Application of egg yolk phospholipoprotein in improving sleep

Abstract

The invention discloses application of egg yolk phospholipoprotein in sleep improvement, and belongs to the field of functional food development. The egg yolk phospholipoprotein is a product obtained after cholesterol and triglyceride are removed from egg yolk, and contains protein and phospholipid components. According to the method, the yolk phospholipoprotein is prepared by adopting a supercritical carbon dioxide method, the supercritical carbon dioxide has relatively high dissolving capacity on cholesterol and triglyceride, but has very low dissolving capacity on protein and phospholipid, in the extraction process, the triglyceride and the cholesterol are extracted together, and the protein and the phospholipid are retained in the raffinate; according to the present invention, the egg yolk phospholipoprotein with triglyceride and cholesterol removed is finally obtained, and the experiment results show that the egg yolk phospholipoprotein can significantly improve the sleep rate and the sleep time, and can significantly improve the activity of sleep.

Description

Technical Field

[0001] The present invention relates to the field of functional food development, and particularly to the application of egg yolk phospholipoprotein in improving sleep. Background Art

[0002] Sleep is an indispensable part of human life activities, playing multiple key roles such as restoring physical strength, consolidating memory, regulating emotions, and maintaining physiological balance. Good sleep quality is an important guarantee for physical and mental health, and has an important role in improving the quality of life, enhancing work efficiency, and maintaining social relationships. However, in the fast-paced modern life, insomnia has become a common health problem, seriously threatening the well-being of a large number of people globally. The causes of insomnia are complex and diverse, including physiological factors such as changes in sleep requirements due to age growth and endocrine disorders; psychological factors such as anxiety, depression, and excessive stress; environmental factors such as noise interference, strong light, and uncomfortable room temperature; and lifestyle factors such as irregular work and rest and excessive use of electronic devices. In addition, certain diseases and medications may also cause insomnia. Therefore, improving sleep is particularly important.

[0003] Currently, the clinical treatment methods for sleep disorders mainly include sedative-hypnotic drugs, antidepressants, and drugs that regulate neurotransmitters, such as benzodiazepines and melatonin receptor agonists. Although these drugs can significantly improve sleep quality in the short term, long-term dependence may bring side effects such as daytime sleepiness, memory loss, and mood swings, causing troubles to the daily life and work of patients. Therefore, it is particularly urgent to develop natural active ingredients that can effectively improve sleep with fewer side effects.

[0004] Sleep regulation is related to neurotransmitters and some receptors related to neural regulation, including GABA (γ-aminobutyric acid), 5-HT (5-hydroxytryptamine), glutamate, dopamine, orexin, etc. One of the mechanisms of sleep regulation is to activate the GABA receptor, which is one of the main multi-subunit proteins in the central nervous system and has two main subtypes. The GABA A R receptor (type A, GABA A R) is a ligand-gated chloride channel receptor, which can induce rapid inhibitory postsynaptic potentials and can be activated by its agonists or positive allosteric modulators. Glutamate is a precursor substance for the synthesis of GABA, so it has potential effects in sleep regulation. The 5-HT signaling pathway is also one of the popular mechanisms for sleep regulation. It has been found that tryptophan is a precursor substance for the synthesis of 5-HT. The synthesis of 5-HT is that tryptophan forms 5-HTP under the action of tryptophan hydroxylase, and then forms 5-HT under the action of 5-hydroxytryptamine decarboxylase. 5-HT will play its role by binding to different receptor subtypes. Among them, when the 5-HT1A receptor binds to 5-HT, it can regulate the transmission of neurotransmitters, thereby affecting sleep.

[0005] Egg yolks are rich in protein, containing all the essential amino acids required by the human body, especially rich in glutamic acid, tyrosine, and tryptophan, and these amino acids have been verified to possibly play a key role in sleep regulation. In the records of "Compendium of Materia Medica", chicken egg yolks have the effect of nourishing blood and calming wind, being able to nourish blood and soothe the nerves, and calming internal wind, and have a certain therapeutic effect on symptoms such as restlessness and insomnia. Therefore, it is of great significance to explore and confirm the sleep-improving activity of egg yolks and its mechanism of action. Summary of the Invention

[0006] The purpose of the present invention is to provide the application of egg yolk phospholipoprotein in improving sleep, so as to solve the problems existing in the above-mentioned prior art. The egg yolk phospholipoprotein obtained by removing triglycerides and cholesterol from egg yolks can significantly improve sleep and enhance sleep quality, which has an important role in applying egg yolk phospholipoprotein to the development of drugs, functional foods, and health products that are helpful for improving sleep.

[0007] To achieve the above purpose, the present invention provides the following solutions:

[0008] The present invention provides the application of egg yolk phospholipoprotein in helping to improve sleep, and the egg yolk phospholipoprotein is obtained by removing triglycerides and cholesterol from egg yolks using the supercritical carbon dioxide method.

[0009] The present invention also provides the application of egg yolk phospholipoprotein in the preparation of products that help to improve sleep, and the egg yolk phospholipoprotein is obtained by removing triglycerides and cholesterol from egg yolks using the supercritical carbon dioxide method.

[0010] The present invention also provides the application of egg yolk phospholipoprotein in increasing the sleep onset rate and / or sleep time, and the egg yolk phospholipoprotein is obtained by removing triglycerides and cholesterol from egg yolks using the supercritical carbon dioxide method.

[0011] The present invention also provides the application of egg yolk phospholipoprotein in the preparation of products for increasing the sleep onset rate and / or sleep time, and the egg yolk phospholipoprotein is obtained by removing triglycerides and cholesterol from egg yolks using the supercritical carbon dioxide method.

[0012] Optionally, the removal of triglycerides and cholesterol from egg yolks using the supercritical carbon dioxide method includes the following steps:

[0013] Place the egg yolk powder in the extraction kettle, set the extraction pressure to 28 - 38 MPa, the extraction temperature to 45 - 55 °C, and the carbon dioxide flow rate to 1000 - 1500 kg / h to promote the carbon dioxide to reach the supercritical state and extract for 3 - 5 h; then reduce the temperature and pressure conditions to rapidly gasify the carbon dioxide and precipitate cholesterol and triglycerides to obtain egg yolk phospholipoprotein.

[0014] The present invention also provides a product helpful for improving sleep. The main active ingredient of the product is egg yolk phospholipoprotein, which is obtained by removing triglycerides and cholesterol in egg yolk using the supercritical carbon dioxide method.

[0015] Optionally, the preparation method of the egg yolk phospholipoprotein includes the following steps: placing egg yolk powder in an extraction kettle, setting the extraction pressure at 28 - 38 MPa, the extraction temperature at 45 - 55 °C, and the carbon dioxide flow rate at 1000 - 1500 kg / h to make carbon dioxide reach the supercritical state, and extracting for 3 - 5 h; then reducing the temperature and pressure conditions to rapidly gasify carbon dioxide and precipitate cholesterol and triglycerides, thus obtaining egg yolk phospholipoprotein.

[0016] Optionally, the product includes drugs, foods, and health products.

[0017] The present invention discloses the following technical effects:

[0018] The present invention first identifies that egg yolk phospholipoprotein has the effect of improving sleep. The function of egg yolk phospholipoprotein in improving sleep activity is evaluated by the pentobarbital sodium - assisted mouse sleep test, and its sleep - promoting mechanism is explored by detecting neurotransmitters through ELISA and detecting the expression level of mRNA through qRT - PCR. The results show that egg yolk phospholipoprotein can improve the sleep onset rate and sleep time of mice, increase the contents of inhibitory neurotransmitters 5 - HT and GABA in the mouse brain tissue, decrease the content of excitatory neurotransmitter DA, and increase the expression level of 5 - HT1A in the mouse brain tissue, having good sleep - improving activity and acting through the 5 - HT pathway.

[0019] The present invention can achieve the effect of efficiently improving sleep only through egg yolk phospholipoprotein. Compared with drugs and complex diet therapy methods, the method is simple, easy to implement, low - cost, and has no side effects, which is conducive to wide promotion and application. Description of the Drawings

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0021] Figure 1 It is the graph of the sleep onset rate result of egg yolk phospholipoprotein in the pentobarbital sodium - induced mouse sleep experiment;

[0022] Figure 2 It is the graph of the sleep time result of egg yolk phospholipoprotein in the pentobarbital sodium - induced mouse sleep experiment;

[0023] Figure 3 It is a detection result diagram of neurotransmitter 5-HT in mouse brain tissue;

[0024] Figure 4 It is a detection result diagram of neurotransmitter GABA in mouse brain tissue;

[0025] Figure 5 It is a detection result diagram of neurotransmitter DA in mouse brain tissue;

[0026] Figure 6 It is a detection result diagram of the expression level of 5-HT1A in mouse brain tissue. Detailed implementation manners

[0027] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0030] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0031] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0032] The present invention uses the supercritical carbon dioxide method to remove triglycerides and cholesterol from egg yolk. Supercritical carbon dioxide has a large solubility for cholesterol and triglycerides, but a very small solubility for proteins and phospholipids. During the extraction process, triglycerides will be extracted together with cholesterol, while proteins and phospholipids remain in the residue, thus achieving the purpose of separation and obtaining egg yolk phospholipid protein.

[0033] The egg yolk powder was purchased from Guangzhou Baiyunshan Pharmaceutical Co., Ltd.

[0034] Example 1 Preparation of Egg Yolk Phospholipid Protein

[0035] Place the egg yolk powder in the extraction kettle, set the extraction pressure to 32 MPa, the extraction temperature to 50 °C, and the CO 2 flow rate to 1200 kg / h to promote the carbon dioxide to reach the supercritical state. It circulates back and forth in the extraction kettle. After contacting with the egg yolk, the cholesterol and triglyceride molecules in the egg yolk dissolve in it. The extraction time is 4 h. After the extraction is completed, the carbon dioxide carrying impurities flows into the separation device. By reducing the temperature and pressure conditions, the carbon dioxide quickly gasifies, and cholesterol and triglycerides precipitate due to the sudden drop in solubility, achieving precise separation and obtaining egg yolk phospholipid protein.

[0036] Example 2 Preparation of Egg Yolk Phospholipid Protein

[0037] Place the egg yolk powder in the extraction kettle, set the extraction pressure to 28 MPa, the extraction temperature to 45 °C, and the CO 2 flow rate to 1000 kg / h to promote the carbon dioxide to reach the supercritical state. It circulates back and forth in the extraction kettle. After contacting with the egg yolk, the cholesterol and triglyceride molecules in the egg yolk dissolve in it. The extraction time is 3 h. After the extraction is completed, the carbon dioxide carrying impurities flows into the separation device. By reducing the temperature and pressure conditions, the carbon dioxide quickly gasifies, and cholesterol and triglycerides precipitate due to the sudden drop in solubility, achieving precise separation and obtaining egg yolk phospholipid protein.

[0038] Example 3 Preparation of Egg Yolk Phospholipid Protein

[0039] Place the egg yolk powder in the extraction kettle, set the extraction pressure to 38 MPa, the extraction temperature to 55 °C, and the CO 2 flow rate to 1500 kg / h to promote the carbon dioxide to reach the supercritical state. It circulates back and forth in the extraction kettle. After contacting with the egg yolk, the cholesterol and triglyceride molecules in the egg yolk dissolve in it. The extraction time is 5 h. After the extraction is completed, the carbon dioxide carrying impurities flows into the separation device. By reducing the temperature and pressure conditions, the carbon dioxide quickly gasifies, and cholesterol and triglycerides precipitate due to the sudden drop in solubility, achieving precise separation and obtaining egg yolk phospholipid protein.

[0040] Comparative Example 1

[0041] Fresh egg yolk without defatting, the obtaining method is as follows: separate the egg yolk and egg white of fresh eggs using an egg yolk separator, roll the egg yolk on filter paper for several circles to completely remove the adhering egg white on the surface, then pierce the film on the surface of the egg yolk to collect the egg yolk liquid, which is the fresh egg yolk without defatting.

[0042] Comparative Example 2

[0043] Completely defatted egg yolk powder, which is the product after removing cholesterol, triglycerides and lecithin from the egg yolk. The obtaining method is as follows: after removing cholesterol and triglycerides by supercritical carbon dioxide treatment shown in Example 1, the obtained egg yolk phospholipoprotein is extracted with an ethanol solution of 80-100% (80% ethanol is selected in this experiment), the ethanol extraction time is 1-3 h (1 h is selected in this experiment), and the extraction times are 2-6 times (3 times is selected in this experiment). Until the mixed ethanol solution of the extracted oil is clear and transparent, the residue is dried to obtain the completely defatted egg yolk powder.

[0044] The sleep-improving activity of egg yolk phospholipoprotein was evaluated through a pentobarbital sodium-assisted sleep animal experiment. The experimental groups were set as a blank control group, a GABA group, a diazepam group, a homogenized egg yolk phospholipoprotein group and two comparative groups (the fresh egg yolk without defatting in Comparative Example 1 and the completely defatted egg yolk powder in Comparative Example 2). Among them, the GABA group and the diazepam group were positive control groups. The pretreatment methods of egg yolk phospholipoprotein and completely defatted egg yolk powder were to be homogenized with pure water for 5 min using a homogenizer, the pretreatment method of fresh egg yolk without defatting was to be diluted with pure water by a factor of one and then gavaged to mice, and the pretreatment method of diazepam was to be dissolved in physiological saline and sonicated for 10 min. During the experiment, the body weight changes of mice were recorded every day, the organ indices were recorded after the mice were dissected, the contents of neurotransmitters γ-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT), and dopamine (DA) in the mouse brain tissues were detected respectively, and qRT-PCR detection was performed on the mouse brain tissues to explore its possible mechanism of action. Specifically as follows:

[0045] (1) Pentobarbital sodium-assisted mouse sleep experiment

[0046] It was determined with reference to the test method for improving sleep in Chapter 6 of "Functional Test and Evaluation Method of Health Food (2023 Edition)". Among them, the blank control group was orally gavaged with 0.1 mL / (10 g·d) of physiological saline, two positive control groups were set, which were GABA at the same dose as the sample group and the anxiolytic drug diazepam at 1.2 mg / kg, and the sample group of egg yolk phospholipoprotein and the two comparative groups were set to be orally gavaged at 800 mg / kg, 0.1 mL / kg.

[0047] The criterion for determining mouse sleep is the disappearance of the righting reflex. After intraperitoneal injection of sodium pentobarbital in each group, when the animals basically stopped moving, their body positions were flipped to the supine position. If the animals remained in the supine position for more than 60 s within 30 min, it was judged that the righting reflex disappeared and they entered the sleep state. After a period of time, when the righting reflex recovered for the first time, they were immediately flipped to the supine position. If it recovered again within 30 s, it was judged that the animals were awake, and the time when the righting reflex first recovered was recorded as the sleep termination time; if it did not recover within 30 s, they were flipped again after the righting reflex recovered again until the end of sleep was determined.

[0048] The results are as Figure 1 and Figure 2 shown. Figure 1 The sleep onset rate of mice is shown in the figure. The sleep onset rate of mice in the blank control group was only 42%, that of the GABA group increased to 62%, that of the egg yolk phospholipoprotein group increased significantly to 92%, and the sleep onset rates of the mice in Comparative Example 1 and Comparative Example 2 were 83% and 67% respectively, both of which were higher than that of the blank control group. The sleep onset rate of the positive drug diazepam group reached 100%. This shows that egg yolk phospholipoprotein can significantly increase the sleep onset rate of mice and play a role in improving sleep. Figure 2 The sleep time of mice is shown in the figure. The sleep time of the GABA group and the two comparative example groups was longer than that of the mice in the blank control group. The sleep time increased by 125.9%, 172.1%, and 135.2% respectively compared with the blank control group. The egg yolk phospholipoprotein group and the positive drug group significantly prolonged the sleep time of mice. The sleep duration of the egg yolk phospholipoprotein group was about 4 times that of the blank control group, and there was no significant difference in the sleep time between the egg yolk phospholipoprotein group and the positive drug group. This shows that egg yolk phospholipoprotein has an obvious effect on prolonging the sleep time of mice, and the effect is close to that of the positive drug diazepam.

[0049] (2) Detection of neurotransmitters 5-HT, GABA, and DA in mouse brain tissue

[0050] The content of neurotransmitters in mouse brain tissue was measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (the final data was presented as the content of neurotransmitters in the brain tissue per unit weight). The treatment method for mouse brain tissue was as follows: The tissue was placed in a centrifuge tube, 2 steel beads and 1 mL of PBS (pre-cooled in advance) were added, and then it was ground with a grinder (the iron block on the grinder needed to be pre-cooled in a -20 °C refrigerator in advance, and attention should be paid to balancing when placing the samples). Grinding was carried out according to the preset parameters (frequency 60 Hz, time 60 s, pause time 5 s, number of times 2). After grinding, it was left to stand at 4 °C for 5 min, then centrifuged at 12,000 r / min for 5 min, and the supernatant was taken for the detection of neurotransmitters.

[0051] The results are as Figures 3 - 5 shown.Figure 3 It is the detection result of the neurotransmitter 5-HT in the brain tissue. It can be seen that compared with the blank control group, the egg yolk phospholipoprotein group and the comparative example 2 group can significantly increase the content of 5-HT in the brain tissue of mice; Figure 4 The detection result of GABA in the brain tissue of mice shows that the GABA group, the egg yolk phospholipoprotein group and the two comparative example groups can all significantly increase the content of GABA in the brain tissue. Especially the effect of egg yolk phospholipoprotein is extremely significant, and it has increased by about 1.35 times compared with the blank control group; From Figure 5 It can be seen from the detection result of DA in the brain tissue that compared with the blank control group, each experimental group can significantly reduce the content of DA in the brain tissue of mice. Among them, the positive drug and GABA have the same effect, while the effect of egg yolk phospholipoprotein is better. There is still an extremely significant difference between the egg yolk phospholipoprotein and other experimental groups, indicating that its ability to reduce the content of DA in the brain tissue of mice is strong and the sleep-promoting effect is good. In summary, it shows that egg yolk phospholipoprotein has a significant sleep-improving effect.

[0052] (3) Determination of the expression level of 5-HT1A in the brain tissue of mice

[0053] The mRNA expression level of 5-HT1A in the brain tissue of mice was analyzed and determined by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) to further explore the mechanism of action of egg yolk phospholipoprotein in regulating sleep. The results are as Figure 6 shown. Compared with the blank control group, there are extremely significant differences in both positive control groups and the egg yolk phospholipoprotein group, and the effect of the egg yolk phospholipoprotein group is better than that of the positive control and has an extremely significant difference. This shows that egg yolk phospholipoprotein can increase the expression level of 5-HT in the brain tissue of mice and inhibit the excitation of neuron cells to achieve the sleep-promoting function and nerve regulation.

[0054] In summary, egg yolk phospholipoprotein can increase the sleep onset rate and sleep time of mice, increase the content of inhibitory neurotransmitters 5-HT and GABA in the brain tissue of mice, decrease the content of excitatory neurotransmitter DA, and increase the expression level of 5-HT1A in the brain tissue of mice, and has good sleep-improving activity.

[0055] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should all fall within the protection scope determined by the claims of the present invention.

Claims

1. The application of egg yolk phospholipid protein in improving sleep, characterized in that: The egg yolk phospholipid protein is obtained by removing triglyceride and cholesterol from egg yolk using a supercritical carbon dioxide method.

2. The use of egg yolk phospholipid protein in preparing a product that helps improve sleep, characterized in that: The egg yolk phospholipid protein is obtained by removing triglyceride and cholesterol from egg yolk using a supercritical carbon dioxide method.

3. The use of egg yolk phospholipid protein in improving the sleep rate and / or sleep time, characterized in that: The egg yolk phospholipid protein is obtained by removing triglyceride and cholesterol from egg yolk using a supercritical carbon dioxide method.

4. The use of egg yolk phospholipid protein in the preparation of a product for improving the rate of falling asleep and / or the duration of sleep, characterized in that: The egg yolk phospholipid protein is obtained by removing triglyceride and cholesterol from egg yolk using a supercritical carbon dioxide method.

5. The use according to any one of claims 1 to 4, characterized in that: The method of removing triglycerides and cholesterol from egg yolk by supercritical carbon dioxide comprises the following steps: Place the egg yolk powder in an extraction kettle, set the extraction pressure to 28-38MPa, the extraction temperature to 45-55°C, the carbon dioxide flow rate to 1000-1500kg / h, make the carbon dioxide reach a supercritical state, and extract for 3-5h; then lower the temperature and pressure conditions to make the carbon dioxide gasify rapidly, precipitate cholesterol and triglycerides, and obtain egg yolk phospholipid protein.

6. A product that helps improve sleep, characterized in that: The main active ingredient of the product is egg yolk phospholipid protein, which is obtained by removing triglyceride and cholesterol from egg yolk using a supercritical carbon dioxide method.

7. The product according to claim 6, characterized in that The preparation method of the egg yolk phospholipid protein comprises the following steps: placing the egg yolk powder in an extraction kettle, setting the extraction pressure to 28-38MPa, the extraction temperature to 45-55°C, and the carbon dioxide flow rate to 1000-1500kg / h, causing the carbon dioxide to reach a supercritical state, and extracting for 3-5h; then lowering the temperature and pressure conditions to rapidly gasify the carbon dioxide, precipitate cholesterol and triglyceride, and obtain the egg yolk phospholipid protein.

8. The product according to claim 6, characterized in that The products include medicines, foods and health products.

Images