Application of phascolosoma esculenta protein peptide in preparation of preparation for improving sleep and / or treating sleep disorder

Abstract

The invention discloses application of phascolosoma esculenta protein peptide in preparation of a preparation for improving sleep and / or treating sleep disorder. The phascolosoma esculenta protein peptide is prepared by sequentially carrying out enzymolysis on phascolosoma esculenta through papain and alkaline protease, and the protein peptide can remarkably prolong the sleep time of caffeine-induced sleep disorder zebra fish and improve abnormal changes of IL-6 and GABA levels in brains of the zebra fish; in addition, the sleep duration of mice induced by pentobarbital sodium can be remarkably prolonged, and relatively high sleep improvement activity is shown. The phascolosoma esculenta protein peptide can be used for preparing a preparation for improving insomnia and treating sleep disorder related diseases, and has a wide application prospect in the field of sleep disorder.

Description

Technical Field

[0001] The present invention relates to the technical field of improving sleep, and in particular to the application of Siempuri odoratus protein peptide in the preparation of a preparation for improving sleep and / or treating sleep disorders. Background Art

[0002] Insomnia refers to a sleep disorder in which patients are dissatisfied with the performance of the sleep process and the length of sleep time under appropriate sleep conditions, thus affecting their normal daytime life. Sleep disorders may appear in different forms, including difficulty falling asleep, sleep interruptions, waking up too early, insufficient sleep time, and feeling tired after waking up. Specific types of such disorders include insomnia, sleep breathing disorders (such as sleep apnea), excessive sleepiness, abnormal sleep behavior, and circadian rhythm disorders. The main feature of sleep disorders is the imbalance between endogenous biological rhythms and external time signals. Its causal mechanism involves multiple factors, and current research is still very limited. Some researchers pointed out that sleep disorders may be closely related to the weakening of melatonin receptor activity, overactivity of the hypothalamus-pituitary-adrenal (HPA) axis, dysregulation of neurotransmitter secretion, and abnormal release of inflammatory cytokines, but it is still unclear what specific pathways or mechanisms are affected.

[0003] At present, the drugs used to improve sleep in clinical practice are mainly chemical drugs, such as benzodiazepines, non-benzodiazepines, etc. However, these chemical drugs often have obvious side effects when treating human sleep disorders, such as mental slackness, drowsiness and excessive sedation, and long-term use will cause great dependence or even addiction. Therefore, exploring effective and safe drugs to improve sleep is of great significance in the development of products for the prevention and treatment of sleep disorders related diseases.

[0004] Edible leather capsule worm ( Phascolosoma esculenta ) is also known as earth nail, earth shoot, mud ding, etc. It belongs to the annelid of the class of siphonophore, and is widely distributed in coastal areas such as Fujian, Guangxi, Guangdong and Hainan in my country. Edible siphonophore has always been considered a healthy food that nourishes yin and nourishes the kidney. It has the functions of nourishing yin and reducing fire, clearing the lungs and replenishing deficiency, promoting blood circulation and strengthening the body, and nourishing the kidney and nourishing the skin. It is also effective for frequent nocturia caused by spleen deficiency and kidney deficiency in young children. Edible siphonophore has antioxidant, antibacterial, lipid-lowering, and therapeutic effects of sweating. It is also rich in lysine and can promote human growth and development. It has important economic value and research value. Studies have shown that protein peptides prepared from edible siphonophore as raw materials can improve the memory of mice. It also has antioxidant effects and can effectively remove superoxide anion free radicals and hydroxyl free radicals. However, although edible siphonophore has made certain progress in disease treatment and biological activity research, there is currently no research report on the effect of edible siphonophore on sleep. Summary of the invention

[0005] In order to overcome the above-mentioned defects and shortcomings in the prior art, the present invention provides the use of sempervivum 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 an application of an edible siphonophore 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: Application of sempervivum protein peptide in the preparation of preparations for improving sleep and / or treating sleep disorders.

[0009] The sleep disorder is insomnia, decreased sleep time, difficulty falling asleep and / or poor sleep quality.

[0010] Preferably, the method for preparing the Sipuncula protein peptide is as follows: subjecting Sipuncula slurry to papain enzymolysis and alkaline protease enzymolysis in sequence, separating the liquid after the enzymolysis, and drying to obtain the Sipuncula protein peptide.

[0011] More preferably, the slurry of Sipuncula edibleis is obtained by mixing Sipuncula edibleis with water and then homogenizing them, and the dosage ratio of the Sipuncula edibleis to water is 1 g: (3-5) mL.

[0012] Further preferably, the dosage ratio of the edible Sipunculus edulis to water is 1 g:4 mL.

[0013] More preferably, the dosage ratio of the edible leather capsule worm to papain is 1 g: (4800-5200) U.

[0014] Further preferably, the usage ratio of the edible leather capsule worm to papain is 1 g:5000 U.

[0015] More preferably, the usage ratio of the edible leather worm to alkaline protease is 1 g: (9800-10200) U.

[0016] Further preferably, the usage ratio of the edible leather worm to alkaline protease is 1 g:10000 U.

[0017] 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.

[0018] More preferably, the enzymatic hydrolysis time of papain is 3 h, and the enzymatic hydrolysis time of alkaline protease is 6 h.

[0019] More preferably, the enzymatic hydrolysis temperature of papain is 55-65°C, and the enzymatic hydrolysis temperature of alkaline protease is 50-60°C.

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

[0021] More preferably, the pH is adjusted to 6.5-7.5 before the papain enzymolysis, and the pH is adjusted to 9.5-10.5 before the alkaline protease enzymolysis.

[0022] More preferably, the pH is adjusted to 7 before the papain hydrolysis, and the pH is adjusted to 10 before the alkaline protease hydrolysis.

[0023] 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.

[0024] More preferably, the enzyme inactivation condition is heating at 90° C. for 30 min.

[0025] More preferably, the separation liquid is centrifuged at 2800-3200 r / min for 8-12 min.

[0026] Further preferably, the separation liquid is centrifuged at 3000 r / min for 10 min.

[0027] Preferably, the formulation increases total sleep time.

[0028] Preferably, the formulation reduces the level of IL-6 in the brain.

[0029] Preferably, the formulation increases the level of gamma-aminobutyric acid in the brain.

[0030] A preparation contains Siempurus esculentus protein peptide. The preparation method of the Siempurus esculentus protein peptide is as follows: subjecting Siempurus esculentus slurry to papain enzymolysis and alkaline protease enzymolysis in sequence, separating the liquid after the enzymolysis, and drying to obtain the Siempurus esculentus protein peptide.

[0031] More preferably, the slurry of Sipuncula edibleis is obtained by mixing Sipuncula edibleis with water and then homogenizing them, and the dosage ratio of the Sipuncula edibleis to water is 1 g: (3-5) mL.

[0032] Further preferably, the dosage ratio of the edible Sipunculus edulis to water is 1 g:4 mL.

[0033] More preferably, the dosage ratio of the edible leather capsule worm to papain is 1 g: (4800-5200) U.

[0034] Further preferably, the usage ratio of the edible leather capsule worm to papain is 1 g:5000 U.

[0035] More preferably, the usage ratio of the edible leather worm to alkaline protease is 1 g: (9800-10200) U.

[0036] Further preferably, the usage ratio of the edible leather worm to alkaline protease is 1 g:10000 U.

[0037] 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.

[0038] More preferably, the enzymatic hydrolysis time of papain is 3 h, and the enzymatic hydrolysis time of alkaline protease is 6 h.

[0039] More preferably, the enzymatic hydrolysis temperature of papain is 55-65°C, and the enzymatic hydrolysis temperature of alkaline protease is 50-60°C.

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

[0041] More preferably, the pH is adjusted to 6.5-7.5 before the papain enzymolysis, and the pH is adjusted to 9.5-10.5 before the alkaline protease enzymolysis.

[0042] More preferably, the pH is adjusted to 7 before the papain hydrolysis, and the pH is adjusted to 10 before the alkaline protease hydrolysis.

[0043] 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.

[0044] More preferably, the enzyme inactivation condition is heating at 90° C. for 30 min.

[0045] More preferably, the separation liquid is centrifuged at 2800-3200 r / min for 8-12 min.

[0046] Further preferably, the separation liquid is centrifuged at 3000 r / min for 10 min.

[0047] As an practicable manner, the preparation includes but is not limited to medicines and health foods, the medicines also include pharmaceutically acceptable excipients, and the health foods also include food-acceptable additives.

[0048] As an practicable manner, the dosage form of the preparation includes but is not limited to tablets, capsules, granules, powders and pills.

[0049] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses the use of a protein peptide of an edible siphonophore in the preparation of a preparation for improving sleep and / or treating sleep disorders. The present invention uses papain and alkaline protease to enzymolyze the edible siphonophore in sequence to prepare a protein peptide of an edible siphonophore, which can not only significantly increase the sleep time of zebrafish with caffeine-induced sleep disorders and improve the abnormal changes in IL-6 and GABA levels in the zebrafish brain, but also significantly prolong the sleep duration of mice induced by sodium pentobarbital, showing a strong activity for improving sleep. The protein peptide of the edible siphonophore 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] 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

[0051] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0052] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0053] Example 1 Preparation of Siempuri edulis protein peptide and determination of amino acid composition 1. Experimental Methods The edible leather worm (purchased from Dongfang Market, Xiashan District, Zhanjiang City, Guangdong Province) was cleaned after removing the internal organs, homogenized, frozen and dried into powder. The edible leather worm powder was mixed with distilled water at a ratio of 1 g: 4 mL for homogenization, the pH value of the homogenate was adjusted to 7.0, papain was added at an enzyme addition amount of 5000 U / g, and enzymolysis was carried out at 60℃ for 3 h.

[0054] After the enzymatic hydrolysis, the pH value of the enzymatic hydrolyzate was adjusted to 10.0, and alkaline protease was added at an enzyme dosage of 10,000 U / g, and the enzymatic hydrolysis was continued in a 55°C constant temperature water bath for 6 h. Stirring was continued during the enzymatic hydrolysis process, and the enzyme was inactivated in a 90°C water bath for 30 min after the enzymatic hydrolysis was completed. After cooling to room temperature, the solution was centrifuged at 3,000 r / min for 10 min, and the upper oil layer was removed. The supernatant was taken for vacuum freeze drying to obtain the edible leather capsule worm protein peptide powder.

[0055] According to the method described in the National Food Safety Standard - Determination of Amino Acids in Foods (GB 5009.124-2016), the amino acid composition of the edible siphonophore protein peptide powder samples was determined using an amino acid analyzer.

[0056] 2. Experimental Results The amino acid composition of the edible leather cyst 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 leather cyst 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%.

[0057] Table 1 Amino acid composition of edible siphonophore protein peptide

[0058] Example 2 Effect of Echinops edibleus protein peptide on the sleep time of caffeine-induced sleep-disordered zebrafish 1. Experimental Methods 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: Control group: fed with ordinary feed for two weeks, twice a day, 0.1 g feed each time; Model group: After two weeks of feeding with ordinary feed (twice a day, 0.1 g feed each time), zebrafish were immersed in a caffeine solution with a concentration of 200 μmol / L for 24 h; Melatonin-positive group: After two weeks of feeding with normal diet (twice a day, 0.1 g of diet each time), zebrafish were immersed in 200 μmol / L caffeine solution for 24 h, and then transferred to 130 mg / L melatonin solution for 2 h; Low-dose intervention group of edible siphonophore protein peptide: 5.0 g / kg of 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 h. High-dose intervention group of edible siphonophore protein peptide: 10.0 g / kg of 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 h.

[0059] After the treatment, 6 zebrafish were randomly selected from each group, and the movement distance of the zebrafish within 10 min was measured using a behavior analyzer. The distance was converted into the total movement distance for 24 h, and then the sleep improvement effect of the edible nematode protein peptide on insomnia zebrafish was evaluated according to the formula: sleep improvement effect = (model group movement distance - intervention group movement distance) / (model group movement distance - control group movement distance) × 100%.

[0060] 2. Experimental Results 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.

[0061] Compared with the model group, the movement distance of zebrafish in the melatonin-positive group (257834±13762 mm) was significantly decreased, and the sleep improvement effect was 60.88%, indicating that melatonin has a significant improvement effect on the sleep of insomnia zebrafish. The movement distances of zebrafish in the low-dose intervention group and the high-dose intervention group of edible nematode protein peptide were 289412±21823 mm and 248733±14245 mm, respectively, which were significantly decreased compared with the model group, and the sleep improvement effects were 44.56% and 65.59%, respectively, indicating that under the experimental dosage conditions, edible nematode protein peptide has a significant improvement effect on the sleep quality of insomnia zebrafish.

[0062] Table 2 Effects of sempervivum protein peptide on the sleep time of zebrafish with caffeine-induced sleep disorder

[0063] Note: Data are expressed as mean ± SEM (standard error), and data among groups were analyzed by one-way ANOVA. * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01.

[0064] Example 3 Effects of Echinops edibleus protein peptide on sleep-related physiological indicators in the brain of zebras with caffeine-induced sleep disorders 1. Experimental Methods IL-6 is a multifunctional cytokine that participates in regulating immune responses and other pathological processes. Modern medicine believes that sleep disorders are closely related to the body's inflammatory state. γ-Aminobutyric acid (GABA) is an inhibitory neurotransmitter that inhibits cerebral cortical cells in the synaptic compartment of the brain and promotes sleep.

[0065] The animals were grouped and treated according to Example 2. After the treatment, the zebrafish were killed and the brain tissue was isolated. The contents of interleukin 6 (IL-6) and gamma-aminobutyric acid (GABA) were detected using an ELISA detection kit.

[0066] 2. Experimental Results The results are as follows Figure 1 As shown in the figure, 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 sepia protein peptide can reduce the IL-6 level to varying degrees, while significantly increasing the GABA level.

[0067] The above results indicate that the edible nematode 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.

[0068] Example 4 Effects of Siempuri edulis protein peptide on the sleep onset time and sleep duration of mice induced by sodium pentobarbital 1. Experimental Methods The mice were randomly divided into 3 groups with 6 mice in each group, half male and half female, and received different treatments: Low-dose group of edible siphonophore protein peptide: The edible siphonophore protein peptide prepared in Example 1 was dissolved in physiological saline, and mice were gavaged with 0.5 g / kg BW of the edible siphonophore protein peptide once a day for 14 consecutive days.

[0069] High-dose group of edible siphonophore protein peptide: The edible siphonophore protein peptide prepared in Example 1 was dissolved in physiological saline, and 1.0 g / kg BW of the edible siphonophore protein peptide was gavaged into the mice once a day for 14 consecutive days.

[0070] Control group: given an equal amount of normal saline once a day for 14 consecutive days.

[0071] 30 min after the last administration, the mice were intraperitoneally injected with sodium pentobarbital (40 mg / kg). The disappearance of the righting reflex of the mice was taken as the sleep onset time, and the time from the disappearance of the righting reflex to the recovery was taken as the sleep duration. The sleep onset time and sleep duration of the mice were recorded respectively.

[0072] 2. Experimental Results 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).

[0073] Table 3 Effects of sempervivum protein peptide on sleep in mice induced by sodium pentobarbital

[0074] Note: Data are expressed as mean ± SEM (standard error), and data among groups were analyzed by one-way ANOVA. * P<0.05.

[0075] 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 equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of edible siphonophore protein peptide in the preparation of preparations for improving sleep and / or treating sleep disorders.

2. The use according to claim 1, characterized in that: The preparation method of the edible leather worm protein peptide comprises the following steps: subjecting the edible leather worm slurry to papain enzymolysis and alkaline protease enzymolysis in sequence, separating the liquid after the enzymolysis is completed, and drying to obtain the edible leather worm protein peptide.

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

4. The use according to claim 2, characterized in that: The dosage ratio of the edible leather worm to papain is 1 g: (4800-5200) U.

5. The use according to claim 2, characterized in that: The dosage ratio of the edible leather siphonophore to alkaline protease is 1 g: (9800-10200) U.

6. The use according to claim 2, 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.

7. The use according to claim 1, characterized in that: The formulation increases total sleep time.

8. The use according to claim 1, characterized in that: The preparation reduces the level of IL-6 in the brain.

9. The use according to claim 1, characterized in that: The preparation increases the level of gamma-aminobutyric acid in the brain.

10. A preparation, characterized in that The preparation contains Siempuri esculenta protein peptide, and the preparation method of the Siempuri esculenta protein peptide is as follows: subjecting Siempuri esculenta slurry to papain enzymolysis and alkaline protease enzymolysis in sequence, separating the liquid after the enzymolysis is completed, and obtaining the Siempuri esculenta protein peptide after drying.

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