GABA (gamma-aminobutyric acid), tremella polysaccharide and theanine composition as well as preparation method and application thereof

Abstract

The invention discloses a composition of GABA (gamma-aminobutyric acid), tremella polysaccharide and theanine as well as a preparation method and application of the composition. The composition comprises a mixture of the tremella polysaccharide and the theanine and the GABA. The mass ratio of tremella polysaccharide to theanine in the mixture is 10: 1; the mass ratio of the mixture of the tremella polysaccharide and the theanine to the GABA is 150: 100. The invention proves that when the GABA and the mixture are compounded according to a specific proportion for use, the GABA and the mixture show a remarkable synergistic effect in the aspects of promoting falling asleep, maintaining sleep, increasing non-rapid eye movement sleep and resisting caffeine-induced awakening, and a solid experimental basis is provided for developing an efficient composite sleep-aiding product.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a composition of GABA, tremella polysaccharide and theanine, and a preparation method and application thereof. BACKGROUND

[0002] Inflammation is a complex defense response of the body to infection, injury or stress. However, excessive or persistent inflammation is the common pathological basis of many chronic diseases such as arthritis, intestinal inflammation, atherosclerosis, etc. At present, the widely used non-steroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids in clinic are effective, but long-term use is often accompanied by serious side effects such as gastrointestinal damage, cardiovascular events and immunosuppression. Therefore, it is an urgent need to develop new anti-inflammatory drugs with safe source, high efficiency and low toxicity.

[0003] Food-derived bioactive peptides have become a new hotspot in the research and development of drugs and functional foods due to their good biocompatibility, low toxicity and diverse physiological functions. These peptides are usually composed of 2-20 amino acids and have no activity in the parent protein, but can exhibit various biological activities including anti-inflammatory after being released by enzymolysis or other methods.

[0004] Mutton is a high-quality source of protein, with a balanced amino acid composition and rich in arginine, leucine and other amino acids with potential anti-inflammatory activity. However, current researches are mostly focused on the nutritional value of mutton, and there are few reports on specific anti-inflammatory peptide sequences from mutton protein sources. In the prior art, there is a lack of a systematic method to efficiently prepare, screen and identify specific peptides with clear anti-inflammatory efficacy from mutton. In particular, there is a lack of research on targeting key inflammation targets (such as inducible nitric oxide synthase, iNOS) for virtual screening through molecular docking technology, and verifying the inhibition of NO activity by in vitro cell models.

[0005] Nitric oxide (NO) is an important inflammatory mediator produced by activated macrophages under inflammatory conditions, and its excessive production is a hallmark event of the inflammatory response. Therefore, substances that can effectively inhibit the production of NO are considered to be highly potential anti-inflammatory candidates. SUMMARY

[0006] The purpose of the present application is to provide a composition of GABA, tremella polysaccharide and theanine, and a preparation method and application thereof.

[0007] The present application is implemented as follows:

[0008] A preparation method of a composition of GABA, tremella polysaccharide and theanine, comprising the following steps:

[0009] Step 1, preparing a tremella strain fermentation broth;

[0010] Step 2: Extract crude polysaccharides from the fermentation broth of the Tremella strain;

[0011] Step 3: Mix the crude polysaccharide of Tremella fuciformis and theanine at a mass ratio of 10:1 to obtain a mixture of Tremella fuciformis polysaccharide and theanine.

[0012] Step 4: Mix the mixture of Tremella polysaccharide and theanine with GABA at a mass ratio of 150:100 to obtain a composition of GABA, Tremella polysaccharide and theanine.

[0013] Further improvements are made to the preparation method of the Tremella fuciformis strain fermentation broth as follows: Tremella fuciformis strain is inoculated into liquid culture medium at an inoculation rate of 5‰, the temperature is controlled at 25±2℃, the stirring speed is 180rpm, the aeration rate is 1:0.8vvm, fermentation is carried out for 10 days, and then the Tremella fuciformis strain fermentation broth is obtained by filtration; wherein the liquid culture medium formula is: glucose 30g / L, soybean peptone 10g / L, yeast extract 5g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L.

[0014] Further improvements were made, and the Tremella fuciformis specimen was deposited at CGMCC 41327, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0015] Further improvements are made, and the specific steps of step two are as follows:

[0016] Add three times the volume of 95% ethanol to the fermentation broth of the *Tremella fuciformis* strain, mix thoroughly, and place in a freezer at 4°C for ethanol precipitation for 1 day. Centrifuge at 4000 rpm for 15 minutes, take the precipitate, add ultrapure water to dissolve the precipitate, and place in a fume hood until the residual ethanol completely evaporates to obtain a crude *Tremella fuciformis* polysaccharide aqueous solution. Freeze the crude *Tremella fuciformis* polysaccharide aqueous solution at -35°C for 24 hours until completely frozen, and then freeze-dry in a freeze dryer to obtain crude *Tremella fuciformis* polysaccharide.

[0017] In a further improvement, the theanine is L-theanine.

[0018] A composition comprising a mixture of tremella polysaccharide and theanine; wherein the mass ratio of tremella polysaccharide to theanine in the mixture is 10:1.

[0019] Further improvements include a mixture of GABA, tremella polysaccharide and theanine, with a mass ratio of 150:100 to GABA.

[0020] One use of the above-described composition is as follows: the composition is used to prepare a medicine for improving sleep.

[0021] A further improvement is that the composition can be used to prepare a drug for prolonging sleep duration.

[0022] Further improvement, the composition is used as a medicine for at least one of promoting sleep, shortening sleep latency, increasing non-rapid eye movement sleep and resisting caffeine-induced wakefulness, in the composition, the mixture of tremella polysaccharide and theanine is used in an amount of 150 mg / kg, and GABA is used in an amount of 100 mg / kg.

[0023] Advantages of the present application:

[0024] The present application proves that γ-aminobutyric acid (GABA) and the mixture of tremella polysaccharide + theanine (ratio 10:1) can effectively shorten sleep latency, prolong sleep time, and improve sleep structure; more importantly, when GABA and the mixture are used in a specific ratio (100 / 150 mg / kg), the compound shows a significant synergistic effect in promoting sleep, maintaining sleep, increasing non-rapid eye movement sleep (NREM), and resisting caffeine-induced wakefulness, the mechanism of which is related to up-regulating the mRNA expression of GABAA receptor and GABAB-R2 receptor, which provides a solid experimental basis for developing high-efficiency composite sleep aid products. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Effects of GABA (A, B) and tremella polysaccharide / theanine mixture (C, D) on sleep latency (A, C) and sleep duration (B, D) of mice treated with hypnotic dose of pentobarbital (42 mg / kg, intraperitoneal injection).

[0026] Figure 2 Effects of GABA / mixture compound preparation on sleep latency (A) and sleep duration (B) of mice treated with hypnotic dose of pentobarbital (42 mg / kg, intraperitoneal injection).

[0027] Figure 3 Effects of GABA, mixture and GABA / mixture compound preparation on sleep quantity and quality.

[0028] Figure 4 Effects of GABA, mixture and GABA / mixture mixture on caffeine (10 mg / kg)-induced wakefulness in rats.

[0029] Figure 5 Effects of GABA / mixture on mRNA expression of GABA and glutamate receptors in mouse brain. DETAILED DESCRIPTION

[0030] Following, the embodiments of the present application will be described in detail by specific examples. Other advantages and effects of the present application can be easily understood by those skilled in the art from the disclosure of this specification. The present application can also be implemented or applied by other different specific embodiments, and various modifications or changes can be made to the details in this specification based on different views and applications without departing from the spirit of the present application.

[0031] Example 1

[0032] Preparation of Tremella strain fermentation broth: The Tremella strain (classified name: Tremella fuciformis, preservation number: CGMCC NO: 41327, preservation address: No. 3, Beichen West Road, Beijing Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences, China General Microbiological Culture Collection Center, preservation date: June 3, 2024) was inoculated in a special liquid medium at an inoculation amount of 5‰ for submerged fermentation. The special liquid medium formula is: glucose 30 g / L, soybean peptone 10 g / L, yeast extract 5 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, pH natural. The fermentation process was carried out in a fermenter, the temperature was controlled at 25±2℃, the stirring speed was 180 rpm, the aeration amount was 1:0.8 vvm, the fermentation period was 10 days, and the Tremella strain fermentation broth was obtained.

[0033] Preparation method of Tremella polysaccharide: alcohol precipitation method was used. The prepared Tremella strain fermentation broth was added with three times the volume of 95% ethanol, and the ethanol and fermentation broth were fully contacted by shaking and stirring. The mixture was placed in a refrigerator at 4°C for alcohol precipitation for 1 day. After alcohol precipitation, the precipitate was obtained by centrifugation at 4000 rpm for 15 min. The precipitate was dissolved in ultrapure water by using a shaker, and the mixture was placed in a fume hood until the residual ethanol was completely volatilized. The obtained crude Tremella polysaccharide was placed in a refrigerator at -35°C for 24 h, and then freeze-dried for 2 days to obtain a freeze-dried powder of crude Tremella polysaccharide. The mechanism of the combination of theanine (L-theanine, purchased from Wuxi Century Bioengineering Co., Ltd.) and Tremella polysaccharide for improving sleep may involve multi-target regulation, but there is little evidence in the current public research directly targeting the sleep mechanism of the combination of the two. The potential mechanism is speculated based on the independent action of the existing ingredients and some combination applications.

[0034] The mechanism of promoting sleep by the combination of tremella polysaccharide and theanine may be based on the synergistic effect of the two: theanine directly improves sleep latency and quality by regulating GABA, inhibiting glutamate excitability, inducing alpha brain waves, and increasing 5-HT / dopamine levels, while tremella polysaccharide indirectly optimizes the sleep environment through immune regulation (such as activating IL-1β and other cytokines) and antioxidant and anti-fatigue effects. The two may complement each other through the dual mechanism of "neurotransmitter regulation + immune-inflammation pathway assistance" to jointly promote the stability of the sleep-wake cycle, but the specific synergistic effect still needs more experimental verification.

[0035] Experimental methods:

[0036] 1.1 Experimental animals

[0037] Male ICR mice (4 weeks old, 18-20 grams) and Sprague-Dawley (SD) rats (8 weeks old, 160-180 grams) were used in this study. All experimental animals were housed in a constant temperature (22±2°C) and humidity (55±5%) environment, and maintained on a 12-hour light-dark cycle. Standard pellet feed and drinking water were freely available during the experiment. All animals underwent a one-week environmental adaptation period before conducting pentobarbital-induced sleep tests and electroencephalogram (EEG) analysis.

[0038] 1.2 Pentobarbital-induced sleep experiment

[0039] The experiment was divided into three groups: gamma-aminobutyric acid (GABA) group; tremella polysaccharide + theanine mixture (group A); gamma-aminobutyric acid + polysaccharide / theanine mixture (group B).

[0040] The ratio of tremella polysaccharide + theanine mixture was 10:1. After 40 minutes of oral administration of different treatments, pentobarbital sodium (42 mg / kg) was injected intraperitoneally into each group of mice. The experimental records were recorded from the injection of compounds to the disappearance of the righting reflex interval (sleep latency), and the duration from the disappearance of the righting reflex to recovery (sleep duration), both measured in seconds. Mice that did not enter sleep within 15 minutes after injection were excluded from the experimental data.

[0041] 1.3 Electrophysiological analysis

[0042] Male SD rats were anesthetized with 2% isoflurane using a gas anesthetic mask within the stereotaxic frame. According to previous literature methods, electroencephalogram recording screw electrodes were implanted into the rat cortex, striatum, and hippocampus. All rats received antibiotic treatment after surgery and were housed individually in a temperature-controlled facility with free access to water and feed. After 7 days of recovery, the rats were randomly divided into control and treatment groups. The experiment was conducted from 10 am to 5 pm for 9 days.

[0043] One hour before EEG signal analysis, different treatment groups were administered medications. The Iox2 system was used to amplify, filter, record, and store the EEG signals. The EEG spectrum was analyzed at 1 Hz intervals and within a standard frequency band. After EEG recording, a Fast Fourier Transform (FFT) was performed every 2 seconds. Based on the average FFT data acquired at 10-second intervals within the 0-30 Hz range, the ecgAUTO3 program was used to calculate wakefulness and sleep time. Caffeine was used to induce wakefulness before the experiment.

[0044] 1.4 Quantitative analysis of receptor mRNA levels

[0045] Using TRIzol TM Total RNA was extracted from mouse brain tissue using reagents and analyzed according to Direct-zol. TM RNA Miniprep Kit Procedure: Remove genomic DNA. Take 1 μg of quality-tested RNA, using oligonucleotide d(T) as a primer, and apply SuperScript... TM cDNA was synthesized using reverse transcriptase III. The resulting cDNA was analyzed by real-time quantitative PCR using a Power Taqman PCR premix kit.

[0046] The qRT-PCR reaction program was set as follows: 50℃ for 2 minutes, 95℃ for 10 minutes; followed by 40 cycles of amplification reactions at 95℃ for 15 seconds and 60℃ for 1 minute. Quantitative calculations were performed using StepOne Plus 2.0 analysis software, and results were normalized using the internal reference gene GAPDH. The target genes used for qRT-PCR are as follows: GABAA receptor, GABAB receptor 1, GABAB receptor 2, GluA1, GluN1, and GluN2A.

[0047] 1.5 Statistical Analysis

[0048] SPSS 12.0 statistical software was used. One-way ANOVA was employed, and Tukey's multiple comparisons test and Bonferroni post-hoc test were used to assess statistical differences in the experimental results. The Tukey multiple comparisons test showed that different letter labels indicated significant differences between groups. All data are expressed as mean ± standard error, and the sample size for each group was n = 6.

[0049] 2. Experimental Results

[0050] 2.1 Effects of Tremella fuciformis polysaccharide mixed with L-theanine on latency and duration of pentobarbital-induced sleep model. Using a pentobarbital-induced sleep model, the sleep latency and duration after administration of GABA or a polysaccharide + theanine mixture were measured to determine the optimal ratio for improving sleep. Figure 1)。With the increase of GABA concentration, sleep latency showed a trend of shortening, while sleep duration showed a trend of prolonging Figure 1 In sleep latency, oral GABA could effectively shorten the sleep latency of mice induced by hypnotic dose of pentobarbital, with significant sleep-promoting effect. In the dose range of 50 to 100 mg / kg, the sleep-promoting effect was significant and stable, but no obvious dose-dependent enhancement trend was observed. Figure 1 In sleep duration, GABA could effectively prolong the sleep time induced by pentobarbital, and the promoting effect was dose-dependent. When the dose reached 75 mg / kg, a significant effect began to occur, and the optimal effect was achieved at 100 mg / kg. At a lower dose of 50 mg / kg, the sleep-promoting effect of GABA did not reach a statistically significant level Figure 1

[0051] The potential sleep-improving effect of tremella polysaccharide / tea amino acid mixture was evaluated by pentobarbital-induced sleep experiment Figure 1 In sleep latency, oral GABA could effectively shorten the sleep latency of mice induced by hypnotic dose of pentobarbital, with significant sleep-promoting effect. In the dose range of 50 to 100 mg / kg, the sleep-promoting effect was significant and stable, but no obvious dose-dependent enhancement trend was observed. Figure 1 In sleep duration, GABA could effectively prolong the sleep time induced by pentobarbital, and the promoting effect was dose-dependent. When the dose reached 75 mg / kg, a significant effect began to occur, and the optimal effect was achieved at 100 mg / kg. At a lower dose of 50 mg / kg, the sleep-promoting effect of GABA did not reach a statistically significant level

[0052] In sleep duration, GABA could effectively prolong the sleep time induced by pentobarbital, and the promoting effect was dose-dependent. When the dose reached 75 mg / kg, a significant effect began to occur, and the optimal effect was achieved at 100 mg / kg. At a lower dose of 50 mg / kg, the sleep-promoting effect of GABA did not reach a statistically significant level

[0053] 2.2 Effect of GABA / mixture compound preparation on sleep latency and duration of pentobarbital-induced sleep model mice

[0054] Comparing the optimal sleep-promoting ratio of GABA / mixture (100 / 150 mg / kg) with single component administration group, it was found that Figure 2 In sleep latency, oral GABA could effectively shorten the sleep latency of mice induced by hypnotic dose of pentobarbital, with significant sleep-promoting effect. In the dose range of 50 to 100 mg / kg, the sleep-promoting effect was significant and stable, but no obvious dose-dependent enhancement trend was observed. ​

[0055] In terms of prolonging sleep duration, the combination of GABA (100 mg / kg) and the mixture (150 mg / kg) achieved the strongest effect in prolonging total sleep time, significantly better than the effect of each single use. Quantitative analysis showed that the combination improved by 46.15% compared to the GABA single use group and by 75.93% compared to the mixture single use group. This strongly proves that GABA and the mixture have a significant synergistic effect in improving sleep maintenance. This result not only confirms the sleep-aiding value of each ingredient, but more importantly reveals the great potential of their combined application, providing a solid experimental basis for the development of efficient and natural composite sleep-aiding products.

[0056] 2.3 Effects of GABA and Tremella Polysaccharide / Tea Amino Acid on Sleep Structure

[0057] By recording electroencephalogram parameters, the synergistic effect of GABA / mixture compound preparation observed in the pentobarbital-induced sleep model was more accurately verified. Figure 3 In this study, the changes in sleep time and sleep structure after single administration of GABA (100 mg / kg), the mixture (150 mg / kg), and GABA / L-mixture compound preparation (100 / 150 mg / kg) were determined. As shown in Fig. A, both GABA and the mixture, whether used alone or in combination, had the effect of prolonging total sleep time and consolidating sleep. The combination of the two achieved the strongest effect in prolonging total sleep time, significantly better than the effect of each single use. Quantitative analysis showed that the combination improved by about 31.46% compared to the GABA single use group and by about 15.95% compared to the mixture single use group. Figure 3 This strongly proves that GABA and the mixture have a significant synergistic effect in improving sleep maintenance. As shown in Fig. B, in terms of wakefulness time, both GABA (100 mg / kg) and the mixture (150 mg / kg) used alone could effectively reduce the wakefulness time of mice, having the effect of promoting sleep maintenance. The data clearly showed that the combination of drugs (GABA / mixture, 100 / 150 mg / kg) produced the strongest effect, with a reduction in wakefulness time (46% lower than the control group) much higher than any single drug (GABA reduced by 23.5%, mixture reduced by 28.5%). Quantitatively, the effect of the combination was about 29.4% higher than that of GABA alone and about 24.5% higher than that of the mixture alone. This suggests that there is a clear trend of synergistic effect of GABA and the mixture in reducing wakefulness and promoting sleep.

[0058] Figure 3 As shown in Fig. C, in terms of sleep latency, both GABA (100 mg / kg) and the mixture (150 mg / kg) used alone could effectively reduce the sleep latency of mice, having the effect of promoting sleep maintenance. The data clearly showed that the combination of drugs (GABA / mixture, 100 / 150 mg / kg) produced the strongest effect, with a reduction in sleep latency (46% lower than the control group) much higher than any single drug (GABA reduced by 23.5%, mixture reduced by 28.5%). Quantitatively, the effect of the combination was about 29.4% higher than that of GABA alone and about 24.5% higher than that of the mixture alone. This suggests that there is a clear trend of synergistic effect of GABA and the mixture in reducing wakefulness and promoting sleep.

[0059] As shown in Fig. D, in terms of sleep latency, both GABA (100 mg / kg) and the mixture (150 mg / kg) used alone could effectively reduce the sleep latency of mice, having the effect of promoting sleep maintenance. The data clearly showed that the combination of drugs (GABA / mixture, 100 / 150 mg / kg) produced the strongest effect, with a reduction in sleep latency (46% lower than the control group) much higher than any single drug (GABA reduced by 23.5%, mixture reduced by 28.5%). Quantitatively, the effect of the combination was about 29.4% higher than that of GABA alone and about 24.5% higher than that of the mixture alone. This suggests that there is a clear trend of synergistic effect of GABA and the mixture in reducing wakefulness and promoting sleep. Figure 3 ​In terms of sleep architecture, the combination of GABA and the mixture significantly increased total sleep time, with the effect mainly reflected in synergistically promoting non-rapid eye movement sleep (NREM). Compared with GABA alone, NREM sleep time was increased by 19.4%; compared with the mixture alone, NREM sleep time was increased by 7.9%. In terms of rapid eye movement sleep (REM), the effect of the combination was equivalent to that of the mixture alone, both of which could maintain a REM sleep level superior to the control group and possibly offset the potential inhibition of REM sleep by GABA alone.

[0060] Electroencephalogram analysis showed that Figure 3 In E and F, the experimental results showed that the effect of the combination of GABA (100 mg / kg) and the mixture (150 mg / kg) on sleep EEG was highly specific. They produced a strong synergistic effect, significantly increasing theta wave activity related to relaxation and sleep initiation, with the effect of the combination far exceeding that of GABA alone (increased by about 41%) and that of the mixture alone (increased by about 16%), proving the strong synergistic effect of the two. However, their effect did not extend to delta wave activity related to deep sleep.

[0061] 2.4 Electroencephalogram acquisition and analysis in a caffeine-induced wakefulness model

[0062] Through electroencephalogram analysis, the sleep-promoting effects of GABA, the mixture, and their combination in a caffeine-induced wakefulness animal model were evaluated. Figure 4 In the caffeine-induced sleep disturbance model, both GABA (100 mg / kg) and the mixture (150 mg / kg) showed significant protective effects, effectively resisting the shortening effect of caffeine on total sleep time Figure 4 A). The combination of GABA and the mixture produced a synergistic effect, with the strongest protective effect, significantly superior to GABA alone (sleep time increased by 13%) and restoring sleep time to near the level of the normal control group. In addition, both GABA (100 mg / kg) and the mixture (150 mg / kg) showed significant sedative effects, effectively antagonizing the caffeine-induced prolongation of wakefulness time Figure 4 B). The combination of GABA and the mixture produced a clear synergistic effect, with an effect of reducing wakefulness time significantly superior to GABA alone and also superior to the mixture alone, almost restoring wakefulness time to the level of the normal control group. This suggests that the two have complementary and enhanced effects in regulating the sleep-wake balance. This provides strong evidence for the development of a combination preparation based on GABA and the mixture for alleviating sleep problems caused by stimulants such as caffeine.

[0063] In the wakefulness model, caffeine severely disrupts sleep structure, leading to a significant reduction in both NREM and REM sleep duration. Both GABA and the mixture effectively counteract this negative effect of caffeine. Crucially, the combined use of GABA and the mixture demonstrates a significant synergistic effect in protecting the most important deep sleep (NREM), significantly outperforming GABA alone and fully restoring sleep depth to normal levels. Figure 4 (C, D). Furthermore, the combined use of GABA (100 mg / kg) and a mixture (15 mg / kg) showed highly specific effects on sleep EEG. They produced a strong synergistic effect, significantly increasing theta wave activity associated with relaxation and sleep initiation. However, its effects did not extend to delta wave activity associated with deep sleep. Figure 4 (E, F). This provides strong scientific evidence for developing compound products based on GABA and mixtures to alleviate sleep disorders and improve sleep quality caused by stimulants such as caffeine.

[0064] 2.5 Effects of combined use of GABA and mixtures on neurotransmitter receptor mRNA levels

[0065] To investigate whether the sleep-promoting mechanism of GABA / mixture mixtures is related to changes in neurotransmitter receptor expression, we assessed the mRNA levels of GABA and glutamate receptors (GABA / mixture mixtures). Figure 5 The combination of GABA and the mixture showed the strongest upregulation of GABAA-R receptor mRNA, increasing expression levels by 45% compared to the control group, and significantly higher than the GABA-only group (32% increase) and the mixture-only group (17% increase). Figure 5 (A). The combined drug group showed a 20% increase in GABAB-R2 expression, which was superior to the monotherapy group. For GABAB-R1, the combined drug group, GABA monotherapy group, and the combination drug monotherapy group all showed significantly higher levels than the control group, but there was no significant difference among the three groups, indicating that the effect at this target is mainly additive rather than synergistic. Figure 5 (B, C). L-Theanine is a major contributor to the regulation of glutamate receptors, and combination therapy ensures that this regulatory effect is achieved at the optimal level. The mixture alone increased GluA1 expression by 21%, while the combination therapy group increased it by 19%, showing comparable efficacy. Figure 5 (D). The combined treatment group showed the highest GluN1 expression level, increasing by 23%, significantly higher than the control group; the single-drug group showed an increase of 15%. This indicates that L-theanine plays a key role in regulating the glutamate system. Figure 5 (E). None of the treatment groups had a significant effect on the mRNA expression of the GluN2A receptor. Figure 5 (F).

[0066] 3. Conclusion

[0067] The experiment proves that γ-aminobutyric acid (GABA) and tremella polysaccharide + theanine mixture (ratio 10:1) can effectively shorten sleep latency, prolong sleep time and improve sleep structure through pentobarbital-induced sleep model, electroencephalogram analysis and quantitative analysis of receptor mRNA level; more importantly, when GABA and the mixture are used in a specific ratio (100 / 150 mg / kg), they show significant synergistic effect in promoting sleep, maintaining sleep, increasing non-rapid eye movement sleep (NREM) and resisting caffeine-induced wakefulness, and the mechanism is related to up-regulation of GABAA receptor and GABAB-R2 receptor mRNA expression, which provides a solid experimental basis for developing high-efficiency composite sleep-aiding products.

[0068] The above merely describes preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a composition of GABA, tremella polysaccharide and theanine, characterized in that, It comprises the following steps: Step one, preparing the tremella strain fermentation liquor; Step two, extracting the tremella crude polysaccharide in the tremella strain fermentation liquor; Step three, mixing the tremella crude polysaccharide and theanine according to a mass ratio of 10:1 to obtain a mixture of tremella polysaccharide and theanine; Step four, mixing the mixture of tremella polysaccharide and theanine with GABA according to a mass ratio of 150:100 to obtain a composition of GABA, tremella polysaccharide and theanine. The preparation method of the tremella strain fermentation liquor is as follows: inoculating the tremella strain into a liquid culture medium at an inoculation amount of 5‰, controlling the temperature to be 25±2℃, the stirring speed to be 180rpm, and the aeration amount to be 1:0.8vvm, and then filtering after 10 days of fermentation to obtain the tremella strain fermentation liquor; wherein the formula of the liquid culture medium is as follows: glucose 30g / L, soybean peptone 10g / L, yeast extract powder 5g / L, potassium dihydrogen phosphate 1g / L, and magnesium sulfate 0.5g / L.

2. The method of claim 1, wherein the preparation of the combination of GABA, tremella polysaccharide and theanine is characterized by, The preservation number of the tremella strain is CGMCC 41327, and the preservation place is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

3. The method of claim 2, wherein the preparation of the combination of GABA, tremella polysaccharide and theanine is characterized by, The specific steps of step two are as follows:

4. The method of claim 1, wherein the preparation of the combination of GABA, tremella polysaccharide and theanine is characterized by, Adding three volumes of 95% ethanol to the tremella strain fermentation liquor, mixing thoroughly, and then placing in a refrigerator at a temperature of 4° for alcohol precipitation for 1 day; using a centrifuge at 4000rpm for centrifugation for 15min; adding ultrapure water to the precipitate to fully dissolve the precipitate; placing in a fume hood until the residual ethanol is completely volatilized to obtain a tremella crude polysaccharide aqueous solution; placing the tremella crude polysaccharide aqueous solution in a-35℃ refrigerator for freezing for 24h to completely freeze solid; and then placing in a freeze dryer to obtain the crude tremella polysaccharide. The theanine is L-theanine.

5. The method of claim 1, wherein the preparation of the combination of GABA, tremella polysaccharide and theanine is characterized by, The mixture comprises the tremella polysaccharide and theanine; and the mass ratio of the tremella polysaccharide to theanine in the mixture is 10:

1.

6. A composition characterized in that, The mixture further comprises GABA; and the mass ratio of the mixture of the tremella polysaccharide and theanine to GABA is 150:

100.

7. The composition of claim 6, wherein The composition is used as a drug for improving sleep.

8. Use of a composition according to any one of claims 1 to 7, characterized in that, The composition is used as a drug for prolonging the sleep duration.

9. Use of a composition according to claim 8, characterized in that, The composition is used as a drug for at least one of promoting falling asleep, shortening the sleep latency, increasing non-rapid eye movement sleep, and resisting caffeine-induced wakefulness; in the composition, the amount of the mixture of the tremella polysaccharide and theanine is 150mg / kg, and the amount of GABA is 100mg / kg.

10. The use of the combination of GABA, tremella polysaccharide and theanine according to claim 8, characterized in that, ​

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