阅读说明：本技术 羟基酪醇醋酸酯和β羟丁酸乙酯的联合使用在制备缓解脑疲劳的保健食品和药物中的应用 (Application of joint use of hydroxytyrosol acetate and beta-hydroxybutyric acid ethyl ester in preparation of health-care food and medicine for relieving brain fatigue ) 是由 龙建纲 胡亚冲 王永耀 王珍 张豫霞 成俊宏 袁小锋 刘健康 于 2019-09-17 设计创作，主要内容包括：本发明公开了一种羟基酪醇醋酸酯和β羟丁酸乙酯的联合使用在制备缓解脑疲劳的保健食品和药物中的应用,本发明通过大鼠水迷宫实验和旷场实验验证了羟基酪醇醋酸酯和β羟丁酸乙酯的联合使用对于脑疲劳学习记忆的改善以及脑疲劳躁狂情绪的改善具有显著作用。(The invention discloses application of joint use of hydroxytyrosol acetate and beta-hydroxybutyric acid ethyl ester in preparation of health-care food and medicines for relieving brain fatigue.)

1. Application of hydroxytyrosol and its derivative esters in preparing health food and medicine for relieving brain fatigue, improving learning and memory ability and mania symptoms is provided.

2. Application of beta-hydroxybutyric acid and various ester compounds derived from the beta-hydroxybutyric acid in preparing health food and medicines for relieving brain fatigue, improving learning and memory ability and mania symptoms is provided.

3. The combined use of the hydroxytyrosol acetate and the beta-hydroxybutyric acid ethyl ester is applied to the preparation of health-care food and medicines for relieving brain fatigue, improving learning and memory abilities and manic symptoms.

4. A health food or a pharmaceutical composition for relieving brain fatigue, improving learning and memory ability and manic symptoms, which is characterized in that the health food or the pharmaceutical composition comprises hydroxytyrosol acetate and beta-hydroxybutyric acid ethyl ester.

5. The health food or pharmaceutical composition for alleviating brain fatigue and improving learning and memory ability and manic symptoms as set forth in claim 4, wherein the effective intake concentration of hydroxytyrosol acetate for adults is 5.6 mg per kg of body weight per day and 3.8 mg of beta-hydroxybutyric acid ethyl ester per kg of body weight per day.

Technical Field

The invention belongs to the technical field of biology and medicine and pharmacology, and relates to application of joint use of hydroxytyrosol acetate and beta-hydroxybutyric acid ethyl ester in preparation of health-care food and medicines for relieving brain fatigue.

Background

In a modern society where life style is becoming busy, more and more people are subjected to continuous work of high intensity for a long time or work in a high stress environment, etc., resulting in brain fatigue. Brain fatigue can impair memory acquisition and consolidation, lead to a decline in attention and alertness, and cause mental symptoms such as mania, depression, and the like. Research shows that sleep can regulate the neuron function of brain in the memory period, sleep deprivation can produce obvious negative effect on various functions of human body and animal body, especially cognition, memory, emotion and other aspects, and the establishment of brain fatigue model by adopting sleep deprivation method is a classic animal model for researching brain fatigue.

The research shows that Hydroxytyrosol (HT) has a molecular formula of C₈H₁₀O₃The relative molecular weight of the compound is 154.16, is a very effective mitochondrial nutrient, and has the effects of resisting inflammation, resisting oxidation and delaying neurodegenerative diseases. On one hand, HT contains rich hydroxyl groups, has certain reducibility, can react with excessive free radicals in cells, and improves DNA damage caused by oxidative stress, thereby achieving the effect of improving the functions of mitochondria; on the other hand, HT also activates mitochondrial biosynthesis, reducing damage by increasing healthy mitochondrial numbersThe ratio of mitochondria ensures the physiological function of cells. It is shown by research that HT protects mitochondria from oxidative damage caused by exercise fatigue. At present, no brain fatigue research report related to the medicine exists.

Beta-hydroxybutyric acid (beta-HB) is one of ketone bodies and has a molecular formula of C₄H₈O₃The molecular weight is 104, which is the main component of ketone body. When sugar supply is insufficient, the liver produces a large amount of ketone bodies to supply energy to peripheral tissues, and beta-HB accounts for about 70% of the total content of ketone bodies in vivo, so that beta-HB is generally considered as a main energy supply substance for the liver to export to peripheral tissues. Besides energy supply, the beta-HB can be used as endogenous bioactive small molecules, and plays an important role in protecting tissues and organs such as nerves, cardiovascular diseases and the like. Thus, β -HB can serve as an important energy-supplying substance for exogenous supplementation. At present, no brain fatigue research report related to the medicine exists.

Esterified form of hydroxytyrosol acetate (HTac) of formula C₁₀H₁₂O₄Molecular weight is 196.2. Esterified form of beta-hydroxybutyrate ethyl beta-Hydroxybutyrate (HBET) with molecular formula C₆H₁₂O₃And the molecular weight is 132. At present, no brain fatigue research report related to the medicine exists. The applicant studies found that these two esterified forms of the compound work significantly more in actual gavage experiments on animals. The applicant further found that the combination of the two has a synergistic effect and can improve the brain fatigue, particularly the learning and memory ability and the manic symptoms.

Disclosure of Invention

The invention aims to provide application of hydroxytyrosol and various ester compounds derived from hydroxytyrosol, beta-hydroxybutyric acid and various ester compounds derived from beta-hydroxybutyric acid or the combined use of hydroxytyrosol acetate (HTac) and beta-hydroxybutyric ethyl ester (HBET) in preparing health-care food and medicines for relieving brain fatigue, improving learning and memory capacity and improving mania and depression related to brain fatigue.

In order to achieve the purpose, the invention adopts the technical scheme that:

application of hydroxytyrosol and its derivative esters in preparing health food and medicine for relieving brain fatigue, improving learning and memory ability and mania symptoms is provided. The hydroxytyrosol and various ester compounds derived from the hydroxytyrosol have obvious effect of improving brain fatigue, can effectively improve learning and memory abilities, and relieve mania.

Application of beta-hydroxybutyric acid and various ester compounds derived from the beta-hydroxybutyric acid in preparing health food and medicines for relieving brain fatigue, improving learning and memory ability and mania symptoms is provided. The beta-hydroxybutyric acid and various ester compounds derived from the beta-hydroxybutyric acid have a remarkable improvement effect on brain fatigue, can effectively improve learning and memory abilities and relieve mania.

The combined use of the hydroxytyrosol acetate and the beta-hydroxybutyric acid ethyl ester is applied to the preparation of health-care food and medicines for relieving brain fatigue, improving learning and memory abilities and manic symptoms. The combined use of beta-hydroxy butyric acid ethyl ester and hydroxytyrosol acetate has a remarkable improvement effect on brain fatigue, can effectively improve learning and memory abilities, and relieves mania.

A health food or a pharmaceutical composition for relieving brain fatigue, improving learning and memory ability and mania symptoms comprises hydroxytyrosol acetate and beta-hydroxybutyrate.

Further, the effective intake concentration of hydroxytyrosol acetate for adults is 5.6 mg per kilogram of body weight per day, and the effective intake concentration of beta-ethyl hydroxybutyrate is 3.8 mg per kilogram of body weight per day. (the conversion ratio of the amount of the drug for rat to human is 1:0.16)

The invention has the beneficial effects that:

(1) the combined use of hydroxytyrosol and various ester compounds derived from hydroxytyrosol, beta-hydroxybutyric acid and various ester compounds derived from beta-hydroxybutyric acid or hydroxytyrosol acetate (HTac) and beta-hydroxybutyric acid ethyl ester (HBET) is found for the first time to relieve brain fatigue, improve learning and memory abilities and improve mania and depression mood related to brain fatigue;

(2) the hydroxytyrosol acetate and the beta-hydroxybutyric acid ethyl ester are jointly used, so that the synergistic effect is achieved on effectively improving the learning and memory capacity reduction caused by the brain fatigue, and meanwhile, the manic mood caused by the brain fatigue can be effectively improved;

(3) the main metabolite of the hydroxytyrosol acetate, namely hydroxytyrosol, is also a metabolite of dopamine circulation, the main metabolite of beta-ethyl hydroxybutyrate, namely beta-hydroxybutyric acid, is one of products of ketone body metabolism, and the two substances have good biocompatibility.

Drawings

FIG. 1 is a graph showing the effect of hydroxytyrosol acetate and beta-hydroxybutyrate on learning and memory improvement by separate use and combined use, wherein the abscissa is a grouping of ingested drugs and the ordinate is a quadrant moving path ratio of a rat water maze inspection test platform;

FIG. 2 is a graph showing the effect of hydroxytyrosol acetate and beta-hydroxybutyrate on learning and memory improvement, respectively and in combination, wherein the abscissa is the grouping of ingested drugs and the ordinate is the ratio of the water maze examination test platform quadrant swimming time of a rat;

FIG. 3 is a graph of the improvement in manic effect of hydroxytyrosol acetate and ethyl beta-hydroxybutyrate, respectively and in combination, in which the abscissa is the group of ingested drugs and the ordinate is the rate of movement of rats;

FIG. 4 is a graph showing the improvement effect on mania by the use of hydroxytyrosol acetate and ethyl beta-hydroxybutyrate, respectively and in combination, in which the abscissa is the group of ingested drugs and the ordinate is the number of movements of rats.

Detailed Description

The technical solutions of the present invention are further described below with reference to specific embodiments and drawings, but it should be understood that the scope of the present invention is not limited by the specific embodiments.

1. Experimental Material

Hydroxytyrosol acetate is available from Santa corporation, USA under CAS number 69039-02-7; beta-hydroxybutyrate is commercially available from Aladdin, USA under CAS number 24915-95-5.

2. Experimental animal feeding and model establishment

250g adult male SD rats aged 8 weeks were selected for this experiment and purchased from the animal center of Shanghai navy, military medical university. Rats were housed in an animal house with controlled temperature (22 ℃ to 28 ℃) and humidity (60%) and light was maintained in the house for a 12 hour day and 12 hour night cycle, with free access to food and water during the experiment. In the experiment, a rat brain fatigue model is established by adopting a sleep deprivation method. The experimental rats were divided into five groups of 10 rats each. The five groups are respectively: (1) normal feeding group is simultaneously perfused with normal saline every day (hereinafter referred to as control group); (2) a brain fatigue model established by a sleep deprivation method is simultaneously infused with normal saline (hereinafter referred to as brain fatigue group) every day (3) the brain fatigue is simultaneously infused with hydroxytyrosol acetate (hereinafter referred to as HTac group, shown in the following table) of 35mg/kg of stomach every day; (4) 23.6mg/kg of beta-hydroxybutyrate (hereinafter referred to as HBET group, shown in the following table) is perfused with the stomach every day while the brain is tired; (5) the patient suffering from brain fatigue was simultaneously gavaged with 35mg/kg of hydroxytyrosol acetate and 23.6mg/kg of beta-hydroxybutyric acid ethyl ester every day (hereinafter referred to as a combination group, as shown in the following table).

Animals are pre-adapted in an animal room for one week, the gastric lavage method is used for medicine intervention for one week during the pre-adaptation period, and then the rats except the control group are subjected to sleep deprivation in a small standing platform water environment method on the second day of acquired training in the water maze experiment to form a brain fatigue stress model. The deprivation device mainly comprises a deprivation box and a platform. The water is contained in the box, the size of the platform can only be used for the rat to stand on the back leg, the platform is connected with the bottom of the box, water is injected around the platform, the platform is higher than the water surface, after the animal is placed on the platform, the animal is frightened because the muscle tension is reduced when the animal is in a sleep state and the gravity center moves forwards to enable the animal to fall into the water, in order to avoid falling into the water, the animal must keep a state of slightly grabbing the edge of the platform, and therefore the aim of causing the brain fatigue of the rat through sleep.

3. Experimental methods

1) Water maze experiment

Description of the Water maze arrangement: the water maze consists of a large circular black water pool with a diameter of 120cm and a height of 50cm, and is divided into four quadrants with equal size on a monitor screen of a computer. Tap water was added to a depth of 30 cm. The water maze was placed in a laboratory at 150lx uniform brightness, 26 ℃. + -. 2 ℃ at room temperature, and contained various visual cues (A4 paper size black geometry). And (3) placing the escape platform with the diameter of 12cm under the water in the middle of the fourth quadrant, and adjusting the distance between the platform surface and the water surface to be 2 cm. Adding non-toxic odorless black dye into water and uniformly mixing to ensure that the platform can not be seen by the rats swimming on the water surface. The water was changed once a day. The water temperature is stable at 26 +/-2 ℃.

Acquired training of water maze experiment: the animal carries out the acquired training of water maze experiment to the rat the first day after animal room pre-adaptation a week, the acquired training of water maze experiment is carried out to the rat to the continuation of the second day, then sleep deprivation is carried out, the third day carries out water maze acquired training to the rat that is carrying out sleep deprivation, then sleep deprivation, the fourth day carries out water maze acquired training to the rat that is carrying out sleep deprivation, then sleep deprivation, the fifth day carries out water maze exploration experiment and open field experiment to the rat that is carrying out sleep deprivation. The drug intervention is administered continuously during the training period.

The operation method of the acquired training comprises the following steps: in the training trial, each rat was required to be trained in one, two, and three quadrants each day, in the order shown in the table below. At the start of the test for each quadrant, the animals were placed in the water so that they faced the side of the pool wall. Once the animal is in the water, allowing 120s to position the escape platform; if not found over 120s, the experimenter gently directs the animal to the platform. Once the animal is on the platform it is allowed to rest for 10s (if less than 10s needs to be redirected) and observe spatial cues on the platform. The time elapsed and distance traveled before the animal found the hidden platform was recorded using a video tracking system. Thereafter, the animals were removed from the water maze and, after training in the first quadrant of the rats was completed, the animals were released by changing the quadrant, during which time more than five minutes of rest was guaranteed for each rat to recover body temperature and physical strength. During the four-day training process, the sequence of the water-entering quadrants every day is ensured to be random.

Quadrant placing sequence of rat positioning navigation test

Water maze exploration experiment: after the sleep deprivation treatment for three days, immediately carrying out a water maze exploration experiment on the rat, taking away the platform, putting the rat into the fourth quadrant, and recording to observe the distance and time of the rat for 120s to swim through the fourth quadrant. During the period, due to the instinct of water avoidance and survival of the rat, the rat can search the escape platform according to the memory of the rat in the acquired experiment, and the escape platform is placed in the middle of the fourth quadrant in the acquired training.

Evaluation indexes are as follows: the proportion of time and distance traveled by the rats in the fourth quadrant. The higher the ratio, the better the learning and memory ability of the rat.

2) Open field experiment

Experimental apparatus: open field experiments are classical behavioral experiments for evaluating the mood of rats. Its principle is based on the natural fears that rodents fear an open field, and therefore spontaneous activity is evasive. The abnormality of avoidance behaviors can indicate the abnormality of the emotion of the rat. Open field experimental apparatus is mainly become by black PVC material open field case and video tracer two parts, and the length and width of open field case is 80cm, and height 50cm has hung video tracer apart from the bottom of the case 1.5 m. The rat can be fully freely moved in the open field case, and the video tracking device is just to the open field case for record rat's activity condition.

Evaluation indexes are as follows: moving speed, moving times. Emotional mania is a manifestation of brain fatigue, and experimental brain-fatigued rats show abnormal excitation, faster movement speed in open field and lower movement times (lower tendency to stop movement) compared to control rats.

3) Statistical analysis

The result is expressed in the form of Mean ± s.e.m, wherein blank group data is the statistical result of 15 rats, other 5 group data is the statistical result of 10 rats, and individual unreasonable behavioural data is removed by a 99.9 percent confidence interval in the statistical process; data analysis using One Way-ANOVA analysis method, significant statistical significance was p <0.05, p <0.01, p <0.001, p < 0.0001.

Example one

The joint use of the hydroxytyrosol acetate and the beta-hydroxybutyric acid ethyl ester has the effect of improving the learning and memory of rats with brain fatigue.

The improvement of the learning and memory ability of the combined drug on a rat brain fatigue model is detected by adopting a water maze experiment method. The indexes of the water maze examination experiment show the influence of brain fatigue caused by sleep deprivation on the learning and memory ability of rats and the improvement effect of medicaments from different angles. In the process of the water maze training experiment, the platform is placed under the water in the fourth quadrant of the water maze, and after the rat learns for four days, the rat is taken away from the underwater platform and is placed in the water pool for a period of time. The higher the proportion of time and distance that the rat dwells in the fourth quadrant indicates that the rat has better learning and memory. Fig. 1 and 2 show the residence time ratio and the swimming distance ratio of rats in the quadrant (i.e., the fourth quadrant) in which the platform was positioned during training in the water maze examination test in the control group, the brain fatigue group, the HTac group, the HBET group, and the combination group, respectively. The results show that in the fourth quadrant residence time index, the brain fatigue groups have significant difference compared with the control group; the combination group and the brain fatigue group have significant difference. On the fourth quadrant swimming distance index, the brain fatigue group has significant difference compared with the control group; the combination group and the brain fatigue group have significant difference. Compared with a control group, the learning and memory abilities of the brain fatigue model group are obviously reduced, the learning and memory abilities of the brain fatigue model rats are not obviously improved by singly using the two medicaments of the hydroxytyrosol acetate and the beta-hydroxybutyric acid ethyl ester, and the learning and memory abilities of the brain fatigue model rats are obviously improved after the two medicaments are jointly used.

Example two

The joint use of the hydroxytyrosol acetate and the beta-hydroxybutyric acid ethyl ester has the effect of improving the manic mood of rats with brain fatigue.

The improvement of the combined drug on the manic mood of the brain fatigue model rat is detected by adopting an open field experiment method. The manic mood is often accompanied by depression mood with different degrees, and the medicine improves the manic mood caused by brain fatigue, so that the medicine has the potential of improving the depression mood caused by the brain fatigue. Various indexes of an open field experiment show the influence of brain fatigue on the mania of rats and the improvement effect of the medicine from different angles. The rats freely move in a square open field with a certain area for a period of time, and some behavioral indexes can intuitively reflect the emotion of the rats. Fig. 3 and 4 show the mean exercise speed and the number of movements in the open field experiment of rats in the control group, brain fatigue group, HTac group, HBET group, and combination group. The faster a manic rat moves in an open field over time, the less the tendency to stop exercising, and the corresponding reduction in total movements. The results show that on the index of the average movement speed, the brain fatigue group has significant difference relative to the control group; the HTac group, HBET group and combination group were significantly different from the brain fatigue group. On the index of the moving times, the brain fatigue group has significant difference compared with a control group; the HTac group and the combined group have significant difference compared with the brain fatigue group. Brain fatigue caused by sleep deprivation can obviously enhance the mania of the rats compared with that of the control group, and the mania of the rats is obviously improved to different degrees after the two medicaments are used independently and jointly.

The above description is only a specific embodiment of the present invention, and not all embodiments, and any equivalent modifications of the technical solutions of the present invention, which are made by those skilled in the art through reading the present specification, are covered by the claims of the present invention.