Application of lycium ruthenicum polysaccharide and medicine or health food for improving sleep

Abstract

The invention discloses application of lycium ruthenicum polysaccharide and a medicine or health food for improving sleep, the lycium ruthenicum polysaccharide is used as a product for improving sleep, and the lycium ruthenicum polysaccharide has no tolerance while improving sleep and has high safety. Besides, as a natural traditional Chinese medicine extract, lycium ruthenicum polysaccharide is safe and green in source, small in side effect, capable of regulating the body in multiple aspects and suitable for long-term administration, has the prospect of being developed into products for improving sleep, provides more choices for improving insomnia clinically, and has important social benefits and economic values.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to the application of a black goji berry polysaccharide and a medicine or health food for improving sleep. Background Technology

[0002] Insomnia is the most common sleep disorder across all age groups, typically manifesting as difficulty falling asleep, frequent awakenings, insufficient sleep depth or frequency, early awakening, and inadequate sleep duration or quality. Prolonged insomnia can lead to chronic fatigue, anxiety, obesity, memory loss, and weakened immunity—a state of "sub-health." This sub-health state not only affects work efficiency and quality of life but also increases the risk of various diseases such as hypertension, diabetes, depression, and cancer. In modern society, the fast pace of life, increased work and study pressure, coupled with irregular sleep patterns, has led to a growing number of people being in a sub-healthy state, with almost everyone experiencing insomnia. Chronic insomnia has a long course, lasting up to 10 years or more, severely impacting people's quality of life and mental and physical health. According to a survey by the World Health Organization (WHO), nearly one-third of the world's population suffers from sleep disorders.

[0003] Therefore, improving sleep and boosting immunity play a crucial role in regulating the body's sub-health state. Currently, clinical treatment of sleep disorders mainly relies on chemical drugs, but long-term use of these drugs may produce side effects, such as low safety, residue, and dependence. Compared with chemical drugs, natural food and medicinal resources have higher safety in improving sleep disorders, are less likely to cause drug resistance and dependence, and have conditioning effects in addition to therapeutic effects, thus gaining increasing popularity among patients. In conclusion, researching and developing a highly effective, safe, and sleep-improving natural drug product has significant social value.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide an application of black goji berry polysaccharide in the preparation of a drug for improving sleep and a medicine or health food for improving sleep, so as to improve the above-mentioned technical problems.

[0006] This invention is implemented as follows:

[0007] In a first aspect, the present invention provides the application of black goji berry polysaccharide in the preparation of a medicine for improving sleep.

[0008] In an optional implementation, the sleep-improving drug is a drug targeting at least one of the following symptoms:

[0009] a. A decrease in the number of brain nerve cells;

[0010] b. Widening of the clefts surrounding brain nerve cells;

[0011] c. Decrease in Nissl bodies.

[0012] In an optional embodiment, the preparation steps of the black goji berry polysaccharide include: water extraction of black goji berries to obtain a crude extract of black goji berries; alcohol precipitation of the crude extract to enrich the black goji berry polysaccharides to obtain a crude polysaccharide extract; and then purification of the crude polysaccharide extract to remove impurities.

[0013] In an optional embodiment, the black goji berries are subjected to water extraction as follows: the dried black goji berries are crushed and then 5 to 30 times the volume of deionized water is added, and the mixture is extracted at 50°C to 80°C for 1 to 4 hours, and the extraction is repeated 2 to 5 times. The extracts are combined to obtain a crude extract of black goji berries; or the fresh black goji berries are crushed, juiced, and the residue is extracted again with water each time, and the extraction is repeated 3 to 4 times. The filtrates obtained from the juice extraction are combined to obtain a crude extract of black goji berries.

[0014] In an optional embodiment, the black goji berry crude extract is enriched by alcohol precipitation by adding 2 to 6 times its volume of 70% to 100% ethanol to the black goji berry crude extract for alcohol precipitation.

[0015] In an optional embodiment, purifying the crude polysaccharide extract includes sequentially performing protein removal, decolorization, and dialysis.

[0016] In an optional embodiment, the protein removal is performed by dissolving the crude polysaccharide extract of black goji berries in ultrapure water and adding Sevag reagent to remove the protein.

[0017] And / or, decolorization is performed by using macroporous resin to decolorize the polysaccharides after protein removal;

[0018] And / or, dialysis involves concentrating the decolorized polysaccharide solution under reduced pressure and then dialyzing it through a dialysis bag to obtain a polysaccharide solution with a molecular weight greater than 3500 Da.

[0019] In optional embodiments, the macroporous resin is a non-polar macroporous adsorption resin, a weakly polar macroporous resin, or a polar macroporous resin. The non-polar macroporous adsorption resin is selected from D101, D101b, DA-201, DM-301, DM-11, HPD-100, HPD-450, HPD-750, D1400, D1300, D3520, D4006, D4020, HP-20, H-103, H107, X-5, ADS-5, ADS-8, BS-55, BS-65, BS- At least one of 80, NKA-2, and NKA-9; the weakly polar macroporous resin is selected from at least one of AB-8, DM130, D860021, DS-401, BS-30, CAD-40, and CAD-45; the polar macroporous resin is selected from at least one of ADS-F8, ADS-21, ADS-7, ADS-17, BS-75, BS-45, S-8, NKA-2, and NKA-9; the elution solvent is ultrapure water, the amount of which is 3 to 5 times the resin volume, and the flow rate is 1 BV / h to 3 BV / h.

[0020] In an optional embodiment, the preparation steps of the black goji berry polysaccharide further include: concentrating the polysaccharide solution obtained after dialysis under reduced pressure, and then freeze-drying it.

[0021] Secondly, the present invention provides a medicine or health food for improving sleep, the active ingredient of which is black goji berry polysaccharide. The medicine is prepared by loading with a pharmaceutically acceptable carrier, and the health food is prepared by loading with a food science-acceptable carrier.

[0022] This invention offers the following beneficial effects: Black goji berry polysaccharides are widely used in sleep improvement products, improving sleep without causing tolerance and exhibiting high safety. Furthermore, as a natural Chinese medicine extract, black goji berry polysaccharides are safe and environmentally friendly, with few side effects. They can regulate the body in multiple ways, are suitable for long-term use, and have the potential to be developed into sleep improvement products. This provides more options for clinical treatment of insomnia, and has significant social and economic value. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The effect of different concentrations of caffeine on the behavioral trajectory of zebrafish (n=8).

[0025] Figure 2 The effects of different concentrations of caffeine on the movement distance, movement time, and resting time of zebrafish ( n=8); compared with the blank control group, * P<0.05.

[0026] Figure 3 The effects of drug treatment on the duration and distance of movement in a zebrafish sleep deprivation model. (n=10); compared with the blank control group, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01.

[0027] Figure 4 The effects of drugs on the number of resting periods, duration, and distance in a zebrafish sleep deprivation model over 24 hours. n=12); compared with the blank control group, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01.

[0028] Figure 5 The effect of drugs on the 24-hour behavioral trajectory of a zebrafish sleep deprivation model (n=12).

[0029] Figure 6 The effects of black goji berry polysaccharide on neurons in the zebrafish brain.

[0030] Figure 7 The effects of black goji berry extract on neurons in the zebrafish brain.

[0031] Figure 8 The effects of black goji berry and ginseng extract tablets on neurons in the brain of zebrafish. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0033] The following is a detailed description of the application of a black goji berry polysaccharide provided by the present invention, as well as its use in medicines or health foods for improving sleep.

[0034] Some embodiments of the present invention provide the application of black goji berry polysaccharide in the preparation of a medicine for improving sleep.

[0035] Black goji berry polysaccharides are bioactive components extracted from black goji berries. They improve sleep without causing tolerance and exhibit high safety. Furthermore, as a natural traditional Chinese medicine extract, black goji berry polysaccharides are safe and green in origin, with few side effects. They can regulate the body in multiple ways, are suitable for long-term use, and have the potential to be developed into sleep-improving products, providing more options for clinical treatment of insomnia and possessing significant social and economic value. Black goji berries grow in a unique environment of high altitude, low temperature, and strong sunlight, resulting in their unique polysaccharide content and composition. Studies have shown that black goji berry polysaccharides are high in arabinose and galactose; this unique monosaccharide composition may endow them with superior biological activity. Compared to other goji berry polysaccharides, black goji berries are also rich in anthocyanins, whose antioxidant capacity is stronger than other pigments in red goji berries. The synergistic effect of anthocyanins and polysaccharides makes black goji berries more effective in reducing oxidative stress damage and maintaining nervous system function, potentially leading to a more pronounced sleep-improving effect.

[0036] Specifically, in some embodiments, black goji berry polysaccharides can be used to prepare drugs having at least one of the following symptoms:

[0037] a. A decrease in the number of brain nerve cells;

[0038] b. Widening of the clefts surrounding brain nerve cells;

[0039] c. Decrease in Nissl bodies.

[0040] Furthermore, the preparation process of black goji berry polysaccharides significantly affects their efficacy. Polysaccharides obtained from different preparation processes vary in purity, structure, and molecular weight distribution, thus affecting their bioactivity and efficacy. Therefore, in some embodiments, to better leverage the sleep-improving effects of black goji berry polysaccharides, the preparation steps include: water extraction of black goji berries to obtain a crude extract; alcohol precipitation to enrich the black goji berry polysaccharides in the crude extract to obtain a crude polysaccharide extract; and then purification and impurity removal of the crude polysaccharide extract.

[0041] Specifically, the water extraction of black goji berries involves: pulverizing dried black goji berries, adding 5-30 times their volume of deionized water, and extracting at 50℃-80℃ for 1-4 hours, repeating the extraction 2-5 times. The extracts are then combined to obtain a crude extract of black goji berries. Alternatively, fresh black goji berries can be crushed, juiced, and the residue can be extracted again with water each time, repeating this process 3-4 times. The filtrates obtained from the juice extraction are then combined to obtain the crude extract of black goji berries. Water extraction utilizes the water-soluble properties of polysaccharides to dissolve them. While hot water extraction of dried fruit is beneficial for polysaccharide extraction, excessively high temperatures or prolonged extraction times can damage the active groups of polysaccharides, weakening their ability to scavenge free radicals. Therefore, the above process allows for rapid extraction of black goji berry polysaccharides without significantly damaging their biological activity.

[0042] Furthermore, in some embodiments, the enrichment of black goji berry polysaccharides by alcohol precipitation of crude black goji berry extract is carried out by adding 2-6 times the volume of 70%-100% ethanol to the crude black goji berry extract for alcohol precipitation. The addition of ethanol alters the polarity of the solvent, reducing the solubility of polysaccharide molecules, causing them to aggregate and precipitate from the solution. This achieves separation from other impurities in the solution (such as small organic molecules, inorganic salts, etc.), thus enriching the polysaccharides. Generally, after adding ethanol, the mixed solution is transferred to a suitable container, sealed, and allowed to stand at a low temperature (generally around 4°C) for a period of time to allow the polysaccharides to fully precipitate. The standing time is usually 12-24 hours, and the specific time can be adjusted according to the polysaccharide precipitation. Then, the precipitate is separated from the supernatant by centrifugation or filtration. The precipitate is then washed with an appropriate amount of ethanol (generally the same concentration as during alcohol precipitation) to remove impurities adsorbed on the precipitate surface, yielding a crude polysaccharide extract.

[0043] Furthermore, in some embodiments, the purification of the crude polysaccharide extract includes sequentially performing protein removal, decolorization, and dialysis.

[0044] For reference, protein removal involves dissolving crude polysaccharide extract from black goji berries in ultrapure water, adding Sevag reagent, and then mixing and shaking a chloroform-n-butanol mixture (typically a volume ratio of 4:1 to 5:1) with the polysaccharide solution. This causes the protein to form a gel-like precipitate at the chloroform-water interface, thus removing it. This method is mild and causes minimal damage to the polysaccharide structure, but requires repeated operations to achieve good protein removal results. For example, 100 mL of black goji berry polysaccharide extract is mixed with 20 mL of Sevag reagent (chloroform:n-butanol = 4:1), shaken on a shaker for 30 minutes, then centrifuged at 3000–5000 rpm for 10–5 minutes, and the upper aqueous phase is collected. This process is repeated 3–5 times.

[0045] Decolorization involves using macroporous resin to decolorize the polysaccharides after protein removal. Based on the difference in charge between the pigments and polysaccharides, a suitable ion exchange resin is selected for adsorption. For example, anion exchange resins can be used for negatively charged pigments. When the wolfberry polysaccharide extract is passed through a column packed with anion exchange resin, the pigments are adsorbed by the resin, while the polysaccharides elute from the column. This method offers good selectivity and relatively low polysaccharide loss. For example, the macroporous resin is a non-polar macroporous adsorption resin, a weakly polar macroporous resin, or a polar macroporous resin. The non-polar macroporous adsorption resin is selected from D101, D101b, DA-201, DM-301, DM-11, HPD-100, HPD-450, HPD-750, D1400, D1300, D3520, D4006, D4020, HP-20, H-103, H107, X-5, ADS-5, ADS-8, BS-55, BS-65, BS-80, N... At least one of KA-2 and NKA-9; the weakly polar macroporous resin is selected from at least one of AB-8, DM130, D860021, DS-401, BS-30, CAD-40 and CAD-45; the polar macroporous resin is selected from at least one of ADS-F8, ADS-21, ADS-7, ADS-17, BS-75, BS-45, S-8, NKA-2 and NKA-9; the elution solvent is ultrapure water, the amount of which is 3 to 5 times the resin volume, and the flow rate is 1 BV / h to 3 BV / h.

[0046] Dialysis involves concentrating the decolorized polysaccharide solution under reduced pressure and then dialyzing it through a dialysis bag to obtain a polysaccharide solution with a molecular weight greater than 3500 Da. The dialysis time is 24–72 h.

[0047] Furthermore, the preparation steps of black goji berry polysaccharide also include: concentrating the polysaccharide solution obtained after dialysis under reduced pressure, and then freeze-drying it to finally obtain black goji berry polysaccharide powder, the total sugar content of which is 0.1-90%.

[0048] Some embodiments of the present invention also provide a medicine or health food for improving sleep, the active ingredient of which is the black goji berry polysaccharide in the above embodiments, wherein the medicine is made by loading with a pharmaceutically acceptable carrier, and the health food is made by loading with a food science-acceptable carrier.

[0049] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0050] Example 1

[0051] 1. Experimental Materials and Instruments

[0052] 1.1 Experimental Animals: Wild-type AB strain zebrafish were used in the experiment. These were purchased from the National Zebrafish Resource Center of the Institute of Hydrobiology, Chinese Academy of Sciences, and housed at the Zebrafish Research Platform of the School of Pharmacy, Xinjiang Medical University. Rearing and breeding were all conducted on the platform. Juvenile fish aged 5 days (5 dpf) after fertilization were used in the experiment. Rearing conditions: The water temperature was (28.0±1)℃, the light-dark cycle was 14h:10h, and the pH of the water was 7.0–8.0. During this period, the zebrafish could obtain nutrients from their own yolk sac and did not require feeding.

[0053] 1.2 Test Drugs:

[0054] Preparation of black goji berry (Lycium ruthenicum Murray, LRM) extract (LRME): Black goji berry powder was added to distilled water at a solid-liquid ratio of 25.00 mL / g, and extracted at 80.0℃ for 2.0 h, repeated 2.0 times. The extracts were combined, concentrated under reduced pressure to obtain crude extract, and then freeze-dried.

[0055] Preparation of crude polysaccharide from black goji berries (LRMP): Black goji berry powder was added to distilled water at a solid-liquid ratio of 25.00 mL / g and extracted at 80.0℃ for 2.0 h, repeated 2.0 times. The extracts were combined and concentrated under reduced pressure to obtain a crude extract. Black goji berry polysaccharides were enriched by alcohol precipitation with 4 times the volume of anhydrous ethanol. The obtained crude polysaccharide extract was dissolved in 200 mL of ultrapure water, and Sevag reagent (chloroform: n-butanol = 4:1) was added for protein removal; this process was repeated multiple times. The polysaccharides were decolorized using AB-8 macroporous resin. The protein-removed polysaccharide solution was adsorbed onto the AB-8 macroporous resin and eluted with 3 column volumes of ultrapure water. The decolorized polysaccharide solution was concentrated under reduced pressure to a certain volume, aliquoted into pre-treated dialysis bags, and dialyzed for 48 h. During dialysis, most of the oligosaccharides were removed, finally yielding a polysaccharide solution with a molecular weight greater than 3500 Da. The polysaccharide solution was then concentrated under reduced pressure and freeze-dried to finally obtain crude polysaccharide powder from black goji berries.

[0056] Black Goji Berry Ginseng Extract Tablets (LGRP): The compound ratio of black goji berries, ginseng, rhizoma polygonati, and polygonatum odoratum is 5:2:2:1.

[0057] The raw material for black goji berries is provided by Xinjiang Black Goji Berry Biotechnology Co., Ltd., and is harvested in Ruoqiang County, Korla City, Xinjiang.

[0058] Melatonin (Beijing Cooler Technology Co., Ltd., batch number: CM34111416); Caffeine (Sigma, USA, batch number: C0750-5G).

[0059] 1.3 Instruments: Zebrafish behavior tracking system (Noldus, Netherlands, model: Danio Vision); Stereo microscope (Olympus, Japan, model: DP74).

[0060] 1.4 Grouping and Modeling: Wild-type AB zebrafish (5 dpf) were selected for the experiment. Normally developed zebrafish were chosen under a microscope and randomly divided into 6 groups: a blank control group, a model group, a melatonin group, and low, medium, and high dose groups of black goji berry polysaccharide, with 12 fish in each group. The drug was administered via in vitro water immersion for 24 hours, followed by incubation at 28℃. Blank control group: Normally fed, no treatment; Model group: Caffeine treated for 24 hours; Drug intervention group: In addition to the model treatment, zebrafish were treated with water-soluble melatonin for 24 hours; Melatonin treatment group: In addition to the model treatment, zebrafish were treated with water-soluble melatonin for 24 hours.

[0061] 1.5 Screening of caffeine concentration for modeling: Zebrafish were treated with five different concentrations of caffeine (300, 350, 400, 450 and 500 μmol / L) for 24 hours. Their behavioral changes were quantitatively analyzed for 1 hour. The movement distance, movement time and resting time of the five groups of zebrafish were detected, with eight zebrafish in each group.

[0062] 2. Half-maximal inhibitory concentration (IC50) of a drug 50 The study investigated the effects of different concentrations of melatonin on the zebrafish. 180 zebrafish were randomly selected and placed in 60mm culture dishes, divided into 12 experimental groups of 15 fish per group. These included a blank control group, a model group, a melatonin group, and a black goji berry polysaccharide group. Preliminary concentrations for each group were 0.1, 0.2, 0.5, 1, and 2 mg / mL (5 groups). Melatonin was preliminarily divided into 0.01, 0.05, 0.1, 0.5, 1, 5, and 10 mg / mL (7 groups). After 24 hours, the zebrafish's responses were observed, and the number and mortality rates of each experimental group were recorded. The IC50 of the drug was calculated. 50 .

[0063] 3. The sleep-improving effect of drugs on a zebrafish sleep deprivation model: based on the half-maximal inhibitory concentration (IC50) of the drug. 50 Based on the results of the investigation, the IC50 of black goji berry polysaccharide was selected. 50 1 / 2 IC 50 and 1 / 4 IC 50 The melatonin was divided into high-dose, medium-dose, and low-dose groups, with an IC50 of 1 / 2. 50Six groups were formed, including a positive control group, a blank control group, and a model group, with 15 zebrafish in each group. After 24 hours of treatment, 10 zebrafish were randomly selected from each experimental group. The time (T) during which the zebrafish were in a state of sleep deprivation (i.e., movement in a dark environment) was quantified using a zebrafish behavior tracking system, with an observation period of 1 hour. The formula for calculating the sleep improvement rate of the zebrafish sleep deprivation model is as follows: Sleep improvement rate = [(T)] 模 [Tmodel group - Tdrug treatment group) / (Tmodel group - Tblank control group)] × 100%.

[0064] 4. Sedative effect of drugs on sleep in a zebrafish sleep deprivation model: Referring to the above study on the sleep improvement effect of drugs in a zebrafish sleep deprivation model, 10 zebrafish were randomly selected from each group. The movement distance (D) of the zebrafish was measured using a zebrafish behavior tracking system, and the sedative effect was calculated. The calculation formula is as follows: Sedative effect = (D... 模型组 -D 药物治疗组 ) / (D 模型组 -D 空白对照组 ).

[0065] 5.24-hour behavioral monitoring: Using a zebrafish behavior analysis system, the trajectory changes of each group of zebrafish were detected over 24 hours and trajectory maps were generated. Resting motion (<5 mm·s) was statistically analyzed at different time periods. -1 (Black), small movement (5-25mm·s) -1 (green), large motion (>25mm·s) -1 The count, time and distance of (red) were recorded, and the differences in their resting motion were analyzed.

[0066] 6. Nissl staining for neuronal morphology: After fixation of the whole brain with 4% paraformaldehyde, the tissue surface was rinsed with PBS, and serial coronal sections were prepared using a microtome. The sections were washed three times with PBS, and then immersed in 1% Nissl staining solution for 10 min. After rinsing with distilled water, the sections were differentiated with a separating solution for 10 s to remove background. The sections were dehydrated with graded ethanol and then cleared with xylene. Finally, the sections were mounted with neutral resin and photographed under a microscope to observe changes in neurons and Nissl bodies.

[0067] SPSS 22.0 software was used for statistical analysis of the data, and the mean ± standard deviation was used for the measurement data. The results indicate that a t-test was used to determine if the distribution conforms to a normal distribution, and a rank-sum test was used to determine if it does not conform to a normal distribution. A p-value < 0.05 indicates a significant difference, a p-value < 0.01 indicates a highly significant difference (statistically significant), and a p-value > 0.05 indicates no statistical significance.

[0068] Test Example 1

[0069] 1. Determination of Caffeine Concentration for Modeling: 5-day-fed zebrafish were immersed in different concentrations of caffeine for 1 hour, and their spontaneous movement behavior was recorded continuously for 1 hour (3600 seconds). Results showed that compared with the blank control group, the zebrafish in the 350 μmol / L caffeine group exhibited significantly increased movement distance and duration, and significantly decreased resting time (P<0.05), demonstrating a more active state. Therefore, a caffeine concentration of 350 μmol / L was selected for modeling. Results are as follows... Figure 1 and Figure 2 As shown.

[0070] 2. Half-maximal inhibitory concentration (IC50) of the drug 50 The mortality rates of zebrafish under different concentrations of black goji berry polysaccharide and melatonin are shown in Tables 1 and 2. The IC50 of black goji berry polysaccharide was calculated. 50 The concentration was 0.6 mg / mL, which is the high-dose group; 1 / 2 IC 50 The IC50 was 0.3 mg / mL, which is the medium-dose group; the 1 / 4 IC50 was 0.15 mg / mL, which is the low-dose group. Melatonin IC50 50 The concentration is 6 mg / mL, and a 1 / 2 IC is set. 50 3 mg / mL served as the positive control group.

[0071] Table 1. Effects of different concentrations of black goji berry polysaccharide on zebrafish.

[0072]

[0073]

[0074] Table 2 Effects of different melatonin concentrations on zebrafish

[0075]

[0076] 3. The sleep-improving and sedative effects of black wolfberry polysaccharides on a zebrafish sleep deprivation model: Within the 1 hour (3600 seconds) observation period, the total duration of insomnia and the total distance of movement in the model group were significantly higher than those in the blank control group (P < 0.01), indicating that the model was successfully established. Compared with the model group, the total duration of insomnia and the total distance of movement in the melatonin group were significantly reduced (P < 0.01), with a sleep improvement rate of 79.89% and a sedative effect of 1.03 times, respectively, indicating that melatonin has a significant improving effect on the zebrafish sleep deprivation model. Compared with the model group, LRMP, LRME, and LGRP all had certain degrees of sleep-improving and sedative effects on the zebrafish sleep deprivation model. The improvement rates of sleep in zebrafish at low, medium, and high doses of LRMP were 58.56%, 73.99%, and 82.69%, respectively, with sedative effects of 0.92-fold, 1.19-fold, and 1.27-fold, respectively. The improvement rates of sleep in zebrafish at low, medium, and high doses of LRME were 54.60%, 69.03%, and 77.97%, respectively, with sedative effects of 0.57-fold, 0.83-fold, and 0.95-fold, respectively. The improvement rates of sleep in zebrafish at low, medium, and high doses of LGRP were 50.89%, 63.39%, and 71.53%, respectively, with sedative effects of 0.60-fold, 0.84-fold, and 0.96-fold, respectively. Figure 3 (Table 3). Conclusion: Sleep improvement and sedative effects: LRMP (polysaccharide) > LRME (extract) > LGRP (black goji berry and ginseng extract tablets).

[0077] Table 3. Sleep improvement and sedative effects of drugs in a zebrafish sleep deprivation model. n=10)

[0078]

[0079] 4. Effects of black goji berry polysaccharides on 24-hour spontaneous activity in a zebrafish sleep deprivation model: Compared with the blank control group, the number of resting movements, duration, and distance were reduced in the model group (P<0.01), indicating reduced sleep activity and successful model establishment. Compared with the model group, the medium- and high-dose drug groups and the melatonin group all prolonged the number of resting movements, duration, and distance in zebrafish (P<0.05). The high-dose drug group showed the greatest increase in the number of movements and distance, followed by the medium-dose group, and the low-dose group showed the least increase. The melatonin group significantly increased the number of resting movements (P<0.01) (see...). Figure 4-5 (Table 4). Conclusion: The number, time and distance of resting exercise were prolonged: LRMP (polysaccharide) > LRME (extract) > LGRP (black goji berry ginseng extract tablets).

[0080] Table 4. Effects of drugs on the number of resting times, duration, and distance in a zebrafish sleep deprivation model over 24 hours. n=12)

[0081]

[0082]

[0083] 5. Nissl staining observation of brain neuron morphology: Nissl staining results showed that the number of neuronal cells in the midbrain tissue of zebrafish in the blank control group was normal, with cells in the tectorial region concentrated in the central gray matter layer, Nissl bodies were abundant and significantly stained, and the nuclei were relatively intact. Compared with the blank control group, the number of neuronal cells in the midbrain of the model group was significantly reduced, the pericellular clefts were widened, Nissl bodies were significantly reduced, and the staining was lighter. After intervention with black goji berry polysaccharide, the cell bodies of zebrafish neurons were more plump, the loss was significantly improved, Nissl bodies were partially visible, and the staining was deepened, with the high-dose group showing better effects than the medium- and low-dose groups. Conclusion: Improvement effect: LRMP (polysaccharide) > LRME (extract) > LGRP (black goji berry ginseng extract tablets). The melatonin group also showed an increase in the number of midbrain neurons and a reduction in damage. Figure 6-8 ).

[0084] In conclusion, black goji berry polysaccharides have sleep-improving and sedative effects on sleep-deprived zebrafish, and can reduce the total duration of insomnia and total movement distance of zebrafish, while increasing the number of resting times, resting time and resting distance, thus having a significant effect on improving sleep.

[0085] 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. Application of black wolfberry polysaccharide in the preparation of drugs to improve sleep.

2. The application according to claim 1, characterized in that, Sleep-improving medications are those that target at least one of the following symptoms: a. A decrease in the number of brain nerve cells; b. Widening of the clefts surrounding brain nerve cells; c. Decrease in Nissl bodies.

3. The application according to claim 1 or 2, characterized in that, The preparation steps of the black goji berry polysaccharide include: water extraction of black goji berries to obtain crude black goji berry extract; alcohol precipitation of the crude black goji berry extract to enrich black goji berry polysaccharides to obtain crude polysaccharide extract; and purification of the crude polysaccharide extract to remove impurities.

4. The application according to claim 3, characterized in that, The black goji berries are extracted by water as follows: the dried black goji berries are crushed and then 5 to 30 times the volume of deionized water is added, and the mixture is extracted at 50℃ to 80℃ for 1 to 4 hours, and the extraction is repeated 2 to 5 times. The extracts are combined to obtain the crude extract of black goji berries; or the fresh black goji berries are crushed and juiced, and the residue is extracted by adding water each time, and the extraction is repeated 3 to 4 times. The filtrates obtained by juicing are combined to obtain the crude extract of black goji berries.

5. The application according to claim 3, characterized in that, The method for ethanol precipitation enrichment of black goji berry polysaccharides from the crude extract of black goji berries is as follows: the crude extract of black goji berries is added to 2 to 6 times its volume of 70% to 100% ethanol for ethanol precipitation enrichment to obtain crude polysaccharide extract.

6. The application according to claim 3, characterized in that, Purification of crude polysaccharide extract involves sequentially performing protein removal, decolorization, and dialysis.

7. The application according to claim 6, characterized in that, The protein removal process involves dissolving the crude polysaccharide extract in ultrapure water and adding Sevag reagent to remove the protein. And / or, decolorization is performed by using macroporous resin to decolorize the polysaccharides after protein removal; And / or, dialysis involves concentrating the decolorized polysaccharide solution under reduced pressure and then dialyzing it through a dialysis bag to obtain a polysaccharide solution with a molecular weight greater than 3500 Da.

8. The application according to claim 7, characterized in that, The macroporous resin is a non-polar macroporous adsorption resin, a weakly polar macroporous resin, or a polar macroporous resin. The non-polar macroporous adsorption resin is selected from D101, D101b, DA-201, DM-301, DM-11, HPD-100, HPD-450, HPD-750, D1400, D1300, D3520, D4006, D4020, HP-20, H-103, H107, X-5, ADS-5, ADS-8, BS-55, BS-65, BS-80, and NK. At least one of A-2 and NKA-9; the weakly polar macroporous resin is selected from at least one of AB-8, DM130, D860021, DS-401, BS-30, CAD-40 and CAD-45; the polar macroporous resin is selected from at least one of ADS-F8, ADS-21, ADS-7, ADS-17, BS-75, BS-45, S-8, NKA-2 and NKA-9; the elution solvent is ultrapure water, the amount of which is 3 to 5 times the resin volume, and the flow rate is 1 BV / h to 3 BV / h.

9. The application according to claim 6, characterized in that, The preparation steps of the black goji berry polysaccharide also include: concentrating the polysaccharide solution obtained after dialysis under reduced pressure, and then freeze-drying it.

10. A medicine or health food for improving sleep, characterized in that, Its active ingredient is black goji berry polysaccharide. The medicine is made by loading with a pharmaceutically acceptable carrier, and the health food is made by loading with a food science-acceptable carrier.

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