Application of sea cucumber phospholipids in the preparation of a preparation for improving sleep and / or treating sleep disorders

Abstract

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 through research that sea cucumber phospholipids can significantly increase the sleep time of zebrafish with sleep disorders induced by caffeine and improve the abnormal changes in the levels of IL-6 and GABA in the brains of zebrafish. At the same time, sea cucumber phospholipids can significantly prolong the sleep duration of mice induced by sodium pentobarbital. After preparing sea cucumber phospholipids into a preparation, it can be used to improve insomnia and treat sleep disorder-related diseases, 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. Specifically, it relates to the application of sea cucumber phospholipids in the preparation of preparations 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 maintenance time. Sleep disorders are often manifested as easy fatigue, listlessness, metabolic disorders, and problems such as decreased learning ability and memory disorders, which have a great impact on physical health and can cause various chronic diseases in the human body.

[0003] The occurrence mechanism of insomnia is relatively complex, and current research is still very limited. Existing research believes that insomnia is closely related to the central nervous system that controls the body's sleep and wakefulness, 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 regions such as the brainstem, hypothalamus, and basal forebrain, and involves 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 is distributed in regions such as the hypothalamus and thalamus. In addition, the body's sleep and wakefulness are also controlled by non-neural mechanisms such as genes, hormones, intercellular ion components, cytokines, and cell metabolites in the body.

[0004] Currently, the drugs used clinically to improve sleep mainly rely on chemical drugs, such as benzodiazepines, non-benzodiazepines, etc. However, these chemical drugs often produce obvious side effects when treating human sleep diseases, such as mental slackness, drowsiness, and excessive sedation. At the same time, long-term use will produce great dependence and even addiction. Therefore, exploring effective and safe sleep-improving drugs is of great significance in the development of products for the prevention and treatment of sleep disorder-related diseases.

[0005] Sea cucumbers have been regarded as a precious nourishing food and medicinal material since ancient times. Ranking first among the "Eight Treasures of the Sea", they have high edible and medicinal values. Traditional Chinese medicine believes that sea cucumbers are warm in nature, sweet and salty in taste, and have the effects of tonifying the kidney and replenishing essence, nourishing yin and blood, etc. Modern medicine shows that sea cucumbers have multiple effects 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 part of phospholipids. Research shows that phospholipids have activities such as improving the body's immunity, antioxidation, regulating lipid metabolism, and enhancing brain function. Chinese invention patent CN110327358A and Chinese invention patent CN110478374A respectively disclose the applications of sea cucumber phospholipids in the preparation of drugs for inhibiting neuroinflammation and in the preparation of drugs for protecting neuron cells. However, there is currently no research report on the effect of sea cucumber phospholipids on sleep. Summary of the Invention

[0006] To overcome the above-mentioned defects and deficiencies in the prior art, the present invention provides the application of sea cucumber phospholipids in the preparation of preparations for improving sleep and / or treating sleep disorders.

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

[0008] The present invention claims the following:

[0009] The application of sea cucumber phospholipids in the preparation of drugs for improving sleep and / or treating sleep disorders.

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

[0011] The active ingredients of the sea cucumber phospholipids include phosphatidylinositol.

[0012] Preferably, the drug increases the total sleep duration.

[0013] Preferably, the drug reduces the content of IL-6 in the brain.

[0014] Preferably, the drug increases the content of γ-aminobutyric acid in the brain.

[0015] Preferably, the preparation method of the sea cucumber phospholipids includes the following steps:

[0016] S1. Add acetone to the sea cucumber sample for extraction, then separate the liquid and dry to obtain a crude product;

[0017] S2. Add acetone to the crude product for extraction, collect the part that is insoluble in acetone, then add ethanol, and collect the part that is insoluble in ethanol to obtain sea cucumber phospholipids.

[0018] More preferably, in step S1, the extraction method is: soak and / or ultrasonically treat the sea cucumber sample with acetone.

[0019] Further preferably, the soaking time is not less than 12 h.

[0020] More preferably, in step S1, the acetone is cold acetone.

[0021] More preferably, in step S1, the method for separating the liquid is suction filtration.

[0022] More preferably, in step S2, the dosage ratio of the crude product to acetone is 1 g:(3 - 5) mL.

[0023] Further preferably, the dosage ratio of the crude product to acetone is 1 g:4 mL.

[0024] More preferably, in step S2, the extraction is carried out at 45 - 55 °C for 55 - 65 min.

[0025] Further preferably, the extraction is carried out at 50 °C for 60 min.

[0026] Further preferably, the number of extraction times is 2 - 4 times.

[0027] Even more preferably, the number of extraction times is 3 times.

[0028] More preferably, in step S2, the volume fraction of the ethanol is 90 - 95%.

[0029] As an implementable way, the drug further includes pharmaceutically acceptable excipients.

[0030] As an implementable way, the dosage forms of the drug include tablets, capsules, granules, powders and pills.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention discloses the application of sea cucumber phospholipids in the preparation of preparations for improving sleep and / or treating sleep disorders. The present invention finds through research that sea cucumber phospholipids can significantly increase the sleep time of zebrafish with sleep disorders induced by caffeine and improve the abnormal changes in the levels of IL-6 and GABA in the brain of zebrafish. At the same time, sea cucumber phospholipids can significantly prolong the sleep duration of mice induced by sodium pentobarbital. After making sea cucumber phospholipids into preparations, they can be used to improve insomnia and treat sleep disorder-related diseases, and have broad application prospects in the field of sleep disorders. Brief Description of the Drawings

[0033] Figure 1 To investigate the effects of sea cucumber phospholipids on the levels of IL-6 and GABA in the brains 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 of the Preferred Embodiments

[0034] The present invention will be further described below in conjunction with specific embodiments, but the embodiments 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 technical field.

[0035] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0036] Example 1 Preparation of Sea Cucumber Phospholipids

[0037] Sea cucumber phospholipids were obtained from the phospholipid ethanol-insoluble part of the dry powder of Holothuria scabra (purchased from Shenzhen Taifeng Biotechnology Co., Ltd.) by stepwise extraction with cold acetone and ethanol. The preparation method was carried out according to the method of "1.2 Extraction of Sea Cucumber Phospholipids" in Example 1 of Chinese Patent No. CN110327358A. The specific operations are as follows:

[0038] 13 kg of Holothuria scabra was taken and crushed into sea cucumber powder. The sea cucumber powder was first soaked in pre-cooled acetone at 4°C overnight, and the volume of acetone was 2 times the volume of the sea cucumber powder. After soaking, filtration was carried out by suction, and the filtrate was rotary evaporated under reduced pressure with a water pump at 50°C to obtain a material with preliminary removal of fat, and then weighed. The filter residue was soaked in anhydrous ethanol with a volume 3 times that of the filter residue overnight, and ultrasonicated at 40°C for 30 min. After ultrasonic treatment, filtration was carried out by suction to obtain the filtrate. The filter residue was continuously extracted 4 times according to this method, and the filtrates obtained from the 4 extractions were combined and rotary evaporated to obtain the crude phospholipids.

[0039] The fat component in the crude phospholipids was removed using acetone: the crude phospholipids and acetone were mixed at a ratio of 1 g:4 mL, and extracted at 50°C for 60 min, with a total of 3 extractions. The acetone-insoluble part was collected, 95% ethanol solution (v / v) was added, the insoluble part was collected, rotary evaporated, and stored at 4°C to obtain the product.

[0040] The prepared sea cucumber phospholipids were white to light yellow powders (i.e., the material of Fr.2-1 part, SCW), and were sent to Wuhan Metware Biotechnology Co., Ltd. for testing. The main components are shown in Table 1.

[0041] Table 1 Main Components of Sea Cucumber Phospholipids

[0042]

[0043] Note: PI indicates phosphatidylinositol, and LPI indicates lysophosphatidylinositol. For example, LPI 19:0 means the phospholipid structure is a straight-chain 19-carbon with an unsaturation of 0; while PI 38:4; PI 18:0-20:0 indicate there are two branched chains of 18-carbon and 20-carbon respectively.

[0044] Example 2 Effect of Sea Cucumber Phospholipids on the Sleep Duration of Zebrafish with Caffeine-Induced Sleep Disorders

[0045] I. Experimental Method

[0046] The zebrafish were randomly divided into 5 groups with 15 fish in each group, and then different treatments were carried out. The specific grouping and treatments are as follows:

[0047] Control group: Fed with ordinary feed for two weeks, twice a day, 0.1 g of feed each time;

[0048] Model group: After feeding with ordinary feed for two weeks (twice a day, 0.1 g of feed each time), the zebrafish were soaked in a caffeine solution with a concentration of 200 μmol / L for 24 h;

[0049] Melatonin positive group: After feeding with ordinary feed for two weeks (twice a day, 0.1 g of feed each time), the zebrafish were soaked in a 200 μmol / L caffeine solution for 24 h, and then transferred to a 130 mg / L melatonin solution and soaked for 2 h;

[0050] Low-dose sea cucumber phospholipid intervention group: The sea cucumber phospholipids prepared in Example 1 were added to the ordinary feed at 50 mg / kg of feed. After feeding for two weeks (twice a day, 0.1 g of feed each time), the zebrafish were soaked in a caffeine solution with a concentration of 200 μmol / L for 24 h;

[0051] High-dose sea cucumber phospholipid intervention group: The sea cucumber phospholipids prepared in Example 1 were added to the ordinary feed at 100 mg / kg of feed. After feeding for two weeks (twice a day, 0.1 g of feed each time), the zebrafish were soaked in a caffeine solution with a concentration of 200 μmol / L for 24 h.

[0052] After the treatment, 6 zebrafish were randomly selected from each group. Using the method measured by a behavior analyzer, the movement distance of the zebrafish within 10 min was measured, and then converted into the total movement distance in 24 h. 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%, the sleep improvement effect of sea cucumber phospholipids on insomniac zebrafish was evaluated.

[0053] II. Experimental Results

[0054] 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.

[0055] 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 an obvious improvement effect on the sleep of insomnia zebrafish.

[0056] The movement distances of zebrafish in the low-dose intervention group and high-dose intervention group of sea cucumber phospholipids were 327463 ± 24327 mm and 274952 ± 17632 mm, respectively. Among them, compared with the model group, the movement distance of zebrafish in the high-dose intervention group of sea cucumber phospholipids was significantly decreased, and the sleep improvement effects were 24.89% and 52.04%, respectively, indicating that sea cucumber phospholipids have an obvious improvement effect on the sleep quality of insomnia zebrafish under the conditions of this experimental dose.

[0057] Table 2 Effects of sea cucumber phospholipids on the sleep time of zebrafish with caffeine-induced sleep disorder

[0058]

[0059] Note: Data are expressed as mean ± SEM (standard error), and data between groups were analyzed by one-way ANOVA. Compared with the control group, * P < 0.05, ** P < 0.01; compared with the model group, # P < 0.05, ## P < 0.01.

[0060] Example 3 Effects of sea cucumber phospholipids on sleep-related physiological indexes in the brains of zebrafish with caffeine-induced sleep disorder

[0061] I. Experimental method

[0062] IL-6 is a multifunctional cytokine that participates in regulating various pathological processes such as immune responses. Modern medicine believes that sleep disorders are closely related to the body's inflammatory state. Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter that plays an inhibitory role on cerebral cortical cells in the synaptic cleft of the brain marrow nerve and can promote sleep.

[0063] According to Example 2 for grouping and treatment, after the treatment was completed, the zebrafish were sacrificed and the brain tissues were isolated, and the contents of interleukin 6 (IL-6) and gamma-aminobutyric acid (GABA) were detected by ELISA detection kits.

[0064] II. Experimental Results

[0065] The results are as Figure 1 shown. Compared with the control group, the level of IL-6 in the brains of zebrafish in the model group was significantly increased, while the level of GABA was significantly decreased. Compared with the model group, the low-dose and high-dose intervention groups of sea cucumber phospholipids could reduce the level of IL-6 to varying degrees and also significantly increase the level of GABA.

[0066] The above results indicate that sea cucumber phospholipids can improve the abnormalities of IL-6 and GABA levels in the brains of zebrafish with sleep disorders, which may be related to its improvement of the sleep quality of zebrafish with insomnia.

[0067] Example 4 Effects of Sea Cucumber Phospholipids on the Sleep Latency and Sleep Duration of Mice Induced by Sodium Pentobarbital

[0068] I. Experimental Methods

[0069] The mice were randomly divided into 3 groups with 6 mice in each group, half male and half female, and were treated differently:

[0070] Low-dose sea cucumber phospholipid group: The sea cucumber phospholipids prepared in Example 1 were dissolved in normal saline, and the mice were gavaged with 50 mg / kg BW of sea cucumber phospholipids once a day for 14 consecutive days.

[0071] High-dose sea cucumber phospholipid group: The sea cucumber phospholipids prepared in Example 1 were dissolved in normal saline, and the mice were gavaged with 100 mg / kg BW of sea cucumber phospholipids once a day for 14 consecutive days.

[0072] Control group: An equal amount of normal saline was given once a day for 14 consecutive days.

[0073] 30 minutes after the last administration, the mice were intraperitoneally injected with sodium pentobarbital (40 mg / kg). The sleep latency was defined as the time when the righting reflex of the mice disappeared, and the sleep duration was defined as the time from the disappearance of the righting reflex to the recovery. The sleep latency and sleep duration of the mice were recorded respectively.

[0074] II. Experimental Results

[0075] The results are shown in Table 3. The low-dose sea cucumber phospholipids had no obvious effect on the sleep latency of mice induced by sodium pentobarbital, but the high-dose sea cucumber phospholipids could significantly prolong the sleep duration of mice (P<0.05).

[0076] Table 3 Effects of Sea Cucumber Phospholipids on the Sleep of Mice Induced by Sodium Pentobarbital

[0077]

[0078] Note: Data are expressed as mean ± SEM (standard error of the mean), and data among groups were analyzed by one-way ANOVA. Compared with the control group, ** P < 0.05.

[0079] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of sea cucumber phospholipids containing phosphatidylinositol and lysophosphatidylinositol in the preparation of a drug for improving sleep and / or treating sleep disorders, characterized in that, The preparation method of the sea cucumber phospholipid comprises the following steps: S1. Add acetone to the sea cucumber sample for extraction, then separate the liquid and dry it to obtain the crude product; S2. Add acetone to the crude product for extraction, collect the part insoluble in acetone, then add ethanol, and collect the part insoluble in ethanol to obtain the sea cucumber phospholipid.

2. The application according to claim 1, wherein The drug increases the total sleep duration.

3. The application according to claim 1, characterized in that The drug reduces the content of IL-6 in the brain.

4. The application according to claim 1, wherein The drug increases the content of γ-aminobutyric acid in the brain.

5. The application according to claim 1, wherein In step S1, the extraction method is: soak and / or ultrasonically treat the sea cucumber sample with acetone.

6. The application according to claim 5, characterized in that, The soaking time is not less than 12 h.

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

8. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

9. The application according to claim 1, characterized in that, The dosage forms of the drug include tablets, capsules, granules, powders or pills. "

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