Spinach hexapeptide for increasing rapid eye movement sleep period and application of spinach hexapeptide

Abstract

The invention belongs to the field of food-borne active peptides, and particularly relates to spinach hexapeptide for increasing a rapid eye movement sleep period and application of spinach hexapeptide. The invention relates to an application of spinach hexapeptide in preparation of a product for increasing a rapid eye movement sleep period. The sequence of the spinach hexapeptide is Tyr-Pro-Leu-Asp-Leu-Phe. The oligopeptide sequence is derived from spinach or synthesized by a chemical synthesis method, and the activity of the oligopeptide in the rapid eye movement sleep period is verified by monitoring natural sleep behaviors of mice through electroencephalogram. The spinach hexapeptide YPLDLF can significantly increase the REM duration of a mouse in the natural sleep period, and can be used for preparing food, health care products or medicines with the sleep aiding effect.

Description

Technical Field

[0001] This invention belongs to the field of food-derived bioactive peptide technology, specifically relating to a spinach hexapeptide that increases REM sleep and its applications. Background Technology

[0002] Sleep is a complex process that includes rapid eye movement (REM) sleep and non-rapid eye movement (NREM) sleep. Normal nighttime sleep consists of multiple alternating NREM-REM cycles, with REM sleep being crucial for mood, memory, and neuroplasticity. Reduced REM sleep is associated with a variety of health problems, while increased REM sleep may be beneficial for certain neuropsychiatric disorders.

[0003] Existing technologies disclose many Western medicines or bioactive peptides that can improve sleep. However, there is no necessary correlation between sleep improvement and an increase in rapid eye movement (REM) sleep. If the primary goal of sleep improvement is to increase deep non-rapid eye movement sleep (N3 stage), such as for physical recovery or growth hormone secretion, then effective interventions (such as certain physical therapies or medications) may compress total REM sleep time, but overall sleep quality will still be improved. Meanwhile, many common sedative-hypnotic drugs (such as benzodiazepines and Z-drugs) and alcohol, while helping with sleep in the short term, typically suppress REM sleep, and long-term use can lead to further disruption of sleep structure. Summary of the Invention

[0004] The purpose of this invention is to provide a spinach hexapeptide that increases REM sleep and its application, thereby overcoming the shortcomings of the prior art. The spinach hexapeptide can increase REM sleep and shows significant advantages in terms of safety, absorption and multi-target regulation.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides the application of spinach hexapeptide in increasing REM sleep, wherein the sequence of the spinach hexapeptide is: Tyr-Pro-Leu-Asp-Leu-Phe, abbreviated as YPLDLF.

[0006] Spinach hexapeptide is a food-derived peptide that exhibits significant advantages in terms of safety, absorption, and multi-target regulation.

[0007] In some other embodiments, the spinach hexapeptide is derived from spinach or chemically synthesized. Current research on food-derived hexapeptides for sleep aid largely focuses on specific hexapeptides derived from casein (milk protein). The spinach hexapeptide of this invention, extracted from or chemically synthesized from spinach, is a novel peptide with activity that increases REM sleep.

[0008] In some other embodiments, the chemically synthesized spinach hexapeptide has a purity >99.26%.

[0009] In some other embodiments, the concentration of the spinach hexapeptide used is 10-30 mg / kg.

[0010] Specifically, the concentrations of spinach hexapeptide used were 10, 15, 20, 25, or 30 mg / kg. With increasing dosage, the effect of YPLDLF in increasing REM sleep duration showed a clear dose-dependent effect. However, when the concentration of spinach hexapeptide was below 10 mg / kg, due to the low concentration, the spinach hexapeptide could not bind to enough target receptors, and therefore could not significantly increase the duration of rapid eye movement sleep.

[0011] In some other implementations, the increase in REM sleep period refers to increasing the duration of REM sleep during natural sleep. Enhanced REM sleep directly contributes to high-value cognitive and emotional functions, avoiding the side effects, dependence, and sleep structure disruption associated with drug intervention.

[0012] In some other embodiments, the product is one or more of food, health products, and pharmaceuticals. Specifically, it can be formulated as tea, hard capsules, soft capsules, oral liquids, pills, tinctures, ointments, beverages, pastries, liquid milk, biscuits, candies, raw material extracts, or compound nutrients.

[0013] In some other embodiments, the product is administered by ingestion, oral administration, gavage, or intramuscular injection.

[0014] Secondly, the present invention provides a drug for increasing REM sleep, containing spinach hexapeptide, the sequence of which is: Tyr-Pro-Leu-Asp-Leu-Phe.

[0015] In some other embodiments, it also includes a pharmaceutically acceptable carrier and / or excipients.

[0016] In some other embodiments, the dosage form of the drug is one or more of the following: oral liquid, capsule, powder, tablet, granule, pill, syrup, suppository, and injection.

[0017] The beneficial effects of this invention are: By using electroencephalography (EEG) monitoring and EEG spectroscopy analysis, the NREM and REM sleep stages were precisely distinguished, revealing the regulatory effect of spinach hexapeptide on different sleep stages. It was found that the spinach hexapeptide described in this invention can significantly increase the duration of REM sleep in mice under natural sleep conditions. Compared with traditional drugs, food-derived peptides show significant advantages in safety, absorption, and multi-target regulation. Attached Figure Description

[0018] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0019] Figure 1 High-performance liquid chromatography chromatogram of spinach hexapeptide Tyr-Pro-Leu-Asp-Leu-Phe, which enhances REM sleep according to this invention; Figure 2 The mass spectrum of the spinach hexapeptide Tyr-Pro-Leu-Asp-Leu-Phe that enhances REM sleep according to this invention; Figure 3 The electroencephalograms of mice in natural sleep experiments in Examples 1 and 2 of this invention are shown. Figure 4 The durations of wakefulness, NREM, and REM sleep in mice during the natural sleep experiment in Example 1 of this invention; Figure 5 The durations of wakefulness, NREM, and REM sleep in mice during the natural sleep experiment in Example 2 of this invention; Figure 6 The present invention describes the interaction site between the spinach hexapeptide Tyr-Pro-Leu-Asp-Leu-Phe, which increases REM sleep, and the melatonin receptor MT1 (PDB ID: 6me5). Detailed Implementation

[0020] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Specific conditions not specified in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Components whose manufacturers are not specified are all commercially available conventional products.

[0021] This invention addresses the various physiological and psychological problems that reduced REM sleep may cause, profoundly impacting health. Specifically: From a psychological perspective, reduced REM sleep is closely related to mood disorders. Depression and anxiety may arise from it, and patients' emotional regulation abilities are significantly weakened. Furthermore, insufficient sleep interferes with emotional processing mechanisms, further increasing the risk of mental illness and posing a serious challenge to individual mental health. In terms of cognitive function, REM sleep plays an indispensable role in consolidating memory. Once REM sleep is reduced, learning and memory abilities are severely impaired, affecting not only daily learning and work efficiency but also potentially having a negative impact on long-term cognitive development. From a physiological health perspective, reduced REM sleep may lead to immune system dysfunction. The number of immune cells may decrease, while the body's inflammatory response may increase accordingly, potentially even prompting the premature onset of autoimmune diseases and placing enormous pressure on the body's defense mechanisms.

[0022] Furthermore, reduced REM sleep is closely linked to metabolic abnormalities. For example, glucose and leptin levels may decrease, while the body's metabolic rate may become abnormally high, potentially leading to weight loss and anorexia, thus affecting overall health. More seriously, long-term REM sleep deprivation may increase the risk of neurodegenerative diseases such as Alzheimer's and Parkinson's. These diseases pose a significant threat to patients' quality of life and safety. In addition, reduced REM sleep may also lead to increased blood pressure and heart rate, significantly increasing the risk of cardiovascular disease and further harming health.

[0023] Increasing REM sleep has positive effects on physical and mental health. For example, in terms of mood regulation, increased REM sleep helps improve mood, especially after experiencing social stress. REM sleep plays a role in consolidating emotional memories; increasing REM sleep helps process and integrate emotional experiences. REM sleep is crucial for brain plasticity; increasing REM sleep may promote the repair and remodeling of neural connections.

[0024] Benzodiazepines are commonly used to treat insomnia and can indeed shorten the time it takes to fall asleep, but they can reduce REM sleep time, and long-term use can lead to further disruption of sleep structure. Compared with traditional drugs, food-derived peptides show significant advantages in terms of safety, absorption, and multi-target regulation.

[0025] (1) Spinach hexapeptide: The spinach hexapeptide of this invention, which has the effect of increasing REM sleep, is derived from spinach or chemically synthesized. The chemical synthesis was specifically entrusted to Qiangyao Biotechnology Co., Ltd., and the purity was 99.2622%.

[0026] The obtained spinach hexapeptide Tyr-Pro-Leu-Asp-Leu-Phe was purified by high performance liquid chromatography. The specific purification process is as follows: The chromatographic column used was a Kromasi L 100-5C18 (4.6 mm × 250 mm, 5 μm), column temperature: 50℃, flow rate: 1 mL / min, detection wavelength: 220 nm, mobile phase: Buffer A (0.1% trifluoroacetic acid acetonitrile solution), Buffer B (0.1% trifluoroacetic acid aqueous solution), elution program: 0.0 min, 32%A, 68%B; 20 min, 57%A, 43%B; 20.1 min, 100%A, 0%B.

[0027] Figure 1 The high-performance liquid chromatogram of spinach hexapeptide Tyr-Pro-Leu-Asp-Leu-Phe is shown below. Figure 1 It can be seen that the main component in the sample is located at 6.966 min, accounting for 99.2622% of the total concentration. The concentrations of the other two components are relatively low, at 0.0832% and 0.6546%, respectively.

[0028] The obtained spinach hexapeptide Tyr-Pro-Leu-Asp-Leu-Phe was detected by mass spectrometry. The specific detection process is as follows: The ion source used was electrospray ionization; the spray voltage was ±(2500~3500) V; the solvent removal temperature was 450℃; and the ion pairs used were [M+H]⁺, [2M+H]⁺, and [M+2H]. 2 ⁺.

[0029] Figure 2 The mass spectrum of spinach hexapeptide Tyr-Pro-Leu-Asp-Leu-Phe is shown below. Figure 2 It can be seen that at a mass-to-charge ratio of 767.58, there is a peak labeled [M+H]⁺ with an ionic intensity of 100%, which is the main component of the sample.

[0030] (2) Screening of mice: Male mice of the same age and similar weight were selected for testing.

[0031] (3) The experimental indicators and methods involved are as follows: Drug administration and natural sleep behavior measurement in mice: Custom-designed EEG / EMG electrodes were first implanted in mice to monitor electroencephalography (EEG) and electromyography (EMG). EEG signals were acquired from the frontal cortex (AP, +2 mm; ML, +2 mm) and occipital cortex (AP, -3.5 mm; ML, +2.5 mm). Simultaneously, a reference electrode was implanted in the cerebellum 1.5 mm posterior to the posterior fontanelle. Two EMG electrodes were inserted into the posterior neck muscles. EEG / EMG signal recording began one week post-surgery.

[0032] Multichannel electrophysiological recording equipment was used to collect electroencephalogram (EEG) data of mice during sleep. Experimental mice were administered 10 mg / kg and 30 mg / kg via gavage, with a gavage volume of 100 μL for each group; control mice received an equal volume of saline via gavage. Signal acquisition was initiated immediately after gavage, with a sampling frequency set to 2000 Hz, and recording continued for 3 hours.

[0033] Mouse sleep is divided into three phases: wakefulness, non-rapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep. Wakefulness is characterized by high-amplitude electromyography (EMG) and low-amplitude electroencephalography (EEG) signals dominated by theta waves (6-9 Hz). During NREM sleep, EEG amplitude increases, with delta waves (0.5-4 Hz) predominating, and low-amplitude EMG. During REM sleep, EEG signals are similar to those during wakefulness, but with low-amplitude EMG.

[0034] In this embodiment of the invention, multiple mice were used for the experiment. During the experiment, the mice were first administered physiological saline by gavage, and their electroencephalogram (EEG) signals were measured immediately afterward to serve as the control group data. Then, at the same time, the mice were administered spinach hexapeptide saline solution by gavage, and their EEG signals were measured again immediately to obtain the experimental group data. The average values ​​of the two groups were then compared and analyzed.

[0035] The specific embodiments and effects of the present invention are described below: Example 1 REM sleep duration test Experimental group: spinach hexapeptide Tyr-Pro-Leu-Asp-Leu-Phe, administered by gavage at a dose of 10 mg / kg, with a gavage solution volume of 100 uL; Control group: administered by gavage with an equal volume of physiological saline.

[0036] Immediately after gavage, EEG signals were collected. The results showed that during the mice's natural 3-hour sleep period, the REM sleep duration in the experimental group increased by 0.72 times compared to the control group.

[0037] Example 2 REM sleep duration test Experimental group: spinach hexapeptide Tyr-Pro-Leu-Asp-Leu-Phe, administered by gavage at a dose of 30 mg / kg, with a gavage solution volume of 100 uL; Control group: administered by gavage with an equal volume of physiological saline.

[0038] Immediately after gavage, EEG signals were collected. The results showed that during the mice's natural 3-hour sleep period, the REM sleep duration in the experimental group was significantly increased by 1.89 times compared to the control group.

[0039] The electroencephalograms, arousal, and rapid eye movement (REM) measurements in the mouse natural sleep experiments of Examples 1-2 were tested respectively, and the results are as follows: Figures 3-5 As shown.

[0040] Figure 3 This is a characteristic electroencephalogram (EEG) pattern of mice during sleep. (The text abruptly ends here.) Figure 3 It was found that, compared with the control group, administration of YPLDLF at a dose of 10 mg / kg to mice increased the REM sleep stage, but this increase was not significant. However, when the dose of YPLDLF was increased to 30 mg / kg, the increase in REM sleep stage became very significant. Furthermore, neither dose of YPLDLF had a significant effect on wakefulness time or NREM sleep in mice. To further elucidate this phenomenon, Figure 4 and Figure 5 The duration of wakefulness, NREM sleep, and REM sleep in mice under different doses of YPLDLF were shown in detail.

[0041] Figure 4 The durations of wakefulness, NREM, and REM sleep in mice during the natural sleep experiment in Example 1 are represented by [data missing]. Figure 4 It can be seen that when YPLDLF was administered by gavage at a dose of 10 mg / kg, there was no statistically significant difference in wakefulness time, NREM sleep time, and REM sleep time between mice administered physiological saline and mice administered by gavage during the natural sleep period of 3 hours. However, YPLDLF showed a trend of increasing REM sleep duration.

[0042] Figure 5 These represent the durations of wakefulness, NREM, and REM sleep in the mouse natural sleep experiment of Example 2. Figure 5 It can be seen that when the gavage dose of YPLDLF is 30 mg / kg, compared with mice that are given saline, the wakefulness time tends to decrease, the NREM sleep time tends to increase, and the REM sleep time increases significantly during the 3-hour natural sleep period of mice.

[0043] It is evident that, compared to 10 mg / kg YPLDLF, the effect of increasing REM sleep duration in mice was more significant when the gavage dose was increased to 30 mg / kg. This indicates that the effect of YPLDLF in increasing REM sleep duration exhibits a clear dose-dependent relationship with increasing dose. Studies have shown that melatonin receptor MT1 is a key regulator of rapid REM sleep, and the MT1 receptor agonist UCM871 can increase REM sleep duration in male rats.

[0044] This invention tested the interaction between the spinach hexapeptide Tyr-Pro-Leu-Asp-Leu-Phe, which increases REM sleep, and the melatonin receptor MT1 (PDB ID: 6me5) using the PepSite2 program. The results are as follows: Figure 6 As shown, the peptide binding residues were found to be Tyr-1, Pro-2, Leu-3, Leu-5, and Phe-6, with a p-value of 0.02396. This suggests that the spinach hexapeptide that increases REM sleep is highly likely to interact with the MT1 receptor, indicating that this spinach hexapeptide may increase REM sleep duration in mice by activating the melatonin receptor MT1.

[0045] YPLDLF exhibits a significant dose-dependent effect on increasing REM sleep duration. This may be because, with increasing dosage, more drug molecules of YPLDLF bind to the MT1 receptor, thereby more effectively activating the MT1 receptor and significantly increasing REM sleep duration in mice.

[0046] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a spinach hexapeptide in the preparation of products that increase REM sleep duration, characterized in that, The sequence of the spinach hexapeptide is: Tyr-Pro-Leu-Asp-Leu-Phe.

2. The application according to claim 1, characterized in that, The spinach hexapeptide is derived from spinach or chemically synthesized.

3. The application according to claim 1, characterized in that, The purity of the chemically synthesized spinach hexapeptide is >99.26%.

4. The application according to claim 1, characterized in that, The concentration of the spinach hexapeptide used is 10-30 mg / kg.

5. The application according to claim 1, characterized in that, The increase in REM sleep period refers to increasing the duration of REM sleep period during natural sleep.

6. The application according to claim 1, characterized in that, The product is one or more of the following: food, health products, and medicine.

7. The application according to claim 1, characterized in that, The product can be taken by ingestion, oral administration, gavage, or intramuscular injection.

8. A drug that increases REM sleep, characterized in that, It contains spinach hexapeptide, the sequence of which is: Tyr-Pro-Leu-Asp-Leu-Phe.

9. The drug for increasing REM sleep according to claim 8, characterized in that, It also includes pharmaceutically acceptable carriers and / or excipients.

10. The drug for increasing REM sleep according to claim 8, characterized in that, The dosage form of the drug is one or more of the following: oral liquid, capsule, powder, tablet, granule, pill, syrup, suppository, and injection.

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