating depression in a subject, comprising administering to the subject an effective amount of bovine spleen peptide powder. According to a tenth aspect, the present invention provides a method for preventing or treating depression in a subject, comprising administering to the subject an effective amount of bovine spleen peptide powder.

[0055] According to an eleventh aspect, the present invention provides a method of improving bowel function in a subject, comprising administering to the subject an effective amount of bovine spleen peptide powder. According to a twelfth aspect, the present invention provides a method for preventing, ameliorating or treating constipation in a subject, comprising administering to the subject an effective amount of bovine spleen peptide powder.

[0056] In some embodiments of any of the above aspects, the bovine spleen peptide powder is produced by a method comprising at least one of the following steps: crushing and homogenizing bovine spleens to obtain a spleen slurry; freezing and thawing the spleen slurry; subjecting the frozen-thawed spleen slurry to solid-liquid separation and recovering the supernatant; filtering the supernatant to obtain a bovine spleen extract; sterilizing the bovine spleen extract; A step of drying the sterilized bovine spleen extract to obtain the bovine spleen peptide powder.

[0057] In some embodiments of any of the above aspects, the production of the bovine spleen peptide powder includes the steps of picking bovine spleen, crushing and homogenizing, freezing and thawing, solid-liquid separation, filtering the supernatant (e.g., by coarse filtration and / or ultrafiltration), sterilizing, and spray drying.

[0058] In some embodiments of any of the above aspects, the content of polypeptide in the bovine spleen peptide powder is 150 to 200 mg / g, e.g., 150, 155, 160, 161, 161.1, 161.2, 161.3, 161.4, 161.5, 161.6, 161.7, 161.8, 161.9, 162, 163, 164, 165, 166, 167, 167.1, 167.2, 167. 3, 167.4, 167.5, 167.6, 167.7, 167.8, 167.9, 170, 175, 180, 185, 190, 191, 192, 193, 194, 195, 195.1, 195.2, 195.3, 195.4, 195.5, 195.6, 195.7, 195.8, 195.9, 196, 197, 198, 199, 200 mg / g, or a range between any two of these values.

[0059] In some embodiments of any of the above aspects, the content of polypeptide in the bovine spleen peptide powder is 195.8 mg / g. In some embodiments of any of the above aspects, the molecular weight of the polypeptide molecules in the bovine spleen peptide powder is less than 17 KD.

[0060] In some embodiments of any of the above aspects, the content of amino acids in the bovine spleen peptide powder is 400 to 450 mg / g, for example, 400, 401, 402, 403, 403.1, 403.2, 403.3, 403.4, 403.5, 403.6, 403.7, 403.8, 403.9, 404, 405, 406, 407, 407.1, 407.2, 407.3, 407.4, 40 7.5, 407.6, 407.8, 407.9, 408, 409, 410, 415, 420, 425, 430, 435, 440, 441, 442, 443, 444, 444.1, 444.2, 444.3, 444.4, 444.5, 444.6, 444.7, 444.8, 444.9, 445, 446, 447, 448, 449, 450 mg / g, or a range between any two of these values.

[0061] In some embodiments of any of the above aspects, the content of amino acids in the bovine spleen peptide powder is 444.5 mg / g.

[0062] In this specification and claims, the words "comprise," "having," and "containing" mean "including but not limited to" and are not intended to exclude other moieties, additives, ingredients, or steps.

[0063] It should be understood that any feature, characteristic, ingredient, or step described in a particular aspect, embodiment, or example of the present invention may be applied to any other aspect, embodiment, or example described herein, unless inconsistent.

[0064] The above disclosure generally describes the present invention, and the following examples further illustrate the present invention and should not be construed as limiting the present invention. The present invention discloses the use of bovine spleen peptide powder in the manufacture of a food product for improving sleep in a subject, alleviating depression in a subject, or improving intestinal function in a subject, or in the manufacture of a pharmaceutical product for preventing or treating sleep disorders or depression in a subject, or improving intestinal function in a subject. Those skilled in the art can achieve their objectives by appropriately modifying process parameters based on the contents of this specification. It should be noted that all similar substitutions and modifications are apparent to those skilled in the art and are considered to be within the scope of the present invention. Those skilled in the art can implement and apply the present invention by making changes or appropriate modifications and combinations to the methods and uses described herein without departing from the spirit, spirit, and scope of the present invention.

[0065] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below in connection with examples. [Example]

[0066] Example 1 Preparation and Characterization of Bovine Spleen Peptide Powder 1. Preparation of bovine spleen peptide powder The method for producing bovine spleen peptide powder includes the following steps (flowchart shown in Figure 1). 1) Crushing and homogenization: fresh bovine spleens are collected, washed, and then the fascia and trachea are removed. The spleens are then cut and crushed in a homogenizer, and purified water in an amount five times the weight of the fresh spleen homogenate is added and mixed uniformly to produce spleen slurry. 2) Freezing and thawing: The spleen slurry is placed in a freezer at -20°C for at least 48 hours, and then thawed in a water bath (the upper limit of thawing temperature is 40°C), and the freezing and thawing is repeated three times. 3) Separation: Separating the supernatant using a sedimentation centrifuge or a tube centrifuge; 4) Coarse filtration: filtering the supernatant obtained in step 3) through a membrane filter having a pore size of 50 μm to obtain a crude filtrate; 5) Ultrafiltration: ultrafiltration of the crude filtrate obtained in step 4) through an ultrafiltration membrane with a pore size of 30 KDa, and obtaining the ultrafiltrate as a bovine spleen extract; 6) Sterilization: filtration through a 0.2 μm sterilizing filter element for sterilization and packaging; 7) A step of spray-drying the packaged solution of step 6) to obtain bovine spleen peptide powder.

[0067] 2. Characterization of Bovine Spleen Peptide Powder 2.1 Detection of peptide content: BCA method Experimental equipment: ultraviolet spectrophotometer (model number: UV1800, manufacturer: TOSOH). Detection reagent: BCA Rapid Detection Kit (product number: A53225, manufacturer: Thermo). Sample preparation: Weigh out 0.05 g of bovine spleen peptide powder sample, dissolve in 10 mL of purified water, and mix well.

[0068] The detection method includes the following steps. 1) serially diluting bovine serum albumin (BSA) standard (included in the kit) to 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL; 2) mixing 100 mL of working solution A and 2 mL of working solution B of the kit to prepare a BCA working solution; 3) placing the diluted standard and test sample into an ELISA plate (20 μL / well), adding 200 μL of BCA working solution, and incubating at 37°C for 30 minutes; 4) A step of reading the absorbance of each well at 562 nm, creating a standard curve, and calculating the test sample concentration.

[0069] The experimental results are shown in Table 2. The peptide content in the bovine spleen peptide powder was 195.8 mg / g.

[0070] [Table 1]

[0071] [Table 2]

[0072] 2.2 Molecular weight detection of polypeptides: SDS-PAGE method Experimental equipment: Mini vertical electrophoresis apparatus (model number: BG-Power600, manufacturer: Beijing Shirai Biotechnology Co., Ltd.), decolorization shaker (model number: WD-9405D, manufacturer: Beijing Liuyi Biotechnology Co., Ltd.).

[0073] Detection reagents: 12% Bis-Tris electrophoresis precast gel (product number: MP0342BOX, manufacturer: Thermo Scientific), electrophoresis buffer (20x) (product number: NP0002, manufacturer: Thermo Scientific), protein stain (product number: 46-5034, manufacturer: Thermo Scientific), sample reducing agent (10x) (product number: NP0009, manufacturer: Thermo Scientific), sample buffer (product number: NP0007, manufacturer: Thermo Scientific), electrophoresis protein molecular weight standards (markers) (product number: 26619, manufacturer: Thermo Scientific).

[0074] Sample preparation: Weigh out 0.1 g of bovine spleen peptide powder sample, dissolve in 10 mL of PBS, and mix well.

[0075] The detection method includes the following steps. 1) taking 50 μL of a test sample, adding 25 μL of sample buffer, 10 μL of a sample reducing agent, and 15 μL of purified water to bring the total volume to 100 μL; 2) heating the mixture obtained in step 1) in boiling water for 3 minutes, then removing it and storing it in a refrigerator at 4°C; 3) Equilibrate the electrophoresis precast gel to room temperature, open it, rinse it, place it in a previously prepared electrophoresis buffer (1x), add the sample (10 μL of test sample, 5 μL of marker), turn on the electrophoresis device, electrophorese at a voltage of 120 V until the bromophenol blue indicator reaches the center of the gel, remove the gel plate, add a staining solution and stain on a shaker for 60 minutes, the quality control product is bovine serum albumin (BSA) with a molecular weight of 66.4 KD; 4) After dyeing, the sample is decolorized using purified water and the molecular weight range is determined.

[0076] The experimental results are shown in Figure 3. The molecular weights of the polypeptides contained in the bovine spleen peptide powder are mainly concentrated at 17 KD or less.

[0077] 2.3 Detection of Amino Acid Content: HPLC Method Experimental equipment: High-performance liquid chromatograph (model number: SPD-20A, manufacturer: Shimadzu Corporation).

[0078] Detection reagents: acetonitrile (product number: 75-05-8, batch number: 203096, manufacturer: Thermo Fisher Scientific (China) Co., Ltd.), methanol (product number: 67-56-1, batch number: 211775, manufacturer: Thermo Fisher Scientific (China) Co., Ltd.), method package for amino acid analysis (product number: AJS-01, manufacturer: Shimadzu Corporation).

[0079] Sample preparation: Weigh 0.03 g of bovine spleen peptide powder sample, add 5 mL of 0.1 mol / L hydrochloric acid, mix uniformly, and then filter through a 0.45 μm filter membrane. Mix 400 μL of the filtrate, 100 mL of 0.1 mol / L hydrochloric acid, and 50 μL of the internal standard, and prepare by uniform mixing.

[0080] Detection method: Mobile phase: Weigh out 9.0 g of disodium hydrogen phosphate dodecahydrate and 9.5 g of sodium tetraborate decahydrate, add 2000 mL of water, adjust the pH to 8.2 with hydrochloric acid, mix uniformly, filter through a 0.45 μm filter membrane, and sonicate to obtain mobile phase A. Take 450 mL of methanol, 450 mL of acetonitrile, and 100 mL of purified water, mix uniformly, filter through a 0.45 μm filter membrane, and sonicate to obtain mobile phase B.

[0081] Wavelength: UV detector 338nm and 262nm. Column temperature: 50°C. Chromatography column: C18. Flow rate: 1.0mL / min. Injection volume: 1 μL.

[0082] Sample Processing: Derivatization 1) 1 μL of a standard mixture of 17 amino acids was precisely measured and injected into a liquid chromatograph, and the chromatogram was recorded. The experimental results are shown in Figure 4. 2) Accurately measure 1 μL of the above sample and inject it into a liquid chromatography column, and record the chromatogram.

[0083] The experimental results are shown in Figure 5 and Table 3. The amino acid content in the bovine spleen peptide powder was 444.5 mg / g.

[0084] [Table 3]

[0085] Example 2: Verification of the sleep-improving effect of bovine spleen peptide powder 1.Detection material 1.1 Sample preparation information Bovine spleen peptide powder was prepared as a 20.0 mg / mL stock solution in standard dilution water, sonicated, and blended prior to immediate use.

[0086] Positive control: Melatonin, white powder, batch number: F1804064, manufactured by Shanghai Aladdin Biochemical Technology Co., Ltd., stored in a cool, dark place. A 50.0 mg / mL mother solution was prepared in DMSO and stored at -20°C.

[0087] 1.2 Experimental animals All zebrafish were kept in culture water at 28°C (water quality: 200 mg of instant sea salt per liter of reverse osmosis water, conductivity 450-550 μS / cm, pH 6.5-8.5, hardness 50-100 mg / L CaCO3). They were provided by the fish breeding center of Hangzhou Huante Biotechnology Co., Ltd., and their experimental animal use permit number was SYXK (Zhejiang) 2022-0004. Breeding procedures conformed to the requirements of the international AAALAC certification (certificate number: 001458).

[0088] Wild-type AB zebrafish were bred by natural pair mating. Five-day-old zebrafish were used for measuring the maximum detectable concentration (MTC) of bovine spleen peptide powder and for evaluating its sleep-improving effects.

[0089] 1.3 Equipment, Consumables and Reagents The equipment included a dissecting microscope (SZX7, OLYMPUS, Japan), a CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China), a precision electronic balance (CP214, OHAUS, USA), a behavioral analysis device (Zebra Lab 3.22.3.31, Viewpoint, France), a 6-well plate (Nest Biotech, China), and a 96-well plate (Nest Biotech, China).

[0090] Pentylenetetrazole (PTZ, batch number: YH0171126, Shanghai Yihe Biotechnology Co., Ltd., China), dimethyl sulfoxide (DMSO, batch number: BCCD8942, Sigma, Switzerland).

[0091] 2. Detection Method 2.1 MTC measurement Five dpf wild-type AB strain zebrafish were randomly selected and placed in a 6-well plate. Thirty zebrafish were placed in each well of the experimental group. Bovine spleen peptide powder aqueous solution was administered at the concentrations shown in Table 4 below. A normal control group (a group raised using conventional methods) was also administered in a 3 mL volume per well. After 24 hours of treatment at 28°C, the MTC of the bovine spleen peptide powder relative to normal zebrafish was measured.

[0092] 2.2 Evaluation of sleep improvement effects Five dpf wild-type AB strain zebrafish were randomly selected and placed in 6-well plates. Experimental groups consisted of 30 zebrafish per well. Bovine spleen peptide powder aqueous solution was administered at the concentrations shown in Table 5 below, and melatonin was administered at 125 μg / mL as a positive control. A normal control group (a group reared using conventional methods) and a model control group (i.e., a zebrafish insomnia model established by PTZ) were also administered. Each well contained 3 mL of solution. After 24 hours of treatment at 28°C, 10 zebrafish were randomly selected from each experimental group and transferred to a 96-well plate at 200 μL per animal, one per well. The experimental groups, except for the normal control group, were administered PTZ aqueous solution to establish the zebrafish insomnia model. A behavioral analysis system was used to analyze the zebrafish's waking activity and waking activity time. Statistical analysis of the above indicators was used to evaluate the sleep-improving effects of bovine spleen peptide powder. The statistical results were expressed as "mean ± standard deviation." SPSS26.0 software was used for statistical analysis, and p<0.05 indicated a statistically significant difference.

[0093] 3. Detection results 3.1 MTC The MTC for evaluating the sleep-improving effect of bovine spleen peptide powder was 250 μg / mL, as shown in Table 4.

[0094] [Table 4]

[0095] 3.2 Evaluation of sleep improvement effects The results in Table 5 and FIGS. 6 to 8 clearly show that bovine spleen peptide powder has a sleep-improving effect, specifically, a decrease in the amount and duration of waking activity was observed.

[0096] [Table 5]

[0097] Example 3: Verification of the antidepressant effect of bovine spleen peptide powder 1.Detection material 1.1 Sample preparation information Bovine spleen peptide powder was prepared as a 20.0 mg / mL stock solution in standard dilution water, sonicated, and blended prior to immediate use.

[0098] Positive control: Zoloft (registered trademark) sertraline hydrochloride tablets (hereafter abbreviated as sertraline), batch number DP1626, manufactured by Pfizer Pharmaceutical Co., Ltd., stored in a cool, dry place. A 5.00 mg / mL mother solution was prepared with ultra-purified water, sonicated, centrifuged, and stored at -20°C.

[0099] 1.2 Experimental animals All zebrafish were kept in culture water at 28°C (water quality: 200 mg of instant sea salt per liter of reverse osmosis water, conductivity 450-550 μS / cm, pH 6.5-8.5, hardness 50-100 mg / L CaCO3) and provided by the fish breeding center of Hangzhou Huante Biotechnology Co., Ltd. The experimental animal use permit number was SYXK (Zhejiang) 2022-0004. Breeding management complied with the requirements of the international AAALAC certification (certification number: 001458).

[0100] Wild-type AB zebrafish were bred by natural pair mating. Five-day-old zebrafish were used for measuring the maximum detectable concentration (MTC) of bovine spleen peptide powder and evaluating its antidepressant effect.

[0101] 1.3 Equipment, Consumables and Reagents A dissecting microscope (SZX7, OLYMPUS, Japan), a CCD camera (VertA1, Shanghai Tumori Vision Technology Co., Ltd., China), a precision electronic balance (CP214, OHAUS, USA), a behavioral analysis device (Zebra Lab 3.22.3.31, Viewpoint, France), a 6-well plate (Nest Biotech, China), and a 96-well plate (Nest Biotech, China).

[0102] Reserpine (Reserpine, batch number: I1905136, Shanghai Aladdin Biochemical Technology Co., Ltd., China), dimethyl sulfoxide (DMSO, batch number: BCCD8942, Sigma, Switzerland).

[0103] 2. Detection Method 2.1 MTC measurement Five dpf wild-type AB strain zebrafish were randomly selected and placed in a 6-well plate. Experimental groups consisted of 30 zebrafish per well. A 3 mL volume of bovine spleen peptide powder solution was administered at the concentrations shown in Table 6. A normal control group (a group raised using conventional methods) and a model control group (a zebrafish depression model established with reserpine) were also included. Each well contained 3 mL of solution. Except for the normal control group, each experimental group was administered with reserpine solution to establish a zebrafish depression model. After 24 hours of treatment at 28°C, the MTC of bovine spleen peptide powder on the model zebrafish was measured.

[0104] 2.2 Evaluation of antidepressant efficacy Five dpf wild-type AB strain zebrafish were randomly selected and placed in six-well plates. Experimental groups consisted of 30 zebrafish per well. Bovine spleen peptide powder solution was administered at the concentrations shown in Table 7 below, along with sertraline (100 ng / mL) as a positive control. A normal control group (conventionally reared) and a model control group were also established. Each well contained 3 mL of solution. Except for the normal control group, each experimental group was administered with reserpine solution to establish a zebrafish depression model. After 24 hours of treatment at 28°C, 10 zebrafish were randomly selected from each experimental group. The percentage of time spent in the bright field and the distance traveled in the bright field were measured using a behavioral analyzer. Statistical analysis of these parameters was used to evaluate the antidepressant effects of the samples. Statistical results were expressed as "mean ± standard deviation." Statistical analysis was performed using SPSS 26.0 software, with p<0.05 indicating statistical significance.

[0105] 3. Detection results 3.1 MTC The MTC for evaluating the antidepressant effect of bovine spleen peptide powder was 250 μg / mL as shown in Table 6.

[0106] [Table 6]

[0107] 3.2 Evaluation of antidepressant efficacy The results in Table 7 and Figures 9-10 show that compared with the depression model control group, the zebrafish administered with bovine spleen peptide powder had improved percentage of light field movement time and light field movement distance, and the dark-taxis state of the depression model zebrafish was improved, demonstrating the antidepressant effect of bovine spleen peptide powder.

[0108] [Table 7]

[0109] Example 4 Regulatory effect of bovine spleen peptide powder on goblet cells in the intestinal tract 1.Detection material 1.1 Sample preparation information Bovine spleen peptide powder was prepared as a 20.0 mg / mL stock solution in standard dilution water, sonicated, and blended prior to immediate use.

[0110] Positive control: prednisone, white powder, batch number: C10016501, manufactured by Shanghai Macklin Biochemical Technology Co., Ltd., stored at 4°C. A 15.0 mg / mL mother solution was prepared in DMSO and stored at -20°C.

[0111] 1.2 Experimental animals All zebrafish were kept in culture water at 28°C (water quality: 200 mg of instant sea salt per liter of reverse osmosis water, conductivity 450-550 μS / cm, pH 6.5-8.5, hardness 50-100 mg / L CaCO3) and provided by the fish breeding center of Hangzhou Huante Biotechnology Co., Ltd. The experimental animal use permit number was SYXK (Zhejiang) 2022-0004. Breeding management complied with the requirements of the international AAALAC certification (certification number: 001458).

[0112] Wild-type AB zebrafish were bred by natural pair mating. Three-day-old (5 dpf) zebrafish were used to measure the maximum detectable concentration (MTC) of bovine spleen peptide powder and evaluate its effect on improving intestinal function.

[0113] 1.3 Equipment, Consumables and Reagents Dissecting microscope (SZX7, OLYMPUS, Japan), CCD camera (VertA1, Shanghai Dosen Visual Technology Co., Ltd., China), microtome (KD2258, Jinhua Kedi Medical Devices Co. Ltd., China), precision electronic balance (CP214, OHAUS, USA), and 6-well plate (Nest Biotech, China).

[0114] Trinitrobenzenesulfonic acid (TNBS, batch number: SLCK4178, Sigma, USA), Alcian blue (batch number: BCBV8028, Sigma, Switzerland), dimethyl sulfoxide (DMSO, batch number: BCCD8942, Sigma, Switzerland), glacial acetic acid (batch number: C10323745, Shanghai McKinney Biochemical Technology Co., Ltd., China), methylcellulose (batch number: G2106167, Shanghai Aladdin Biochemical Technology Co., Ltd., China), 4% tissue and cell fixative (batch number: 20210828, Beijing Solarbio Technology Co., Ltd., China), xylene (batch number: 20201113, Sinopharm Chemical Reagent Co., Ltd., China). Ltd., China), PBS buffer solution (batch number: 70115000, Biosharp, China), Mayer's hematoxylin staining solution (batch number: 20220120, Shanghai Yihe Biological Technology Co., Ltd., China), eosin staining solution (batch number: 20220120, Shanghai Yihe Biological Technology Co., Ltd., China), neutral gum (batch number: 330A021, Solarbio, China), Huabao high-efficiency sectioning paraffin (batch number: 20210828, Shanghai Huayong Paraffin Co., Ltd., China), and absolute ethanol (batch number: 20210107, Sinopharm Group Chemical Reagents Co., Ltd., China).

[0115] 2. Detection Method 2.1 MTC measurement Three dpf wild-type AB zebrafish were randomly selected and placed in a 6-well plate. Experimental groups were established with 30 zebrafish per well. Each experimental group, except for the normal control group (conventionally reared), was administered TNBS solution to establish a zebrafish gastrointestinal mucosal injury model. After two days of treatment at 28°C, TNBS was removed, and bovine spleen peptide powder solution was administered at the concentrations shown in Table 8 below. A normal control group and a model control group were also established, with each well containing 3 mL of solution. After two consecutive days of treatment at 28°C, the MTC of bovine spleen peptide powder in the zebrafish gastrointestinal mucosal injury model was measured.

[0116] 2.2 Evaluation of the regulatory effect on intestinal goblet cells Three dpf wild-type AB zebrafish were randomly selected and placed in a 6-well plate. Thirty zebrafish were placed in each well to form experimental groups. Each experimental group, except for the normal control group (conventionally reared), was treated with TNBS solution to establish a zebrafish gastrointestinal mucosal injury model. After two days of treatment at 28°C, TNBS was removed and bovine spleen peptide powder solution was administered at the concentrations shown in Table 9. A positive control, prednisone, was administered at a concentration of 15.0 μg / mL. A normal control group and a model control group (i.e., a zebrafish gastrointestinal mucosal injury model established by TNBS), were also established. Each well contained 3 mL of solution. After two consecutive days of incubation at 28°C, zebrafish were stained with Alcian blue. Ten zebrafish were randomly selected from each experimental group and photographed under a dissecting microscope. Data were collected using NIS-Elements D 3.20 advanced image processing software to analyze the number of intestinal goblet cells in the zebrafish. Statistical analysis of this index was used to evaluate the effect of bovine spleen peptide powder on intestinal goblet cell regulation. Statistical results were expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 26.0 software, with p<0.05 indicating statistical significance.

[0117] 3. Detection results 3.1 MTC The maximum detectable concentration (MTC) of the regulatory effect of bovine spleen peptide powder on goblet cells was 250 μg / mL, as detailed in Table 8.

[0118] [Table 8]

[0119] 3.2 Regulatory effects of bovine spleen peptide powder on intestinal goblet cells The results in Table 9 and Figures 11 to 12 show that bovine spleen peptide powder has a regulatory effect on goblet cells and can increase the number of intestinal goblet cells.

[0120] [Table 9]

[0121] Example 5 Evaluation of the constipation prevention effect of bovine spleen peptide powder 1.Detection material 1.1 Sample preparation information Bovine spleen peptide powder was prepared as a 20.0 mg / mL stock solution in standard dilution water, sonicated, and blended prior to immediate use.

[0122] Positive control: Spirulina alfalfa powder, white powder, batch number 145611, manufactured by Perfect (China) Co., Ltd., stored in a cool, shaded area. A 2.00 mg / mL stock solution was prepared with standard dilution water and used immediately after blending.

[0123] 1.2 Experimental animals All zebrafish were kept in culture water at 28°C (water quality: 200 mg of instant sea salt per liter of reverse osmosis water, conductivity 450-550 μS / cm, pH 6.5-8.5, hardness 50-100 mg / L CaCO3) and provided by the fish breeding center of Hangzhou Huante Biotechnology Co., Ltd. The experimental animal use permit number was SYXK (Zhejiang) 2022-0004. Breeding management complied with the requirements of the international AAALAC certification (certification number: 001458).

[0124] Wild-type AB strain zebrafish were bred by natural pair mating. Five-day-old zebrafish were used for measuring the maximum detectable concentration (MTC) of bovine spleen peptide powder and evaluating its efficacy in preventing constipation.

[0125] 1.3 Equipment, Consumables and Reagents Dissecting microscope (SZX7, OLYMPUS, Japan), CCD camera (VertA1, Shanghai Tsumori Visual Technology Co., Ltd., China), motorized focusing continuously variable magnification fluorescence microscope (AZ100, Nikon, Japan), precision electronic balance (CP214, OHAUS, USA), 6-well plate (Nest Biotech, China).

[0126] Methylcellulose (batch number: C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China), aluminum sulfate (batch number: D1909026, Shanghai Aladdin Biochemical Technology Co., Ltd., China), and Nile red (batch number: SLBP9326V, Sigma, India).

[0127] 2. Detection Method 2.1 Measuring MTC Five dpf wild-type AB strain zebrafish were randomly selected in six-well plates, with 30 fish per well used as experimental groups. A 3 mL volume of bovine spleen peptide powder solution was administered at the concentrations shown in Table 10 below to establish a normal control group (a group raised using conventional methods) and a model control group (a group established as a constipation model using aluminum sulfate). After two days of treatment at 28°C, the bovine spleen peptide powder was removed, and each experimental group was administered Nile Red solution to stain the gastrointestinal tract. After staining, all experimental groups except the normal control group were administered aluminum sulfate solution to establish the constipation model. After 6 hours of treatment with aluminum sulfate, the MTC of bovine spleen peptide powder on the model zebrafish was measured.

[0128] 2.2 Evaluation of constipation prevention effect Five dpf wild-type AB zebrafish were randomly selected and distributed into six-well plates, with 30 zebrafish per well for each experimental group. The bovine spleen peptide powder solution was used at a concentration of 667 μg / mL (Table 11), along with a positive control, Spirulina / Alfalfa powder, at a concentration of 667 μg / mL. A normal control group (a group raised using conventional methods) and a model control group (a group established as a constipation model using aluminum sulfate) were also included. Each well contained 3 mL of solution. After 24 hours of treatment at 28°C, the bovine spleen peptide powder was removed, and each experimental group was administered Nile Red solution to stain the gastrointestinal tract. After staining, all experimental groups except the normal control group were administered aluminum sulfate solution to establish the constipation model. After 6 hours of aluminum sulfate treatment, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. Data were then collected using the advanced image processing software NIS-Elements D 3.20. Fluorescence intensity in the gastrointestinal tract of the zebrafish was analyzed, and statistical analysis of this index was used to evaluate the efficacy of bovine spleen peptide powder in preventing constipation. Statistical results were expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 26.0 software, with p<0.05 indicating statistical significance.

[0129] 3. Test Results 3.1 MTC The maximum detectable concentration (MTC) of bovine spleen peptide powder for its constipation prevention effect was 250 μg / mL, as shown in Table 10.

[0130] [Table 10]

[0131] 3.2 Evaluation of constipation prevention effect The results in Table 11 and Figures 13 and 14 show that bovine spleen peptide powder has a constipation prevention effect.

[0132] [Table 11]

[0133] All publications and patent documents cited herein are indicated as if each publication or patent were expressly incorporated herein by reference. Various modifications and equivalent substitutions may be made to the various embodiments disclosed herein without departing from the true spirit and scope of the invention. Any feature, step, or embodiment of an embodiment of the invention may be used in combination with any other feature, step, or embodiment, unless the context dictates otherwise.

Claims

1. 10. Use of bovine spleen peptide powder in the manufacture of a food product for improving sleep in a subject, or in the manufacture of a pharmaceutical product for preventing or treating a sleep disorder in a subject.

2. 10. Use of bovine spleen peptide powder in the manufacture of a food product for alleviating depression in a subject, or in the manufacture of a medicament for preventing or treating depression in a subject.

3. 10. Use of bovine spleen peptide powder in the manufacture of a food product for improving intestinal function in a subject, or in the manufacture of a pharmaceutical product for improving intestinal function in a subject.

4. The use according to any one of claims 1 to 3, wherein the bovine spleen peptide powder is used at a concentration of 250 μg / mL or less, preferably 62.5 to 250 μg / mL.

5. The use of any one of claims 1 to 4, wherein the bovine spleen peptide powder or the bovine spleen peptide powder is manufactured into a food product together with a food supplement, preferably the food product is a special medical compound food or a health food, more preferably the food product is a tablet, powder, granule, tea, hard capsule, soft capsule, oral liquid, pill, tincture, ointment, beverage, confectionery, liquid milk, biscuit, confectionery, raw material extract, or nutritional premix.

6. The use according to any one of claims 1 to 4, wherein the bovine spleen peptide powder is formulated into a pharmaceutically acceptable dosage form together with a pharmaceutically acceptable carrier, and preferably the pharmaceutically acceptable dosage form is an oral liquid, capsule, powder, tablet, granule, pill, syrup, suppository, or injection.

7. The use according to any one of claims 1 to 4 and 6, wherein the medicament is administered orally, subcutaneously, intramuscularly or intraperitoneally, preferably the medicament is administered orally.

8. The use according to any one of claims 1 to 7, wherein the subject is a vertebrate.

9. The use according to any one of claims 1 to 8, wherein the subject is a fish, a mammal, a cyclostome, an amphibian, a reptile, or a bird, preferably the subject is a fish or a mammal, more preferably the subject is a zebrafish or a human.

10. The use of claim 1, wherein the subject suffers from a sleep disorder.

11. 2. The use of claim 1, wherein the sleep disorder is one or more selected from the group consisting of insomnia, sleep-disordered breathing, central hypersomnia, circadian rhythm sleep-wake disorder, parasomnia, and sleep movement disorder.

12. 2. The use of claim 1, wherein improving the subject's sleep or treating a sleep disorder in the subject comprises one or more of increasing the subject's sleep time, increasing the subject's sleep efficiency, shortening the subject's sleep onset latency, shortening the subject's amount of wakefulness activity after sleep onset, and shortening the subject's wakefulness activity time after sleep onset.

13. 4. The use of claim 3, wherein improving bowel function in the subject comprises lubricating the intestinal tract and / or preventing, ameliorating or treating constipation.

14. The bovine spleen peptide powder is subjected to the following steps: crushing and homogenizing bovine spleens to obtain a spleen slurry; freezing and thawing the spleen slurry; subjecting the frozen-thawed spleen slurry to solid-liquid separation and recovering the supernatant; filtering the supernatant to obtain a bovine spleen extract; sterilizing the bovine spleen extract; drying the sterilized bovine spleen extract to obtain the bovine spleen peptide powder; The use according to any one of claims 1 to 13, wherein the composition is produced by a method comprising at least one of the following steps:

15. The use according to any one of claims 1 to 14, wherein the content of polypeptide in the bovine spleen peptide powder is 150 to 200 mg / g.

16. The use according to any one of claims 1 to 15, wherein the molecular weight of the polypeptide molecules in the bovine spleen peptide powder is less than 17 KD.

17. The use according to any one of claims 1 to 16, wherein the content of amino acids in the bovine spleen peptide powder is 400 to 450 mg / g.

18. 1. A method for improving sleep in a subject, comprising administering to the subject an effective amount of bovine spleen peptide powder.

19. A method for preventing or treating a sleep disorder in a subject, comprising administering to the subject an effective amount of bovine spleen peptide powder.

20. 1. A method for alleviating depression in a subject, comprising administering to the subject an effective amount of bovine spleen peptide powder.

21. A method for preventing or treating depression in a subject, comprising administering to the subject an effective amount of bovine spleen peptide powder.

22. 1. A method for improving bowel function in a subject, comprising administering to the subject an effective amount of bovine spleen peptide powder.

23. A method for preventing, ameliorating, or treating constipation in a subject, comprising administering to the subject an effective amount of bovine spleen peptide powder.

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