Composition, medicine and preparation, detection, positioning method and use thereof

Abstract

The present invention discloses a composition, a drug, and a preparation, detection, positioning method and use thereof, and relates to the technical field of biomedicine research and development. The composition comprises phloretin and taurine. The present invention prepares the drug by adding a medically acceptable auxiliary material to the composition, and combines phloretin with taurine for the first time. It is unexpectedly found that the phloretin and taurine compound has a significant improvement effect on cognitive function and learning and memory disorders induced by sleep deprivation, which is beneficial to improving the physical condition of personnel undergoing high-intensity continuous operations.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical research and development, and more specifically to a composition, a drug, and methods and uses thereof for preparing, detecting, and locating. Background Art

[0002] Insufficient sleep has become a major global problem, adversely affecting the lives of millions of people around the world. Sleep deprivation (SD) refers to insufficient sleep in individuals, leading to various physiological, psychological and behavioral abnormalities, including cognitive impairment. Patients with insufficient sleep are prone to daytime inattention, impaired learning and memory functions, and reduced work and social participation. For individuals who inevitably encounter SD problems (such as medical staff or military personnel responding to epidemics in emergency situations), the judicious and moderate use of certain psychostimulants may be appropriate and reasonable. However, emerging evidence suggests that there is a potential association between these drugs and cognitive deficits, addiction, insomnia or neurosis caused by psychostimulants. Therefore, it is necessary to develop a safe, efficient and convenient compound preparation to effectively intervene in cognitive impairment after sleep deprivation.

[0003] Neuroinflammation plays a key role in the development and progression of cognitive impairment. The chronic neuroinflammatory state maintained by a variety of proinflammatory factors such as tumor necrosis factor-α (TNF-α) has an adverse effect on neurogenesis and spatial memory. Interleukin-1β (IL-1β) and TNF-α have an inhibitory effect on memory formation and impair learning and memory functions. In addition, the release of inflammatory factors can adversely affect synaptic plasticity and hinder the expression or activity of the glutamate system, leading to impaired cognitive functions related to learning and memory. Existing evidence shows that SD can cause changes in immune responses, affect the levels of proinflammatory and anti-inflammatory molecules, and thus lead to cognitive impairment.

[0004] Phloretin (PHL), also known as trihydroxyphenol acetone, belongs to the flavonoid compound. It is a natural active substance found in fruits such as apples and pears and in the juice of various vegetables. It can protect plants from ultraviolet damage. Phloretin has a wide distribution range and is named after it is found in the root bark of fruit trees such as apples. It has been discovered and isolated for more than 100 years. Previous studies have shown that phloretin can effectively penetrate the blood-brain barrier. Phloretin has multiple pharmacological activities such as antioxidant, anti-inflammatory, anti-cancer, anti-apoptosis and neuroprotection. In addition to the above biological activities, phloretin can also act as a positive allosteric modulator of α-7 nicotinic acetylcholine receptors, indicating that it can enhance cognitive function through the cholinergic system. Therefore, phloretin has the potential to be a natural therapy for improving cognitive impairment caused by sleep deprivation.

[0005] Taurine (TAU), also known as β-aminoethanesulfonic acid, exists in mammals. Taurine is a sulfur-containing non-protein amino acid that exists in the body in a free state. It has multiple physiological effects such as analgesia, anti-inflammatory, promoting fat metabolism, protecting heart health, enhancing immunity and improving cognitive function. Previous studies have found that taurine can activate GABA and glycine receptors, inhibit the excitotoxicity of glutamate on neurons, and thus play a neuroprotective role. Taurine is an endogenous amino acid in the human body and is expected to become an effective drug for treating cognitive impairment caused by sleep deprivation.

[0006] However, the effectiveness of phloretin and taurine in treating cognitive impairment caused by sleep deprivation needs to be improved. Summary of the invention

[0007] In view of this, the present invention is proposed.

[0008] The first object of the present invention is to provide a composition.

[0009] The second object of the present invention is to provide a medicine prepared from the above composition.

[0010] The third object of the present invention is to provide a method for preparing the above-mentioned medicine.

[0011] The fourth object of the present invention is to provide a method for detecting the above-mentioned drug.

[0012] The fifth object of the present invention is to provide a method for locating the above-mentioned drug target.

[0013] The sixth object of the present invention is to provide the use of the above-mentioned medicine.

[0014] In order to achieve the above object, the present invention adopts the following technical solution:

[0015] The first aspect of the present invention provides a composition comprising any one of the following 1) to 4):

[0016] 1) Taurine and phloretin;

[0017] 2) Taurine derivatives and phloretin;

[0018] 3) taurine derivatives and phloretin derivatives;

[0019] 4) Taurine and phloretin derivatives;

[0020] The composition is used for treating cognitive impairment caused by sleep deprivation.

[0021] In a preferred embodiment, the structural formula of phloretin is shown in Formula I; the structural formula of taurine is shown in Formula II;

[0022]

[0023] In a preferred embodiment,

[0024] The mass ratio of phloretin to taurine is 1:20; or,

[0025] The mass ratio of phloretin to the derivative of taurine is 1:20; or,

[0026] The mass ratio of the phloretin derivative to the taurine derivative is 1:20; or,

[0027] The mass ratio of the phloretin derivative to taurine is 1:20.

[0028] The second aspect of the present invention is to provide a medicine comprising the above-mentioned composition.

[0029] In a preferred embodiment, phloretin and taurine are packaged separately or mixed; or, phloretin and taurine derivatives are packaged separately or mixed; or, phloretin derivatives and taurine derivatives are packaged separately or mixed; or, phloretin derivatives and taurine are packaged separately or mixed.

[0030] In a more preferred embodiment, phloretin / phloretin derivatives and taurine / taurine derivatives are each diluted with a solvent to prepare an injection solution and packaged separately, or phloretin and taurine are mixed and diluted with a solvent, or phloretin and a taurine derivative are mixed and diluted with a solvent, or a phloretin derivative and a taurine derivative are mixed and diluted with a solvent, or a phloretin derivative and taurine are mixed and diluted with a solvent.

[0031] In a more preferred embodiment, the solvent is physiological saline.

[0032] In a preferred embodiment, the drug is a packaged product, which contains instructions for use of the drug or has the dosage of the drug recorded on the outer surface of the drug package.

[0033] The dosage of phloretin / phloretin derivatives and taurine / taurine derivatives in the medicine is 2.5 mg / kg of phloretin / phloretin derivatives: 50 mg / kg of taurine / taurine derivatives or more.

[0034] In a more preferred embodiment, the dosage is from (phloretin / phloretin derivative 2.5 mg / kg: taurine / taurine derivative 50 mg / kg) to (phloretin / phloretin derivative 5 mg / kg: taurine / taurine derivative 100 mg / kg).

[0035] In a more preferred embodiment, the dosage is 2.5 mg / kg of phloretin: 50 mg / kg of taurine. More preferably, the drug is in the form of tablets, pills, pellets, capsules, granules, powders, solutions, sprays, drinks, candies or jelly.

[0036] In a preferred embodiment, the drug further comprises an auxiliary material; wherein the auxiliary material comprises one or any combination of a carrier, a diluent, an excipient, a disintegrant or a dispersant.

[0037] In a more preferred embodiment, the disintegrant comprises sodium carboxymethyl cellulose, sodium starch glycolate, and pregelatinized starch.

[0038] In a more preferred embodiment, the filler includes any one of lactose, starch, and microcrystalline cellulose.

[0039] In a more preferred embodiment, the auxiliary material further includes an antioxidant, wherein the antioxidant can be sodium sulfite, sodium bisulfite, or sodium pyrosulfite.

[0040] The third aspect of the present invention is to provide a method for preparing the drug using two substances in any one of 1) to 4) as active ingredients.

[0041] In a preferred embodiment, the preparation method can be that taurine / taurine derivatives are mixed with phloretin / phloretin derivatives, and then auxiliary materials are added to prepare the drug.

[0042] The fourth aspect of the present invention is to provide a method for detecting the drug prepared by the method.

[0043] In a preferred embodiment, the detection method can be the detection of the content of taurine / taurine derivatives and phloretin / phloretin derivatives in the drug, or the detection of the drug effect and activity.

[0044] The fifth aspect of the present invention is to provide a method for locating the drug target prepared by the method.

[0045] In a preferred embodiment, the method for localizing the target can be a method for localizing the target based on taurine / taurine derivatives, phloretin / phloretin derivatives by network pharmacology analysis.

[0046] The sixth aspect of the present invention is to provide the use of the composition and the medicine for preparing a medicine for treating cognitive impairment caused by sleep deprivation.

[0047] In a preferred embodiment, the two components in the composition are used in combination to synergistically reverse cognitive impairment in sleep deprived mice.

[0048] In a preferred embodiment, the composition improves novel object recognition ability in sleep-deprived mice.

[0049] In a preferred embodiment, the composition improves memory performance in sleep-deprived mice.

[0050] In a preferred embodiment, the composition improves cognitive performance in sleep-deprived mice.

[0051] In a preferred embodiment, the composition improves the spatial learning and memory ability of sleep-deprived mice. In a preferred embodiment, the composition reverses the phenomenon of decreased percentage of time in the target quadrant and decreased number of platform crossings in sleep-deprived mice after a water maze test.

[0052] In a preferred embodiment, the composition reduces the expression level of pro-inflammatory factors in the hippocampus tissue of sleep deprived mice.

[0053] In a more preferred embodiment, the pro-inflammatory factors are Tnf-α, Il-6, Nos2 and Il-1β.

[0054] Through the above technical scheme, it can be known that compared with the prior art, the present invention discovers for the first time that the compound of phloretin and taurine can significantly improve the learning and memory function and inflammatory response of sleep-deprived mice, and the effect is better than that of the single-use group of phloretin and taurine, indicating that the two have a certain synergistic effect. Therefore, it is unexpectedly found that the compound of phloretin and taurine has a significant improvement effect on cognitive function and learning and memory disorders induced by sleep deprivation, which is beneficial to improving the physical condition of people undergoing high-intensity continuous operations. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0056] Attached Figure 1 To investigate the effects of different doses of phloretin and taurine alone and in combination on learning and memory;

[0057] Attached Figure 2 The effect of the optimal combination of phloretin and taurine on learning and memory in SD mice in the process of novel object recognition;

[0058] Attached Figure 3 To investigate the effects of phloretin and taurine compound on spatial learning and memory of SD mice in Morris water maze;

[0059] Attached Figure 4 This is the effect of phloretin and taurine compound on inflammatory factors in the hippocampal tissue of SD mice.

[0060] In the above figures, the data are expressed as mean ± standard error, and compared with the SD control group, *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0061] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0062] The structural formula of phloretin used in the article is shown in Formula I; the structural formula of taurine is shown in Formula II;

[0063]

[0064] Experimental animals used in the embodiment: SPF male C57BL / 6J mice (8-9 weeks old, 4 per cage) were kept at 22±2°C, with free access to food and water, and a 12h light / dark cycle (lights on at 8 am). All experiments were conducted in accordance with the requirements of the China Animal Protection Association and in accordance with professional and ethical standards. Mouse behavioral experiments were only performed during the light period. Before the behavioral experiments, the mice were familiar with the acclimatization environment for 1 week to reduce the stress associated with the operation.

[0065] All data in the embodiment were analyzed using GraphPadPrism8.2 (GraphPadSoftware) and expressed as mean ± standard error (Mean ± SEM). Two-tailed t-test or Bonferroni-corrected multiple t-test (single or two-way ANOVA, Bonferroni test was used for comparison of more than two groups) were used for comparison between the two groups. Repeated measures ANOVA was used to analyze individual data at different times. p < 0.05 was considered significant, and significance was set at 95% confidence interval.

[0066] Example 1 Preparation of sleep deprivation mouse model and drug administration method

[0067] In this example, a composition of phloretin and taurine was prepared, diluted with physiological saline, and injected into mice.

[0068] S1. Establishment of sleep deprivation model mice

[0069] A sleep deprivation model was induced in a water tank using a modified multi-platform method for 72 hours. In short, mice in the SD group were placed on a small platform (3 cm in diameter) surrounded by water, and were allowed to move freely between platforms. If they entered the sleep period, they would fall into the water due to the relaxation of muscle tension, and the mice could only regain their spirits and climb onto the platform, thus achieving the effect of sleep deprivation. Mice in the control group were placed on a platform with a diameter of 10 cm. A 12-hour light / dark cycle was given in the mouse activity space, and SD started in the dark period. Food pellets and water were freely available in each group.

[0070] S2. Drug sources and administration methods

[0071] Phloretin (Cat. No. P7912) and taurine (Cat. No. T0625) were purchased from SigmaAldrich. After the mice were adapted for 1 week, each group was intraperitoneally injected with drugs, including phloretin group (2.5 mg / kg, 5 mg / kg, 10 mg / kg, ip), taurine group (50 mg / kg, 100 mg / kg, 200 mg / kg, ip), compound group (26.25 mg / kg, 52.5 mg / kg, 105 mg / kg, the mass ratio of phloretin to taurine was 1:20, ip), and the control group was intraperitoneally injected with an equal amount of saline once a day for 3 days of pre-administration; then sleep deprivation was performed for 72 hours, and during the SD period, drugs were administered once a day (at the same time in the morning) for 3 consecutive days. Behavioral experiments were carried out 72 hours later, and SD modeling and drug administration were performed simultaneously during behavioral testing.

[0072] Example 2 Determining the most appropriate dosage

[0073] The mice were randomly divided into 11 groups, with 10 mice in each group: normal sleep + saline group (normal sleep group), SD + saline group (SD control group), SD + phloretin 2.5 mg / kg, SD + phloretin 5 mg / kg (phloretin group), SD + phloretin 10 mg / kg, SD + taurine 50 mg / kg, SD + taurine 100 mg / kg (taurine group), SD + taurine 200 mg / kg, SD + compound 26.25 mg / kg (phloretin 1.25 mg / kg + taurine 25 mg / kg), SD + compound 52.5 mg / kg (compound group, phloretin 2.5 mg / kg + taurine 50 mg / kg), SD + compound 105 mg / kg (phloretin 5 mg / kg + taurine 100 mg / kg).

[0074] Behavioral testing methods

[0075] S1. Novel object recognition test (NOR)

[0076] The novel object recognition test is a commonly used behavioral test used to study learning and memory in mice under various environments. The NOR test has three phases: habituation, learning, and testing. In the habituation phase, mice were placed in a 40×40×35 cm (length×width×height) box, where they explored freely for 5 min to adapt to the test environment. During the learning process, mice explored two identical objects (O), and the time spent exploring each object was recorded during a 5-min observation period. On the test day, a new training object (N) was introduced to replace the old training object, and the analysis was performed as before. Between each experimental mouse, the apparatus was thoroughly cleaned with 75% ethanol. Exploration was defined as walking within 2-3 cm of the object while sniffing or scanning the object's tentacles, and the time spent sitting on the object was not counted. In rodents, novelty is innate, so rodents that remember familiar objects will spend more time exploring new objects. Memory was evaluated by calculating the percentage of time spent exploring new objects to the total exploration time (discrimination index) for each group.

[0077] S2. Morris water maze test

[0078] The water maze consists of a circular water tank The maze is actually divided into four quadrants, one of which contains a hidden platform. Located 1 cm below the water surface. Mice were trained to locate the platform by utilizing three asymmetrically positioned external surprise cues as spatial references. During the acquisition phase, animals were introduced into the water in a quasi-random manner to prevent the acquisition of strategy learning. Mice were given a 90-s period to locate the platform; if the mouse failed to find the platform within 90 s, it was gently guided to the platform and remained there for 30 s before the subsequent trial began. After completing four trials, the mice were thoroughly dried and subsequently returned to their respective home cages. Mice received four training sessions per day for six consecutive days. Escape latency was quantified as the time required for the subject to successfully locate the hidden platform within the target quadrant Q1. Spatial exploration experiment: 24 h after the last training session (day 6), the platform was removed and the mice were allowed to swim for 90 s. The landing position was located at the border between the third and fourth quadrants, and the mouse initially faced the wall at the beginning. Data are presented as the percentage of time spent in quadrant Q1, the number of platform crossings, and the swimming speed. Swimming behavior was recorded using a video camera, and the video signal was analyzed using ethvisionXT video tracking software (Noldus Information Technology).

[0079] In the above experiment, the dosages of phloretin and taurine used alone and in combination are shown in Table 1.

[0080] Table 1

[0081]

[0082] The results are as follows Figure 1 As shown in the figure, the novel object recognition behavioral experiment showed that phloretin, taurine, and the compound were all dose-dependent. As the dose increased, the discrimination index percentage of each group increased significantly, and the discrimination index percentage of the compound was significantly higher than that of the phloretin alone group and the taurine alone group. At the same time, the platform quadrant residence time and crossing times of the water maze experiment also verified this conclusion. This shows that the phloretin and taurine compound has a better effect on improving the memory and cognitive ability of SD mice, indicating that phloretin and taurine have a synergistic effect. The optimal dose of the phloretin and taurine compound is 52.5 mg / kg, and the mass ratio of phloretin to taurine is 1:20, that is, phloretin 2.5 mg / kg + taurine 50 mg / kg.

[0083] Example 3 Effects of Phloretin and Taurine Compound on Learning and Memory in New Object Recognition in Sleep Deprived Mice

[0084] From the above experiments, it was determined that the optimal dose of phloretin alone was 5 mg / kg, the optimal dose of taurine alone was 100 mg / kg, and the optimal dose of phloretin and taurine compound was 52.5 mg / kg (phloretin 2.5 mg / kg + taurine 50 mg / kg). Subsequent experiments were all conducted using the optimal dose. The above experimental results show that the practical dose of phloretin and taurine when used in combination can be significantly less than the dose used alone, which is beneficial to reduce the side effects of the drug and enhance the safety of the drug. The experimental process is the same as S1 in Example 2;

[0085] Open field test process:

[0086] The mice were placed in a 40×40×30 cm (length×width×height) box with a bright light source emitting 650 lux in the center of the box, specifically targeting the central area. The mice were allowed to explore freely for 30 min. The total distance traveled by the mice during the whole process was recorded and analyzed using ethvisionXT video tracking software to evaluate the mice's motor ability.

[0087] The results are as follows Figure 2 As shown in the open field test data, there was no difference in the movement distance of each group, indicating that sleep deprivation and drugs had no significant effect on the motor ability of mice. The novel object recognition behavioral experiment showed that compared with the SD control group, the discrimination index percentages of the other groups increased significantly, among which the discrimination index percentage of the compound group was significantly higher than that of the phloretin group and the taurine group, indicating that the phloretin and taurine compound has a better effect on improving the memory and cognitive ability of SD mice.

[0088] Example 4 Effects of phloretin and taurine compound on spatial learning and memory in Morris water maze of sleep-deprived mice.

[0089] The experiment was carried out using the optimal dosage, and the experimental process was the same as S2 in Example 2;

[0090] The results are as follows Figure 3 As shown in Figure 2, during the Morris water maze training, compared with the SD control group, the latency of the mice in the other groups to reach the platform was significantly shortened, and the latency of the compound group was significantly lower than that of the phloretin group and the taurine group ( Figure 3 a), indicating that the compound of phloretin and taurine improves the spatial learning of SD mice. In the spatial exploration test phase, the percentage of time that the SD control group mice spent in the target quadrant ( Figure 3 b) and the number of times the platform is crossed ( Figure 3 c) decreased, while the phloretin group, taurine group and compound group significantly reversed this phenomenon, and the compound group mice performed even better than the phloretin group and taurine group. These results show that the phloretin and taurine compound can significantly improve the spatial learning and memory ability of SD mice.

[0091] Example 5 Effect of phloretin and taurine compound on inflammatory factors in hippocampal tissue of SD mice

[0092] After the behavioral experiment, the mice were anesthetized with sodium pentobarbital (75 mg / kg), and the hippocampal tissue was removed on ice and quickly frozen in liquid nitrogen. RNA was extracted from the tissue using the RNAisoPlus (Takara, Japan) kit according to the instructions. The total RNA was quantified using a spectrophotometer and then reverse transcribed into complementary DNA using the Prime ScriptTMRT kit (Takara, Japan). Real-time quantitative PCR was performed using the SYBR PremixExTaqTM (Takara, Japan) kit on a 7500 real-time fluorescence quantitative PCR instrument (ThermoFisherScientific) to detect the mRNA levels of inflammatory factors (Tnf-α, Il-6, Nos2, and Il-1β). The samples were tested in triplicate. The primer sequences are shown in Table 2, where the ribosomal protein Rps18 gene was used as a housekeeping reference. 2 -ΔΔCt Methods The relative expression level of each amplicon was determined.

[0093] Table 2 Primer sequences

[0094]

[0095] Sleep deprivation aggravates neuroinflammation in the central nervous system through hippocampal damage or central neurotransmitters, thereby impairing cognitive function and memory ability. Figure 4As shown, the expression levels of pro-inflammatory factors (Tnf-α, Il-6, Nos2 and Il-1β) in the hippocampus of mice in the SD control group were significantly increased, while the phloretin group, taurine group and compound group significantly reduced the expression of pro-inflammatory factors. The mice in the compound group performed better than the phloretin group and taurine group, and even recovered to the level of the normal sleep group. These results indicate that the phloretin and taurine compound can antagonize SD-induced neuroinflammation, thereby reducing its effects on cognition and memory.

[0096] Each embodiment of this specification focuses on the differences from other embodiments, and the same or similar parts between the embodiments can be referenced to each other.

[0097] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of a combination of phloretin and taurine as an effective ingredient in the preparation of a composition or a drug for treating and / or preventing cognitive impairment caused by sleep deprivation; wherein: The mass ratio of phloretin to taurine is 1:

20.

2. The use according to claim 1, characterized in that: The structural formula of the phloretin is shown in Formula I; the structural formula of taurine is shown in Formula II; ； I; II.

3. The use according to claim 1, characterized in that: The phloretin and the taurine are packaged separately or mixedly.

4. The use according to claim 1, characterized in that: The drug is a packaged product, which contains instructions for use of the drug or has the dosage of the drug recorded on the outer surface of the drug package; The dosage of the phloretin and the taurine in the medicine is 2.5 mg / kg or more of phloretin and 50 mg / kg or more of taurine.

5. The use according to claim 4, characterized in that: The dosage is phloretin 2.5mg / kg ~5mg / kg: taurine 50 mg / kg ~100 mg / kg.

6. The use according to claim 5, characterized in that: The dosage is phloretin 2.5 mg / kg: taurine 50 mg / kg.

7. The use according to claim 1, characterized in that: The phloretin and the taurine act synergistically.

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