Composition with sleep-improving effect and use thereof

Abstract

The present application belongs to the technical field of pharmaceutical compositions, and particularly relates to a composition with sleep improvement effect and application thereof.The composition comprises gamma-aminobutyric acid, Poria cocos extract, and can further comprise Rhizoma Polygonati extract and phosphatidylserine.The composition can achieve the effect of sleep improvement within a specific ratio range.The composition takes into account functionality and nutrition, and exhibits broad application prospects in the aspect of sleep improvement function.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical compositions, and particularly relates to a composition with sleep improvement effect and application thereof. BACKGROUND

[0002] Sleep is a necessary natural physiological process that promotes the clearance of neurotoxic waste and maintains synapses related to memory and learning. Therefore, sleep is crucial to brain function, especially cognitive function. Insufficient sleep is associated with various body dysfunctions, including endocrine, metabolic, immune, cardiovascular, cortical, and nervous system disorders. Short-term insomnia affects memory and attention, while long-term insomnia leads to neurological dysfunction, exacerbates existing diseases, and even death in severe cases. Insufficient sleep has become a widespread concern, affecting people's physical and mental health.

[0003] Traditional hypnotics increase total sleep time at night and reduce sleep latency, but have risks such as easy overdose, increased tolerance and addiction, daytime sedation, and withdrawal reactions. Insomnia, as a public health problem, has not been adequately and effectively treated. Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter mainly present in the central nervous system. Its main function is to regulate neuronal excitability and help balance brain activity. In recent years, research has found that GABA can promote relaxation and sedation by binding to its receptors, thereby helping to reduce anxiety and stress, and is also closely related to the improvement of sleep quality.

[0004] Poria cocos extract contains various active ingredients such as polysaccharides and triterpenoids, which work together to help improve sleep quality. In clinical applications, many patients report that their sleep time is extended and they feel energetic and happy after taking Poria cocos extract.

[0005] Although GABA shows certain potential in improving sleep, there are some limitations in its functional effect when used alone. On the one hand, due to the presence of the blood-brain barrier in the human body, the amount of GABA that can effectively cross the blood-brain barrier into the brain and exert its effect after oral administration is relatively limited, making it difficult to achieve the desired ideal state of sleep improvement. On the other hand, when GABA is used alone, its duration of action is short, making it difficult to achieve a persistent good sleep state. Therefore, introducing other substances to work together with GABA to improve sleep effect is necessary for developing products with better sleep improvement effect and more optimal nutritional function. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application aims to provide a composition for improving sleep and its application, which can achieve a better sleep improvement effect by reasonably compounding GABA with other functional components. The composition takes into account functionality and nutrition, and has broad application prospects in improving sleep function.

[0007] To achieve the above-mentioned purpose, in one aspect, the present application provides a composition for improving sleep, which comprises gamma-aminobutyric acid and Poria cocos extract, and the mass ratio of the gamma-aminobutyric acid and Poria cocos extract is 55-85:15-45, for example, 85:15, 70:30, 55:45, 66:45 or 77:23.

[0008] Further, the mass ratio of the gamma-aminobutyric acid and Poria cocos extract is 11:9.

[0009] In a second aspect, the present application provides a composition for improving sleep, which comprises gamma-aminobutyric acid and Poria cocos extract, and the composition further comprises Poria cocos extract and phosphatidylserine, and the weight ratio of the gamma-aminobutyric acid, Poria cocos extract, Poria cocos extract and phosphatidylserine is 55-85:15-45:30-40:2-15, for example, 85:15:40:5, 70:30:40:5, 55:45:40:5, 66:45:40:5, 77:23:40:5, 55:45:30:15, 55:45:43:2.

[0010] Further, the weight ratio of the gamma-aminobutyric acid, Poria cocos extract, Poria cocos extract and phosphatidylserine is 11:9:8:1.

[0011] In a third aspect, the present application provides a product comprising the composition of the first aspect or the second aspect.

[0012] Further, the product is a health food or a drug, and the product further comprises a health food or a drug acceptable adjuvant.

[0013] In a fourth aspect, the present application provides the use of the composition of the first aspect in the preparation of a product having the effect of improving sleep, and the product is a health food or a drug.

[0014] Further, the use is the use in the preparation of a product capable of prolonging sleep time.

[0015] Further, the use is one or more of the following:

[0016] (1) the use in the preparation of a product capable of improving the decrease of GABA secretion level caused by insomnia;

[0017] (2) in the preparation of insomnia caused by the decrease of 5-HT level of the product can improve the application;

[0018] (3) in the preparation of insomnia caused by the increase of ACTH content of the product can improve the application;

[0019] (4) in the preparation of insomnia caused by the increase of CORT content of the product can improve the application;

[0020] (5) in the preparation of insomnia caused by oxidative damage of the product can improve the application;

[0021] (6) in the preparation of insomnia caused by the increase of inflammation level of the product can improve the application;

[0022] (7) in the preparation of insomnia caused by the damage of neuron cell of the product can improve the application.

[0023] Compared with the prior art, the present application has the following advantages,

[0024] (1) the present application proves that the combination of gamma-aminobutyric acid and poria cocos extract can help improve sleep, and the two components have synergistic effect;

[0025] (2) the present application proves that the combination of gamma-aminobutyric acid, poria cocos extract, polygonatum extract and phosphatidylserine can help improve sleep, and the four components have synergistic effect.

[0026] (3) the composition of the present application can improve sleep, can prolong the sleep time of insomnia patients, and has the functions of improving the decrease of GABA secretion level caused by insomnia, improving the decrease of 5-HT level caused by insomnia, improving the increase of ACTH content caused by insomnia, improving the increase of CORT content caused by insomnia, improving oxidative damage caused by insomnia, improving the increase of inflammation level caused by insomnia, improving the damage of neuron cell caused by insomnia and the like. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The results of the composition on pentobarbital sodium induced sleep latency of mice are shown.

[0028] Figure 2 The results of the composition on pentobarbital sodium induced sleep duration of mice are shown.

[0029] Figure 3 The effects of the composition on serum and brain GABA content and 5-HT content of mice are shown, wherein A is serum GABA content; B is brain GABA content; C is serum 5-HT content; D is brain 5-HT content.

[0030] Figure 4The effects of the composition on the serum ACTH content and CORT content of mice are shown, wherein A represents the serum ACTH content; and B represents the serum CORT content.

[0031] Figure 5 The effects of the composition on the serum SOD, CAT and GSH contents of mice are shown, wherein A represents the serum SOD content; B represents the serum CAT content; and C represents the serum GSH content.

[0032] Figure 6 The effects of the composition on the serum TNF-α, IL-1β and IL-6 contents of mice are shown, wherein A represents the serum TNF-α content; B represents the serum IL-1β content; and C represents the serum IL-6 content.

[0033] Figure 7 The effects of the composition on the histopathological examination results of the hippocampus of mice are shown.

[0034] Figure 8 The effects of the composition on the c-Fos positive cell density of the hippocampus of mice are shown. DETAILED DESCRIPTION

[0035] The concept and the technical effects of the present application are further described below in combination with specific examples, so as to fully understand the purposes, features and effects of the present application. The methods are all conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative examples of the present application and the descriptions thereof are used to explain the present application and do not constitute an improper limitation on the present application. It should be noted that the examples in the present application and the features in the examples can be combined with each other without conflict.

[0036] Example 1: Screening of raw materials of the composition

[0037] GABA and 5-HT, as important neurotransmitters in the central nervous system, play a key role in regulating the sleep-wake cycle and improving sleep quality. In the present application, the expression levels of GABA mRNA and 5-HT mRNA are used to characterize the potential activity of each substance in improving sleep, and the specific steps are as follows:

[0038] Each test substance is dissolved in DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin), and filtered through a 0.22 µm sterile needle filter to prepare a solution with a concentration of 250~2000 µg / mL. The SH-SY5Y cells are seeded in a 96-well plate at a density of 5×10 4The SH-SY5Y cells were inoculated in the 6-well plates at a density of 1.5 x 105 / mL and cultured overnight at 37 °C in a 5% CO2 environment. After the cells adhered to the wall, the original culture medium was discarded, and the SH-SY5Y cells were intervened with 3 mL of the test substance of different components. The blank control group was continuously cultured with the original culture medium. After being cultured at 37 °C in a 5% CO2 environment for 48 h, the culture medium in the 6-well plates was discarded, 1 mL of Trizol was added to each well, and the solution in each well was transferred to a 1.5 mL Eppendorf tube. After 200 μL of chloroform was added and the solution was shaken until it became a uniform milky white color, the solution was centrifuged at 12,000 rpm for 15 min at 4 °C, and the upper aqueous phase was transferred to another clean centrifuge tube. An equal volume of isopropanol was added, mixed well by inverting, and then left to stand at room temperature for 30 min. After being centrifuged at 12,000 rpm for 15 min at 4 °C, the supernatant was discarded, and 1 mL of 75% ethanol (DEPC water) pre-cooled at -20 °C was added to wash the precipitate (white). The mixture was centrifuged at 5,000 rpm for 5 min at 4 °C, and the supernatant was carefully discarded. The precipitate was air-dried and dissolved in 30 μL of DEPC water, and then stored at -80 °C.

[0039] 10 μL of the RNA was taken into a 200 μL enzyme-free EP tube, 4 μL of 5x All-In-One RT MasterMix and 6 μL of Nuclease-free H2O were added. After a short centrifugation in a palm centrifuge, the cDNA was synthesized according to the following program: 25 °C for 10 min, 42 °C for 50 min, and 85 °C for 5 min.

[0040] 1 μL of cDNA, 0.2 μL of upstream primer, 0.2 μL of downstream primer, 10 μL of SYBR Premix Ex Taq and 8.6 μL of sterilized water were mixed in an eight-tube tube. The program was set as follows: pre-denaturation at 95 °C for 180 s, amplification at 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s, and 40 cycles. GAPDH was used as an internal reference for PCR amplification. ® Premix Ex Taq ™ 1 μL of cDNA, 0.2 μL of upstream primer, 0.2 μL of downstream primer, 10 μL of SYBR Premix Ex Taq and 8.6 μL of sterilized water were mixed in an eight-tube tube. The program was set as follows: pre-denaturation at 95 °C for 180 s, amplification at 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s, and 40 cycles. GAPDH was used as an internal reference for PCR amplification.

[0041] The mRNA expression amount of the blank control group was taken as 1, and the ratio of the mRNA expression amount of each intervention group to that of the blank control group was taken as the final result.

[0042] As shown in Table 1, the screened raw materials GABA (purchased from Tianjin Shijieweikang Biological Technology Co., Ltd.), tea theanine (purchased from Tianjin Shijieweikang Biological Technology Co., Ltd.), Poria cocos extract (purchased from Shaanxi Duxing Pharmaceutical Technology Co., Ltd.), lily extract (purchased from Xi'an Fenghe Biological Technology Co., Ltd.), polygonatum extract (purchased from Shaanxi Yunhe Biological Technology Co., Ltd.), and phosphatidylserine (purchased from Xi'an Muyuan Biological Technology Co., Ltd.) all have the activity of up-regulating the relative expression of GABA mRNA and 5-HT mRNA.

[0043] When formulating a food, multiple dimensions such as bioactive function, economic cost, sensory characteristics, and safety need to be comprehensively evaluated. Specific requirements include the interaction between the nutritional components and functional components of the raw materials, the stability and seasonal fluctuations in price, and the storage stability; at the same time, attention should also be paid to the sensory characteristics such as texture, taste, aroma, appearance, and color; in terms of safety, potential allergenic risk and disease prevention ability should be evaluated; in addition, factors such as sustainability, ecological friendliness, social responsibility, recommended intake, and processing methods should also be considered. By comprehensively considering the above factors, a high-quality and safe food formulation raw material selection guideline that meets the standard is developed, and then GABA and Poria cocos extract are selected as the basic raw materials in the formulation, and polygonatum extract and phosphatidylserine are further added as raw materials for further research on the specific amount of addition in the formulation.

[0044] Table 1 Effect of formulation raw materials on the relative expression of GABA mRNA and 5-HT mRNA

[0045]

[0046] Example 2 Effect of composition on the relative expression of GABA mRNA and 5-HT mRNA

[0047] After each raw material is weighed according to the following formulation, the raw materials are mixed in a double-helical conical mixer for 15-30 min to obtain a composition.

[0048] The raw materials of composition I include, by weight: GABA 85 g, Poria cocos extract 15 g, polygonatum extract 40 g, and phosphatidylserine 5 g.

[0049] The raw materials of composition II include, by weight: GABA 70 g, Poria cocos extract 30 g, polygonatum extract 40 g, and phosphatidylserine 5 g.

[0050] The raw materials of composition III include, by weight: GABA 55 g, Poria cocos extract 45 g, polygonatum extract 40 g, and phosphatidylserine 5 g.

[0051] The raw materials of Composition IV include, by weight parts: GABA 66 g, Poria cocos extract 34 g, Rhizoma polygonati extract 40 g, and phosphatidylserine 5 g.

[0052] The raw materials of Composition V include, by weight parts: GABA 77 g, Poria cocos extract 23 g, Rhizoma polygonati extract 40 g, and phosphatidylserine 5 g.

[0053] The raw materials of Composition VI include, by weight parts: GABA 55 g, Poria cocos extract 45 g, Rhizoma polygonati extract 30 g, and phosphatidylserine 15 g.

[0054] The raw materials of Composition VII include, by weight parts: GABA 55 g, Poria cocos extract 45 g, Rhizoma polygonati extract 43 g, and phosphatidylserine 2 g.

[0055] The raw materials of Composition VIII include, by weight parts: GABA 79.75 g, Poria cocos extract 65.25 g.

[0056] The raw materials of Comparative Example 1 include, by weight parts: Poria cocos extract 145 g.

[0057] The raw materials of Comparative Example 2 include, by weight parts: GABA 145 g.

[0058] The raw materials of Comparative Example 3 include, by weight parts: Rhizoma polygonati extract 145 g.

[0059] The raw materials of Comparative Example 4 include, by weight parts: phosphatidylserine 145 g.

[0060] The effects of Comparative Examples 1-4 and Compositions I-VII on the relative expression amounts of GABA mRNA and 5-HT mRNA were determined by the method in Example 1 to characterize the potential sleep improvement activity of the compositions, and the results are shown in Table 2. The results show that the up-regulation activity of the compositions with the addition of GABA and Poria cocos extract on GABA mRNA and 5-HT mRNA is greatly improved, and is much higher than the activity when GABA and Poria cocos extract exist separately. When GABA and Poria cocos extract exist simultaneously, the relative expression amounts of GABA mRNA and 5-HT mRNA are the highest, reaching 16.897 ± 1.123 and 22.150 ± 0.710, respectively. It can be seen that GABA and Poria cocos extract synergistically exert the sleep improvement effect. Although the up-regulation activity of the compositions on GABA mRNA and 5-HT mRNA increases after the amount of the compositions is increased, according to the comprehensive sensory evaluation results, it is ultimately determined that the potential sleep improvement activity of Composition III is the best, and the ratio of GABA, Poria cocos extract, Rhizoma polygonati extract, and phosphatidylserine is 11:9:8:1.

[0061] Table 2 Effects of the composition on the relative expression of GABA mRNA and 5-HT mRNA

[0062]

[0063] Example 3. Animal test proves the sleep improvement effect of the composition

[0064] Seventy ICR male mice were selected and raised under the conditions of 23±3 ℃; 65±5% humidity, light-dark cycle of 12 h, and good ventilation. Before the experiment, the mice were raised for 1 week to adapt to the laboratory environment. The mice were randomly divided into a blank control group, a model group, a low-dose formula group, a medium-dose formula group, a high-dose formula group, a comparative example 1 group, a comparative example 2 group, a comparative example 3 group, a comparative example 4 group, and a positive drug group (10 mice in each group). Among them, the blank control group was injected with normal saline; the rest of the groups were injected with p-chlorophenylalanine (PACA) (450 mg / kg·bw) intraperitoneally for 3 days to establish an insomnia model, except for the blank control group.

[0065] After the modeling period, the blank control group and the model group were given normal saline by gavage, the low-dose formula group was given low-dose composition III (76 mg / kg·bw) by gavage, the medium-dose formula group was given medium-dose composition III (95 mg / kg·bw) by gavage, the high-dose formula group was given high-dose composition III (114 mg / kg·bw) by gavage, the comparative example 1 group was given Poria cocos extract (95 mg / kg·bw) by gavage, the comparative example 2 group was given GABA (95 mg / kg·bw) by gavage, the comparative example 3 group was given Rhizoma Polygonati extract (95 mg / kg·bw) by gavage, the comparative example 4 group was given phosphatidylserine (95 mg / kg·bw) by gavage, and the positive drug group was given estazolam solution (2 mg / kg·bw) by gavage.

[0066] After 30 days of administration, pentobarbital sodium (45 mg / kg·bw) was injected into the mice after 30 min of gavage, and the state of the righting reflex of the mice was observed. The mice were placed in a dorsal recumbent position, and if the abdominal part remained upward at all times within 30-60 s, it was considered that the righting reflex was lost. If the mouse successfully flipped over more than 3 times within 30 s, it was considered that the righting reflex was restored. The time from injection to loss of righting reflex was recorded as the sleep latency, and the time from loss of righting reflex to restoration of righting reflex was recorded as the sleep duration.

[0067] As Figure 1Compared with the blank control group, the sleep latency of the model group was significantly increased, indicating that the modeling was successful. Compared with the model group, the sleep latency induced by sodium pentobarbital in the positive drug group, the low-dose formula group, the medium-dose formula group, and the low-dose formula group was significantly shortened, which was (93±15) s, (117±11) s, (106±18) s, and (104±14) s, respectively. The formula group played a role in shortening the sleep latency of mice, which was shortened by 19.15%, 24.82%, and 26.24%, respectively. Among them, the medium-dose formula group and the high-dose formula group had the best effect on shortening the sleep latency of mice.

[0068] The length of sleep time is directly related to the quality of sleep. As Figure 2 Compared with the blank control group, the sleep time of the model group was significantly decreased, indicating that the modeling was successful. Compared with the model group, the sleep time induced by sodium pentobarbital in the positive drug group, the low-dose formula group, the medium-dose formula group, and the low-dose formula group was significantly prolonged, which was (4055±252) s, (2283±434) s, (2900±340) s, and (2784±225) s, respectively. Among them, the medium-dose formula group and the high-dose formula group had the best effect on prolonging the sleep time of mice. Compared with the medium-dose formula group, the effect of the comparative examples 1-4 on prolonging the sleep time of mice was significantly weakened, which was (2107±206) s, (2354±169) s, (1875±126) s, and (1563±117) s, respectively. This indicates that compared with the same dose of composition III, the intervention effect of each single component is better, i.e., there is a synergistic effect of improving sleep among the substances.

[0069] The GABA content in the serum was detected by ELISA kit, and the results are shown in Figure 3 (A). The GABA content in the serum of the model group (1.202±0.092 µmoL / L) was significantly lower (P<0.05) than that of the control group (3.404±0.078 µmoL / L), indicating that the PACA modeling was successful and inhibited the secretion of inhibitory neurotransmitter GABA in ICR mice. Compared with the model group, the GABA content in the serum of the low-dose formula group, the medium-dose formula group, the high-dose formula group, and the positive drug group was significantly improved (P<0.05), which was (2.761±0.219) µmoL / L, (3.358±0.096) µmoL / L, (3.028±0.085) µmoL / L, and (3.279±0.104) µmoL / L, respectively. The GABA content in the brain of mice is shown in Figure 3(B) showed that the brain GABA content of the model group (2.830±0.153 µmoL / L) was significantly lower than that of the control group (4.502±0.122 µmoL / L). Compared with the model group, the brain GABA content of the low-dose formula group, the medium-dose formula group, the high-dose formula group, and the positive drug group was significantly improved (P<0.05), which was (3.233±0.087) µmoL / L, (4.060±0.070) µmoL / L, (3.826±0.067) µmoL / L, and (4.094±0.088) µmoL / L, respectively. This indicated that the formula intervention could significantly inhibit the decrease of GABA secretion level caused by insomnia, and the medium-dose formula group had the best effect on improving the serum GABA content.

[0070] The 5-HT concentration in the serum of mice was detected by ELISA kit, and the results were shown in Figure 3 (C). Compared with the control group, the 5-HT concentration in the serum of mice (108.942±6.911) pg / mL was significantly decreased after PCPA modeling (P<0.05), indicating that PCPA had an inhibitory effect on the synthesis of 5-HT in mice. Compared with the model group, the 5-HT content in the serum of the low-dose formula group, the medium-dose formula group, the high-dose formula group, and the positive drug group was significantly improved (P<0.05), which was (131.378±5.524) pg / mL, (145.321±8.456) pg / mL, (140.673±8.526) pg / mL, and (150.687±4.137) pg / mL, respectively. The brain 5-HT content of mice was shown in Figure 3 (D). The brain 5-HT content of the model group (95.577±10.533 pg / mL) was significantly lower than that of the control group (203.750±13.358 pg / mL). Compared with the model group, the serum 5-HT content of the low-dose formula group, the medium-dose formula group, the high-dose formula group, and the positive drug group was significantly improved (P<0.05), which was (139.327±6.731) pg / mL, (168.173±5.742) pg / mL, (176.827±6.911) pg / mL, and (225.865±7.752) pg / mL, respectively. This indicated that the formula treatment could significantly improve the decrease of 5-HT level caused by insomnia, and the medium-dose and high-dose had the best effect.

[0071] Example 4 Efficacy of the composition on relieving stress in mice

[0072] Adrenocorticotropic hormone (ACTH) is a polypeptide hormone secreted by the pituitary gland of vertebrates, which can promote the growth of adrenal cortex and the production and secretion of cortical hormones. When the body is in a high stress state due to lack of sleep, the synthesis of ACTH will increase. The concentration of ACTH in the serum of mice was detected by ELISA kit, and the results are shown in Figure 4 (A). Compared with the control group, the concentration of ACTH in the serum of mice after PCPA modeling was significantly increased (86.752±2.229) ng / L (P<0.05), indicating that PCPA increased the stress level of mice. Compared with the model group, the low-dose formula group, the middle-dose formula group, the high-dose formula group and the positive drug group, the content of ACTH in the serum was significantly decreased (P<0.05), which was (64.58±3.95) ng / L, (61.90±1.89) ng / L, (72.49±3.57) ng / L and (64.39±1.53) ng / L, respectively. It is proved that the formula can significantly improve the increase of ACTH content caused by insomnia, and the middle-dose group has the best improvement effect.

[0073] Corticosterone (CORT) is a steroid hormone produced in the adrenal cortex, which can bind to glucocorticoid and mineralocorticoid receptors. It is produced in response to ACTH and is the precursor of aldosterone synthesis. Due to the increase in the production of glucocorticoids due to stress, it is often used as a biomarker of stress. The concentration of CORT in the serum of mice was detected by ELISA kit, and the results are shown in Figure 4 (B). Compared with the control group, the concentration of CORT in the serum of mice after PCPA modeling was significantly increased (56.397±3.524) µg / L (P<0.05), indicating that PCPA increased the stress level of mice. Compared with the model group, the low-dose formula group, the middle-dose formula group, the high-dose formula group and the positive drug group, the content of CORT in the serum was significantly decreased (P<0.05), which was (47.73±3.34) µg / L, (40.62±2.54) µg / L, (43.9±3.45) µg / L and (42.35±1.20) µg / L, respectively. It is proved that the formula can significantly improve the increase of CORT content caused by insomnia, and the middle-dose group has the best improvement effect.

[0074] Oxidative stress refers to a state of imbalance between oxidation and antioxidant effects in the body, leading to accumulation of oxygen active substances (ROS) in the body and causing oxidative damage to tissues and organs. Oxidative stress is a negative effect caused by free radicals in the body and is considered an important factor leading to aging and disease. In the antioxidant defense system, superoxide dismutase (SOD) and catalase (CAT) are the main antioxidant enzymes, playing an irreplaceable role in the dismutation of superoxide radicals and hydrogen peroxide (H2O2), effectively counteracting the harmful effects of ROS, assisting in the removal of free radicals, and reducing and eliminating oxidative damage.

[0075] The SOD concentration in the serum of mice was detected by ELISA kit, and the results are shown in Figure 5 (A). Compared with the control group, the SOD concentration in the serum of mice after PCPA modeling was significantly decreased (2.855±0.221) U / mL (P<0.05), indicating that insomnia caused oxidative damage in mice. Compared with the model group, the SOD content in the serum of the low-dose formula group, the medium-dose formula group, the high-dose formula group, and the positive drug group was significantly increased (P<0.05), respectively (5.415±0.304) U / mL, (6.238±0.228) U / mL, (5.706±0.191) U / mL, and (7.602±0.312) U / mL. The CAT content in the serum of mice is shown in Figure 5 (B). Compared with the control group, the CAT concentration in the serum of mice after PCPA modeling was significantly decreased (6.724±0.221) µmoL / mL (P<0.05). Compared with the model group, the CAT content in the serum of the low-dose formula group, the medium-dose formula group, the high-dose formula group, and the positive drug group was significantly increased (P<0.05), respectively (9.992±1.043) µmoL / mL, (13.001±1.228) µmoL / mL, (11.986±0.624) µmoL / mL, and (11.833±0.781) µmoL / mL. The GSH content in the serum of mice is shown in Figure 5 (C). Compared with the control group, the GSH concentration in the serum of mice after PCPA modeling was significantly decreased (3.587±0.856) µmoL / L (P<0.05). Compared with the model group, the CAT content in the serum of the low-dose formula group, the medium-dose formula group, the high-dose formula group, and the positive drug group was significantly increased (P<0.05), respectively (13.659±2.081) µmoL / L, (15.438±1.266) µmoL / L, (15.323±1.263) µmoL / L, and (21.980±0.820) µmoL / L. In summary, the formula group can significantly reduce the oxidative damage caused by insomnia, and the medium-dose group is the best.

[0076] Sleep deprivation can also cause the body's inflammation risk to rise, and thus cause adverse reactions such as cardiovascular disease, hypertension, diabetes, etc. To evaluate the effect of sleep quality change on the body's inflammation level, the content of several inflammatory factors in the serum of mice was detected. The concentration of TNF-α in the serum of mice is shown in Figure 6 (A). Compared with the control group, the concentration of TNF-α in the serum of mice after PCPA modeling was significantly increased (601.250±14.387) pg / mL (P<0.05), indicating that insomnia increased the content of pro-inflammatory factors in the body of mice. Compared with the model group, the content of TNF-α in the serum of low-dose formula group, medium-dose formula group, high-dose formula group and positive drug group was significantly decreased (P<0.05), which was (519.487±13.483) pg / mL, (503.170±12.830) pg / mL, (489.100±15.927) pg / mL and (459.286±15.290) pg / mL, respectively. The content of IL-1β in the serum of mice is shown in Figure 6 (B). Compared with the control group, the concentration of IL-1β in the serum of mice after PCPA modeling was significantly increased (64.905±1.597) pg / mL (P<0.05). Compared with the model group, the content of IL-1β in the serum of low-dose formula group, medium-dose formula group, high-dose formula group and positive drug group was significantly decreased (P<0.05), which was (61.237±0.673) pg / mL, (56.328±1.977) pg / mL, (54.643±1.275) pg / mL and (53.226±1.779) pg / mL, respectively. The content of IL-6 in the serum of mice is shown in Figure 6 (C). Compared with the control group, the concentration of IL-6 in the serum of mice after PCPA modeling was significantly increased (108.327±5.297) pg / mL (P<0.05). Compared with the model group, the content of IL-6 in the serum of low-dose formula group, medium-dose formula group, high-dose formula group and positive drug group was significantly decreased (P<0.05), which was (88.329±4.936) pg / mL, (82.033±3.275) pg / mL, (93.927±1.328) pg / mL and (77.832±4.972) pg / mL, respectively. In summary, the formula group can significantly alleviate the increase of inflammation level caused by insomnia, and the medium-dose group is the best.

[0077] The effect of formula intervention on the neurons of hippocampus of mice was analyzed by HE staining, and the results are shown in Figure 7The number of neurons in the hippocampus of the blank control group was abundant, the cell morphology was full and round, the staining was light, and the arrangement was neat. The neurons in the insomnia model group were arranged in disorder, the cells were degenerated and atrophied, the shape was irregular, and deep staining appeared. After positive drug intervention, the cell morphology returned to normal. Only a few cells in the goat milk group were in normal shape, most of the cells were still in atrophic state, and the number of cells was less. After intervention by the formula group, the damage of the neurons was repaired to different degrees. In the medium dose and high dose groups, more normal cells were observed, most of the cells were arranged in order, the morphology was full, and the staining was light.

[0078] C-Fos protein is a nuclear phosphoprotein, which is the product of immediate early gene (IEG) and is involved in the regulation of neuronal excitability and survival. The expression of c-Fos is closely related to stress response. After stress stimulation, it has been proved that the expression of c-Fos protein in the hypothalamus, hippocampus and other regions related to cognition and anxiety-like behavior increases. By using c-Fos antibody to specifically immunofluorescently stain the neurons in the hippocampus of mice to compare the stress levels of different treatment groups, the results are shown in Figure 8 The results show that compared with the blank control group, a large number of c-Fos+ neurons appear in the insomnia model group, indicating that insomnia induces neuronal activation and up-regulates the body's stress response. After intervention by the formula group, the c-Fos + The density of neurons in the hippocampus of the mice decreased significantly, indicating that the formula intervention effectively reversed the stress caused by insomnia. The c-Fos + The density of neurons in the hippocampus of the mice decreased significantly, indicating that the formula intervention effectively reversed the stress caused by insomnia. The c-Fos

[0079] The present application proves by RT-PCR that the composition can up-regulate the expression levels of GABAAR mRNA and 5-HT1AR mRNA in SH-SY5Y neuronal cells. Further, by pentobarbital sodium-induced sleep experiment, it is proved that the composition can shorten the sleep latency and prolong the sleep time, and the contents of GABA and 5-hydroxytryptamine (5-HT) in serum and brain tissue are detected, confirming that the composition can promote the release of neurotransmitters related to sleep improvement. In addition, by analyzing the levels of adrenocorticotropic hormone (ACTH), corticosterone (CORT), superoxide dismutase (SOD), catalase (CAT) and reduced glutathione (GSH) in serum, it is proved that the composition has the effect of relieving oxidative stress damage of the body; by detecting interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor (TNF-α) in serum, it is found that the composition can effectively down-regulate the secretion of pro-inflammatory factors. In addition, the composition can inhibit the expression of c-Fos protein in the hippocampus related to neuronal stress, and relieve the atrophy of hippocampal neurons caused by insomnia.

[0080] The above described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present application.

Claims

1. A composition for helping to improve sleep, characterized by, The composition is gamma-aminobutyric acid, Poria cocos extract, Rhizoma Polygonati extract and phosphatidylserine, and the weight ratio of the gamma-aminobutyric acid, Poria cocos extract, Rhizoma Polygonati extract and phosphatidylserine is 11:9:8:1, and the composition is prepared by mixing gamma-aminobutyric acid, Poria cocos extract, Rhizoma Polygonati extract and phosphatidylserine.

2. A product characterized by, The product comprises the composition of claim 1, and the product is a health food or a medicine, and the product further comprises a health food or a medicine acceptable adjuvant.

3. Use of the composition according to claim 1 for the manufacture of a product having an improved sleep effect, characterized in that, The product is a health food or a medicine.

4. Use according to claim 3, characterized in that, The application is the application in the preparation of a product capable of prolonging sleep time.

5. Use according to claim 4, characterized in that, The application is the application in the preparation of a product capable of improving oxidative damage caused by insomnia.

6. Use according to claim 4, characterized in that, The application is the application in the preparation of a product capable of improving the rise of inflammation level caused by insomnia.

7. Use according to claim 4, characterized in that, The application is the application in the preparation of a product capable of improving the damage of neuron cells caused by insomnia.

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