Application of esculent siphonophore protein peptide in preparing preparations for improving sleep and / or treating sleep disorders

Abstract

The present invention discloses the application of a protein peptide from the worm Siegesneria oleracea in the preparation of a preparation for improving sleep and / or treating sleep disorders. The present invention uses papain and alkaline protease to hydrolyze the worm Siegesneria oleracea in sequence to prepare a protein peptide from the worm Siegesneria oleracea. The protein peptide can not only significantly increase the sleep time of zebrafish with caffeine-induced sleep disorders and improve abnormal changes in IL-6 and GABA levels in the zebrafish brain, but can also significantly prolong the sleep duration of mice induced by sodium pentobarbital, showing strong sleep-improving activity. The protein peptide from the worm Siegesneria oleracea of ​​the present invention can be used to prepare a preparation for improving insomnia and treating diseases related to sleep disorders, and has broad application prospects in the field of sleep disorders.

Description

Technical Field

[0001] The present invention relates to the technical field of improving sleep, and in particular to the use of Siempuri odorata protein peptides in preparing preparations for improving sleep and / or treating sleep disorders. Background Art

[0002] Insomnia refers to a sleep disorder characterized by dissatisfaction with sleep performance and duration, even under appropriate sleep conditions, which can impact daytime function. Sleep disorders can manifest in various ways, including difficulty falling asleep, interrupted sleep, premature awakening, insufficient sleep duration, and fatigue after waking. Specific types of these disorders include insomnia, sleep-disordered breathing (such as sleep apnea), excessive sleepiness, abnormal sleep behavior, and circadian rhythm deregulation. The primary characteristic of sleep disorders is a misalignment between endogenous circadian rhythms and external time cues. The underlying mechanisms are multifactorial, and research remains limited. Some researchers suggest that sleep disorders may be closely related to decreased melatonin receptor activity, overactive hypothalamic-pituitary-adrenal (HPA) axis, dysregulated neurotransmitter secretion, and abnormal release of inflammatory cytokines. However, the specific pathways or mechanisms at play remain unclear.

[0003] Currently, the drugs used clinically to improve sleep are primarily chemical drugs, such as benzodiazepines and non-benzodiazepines. However, these drugs often produce significant side effects when treating sleep disorders, such as mental fatigue, drowsiness, and excessive sedation. Long-term use can also lead to extreme dependence and even addiction. Therefore, the search for effective and safe sleep-improving drugs is crucial for the development of products to prevent and treat sleep disorders.

[0004] Edible leather capsule worm ( Phascolosoma esculenta Siegespermum ediblei, also known as earth nails, earth shoots, and mud ding, belongs to the class Siegespermum, phylum Siegespermum, and is widely distributed in coastal areas of my country, including Fujian, Guangxi, Guangdong, and Hainan. Siegespermum ediblei has long been considered a healthy food that nourishes yin and tonifies the kidneys, boasting benefits such as nourishing yin and reducing heat, clearing the lungs and replenishing deficiency, promoting blood circulation and strengthening the body, and nourishing the kidneys and enhancing beauty. It is also effective for treating frequent nocturnal urination in young children caused by spleen and kidney deficiency. Siegespermum ediblei has antioxidant, antibacterial, lipid-lowering, and therapeutic effects, and is rich in lysine, which can promote human growth and development. It holds significant economic and research value. Studies have shown that protein peptides prepared from Siegespermum ediblei can improve memory in mice and also possess antioxidant properties, effectively scavenging superoxide anion radicals and hydroxyl radicals. However, despite progress in disease treatment and biological activity research using Siegespermum ediblei, there are currently no reports on its effects on sleep. Summary of the Invention

[0005] In order to overcome the above-mentioned defects and deficiencies in the prior art, the present invention provides the use of Siempuri odorata protein peptide in the preparation of a preparation for improving sleep and / or treating sleep disorders.

[0006] The first object of the present invention is to provide a use of Siempuri protein peptide in the preparation of a preparation for improving sleep and / or treating sleep disorders.

[0007] The second object of the present invention is to provide a preparation.

[0008] The present invention claims the following:

[0009] Application of Siempuri odorata protein peptide in the preparation of medicines for improving sleep and / or treating sleep disorders.

[0010] The sleep disorder is insomnia, decreased sleep time, difficulty falling asleep and / or poor sleep quality.

[0011] Preferably, the preparation method of the Sipuncula esculenta protein peptide is as follows: subjecting Sipuncula esculenta slurry to papain hydrolysis and alkaline protease hydrolysis in sequence, separating the liquid after the hydrolysis, and drying to obtain the Sipuncula esculenta protein peptide.

[0012] More preferably, the slurry of Sipuncula edibleis is obtained by mixing Sipuncula edibleis with water and then homogenizing the mixture, and the usage ratio of Sipuncula edibleis to water is 1 g: (3-5) mL.

[0013] Further preferably, the dosage ratio of the edible Sipunculus edulis to water is 1 g:4 mL.

[0014] More preferably, the usage ratio of the edible siphonophore to papain is 1 g: (4800-5200) U.

[0015] Further preferably, the usage ratio of the edible leather capsule worm to papain is 1 g:5000 U.

[0016] More preferably, the usage ratio of the edible siphonophore to alkaline protease is 1 g: (9800-10200) U.

[0017] Further preferably, the usage ratio of the edible leather capsule worm to alkaline protease is 1 g:10000 U.

[0018] More preferably, the enzymatic hydrolysis time of papain is 2.5 to 3.5 h, and the enzymatic hydrolysis time of alkaline protease is 5.5 to 6.5 h.

[0019] More preferably, the enzymatic hydrolysis time of papain is 3 h, and the enzymatic hydrolysis time of alkaline protease is 6 h.

[0020] More preferably, the temperature for enzymatic hydrolysis of papain is 55-65°C, and the temperature for enzymatic hydrolysis of alkaline protease is 50-60°C.

[0021] More preferably, the enzymatic hydrolysis temperature of papain is 60°C, and the enzymatic hydrolysis temperature of alkaline protease is 55°C.

[0022] More preferably, the pH is adjusted to 6.5-7.5 before the enzymatic hydrolysis by papain, and the pH is adjusted to 9.5-10.5 before the enzymatic hydrolysis by alkaline protease.

[0023] Further preferably, the pH is adjusted to 7 before the enzymatic hydrolysis by papain, and the pH is adjusted to 10 before the enzymatic hydrolysis by alkaline protease.

[0024] More preferably, the enzyme is inactivated before the liquid is separated, and the enzyme inactivation condition is heating at 85-95° C. for 25-35 min.

[0025] More preferably, the enzyme inactivation condition is heating at 90° C. for 30 min.

[0026] More preferably, the separation liquid is centrifuged at 2800-3200 r / min for 8-12 min.

[0027] Further preferably, the separation liquid is centrifuged at 3000 r / min for 10 min.

[0028] Preferably, the medicament increases total sleep time.

[0029] Preferably, the medicament reduces the level of IL-6 in the brain.

[0030] Preferably, the drug increases the level of gamma-aminobutyric acid in the brain.

[0031] A medicine contains Siempuri esculenta protein peptide. The preparation method of the Siempuri esculenta protein peptide comprises the following steps: subjecting Siempuri esculenta slurry to papain enzymolysis and alkaline protease enzymolysis in sequence, separating the liquid after the enzymolysis, and drying to obtain the Siempuri esculenta protein peptide.

[0032] More preferably, the slurry of Sipuncula edibleis is obtained by mixing Sipuncula edibleis with water and then homogenizing the mixture, and the usage ratio of Sipuncula edibleis to water is 1 g: (3-5) mL.

[0033] Further preferably, the dosage ratio of the edible Sipunculus edulis to water is 1 g:4 mL.

[0034] More preferably, the usage ratio of the edible siphonophore to papain is 1 g: (4800-5200) U.

[0035] Further preferably, the usage ratio of the edible leather capsule worm to papain is 1 g:5000 U.

[0036] More preferably, the usage ratio of the edible siphonophore to alkaline protease is 1 g: (9800-10200) U.

[0037] Further preferably, the usage ratio of the edible leather capsule worm to alkaline protease is 1 g:10000 U.

[0038] More preferably, the enzymatic hydrolysis time of papain is 2.5 to 3.5 h, and the enzymatic hydrolysis time of alkaline protease is 5.5 to 6.5 h.

[0039] More preferably, the enzymatic hydrolysis time of papain is 3 h, and the enzymatic hydrolysis time of alkaline protease is 6 h.

[0040] More preferably, the temperature for enzymatic hydrolysis of papain is 55-65°C, and the temperature for enzymatic hydrolysis of alkaline protease is 50-60°C.

[0041] More preferably, the enzymatic hydrolysis temperature of papain is 60°C, and the enzymatic hydrolysis temperature of alkaline protease is 55°C.

[0042] More preferably, the pH is adjusted to 6.5-7.5 before the enzymatic hydrolysis by papain, and the pH is adjusted to 9.5-10.5 before the enzymatic hydrolysis by alkaline protease.

[0043] Further preferably, the pH is adjusted to 7 before the enzymatic hydrolysis by papain, and the pH is adjusted to 10 before the enzymatic hydrolysis by alkaline protease.

[0044] More preferably, the enzyme is inactivated before the liquid is separated, and the enzyme inactivation condition is heating at 85-95° C. for 25-35 min.

[0045] More preferably, the enzyme inactivation condition is heating at 90° C. for 30 min.

[0046] More preferably, the separation liquid is centrifuged at 2800-3200 r / min for 8-12 min.

[0047] Further preferably, the separation liquid is centrifuged at 3000 r / min for 10 min.

[0048] As an practicable manner, the drug further includes a pharmaceutically acceptable excipient.

[0049] As an practicable manner, the dosage form of the drug includes but is not limited to tablets, capsules, granules, powders and pills.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] The present invention discloses the use of a protein peptide from Siempuri odoratus in the preparation of a preparation for improving sleep and / or treating sleep disorders. The present invention sequentially enzymatically hydrolyzes Siempuri odoratus using papain and alkaline protease to prepare a protein peptide from Siempuri odoratus. This protein peptide can not only significantly increase the sleep time of zebrafish with caffeine-induced sleep disorders and improve abnormal changes in IL-6 and GABA levels in the zebrafish brain, but can also significantly prolong the sleep duration of mice induced by sodium pentobarbital, demonstrating strong sleep-improving activity. The protein peptide from Siempuri odoratus can be used to prepare preparations for improving insomnia and treating sleep-related diseases, and has broad application prospects in the field of sleep disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The delicious nematode protein peptide improved the IL-6 and GABA levels in the zebrafish brain induced by caffeine-induced sleep disturbance. * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01. DETAILED DESCRIPTION

[0053] The present invention is further described below with reference to specific examples, which, however, are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0054] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0055] Example 1 Preparation of Sipuncula edulis protein peptide and determination of amino acid composition

[0056] 1. Experimental Methods

[0057] Silurus esculentus (purchased from Dongfang Market in Xiashan District, Zhanjiang City, Guangdong Province) was eviscerated, washed, homogenized, freeze-dried, and powdered. Silurus esculentus powder was mixed with distilled water at a ratio of 1 g:4 mL and homogenized. The pH of the homogenate was adjusted to 7.0, and papain was added at 5000 U / g. Enzymatic hydrolysis was performed at 60°C for 3 h.

[0058] After enzymatic hydrolysis, the pH of the hydrolyzate was adjusted to 10.0, and alkaline protease was added at a concentration of 10,000 U / g. Enzymatic hydrolysis was continued in a 55°C water bath for 6 h. Stirring was continued during the hydrolysis process. After completion, the enzyme was inactivated in a 90°C water bath for 30 min. After cooling to room temperature, the solution was centrifuged at 3,000 rpm for 10 min. The upper oil layer was removed, and the supernatant was vacuum freeze-dried to obtain the sepia protein peptide powder.

[0059] According to the method described in the National Food Safety Standard - Determination of Amino Acids in Food (GB 5009.124-2016), the amino acid composition of the edible siphonophore protein peptide powder samples was determined using an amino acid analyzer.

[0060] 2. Experimental Results

[0061] The amino acid composition of the edible siphonophore protein peptide is shown in Table 1. A total of 9 essential amino acids (EAA) and 8 non-essential amino acids (NEAA) were detected from the edible siphonophore protein peptide, including functional amino acids such as glutamic acid (Glu), aspartic acid (Asp), glycine (Gly), and arginine (Arg). The total amino acid (TAA) content reached 56.43%.

[0062] Table 1 Amino acid composition of Sipuncula edible protein peptide

[0063]

[0064] Example 2 Effects of Siempuri odoratus protein peptides on the sleep duration of caffeine-induced sleep-disordered zebrafish

[0065] 1. Experimental Methods

[0066] The zebrafish were randomly divided into 5 groups, 15 in each group, and then subjected to different treatments. The specific groups and treatments are as follows:

[0067] Control group: fed with ordinary feed for two weeks, twice a day, 0.1 g feed each time;

[0068] Model group: After two weeks of feeding with ordinary diet (twice a day, 0.1 g of diet each time), zebrafish were immersed in a 200 μmol / L caffeine solution for 24 h;

[0069] Melatonin-positive group: After two weeks of feeding with a normal diet (twice a day, 0.1 g of diet each time), zebrafish were immersed in a 200 μmol / L caffeine solution for 24 h, and then transferred to a 130 mg / L melatonin solution for 2 h.

[0070] Low-dose intervention group of edible siphonophore protein peptide: 5.0 g / kg feed of the edible siphonophore protein peptide prepared in Example 1 was added to ordinary feed. After two weeks of feeding (twice a day, 0.1 g feed each time), zebrafish were soaked in a caffeine solution with a concentration of 200 μmol / L for 24 hours.

[0071] High-dose intervention group of edible siphonophore protein peptide: 10.0 g / kg of feed of the edible siphonophore protein peptide prepared in Example 1 was added to ordinary feed. After two weeks of feeding (twice a day, 0.1 g of feed each time), zebrafish were soaked in a caffeine solution with a concentration of 200 μmol / L for 24 hours.

[0072] After the treatment, 6 zebrafish were randomly selected from each group. The movement distance of the zebrafish within 10 minutes was measured using a behavior analyzer. The total movement distance was converted into 24 hours. The sleep improvement effect of the edible nematode protein peptide on insomnia zebrafish was evaluated according to the formula: sleep improvement effect = (movement distance of the model group - movement distance of the intervention group) / (movement distance of the model group - movement distance of the control group) × 100%.

[0073] 2. Experimental Results

[0074] The results are shown in Table 2. Compared with the movement distance of zebrafish in the control group (182162±10431 mm), the movement distance of zebrafish in the model group (375621±18531 mm) was significantly increased (P<0.01), indicating that the model was successfully established.

[0075] Compared with the model group, the melatonin-positive zebrafish had a significantly decreased movement distance (257,834 ± 13,762 mm), and a sleep improvement effect of 60.88%, indicating that melatonin significantly improves the sleep of insomniac zebrafish. The movement distances of zebrafish in the low-dose and high-dose sepia peptide intervention groups were 289,412 ± 21,823 mm and 248,733 ± 14,245 mm, respectively, both significantly decreased compared with the model group, and sleep improvement effects of 44.56% and 65.59%, respectively, indicating that sepia peptide significantly improves the sleep quality of insomniac zebrafish under this experimental dose.

[0076] Table 2 Effects of Sipuncula edible protein peptides on the sleep time of zebrafish with caffeine-induced sleep disorder

[0077]

[0078] Note: Data are expressed as mean ± SEM (standard error), and data between groups were analyzed by one-way ANOVA.* P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01.

[0079] Example 3 Effects of Siempuri odoratum protein peptides on sleep-related physiological indicators in the brain of zebras with caffeine-induced sleep disorder

[0080] 1. Experimental Methods

[0081] IL-6 is a multifunctional cytokine involved in regulating various pathological processes, including immune responses. Modern medicine believes that sleep disorders are closely linked to inflammatory states. γ-Aminobutyric acid (GABA) is an inhibitory neurotransmitter that inhibits cerebral cortical cells within the synaptic compartments of the brain, promoting sleep.

[0082] The zebrafish were grouped and treated according to Example 2. After the treatment, the zebrafish were killed and the brain tissues were isolated. The levels of interleukin-6 (IL-6) and gamma-aminobutyric acid (GABA) were detected using ELISA kits.

[0083] 2. Experimental Results

[0084] The results are as follows Figure 1 As shown in the results, compared with the control group, the IL-6 level in the zebrafish brain of the model group was significantly increased, while the GABA level was significantly decreased. Compared with the model group, the protein peptides from the worm can reduce the IL-6 level to varying degrees, while significantly increasing the GABA level.

[0085] The above results indicate that the edible siphonophore protein peptide can improve the abnormal IL-6 and GABA levels in the brain of zebrafish with sleep disorders, which may be related to its improvement of the sleep quality of insomnia zebrafish.

[0086] Example 4 Effects of Siempuri edulis protein peptides on sleep onset time and sleep duration in mice induced by sodium pentobarbital

[0087] 1. Experimental Methods

[0088] The mice were randomly divided into three groups of 6 mice each, half male and half female, and received different treatments:

[0089] Low-dose group of Silurian worm protein peptide: The Silurian worm protein peptide prepared in Example 1 was dissolved in physiological saline, and mice were gavaged with 0.5 g / kg BW of the Silurian worm protein peptide once a day for 14 consecutive days.

[0090] High-dose group of Silurian worm protein peptide: The Silurian worm protein peptide prepared in Example 1 was dissolved in physiological saline, and mice were gavaged with 1.0 g / kg BW of the Silurian worm protein peptide once a day for 14 consecutive days.

[0091] Control group: administered an equal amount of normal saline once a day for 14 consecutive days.

[0092] Thirty minutes after the last administration, the mice were intraperitoneally injected with sodium pentobarbital (40 mg / kg). The disappearance of the righting reflex was defined as the time of sleep onset, and the time from the disappearance of the righting reflex to the recovery was defined as the sleep duration. The sleep onset time and sleep duration of the mice were recorded respectively.

[0093] 2. Experimental Results

[0094] The results are shown in Table 3. The edible nematode protein peptide had no significant effect on the sleep time of mice induced by sodium pentobarbital, but a high dose of edible nematode protein peptide could significantly prolong the sleep duration of mice (P<0.05).

[0095] Table 3 Effects of Siempuri odoratus protein peptides on sleep in mice induced by sodium pentobarbital

[0096]

[0097] Note: Data are expressed as mean ± SEM (standard error), and data between groups were analyzed by one-way ANOVA. * P<0.05.

[0098] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. The use of a protein peptide from Echinops esculentus in the preparation of a medicament for treating sleep disorders, characterized in that: The preparation method of the edible siphonophore protein peptide comprises the following steps: subjecting the edible siphonophore slurry to papain enzymolysis and alkaline protease enzymolysis in sequence, separating the liquid after the enzymolysis is completed, and drying to obtain the edible siphonophore protein peptide; The sleep disorder is insomnia caused by caffeine.

2. The use according to claim 1, characterized in that The edible leather cyst slurry is obtained by mixing the edible leather cyst with water and then homogenizing the mixture. The dosage ratio of the edible leather cyst to water is 1 g: (3-5) mL.

3. The use according to claim 1, characterized in that The dosage ratio of the edible leather worm and papain is 1 g: (4800-5200) U.

4. The use according to claim 1, characterized in that The dosage ratio of the edible leather worm and alkaline protease is 1 g: (9800-10200) U.

5. The use according to claim 1, characterized in that The enzymatic hydrolysis time of the papain is 2.5 to 3.5 hours, and the enzymatic hydrolysis time of the alkaline protease is 5.5 to 6.5 hours.

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