Oral sustained-release preparation capable of improving intracerebral 5-HT level and application of oral sustained-release preparation in improvement of insomnia

Abstract

The invention discloses an oral preparation capable of improving the level of 5-HT in the brain and application of the oral preparation in improvement of insomnia, and belongs to the field of food science. According to the oral preparation, the starch and the beta-cyclodextrin are used as nanoparticle carriers to wrap the 5-HTP, the release rate of the 5-HTP can be effectively controlled, the 5-HTP is slowly released in gastrointestinal tracts, and the defect that the half-life period of the 5-HTP is short is overcome. The preparation can significantly increase the content of 5-HT in the brain and improve the metabolic dynamic characteristics of 5-HT, thereby effectively improving the insomnia symptom. Experimental data show that compared with the traditional 5-HTP, the sustained release preparation disclosed by the invention has better bioavailability and longer intracerebral drug effect duration. The innovative preparation not only has remarkable advantages in technology, but also opens up a new, safe and effective way for treatment of 5-HT related nervous system diseases, and has important application value and research significance.

Description

Technical Field

[0001] The invention relates to the preparation and functional application of a nanoparticle-embedded 5-HTP oral sustained-release preparation, and belongs to the field of food science. Background Art

[0002] With the accelerated pace of modern society and the increase of life pressure, insomnia has seriously affected people's quality of life and health. Insomnia not only causes daytime sleepiness and lack of concentration, but may also cause a series of psychological and physiological problems, such as depression, anxiety, cardiovascular disease, etc. 5-HT is an important neurotransmitter widely distributed in the central nervous system and peripheral nervous system. Studies have shown that 5-HT plays a key role in regulating emotions, sleep and appetite. Especially in terms of sleep regulation, 5-HT is closely related to the synthesis of melatonin, which can promote the cyclical change of rapid eye movement (REM) and non-rapid eye movement (NREM) sleep, thereby regulating sleep structure and sleep quality. Existing studies have shown that the reduction of 5-HT levels in the brain is closely related to the occurrence of insomnia, but peripheral 5-HT cannot pass through the blood-brain barrier into the brain.

[0003] Currently, drugs used to treat insomnia are mainly divided into two categories: one is traditional sleeping pills, such as benzodiazepines There are two types of drugs, which can induce sleep quickly, but long-term use can easily lead to drug resistance and dependence, and may also bring residual sedation effects the next day; the other type is selective 5-HT reuptake inhibitors (SSRIs), which improve mood and sleep by blocking the reuptake of 5-HT in the synaptic cleft and increasing the concentration of 5-HT in the synaptic cleft, but these drugs are only effective for one-third of patients. 5-HTP can be quickly converted into 5-HT after passing through the blood-brain barrier, increasing the level of 5-HT in the brain. Therefore, in comparison, directly supplementing 5-HT's precursor 5-HTP is considered to be a safer, faster-acting and more universal alternative. However, 5-HTP is metabolized rapidly in the body, and the peak plasma concentration is usually reached within 1-2 hours after oral administration, but then rapidly decreases, resulting in a shorter duration of action, which makes it less than ideal as a drug for the treatment of insomnia. When 5-HTP is co-administered with a peripheral amino acid decarboxylase inhibitor (DCI), the half-life can be doubled, but DCI still cannot cross the blood-brain barrier and enter the brain, and still acts on the periphery.

[0004] In order to better prolong the half-life of 5-HTP and improve sleep quality, it is urgent to develop related sustained-release preparations of 5-HTP. Summary of the invention

[0005] The present invention aims at the above-mentioned short half-life of 5-HTP in vivo and provides a 5-HTP nano oral sustained-release preparation prepared by combining starch and β-cyclodextrin, which has the advantages of high encapsulation rate, slow release, stabilization of 5-HT level in the brain, prevention and / or relief of sleep disorders, etc.

[0006] The first technical solution provided by the present invention is a method for preparing a 5-HTP oral sustained-release preparation, comprising the following steps:

[0007] S1, 5-HTP and corn starch are dissolved in water to form a mixed solution, and an ethanol solution is added dropwise to the mixed solution while stirring, followed by ultrasonication and rotary evaporation of ethanol to obtain a complex;

[0008] S2, adding β-cyclodextrin to the complex of step S1, stirring overnight, ultrasonicating, centrifuging, collecting the supernatant, and freeze-drying to obtain a 5-HTP oral sustained-release preparation modified with starch and β-cyclodextrin nanoparticles.

[0009] In one embodiment, in step S1, the mass ratio of 5-HTP to corn starch is 10:1 to 1:500, preferably, the mass ratio of 5-HTP to corn starch is 1:2.

[0010] In one embodiment, in step S1, in the mixed solution, the concentration of 5-HTP is 0.1-10 mg / mL, and the concentration of corn starch is 1-50 mg / mL; preferably, the concentration of 5-HTP is 5 mg / mL, and the concentration of corn starch is 10 mg / mL.

[0011] In one embodiment, in step S1, the concentration of the ethanol solution is 70-75%, and the volume ratio of the ethanol solution to the mixed solution is 1:2-2:1. Preferably, the volume ratio of the ethanol solution to the mixed solution is 1:1.

[0012] In one embodiment, in step S1, the dropping speed of the ethanol solution is 0.1-1 mL / min, and optionally, the dropping speed of the ethanol solution is 0.4 mL / min.

[0013] In one embodiment, in step S1, the ultrasonication time is 1 hour.

[0014] In one embodiment, in step S2, the mass ratio of β-cyclodextrin to corn starch is 1:10 to 10:1. Optionally, the mass ratio of β-cyclodextrin to corn starch is 1:1.

[0015] In one embodiment, in step S2, the parameters for stirring overnight are as follows: magnetic stirring at 700 rpm / min at 50°C overnight.

[0016] In one embodiment, in step S2, the ultrasonication time is 1 hour.

[0017] In one embodiment, in step S2, centrifugation is performed at 12000 g for 15 min.

[0018] The second technical solution provided by the present invention is a 5-HTP oral sustained-release preparation, wherein the sustained-release agent includes a nanocarrier, wherein the nanocarrier is embedded with 5-HTP, and the nanocarrier is composed of starch and β-cyclodextrin in a mass ratio of 1:10 to 10:1, and the embedding rate of the nanocarrier for 5-HTP is 84.58 to 94.27%, and the loading rate is 62.65 to 69.83%.

[0019] The third technical solution provided by the present invention is a product containing the 5-HTP oral sustained-release preparation described in the second technical solution.

[0020] In one embodiment, the product includes, but is not limited to, a medicine, a functional food, a fermented food, a nutritional supplement, a food additive, or a health product.

[0021] In one embodiment, the product is a food produced using a 5-HTP oral sustained-release preparation, and the food includes solid food, liquid food, and semi-solid food.

[0022] The fourth technical solution provided by the present invention is the use of the method described in the first technical solution, or the oral sustained-release preparation described in the second technical solution in the preparation of a product for preventing, improving and / or treating sleep disorders.

[0023] In one embodiment, the product includes, but is not limited to, a medicine, a functional food, a fermented food, a nutritional supplement, a food additive, or a health product.

[0024] In one embodiment, the product is a food produced using a 5-HTP oral sustained-release preparation, and the food includes solid food, liquid food, and semi-solid food.

[0025] In one embodiment, the sleep disorder includes but is not limited to abnormal sleep amount and abnormal behavior during sleep.

[0026] In one embodiment, the use further comprises preventing and / or alleviating behavioral abnormalities caused by sleep disorders.

[0027] In one embodiment, the application also includes regulating abnormal emotions, protecting cognitive and memory abilities, restoring the ability of central nervous system fibers to normally release receptors, etc.

[0028] The technical effects of the present invention are as follows:

[0029] The present invention provides a 5-HTP nano oral sustained-release preparation for preventing and / or alleviating sleep disorders prepared by combining starch and β-cyclodextrin. By encapsulating 5-HTP in a nanocarrier composed of starch and β-cyclodextrin, a high encapsulation rate and slow release are achieved, the 5-HT level in the brain is effectively increased, the 5-HT level is stably acted on the nervous system, and sleep disorders such as insomnia caused by insufficient 5-HT levels are prevented and / or alleviated. The sustained-release preparation achieves long-term release through an oral route, reduces the frequency of medication, and improves patient compliance. Starch and β-cyclodextrin are natural food materials with good biocompatibility to ensure that the preparation is safe and non-toxic. By stably increasing the 5-HT level in the body, the mood and sleep quality are significantly improved, providing a safe, effective, and non-dependent treatment option for insomnia patients. The present invention expands the application of 5-HTP, is of great significance to the research and development of functional foods and drugs, and opens up new ways and solutions for preventing and treating insomnia and related emotional disorders using dietary strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The 5-HTP oral sustained-release preparation modified with starch and β-cyclodextrin nanoparticles shows the changes in 5-HTP encapsulation capacity and functional groups as the mass ratio of starch to β-cyclodextrin decreases. (A) Encapsulation efficiency and loading rate; (B) Fourier transform infrared spectrum.

[0031] Figure 2 The 5-HTP oral sustained-release preparation modified with starch and β-cyclodextrin nanoparticles shows the change in particle size as the mass ratio of starch to β-cyclodextrin decreases. (A) Particle size; (B) Zeta potential.

[0032] Figure 3 This is the release experiment of 5-HTP oral sustained-release preparation modified by starch and β-cyclodextrin nanoparticles. (A) Release after simulated digestion by gastric juice; (B) Release after simulated digestion by intestinal juice; the 0 time point of intestinal juice digestion is calculated from 2 hours after gastric juice digestion.

[0033] Figure 4 Comparison of oral in vivo pharmacokinetics of 5-HTP sustained-release preparation and 5-HTP. (A) Serum 5-HTP concentration-time curve; (B) Area under the curve / maximum concentration / high concentration duration; The area under the curve is based on 0μg / L as the baseline, the maximum concentration refers to the highest concentration of 5-HTP in serum, and the high concentration refers to more than 500μg / L. ***P<0.001; ****P<0.0001.

[0034] Figure 5Comparison of the brain metabolic kinetics of 5-HTP sustained-release preparations and oral 5-HTP. (A) Changes of 5-HT content in the brain with time points; (B) Changes of NAS content in the brain with time points. Among them, NAS is a downstream metabolite of 5-HT.

[0035] Figure 6 The performance of mice in the behavioral experiment. (A) Sleep amount; (B) Exploration time of the central area of ​​the open field; (C) Difference score of novel object recognition. ****P<0.0001. DETAILED DESCRIPTION

[0036] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0037] Test method:

[0038] 1. Detection of embedding rate and loading rate: The free 5-HTP content in the supernatant of 5-HTP oral sustained-release preparations modified with starch and β-cyclodextrin nanoparticles was detected by HPLC. Take 1 mL of nanoparticle suspension, centrifuge, wash the precipitate three times with an equal volume of ultrapure water, collect all the supernatant and centrifuge at 12000g for 15 minutes, filter the supernatant through a 0.22μm filter membrane, add it to the liner tube, place it in a liquid phase vial and test it on the machine. The standard is diluted with the supernatant of empty nanoparticles. The calculation method of embedding rate and loading rate is as follows:

[0039]

[0040] Wherein: M1 represents the total amount of added 5-HTP (mg), M2 represents the amount of free (unencapsulated) 5-HTP (mg), and M3 represents the mass of the empty sustained-release preparation (mg).

[0041] 2. Fourier transform infrared spectroscopy: After the nanoparticle preparation was freeze-dried, the FTIR spectrum from 4000 cm-1 to 650 cm-1 was detected at room temperature using a Fourier transform infrared spectrometer.

[0042] 3. Nanoparticle size detection: 1 mL of the nanoparticle solution before centrifugation was aspirated, diluted 10 times with ultrapure water, and the particle size and Zeta potential were detected using a Zetasizer nano ZS instrument.

[0043] 4. In vitro release rate detection: Use simulated gastrointestinal fluid to detect the release rate of 5-HTP oral sustained-release preparations. Simulated gastric fluid (SGF, pH 3.0) and simulated intestinal fluid (SIF, pH 7.4) were prepared according to the INFOGEST consensus protocol. Weigh 1 mg of freeze-dried sample and add it to 10 mL of simulated gastrointestinal fluid, and continue to incubate at 37°C with shaking. The gastric and intestinal fluid environments are separated by 15 minutes and 30 minutes, respectively. Take 20 μL of sample solution, centrifuge at 12000g for 15 minutes, filter the supernatant through a 0.22 μm organic filter membrane, add the filtrate to the liner, and use HPLC to detect the 5-HTP content in the solution. The standard is diluted with the supernatant of the blank preparation digestion. The formula for calculating the in vitro release rate is as follows:

[0044]

[0045] 5. Serum 5-HTP content detection: Take 100μL serum sample, add 800μL methanol, vortex for 15s, and homogenize 3 times at 65Hz. Centrifuge the mixed sample at 15000g for 10min, take the supernatant and evaporate it in vacuum concentration for 2h, add 100μL 10% methanol solution, shake well, centrifuge at 15000g for 10min, transfer the supernatant to a liquid phase vial containing an inner liner tube, and wait for detection on the machine.

[0046] 6. Detection of 5-HT and its metabolite NAS content in the brain: Weigh a certain amount of sample, place it in an EP tube containing 3mm diameter zirconium beads, and add 900μL 50% methanol solution. Vortex and shake for 15s, set to 65Hz, homogenize 3 times, and place at -20℃ for 1h. Centrifuge at 15000g for 10min, take the supernatant and vacuum concentrate at 45℃ for 2-4h. Add 100μL 10% methanol solution to the precipitate, shake and resuspend, place at -20℃ for 2h, centrifuge at 15000g for 10min at 4℃, and transfer the supernatant to a liquid phase vial containing an inner liner for detection.

[0047] 7. HPLC parameter setting: Use Waters Atlantis T3 column (250mm×4.6mm, 5μm) for material separation, mobile phase A is 0.01mol / L PBS, use phosphoric acid to adjust pH to 4.0, mobile phase B is chromatographic grade acetonitrile. Gradient elution method is 5% B, 0-1min; 95% B, 1-12min; 95% B, 12-15min; 5% B, 15-16min; 5% B, 16-20min. The flow rate is 1mL / min, and the column temperature is set to 35℃. The injection volume is 10μL, and the fluorescence detector is used for detection, the excitation wavelength is 280nm, and the emission wavelength is 360nm. Use external standard method for quantification.

[0048] 8. LC-MS parameter setting: DIONEX UltiMate 3000 HPLC system was used in conjunction with Q-Exactive mass spectrometry, and the separation column was ACQUITY BEH C18 column (1.7μm, 100mm×2.10mm). The injection volume was 2μL, the mobile phase A was 0.1% formic acid water, and the mobile phase B was acetonitrile. The column temperature was 35℃, the sample chamber temperature was 4℃, and the flow rate was 0.3mL / min. The gradient elution method was 5% B, 0-3min; 30% B, 3-9min; 100% B, 9-12min; 100% B flush to 13.5min; 5% B, 13.5-15min. The mass spectrometry conditions were positive ion scanning mode, spray voltage 3500V, ion source temperature 320℃, and nitrogen as the protective gas and auxiliary gas for mass spectrometry.

[0049] The raw materials used in the embodiment:

[0050] 1. Corn starch (CAS: 9005-25-8) and β-cyclodextrin (CAS: 7585-39-9, molecular weight 1134.98, purity ≥ 98%) were both obtained from Shanghai Aladdin Reagent Co., Ltd.

[0051] 2. The rest of the reagents are from Sinopharm Group.

[0052] 3. Wistar rats were obtained from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., 8 weeks old, weighing ∼400 g.

[0053] 4. C57BL / 6J mice were obtained from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., 8 weeks old, weighing ∼25 g.

[0054] Example 1: Preparation of 5-HTP nanoparticle modified oral formulation

[0055] The preparation steps of the 5-HTP oral preparation modified with starch and β-cyclodextrin nanoparticles are as follows:

[0056] After 5-HTP and corn starch (w:w=1:2, 10 mg / mL) were dissolved in ultrapure water, 70-75% ethanol solution (v:v=1:1) was added dropwise at 0.4 mL / min, stirring was performed while adding, ultrasonication was performed for 1 hour, and ethanol was evaporated. β-cyclodextrin was added and magnetic stirring was performed at 700 rpm / min at 50°C overnight. Ultrasonication was performed for 1 hour, centrifugation was performed at 12000g for 15 minutes, the supernatant was removed, and freeze-dried to obtain a 5-HTP oral sustained-release preparation modified with starch and β-cyclodextrin nanoparticles.

[0057] Among them, different preparations were obtained according to seven different mass ratios of starch and β-cyclodextrin, namely 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, and 1:10. Single-factor process optimization experiments were carried out to study the effect of this ratio on the encapsulation efficiency, particle size, and chemical structure of nanoparticles.

[0058] (1) Determination of 5-HTP encapsulation capacity of nanoparticle-modified oral preparations

[0059] The encapsulation efficiency and loading efficiency of the 5-HTP nanoparticle modified oral preparation prepared in Example 1 were determined by high performance liquid chromatography (HPLC). Figure 1 As shown in (A), as the mass ratio of starch to β-cyclodextrin decreases, the embedding efficiency and loading rate show significant changes, and both reach the highest level when the ratio is 1:1. Further, the chemical bond formation between 5-HTP in the preparation and the carrier is observed by Fourier transform infrared spectroscopy (FTIR), as shown in Figure 1 (B), 3273cm -1 The strong absorption band near is caused by the stretching of -OH of starch, which promotes the intermolecular and intramolecular bonding of nanoparticles. The peak width indicates the degree of intermolecular and intramolecular hydrogen bond formation. At the same time, the stretching vibration of β-cyclodextrin on the double carbon conjugated system is at 1020cm -1 The results showed that with the change of the mass ratio of starch to β-cyclodextrin, the hydrogen bond absorption peak band migrated to varying degrees, further verifying that 5-HTP was more effectively encapsulated when the ratio was 1:1, and the migration degree of hydrogen bonds was more conducive to the formation of nanoformulations.

[0060] (2) Particle size analysis of nanoparticle-modified oral preparations

[0061] The particle size of the nanoparticle-modified oral sustained-release preparation prepared in Example 1 was measured using a laser particle size analyzer, and its surface charge was measured using a Zeta potential analyzer. Figure 2 As shown in (A) and (B), as the mass ratio of starch to β-cyclodextrin decreases, the particle size of the nanoparticles decreases first and then increases, reaching the minimum value when the mass ratio is 1:1; the Zeta potential shows that the surface of the nanoparticles is negatively charged, and its absolute value decreases first and then remains unchanged as the ratio decreases. This shows that the addition of β-cyclodextrin causes the nanoparticles to condense, and the agglomeration of the nanoparticles reaches an optimal level at 1:1. At the same time, due to the adsorption of more 5-HTP, its -OH increases the surface negative charge, which indicates that 5-HTP can be better encapsulated; when the mass is greater than corn starch, the particle size tends to increase due to its cavity structure, but the charge does not change significantly. Different carrier ratios have an important influence on the physicochemical properties of nanoparticles, which may affect the release characteristics and bioavailability of drugs.

[0062] Example 2: Nanoparticle-modified oral formulation can achieve slow release of 5-HTP

[0063] The 5-HTP nanoparticles modified oral sustained-release preparation with a starch to β-cyclodextrin mass ratio of 1:1 in Example 1 were subjected to in vitro release experiments in simulated gastrointestinal fluid, digested in a 37°C constant temperature shaker, and samples were taken at regular time intervals to determine the release of 5-HTP using HPLC. The results are shown in Figure 1. Figure 3 (A) and Figure 3 As shown in (B), the unencapsulated 5-HTP is released immediately in the gastric fluid environment, while the 5-HTP sustained-release preparation is pH-responsive, with a release rate of about 20% in the gastric environment after 2 hours, and a maximum release rate of about 40% after 6 hours, indicating that the nano-modification makes it resistant to gastric digestion. After digestion in intestinal fluid, 5-HTP can be completely released, and the release rate is slow. It can be completely digested after 8 hours, which significantly improves the stability and bioavailability of 5-HTP in the simulated gastrointestinal tract, indicating that the preparation may have a good sustained-release effect in vivo.

[0064] Furthermore, in vivo pharmacokinetic experiments were performed to compare the results. Twelve rats were divided into two groups and given regular 5-HTP and the 5-HTP sustained-release preparation prepared in Example 1 by gavage, respectively. Blood was collected from the tail vein at different time points, and serum samples were separated by centrifugation at 4000 g for 15 min. The concentration of 5-HTP in serum was determined by LC-MS. The results are shown in Table 1. Figure 4 As shown in (A) and (B), the 5-HTP in the sustained-release preparation group can maintain a high serum concentration for 10.0 to 11.7 hours, while the ordinary 5-HTP group can only maintain it for 1.9 to 2.8 hours. At the same time, its area under the curve is significantly higher than that of the ordinary 5-HTP preparation by 24 to 2.7 times, indicating that the 5-HTP sustained-release preparation has a good sustained release effect, which helps to maintain the long-term effect of 5-HTP and improve its short half-life defect. In addition, the maximum concentration is significantly lower than that of the ordinary 5-HTP group by 3 / 5 to 4 / 5 times, indicating that the 5-HTP sustained-release preparation can avoid the acute release of 5-HTP after ingestion and effectively prevent the occurrence of side effects such as serotonin syndrome.

[0065] Example 3: 5-HTP sustained-release preparation can improve brain metabolic dynamics

[0066] Thirty-six mice were divided into two groups and gavaged with ordinary 5-HTP and the 5-HTP sustained-release preparation prepared in Example 1 with a starch to β-cyclodextrin mass ratio of 1:1, respectively. Brain tissue samples were collected at six different time points, 0, 1, 2, 4, 6, and 10 h, and the contents of 5-HT and its metabolite NAS in the brain were determined by HPLC.

[0067] The results are as follows Figure 5 As shown in (A), the 5-HT content in the brain of mice in the ordinary 5-HTP group began to increase significantly 1 hour after oral administration, reached a peak at 2 hours, then gradually decreased, and returned to normal physiological levels at 4 hours; in contrast, the 5-HT content in the 5-HTP sustained-release preparation group showed a more stable change trend throughout the monitoring period, with the peak delayed to 4 hours, and remained at a high level at 6 hours and 10 hours, indicating that the sustained-release preparation can effectively prolong the existence time of 5-HT in the brain and improve its metabolic kinetic characteristics. In addition, NAS, as a downstream metabolite of 5-HT, its content changes were also monitored, and the experimental results are shown in Figure 2. Figure 5 As shown in (B), the trend of NAS content change is similar to that of 5-HT. The NAS in the 5-HTP sustained-release preparation group gradually increased in 2 hours and reached a peak at about 5 hours. Compared with the pure 5-HTP group, the metabolic rate of NAS was slower, and the high level could last until 10 hours. This shows that the sustained-release preparation can not only prolong the action time of 5-HT in the brain, but also have a positive effect on its metabolic process, thereby helping to improve related neurophysiological functions.

[0068] Example 4: 5-HTP sustained-release preparation can improve behavioral abnormalities in insomnia mice

[0069] 28 mice were selected to ensure that the mice were exposed to a 12h:12h day and night cycle. The animal groups and treatment methods are shown in Table 1.

[0070] Table 1 Animal experimental groups and treatment methods

[0071]

[0072] Insomnia model: After one week of adaptation, the mice were subjected to sleep deprivation stress for 20 hours every day for 15 days. The mice in the blank control group remained undisturbed. From the 16th day, the mice were subjected to behavioral tests, including sleep monitoring, open field test and novel object recognition test.

[0073] Sleep monitoring: The mice were subjected to sleep / wake analysis. The experimental mice were placed in cages with the same environment and the duration of sleep and wakefulness of the mice was recorded within 24 hours. The 12h:12h day and night alternation pattern was maintained within 24 hours. The experimental results are as follows Figure 6 (A) Sleep deprivation induced mice to stay awake for 190 minutes a day, accounting for about 87% of the total time. Oral administration of 5-HTP sustained-release preparations can make the mice sleep for 52% of the total time every day, effectively regulating the abnormal amount of sleep. At the same time, oral 5-HTP can only reach 22%, which is not effective in improving insomnia.

[0074] Open field test: Before the experiment, the experimental mice were acclimatized to the environment. The experimental mice were placed in the center of a 40 cm × 40 cm open field and explored for 6 min. The exploration time of the mice in the center area of ​​the open field during this period was recorded. Center area exploration time (%) = center area stay time / total time × 100. The experimental results are as follows Figure 6 (B) Sleep deprivation induced a significant decrease in the total time that mice spent in the central area, impaired their exploratory ability, and showed hyperactivity and anxiety-like behaviors. Intervention with 5-HTP sustained-release preparations can alleviate these emotional abnormalities and improve sleep quality, while 5-HTP intervention has no such effect.

[0075] New object recognition experiment: Place two different objects on the left and right ends of the side wall, place the mouse in the center of the box, and learn for 10 minutes. After 24 hours, one object is replaced by another new object, which is obviously different in shape from the old object). Then put the mouse into the box in the same way and test it for 10 minutes. Record the time the mouse explores the new object. Recognition difference score = (new object exploration time - familiar object exploration time) / (new object exploration time + familiar object exploration time). A positive difference score indicates that more time is spent exploring the new object; a negative value indicates that more time is spent exploring the old object; zero means that the same time is spent on both objects. The experimental results are as follows. Figure 6 As shown in (C), the cognitive memory ability of stressed mice for new objects was impaired, while taking a 5-HTP sustained-release preparation was able to reduce the damage to their cognitive memory and help recovery, while taking 5-HTP had no such effect.

[0076] In summary, the 5-HTP nanoparticle modified oral sustained-release preparation of the present invention has the following biological characteristics:

[0077] (1) Nanoparticle characteristics: The nanoparticles in this preparation are spherical, with a uniform particle size of about 200 nm and a negative surface charge, which helps to improve in vivo stability and bioavailability.

[0078] (2) Release characteristics: The preparation exhibits good sustained-release effect in simulated gastrointestinal fluid and can maintain effective drug concentration in the body for a long time.

[0079] (3) Pharmacokinetic properties: In vivo experiments showed that the 5-HTP in the sustained-release preparation exhibited a longer half-life in serum, and the area under the curve was significantly higher than that of ordinary 5-HTP preparations, indicating that it has a better sustained release effect, indicating that the preparation can improve the absorption and utilization of the drug.

[0080] (4) Regulation of 5-HT levels in the brain: In animal models, oral administration of this sustained-release preparation can significantly increase 5-HT levels in the brain and maintain a high-efficiency state for a longer period of time, thereby effectively improving insomnia symptoms.

[0081] (5) Neuroprotective effect: In insomnia model mice, the sustained-release preparation can significantly improve the sleep quality of mice and promote the recovery of normal function of the nervous system by increasing the level of 5-HT in the brain, thereby protecting neurons from stress damage.

[0082] (6) Biosafety: Mice that took the sustained-release preparation orally for a long time showed no obvious adverse reactions, indicating that it has good safety and tolerability.

[0083] (7) Anti-stress ability: In stress model mice, the sustained-release preparation can significantly alleviate the negative behavioral responses caused by stress and promote the improvement of the behavioral performance of mice.

[0084] (8) Pleiotropic synergistic effects: Through nanoparticle modification, 5-HTP forms a stable complex with the carrier, which enables the drug to exert pleiotropic synergistic effects in the body, thereby improving the efficacy of the drug and reducing side effects.

[0085] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing a 5-HTP oral sustained-release preparation, characterized in that: The following steps are involved: S1, 5-HTP and corn starch are dissolved in water to form a mixed solution, and an ethanol solution is added dropwise to the mixed solution while stirring, followed by ultrasonication and rotary evaporation of ethanol to obtain a complex; S2, adding β-cyclodextrin to the complex of step S1, stirring overnight, ultrasonicating, centrifuging, collecting the supernatant, and freeze-drying to obtain a 5-HTP oral sustained-release preparation modified with starch and β-cyclodextrin nanoparticles.

2. The method according to claim 1, characterized in that In step S1, the mass ratio of 5-HTP to corn starch is 10:1 to 1:500; In the mixed solution, the concentration of 5-HTP is 0.1-10 mg / mL, and the concentration of corn starch is 1-50 mg / mL.

3. The method according to claim 1 or 2, characterized in that: In step S1, the concentration of the ethanol solution is 70-75%, the volume ratio of the ethanol solution to the mixed solution is 1:2-2:1; and the dropping speed of the ethanol solution is 0.1-1 mL / min.

4. The method according to any one of claims 1 to 3, characterized in that: In step S2, the mass ratio of β-cyclodextrin to corn starch is 1:10 to 10:

1.

5. A 5-HTP oral sustained-release preparation, characterized in that: The sustained-release agent comprises a nanocarrier, wherein the nanocarrier is embedded with 5-HTP, and the nanocarrier is composed of corn starch and beta-cyclodextrin in a mass ratio of 1:10 to 10:1, and the embedding rate of the nanocarrier to 5-HTP is 84.58 to 94.27%, and the loading rate is 62.65 to 69.83%.

6. A product containing the 5-HTP oral sustained-release preparation according to claim 5.

7. The product according to claim 6, characterized in that The product is a medicine, functional food, fermented food, nutritional supplement, food additive or health product.

8. Use of the method according to any one of claims 1 to 4, or the oral sustained-release preparation according to claim 5, in the preparation of a product for preventing, improving and / or treating sleep disorders.

9. The use according to claim 8, characterized in that: The sleep disorder includes but is not limited to abnormal sleep amount and abnormal behavior during sleep.

10. The use according to claim 8, characterized in that: The applications also include: (1) preventing and / or alleviating behavioral abnormalities caused by sleep disorders; (2) Regulate emotional abnormalities caused by sleep disorders, protect the cognitive and memory abilities of individuals with sleep disorders, and restore the ability of central nervous system fibers to release receptors normally in individuals with sleep disorders.

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