Application of sea cucumber phospholipid in preparation of preparation for improving sleep and / or treating sleep disorder

Abstract

The invention discloses an application of sea cucumber phospholipid in preparation of a preparation for improving sleep and / or treating sleep disorders. Researches find that the sea cucumber phospholipid can significantly prolong the sleep time of the zebrafish with sleep disorder induced by caffeine and improve abnormal changes of IL-6 and GABA levels in brains of the zebrafish. Meanwhile, the sea cucumber phospholipid can obviously prolong the sleep duration of the mice induced by pentobarbital sodium. After being prepared into a preparation, the sea cucumber phospholipid can be used 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 sea cucumber phospholipids in preparing a preparation for improving sleep and / or treating sleep disorders. Background Art

[0002] Insomnia is the main common symptom of sleep disorders, often characterized by difficulty falling asleep and short sleep duration. Sleep disorders often manifest as fatigue, lack of energy, metabolic disorders, and are prone to problems such as decreased learning ability and memory impairment, which have a great impact on physical health and cause a variety of chronic diseases in the human body.

[0003] The mechanism of insomnia is relatively complex, and current research is still very limited. Existing studies believe that insomnia is closely related to the central nervous system that controls the body's sleep and wakefulness, as well as related neurotransmitters and inflammatory factors. Some researchers have proposed that an imbalance between sleep induction and wakefulness induction will lead to insomnia. The body's wakefulness control system is distributed in various areas such as the brainstem, hypothalamus, and basal forebrain, involving substances that promote wakefulness such as norepinephrine, 5-hydroxytryptamine, dopamine, glutamate, choline, histamine, acetylcholine, and glutamate; the body's sleep-promoting control system is mainly regulated by inhibitory neurotransmitters such as γ-aminobutyric acid, which are distributed in the hypothalamus, thalamus and other areas. In addition, the body's sleep and wakefulness are also controlled by non-neural mechanisms such as genes, hormones, interstitial ion components, cytokines, and cellular metabolites in the body.

[0004] 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.

[0005] Sea cucumbers have been considered a precious nourishing food and medicinal material since ancient times. They are ranked first among the "Eight Treasures of the Sea" and have high edible and medicinal value. Traditional Chinese medicine believes that sea cucumbers are warm in nature, sweet and salty in taste, and have the effects of nourishing the kidney and essence, nourishing yin and blood. Modern medicine shows that sea cucumbers have multiple functions such as immune regulation. Sea cucumbers contain many active substances, such as sea cucumber polysaccharides, sea cucumber phospholipids and sea cucumber cerebrosides. Among them, sea cucumber phospholipids are one of the substances with high biological activity. The body wall of sea cucumbers is rich in phospholipids, and sea cucumber phospholipids are mainly phosphatidylcholine and phosphatidylethanolamine. Among them, phosphatidylcholine (PC), also known as lecithin, is an important component of phospholipids. Studies have shown that phospholipids have the activity of improving immunity, anti-oxidation, regulating lipid metabolism, and enhancing brain function. Chinese invention patents CN110327358A and CN110478374A respectively disclose the use of sea cucumber phospholipids in the preparation of drugs for inhibiting neuroinflammation and in the preparation of neuronal cell protection drugs. However, there are currently no reports on the effects of sea cucumber phospholipids on sleep. Summary of the invention

[0006] In order to overcome the above-mentioned defects and deficiencies in the prior art, the present invention provides the use of sea cucumber phospholipids in the preparation of a preparation for improving sleep and / or treating sleep disorders.

[0007] The first object of the present invention is to provide the use of sea cucumber phospholipids in preparing a preparation for improving sleep and / or treating sleep disorders.

[0008] The present invention claims the following: Application of sea cucumber phospholipids in preparing 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] The active ingredients of the sea cucumber phospholipids include phosphatidylinositol.

[0011] Preferably, the formulation increases total sleep time.

[0012] Preferably, the formulation reduces the level of IL-6 in the brain.

[0013] Preferably, the formulation increases the level of gamma-aminobutyric acid in the brain.

[0014] Preferably, the method for preparing the sea cucumber phospholipids comprises the following steps: S1. Adding acetone to the sea cucumber sample for extraction, and then separating the liquid and drying to obtain a crude product; S2. Add acetone to the crude product for extraction, collect the acetone insoluble part, then add ethanol, collect the ethanol insoluble part, and obtain sea cucumber phospholipids.

[0015] More preferably, in step S1, the extraction method is: soaking and / or ultrasonicating the sea cucumber sample in acetone.

[0016] Further preferably, the soaking time is not less than 12 h.

[0017] More preferably, in step S1, the acetone is cold acetone.

[0018] More preferably, in step S1, the method of separating the liquid is suction filtration.

[0019] More preferably, in step S2, the usage ratio of the crude product to acetone is 1 g: (3-5) mL.

[0020] Further preferably, the usage ratio of the crude product and acetone is 1 g:4 mL.

[0021] More preferably, in step S2, the extraction is performed at 45-55° C. for 55-65 min.

[0022] More preferably, the extraction is performed at 50° C. for 60 min.

[0023] More preferably, the extraction is performed 2 to 4 times.

[0024] More preferably, the extraction is performed three times.

[0025] More preferably, in step S2, the volume fraction of ethanol is 90-95%.

[0026] As an practicable manner, the preparation includes a medicine and a health food, the medicine further includes a pharmaceutically acceptable excipient, and the health food further includes a food-acceptable additive.

[0027] As an practicable manner, the dosage form of the preparation includes tablets, capsules, granules, powders and pills.

[0028] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses the application of sea cucumber phospholipids in the preparation of a preparation for improving sleep and / or treating sleep disorders. The present invention has found that sea cucumber phospholipids can 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. At the same time, sea cucumber phospholipids can significantly prolong the sleep duration of mice induced by sodium pentobarbital. After sea cucumber phospholipids are made into preparations, they can be used to improve insomnia and treat diseases related to sleep disorders, and have broad application prospects in the field of sleep disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Sea cucumber phospholipids improved the levels of IL-6 and GABA in the brain of zebrafish with caffeine-induced sleep disorders. Compared with the control group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01. DETAILED DESCRIPTION

[0030] 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.

[0031] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0032] Example 1 Preparation of sea cucumber lecithin Sea cucumber lecithin is the phospholipid ethanol-insoluble part obtained by gradually extracting dry powder of southern rough sea cucumber (purchased from Shenzhen Taifeng Biotechnology Co., Ltd.) with cold acetone and ethanol. The preparation method is carried out according to the method of "1.2 Extraction of sea cucumber lecithin" in Example 1 of Chinese invention patent CN110327358A. The specific operation is as follows: Take 13 kg of rough sea cucumber and crush it to make sea cucumber powder. The sea cucumber powder is first soaked in 4℃ precooled acetone overnight, and the volume of acetone is twice the volume of the sea cucumber powder. After soaking, filter and dry the filtrate under reduced pressure with a water pump at 50℃ to obtain a material with preliminary fat removal, which is weighed. Soak the filter residue in 3 times the volume of anhydrous ethanol overnight, ultrasonicate at 40℃ for 30 min, and filter the filtrate after the ultrasonication. Extract the filter residue 4 times continuously according to this method, and combine the filtrates extracted 4 times and dry them and weigh them to obtain crude phospholipids.

[0033] Use acetone to remove the fat components in the crude phospholipids: Mix the crude phospholipids and acetone in a ratio of 1 g: 4 mL, extract at 50°C for 60 min, and extract 3 times in total. Collect the acetone-insoluble part, add 95% ethanol solution (v / v), collect the insoluble part, spin dry, and store at 4°C.

[0034] The prepared sea cucumber phospholipids were white to light yellow powder (i.e., material Fr.2-1 part, SCW), which was sent to Wuhan Mattwell Biotechnology Co., Ltd. for testing. The main components are shown in Table 1.

[0035] Table 1 Main components of sea cucumber phospholipids

[0036] Note: PI stands for phosphatidylinositol, and LPI stands for lysophosphatidylinositol. For example, LPI 19:0 means that the phospholipid structure is a straight chain of 19 carbons and the degree of unsaturation is 0; while PI 38:4; PI 18:0-20:0 means that there are two branched chains with 18 carbons and 20 carbons respectively.

[0037] Example 2 Effect of sea cucumber phospholipids 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; Sea cucumber lecithin low-dose intervention group: 50 mg / kg of the sea cucumber lecithin prepared in Example 1 was added to the 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; Sea cucumber lecithin high-dose intervention group: 100 mg / kg of the sea cucumber lecithin 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.

[0038] 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 sea cucumber phospholipids 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%.

[0039] 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.

[0040] 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 insomniac zebrafish.

[0041] The movement distances of zebrafish in the low-dose and high-dose sea cucumber phospholipid intervention groups were 327463±24327 mm and 274952±17632 mm, respectively. Compared with the model group, the movement distances of zebrafish in the high-dose sea cucumber phospholipid intervention group were significantly decreased, and the sleep improvement effects were 24.89% and 52.04%, respectively, indicating that sea cucumber phospholipids have a significant improvement effect on the sleep quality of insomnia zebrafish under the experimental dosage conditions.

[0042] Table 2 Effects of sea cucumber phospholipids on the sleep time of zebrafish with caffeine-induced sleep disorder

[0043] 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.

[0044] Example 3 Effects of sea cucumber phospholipids on sleep-related physiological indicators in the brain of zebrafish 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.

[0045] 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.

[0046] 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 low-dose sea cucumber phospholipid intervention group and the high-dose sea cucumber phospholipid intervention group could reduce the IL-6 level to varying degrees, and also significantly increase the GABA level.

[0047] The above results indicate that sea cucumber phospholipids can improve the abnormal levels of IL-6 and GABA in the brain of zebrafish with sleep disorders, which may be related to its improvement of the sleep quality of insomnia zebrafish.

[0048] Example 4 Effects of sea cucumber phospholipids on 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: Sea cucumber lecithin low-dose group: The sea cucumber lecithin prepared in Example 1 was dissolved in physiological saline, and 50 mg / kg BW of sea cucumber lecithin was administered orally to mice once a day for 14 consecutive days.

[0049] Sea cucumber lecithin high-dose group: The sea cucumber lecithin prepared in Example 1 was dissolved in physiological saline, and 100 mg / kg BW of sea cucumber lecithin was gavaged into the mice once a day for 14 consecutive days.

[0050] Control group: given an equal amount of normal saline once a day for 14 consecutive days.

[0051] 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.

[0052] 2. Experimental Results The results are shown in Table 3. Low-dose sea cucumber phospholipids had no significant effect on the sleep time of mice induced by sodium pentobarbital, but high-dose sea cucumber phospholipids could significantly prolong the sleep duration of mice (P<0.05).

[0053] Table 3 Effects of sea cucumber phospholipids on sleep in mice induced by sodium pentobarbital

[0054] Note: Data are expressed as mean ± SEM (standard error), and data among groups were analyzed by one-way ANOVA. ** P<0.05.

[0055] 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 sea cucumber phospholipids in the preparation of preparations for improving sleep and / or treating sleep disorders.

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

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

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

5. The use according to claim 1, characterized in that: The preparation method of the sea cucumber phospholipids comprises the following steps: S1. Adding acetone to the sea cucumber sample for extraction, and then separating the liquid and drying to obtain a crude product; S2. Add acetone to the crude product for extraction, collect the acetone insoluble part, then add ethanol, collect the ethanol insoluble part, and obtain sea cucumber phospholipids.

6. The use according to claim 5, characterized in that: In step S1, the extraction method is: soaking and / or ultrasonicating the sea cucumber sample in acetone.

7. The use according to claim 6, characterized in that The soaking time is not less than 12 h.

8. The use according to claim 5, characterized in that In step S2, the usage ratio of the crude product and acetone is 1 g: (3-5) mL.

9. The use according to claim 1, characterized in that: The preparation includes medicine and health food, the medicine also includes pharmaceutically acceptable excipients, and the health food also includes food-acceptable additives.

10. The use according to claim 1, characterized in that: The dosage forms of the preparation include tablets, capsules, granules, powders and pills.

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