阅读说明：本技术 化合物3-羟基-3’,4’-二羟基-丁酸苯乙酯的制备方法和用途 (Preparation method and application of compound 3-hydroxy-3 ', 4' -dihydroxy-butyric acid phenethyl ester ) 是由 龙建纲 胡亚冲 王永耀 王珍 马庆庆 张豫霞 蒋庆林 许小红 张婷华 刘健康 于 2019-09-17 设计创作，主要内容包括：本发明公开了一种式Ⅰ所示的化合物3-羟基-3’,4’-二羟基-丁酸苯乙酯的制备方法和用途,其中具体的,公开了一种化合物,其结构如式Ⅰ所示；公开了式Ⅰ所示化合物、化合物光学纯的异构体、对映异构体任意比例混合物或其药学上可接受的盐在制备缓解脑疲劳、改善学习记忆能力及躁狂症状的保健食品和药物中的应用。(The invention discloses a preparation method and application of a compound 3-hydroxy-3 ', 4' -dihydroxy-butyric acid phenethyl ester shown in a formula I, wherein the structure of the compound is shown in the formula I; discloses the application of a compound shown as a formula I, optically pure isomers of the compound, mixtures of enantiomers in any proportion or pharmaceutically acceptable salts thereof in preparing health-care foods and medicines for relieving brain fatigue, improving learning and memory abilities and manic symptoms.)

1. A compound of formula i having the formula:

2. a process for the preparation of a compound of formula i as claimed in claim 1, comprising the steps of:

s1 synthesis of beta-benzyloxy butyric acid

Weighing crotonic acid in a reaction vessel, adding benzyl alcohol, adding mercuric acetate, and stirring at normal temperature overnight; cooling the reaction container in a low-temperature condensation tank to 0 deg.C, adding 3N sodium hydroxide within 5-10min, adding 0.5M sodium borohydride 3N sodium hydroxide aqueous solution, and maintaining the solution at 0 deg.C for 3-10 min; taking out the reaction vessel, stirring at room temperature for 1-2h, filtering to obtain filtrate, and extracting with diethyl ether for 3-4 times to remove excessive benzyl alcohol; acidifying the water layer with 10% hydrochloric acid until the pH value is 2, separating out a large amount of white solid, and filtering to obtain beta-benzyloxy butyric acid;

synthesis of S2 and 3, 4-dibenzyloxy phenethyl alcohol

Weighing 3, 4-dihydroxy phenethyl alcohol and potassium carbonate in a reaction container, adding anhydrous acetone for dissolving, adding benzyl bromide, stirring and refluxing for 4-5h at 70 ℃, detecting by TLC (thin layer chromatography) for complete reaction, filtering out potassium carbonate, concentrating, mixing samples, and carrying out column chromatography to obtain 3, 4-dibenzyloxy phenethyl alcohol;

synthesis of S3, 3-benzyloxy-3 ', 4' -dibenzyloxy-phenethylbutyrate

Weighing beta-benzyloxy butyric acid prepared by S1, adding THF (tetrahydrofuran) for dissolving, adding 3, 4-dibenzyloxy phenethyl alcohol prepared by S2, EDCI and DMAP (dimethyl formamide) and stirring in an oil bath at 30 ℃ for reacting for 3-4h, detecting by TLC (thin layer chromatography), and stopping the reaction when the 3-benzyloxy butyric acid disappears; concentrating, dissolving and washing with EA for 2-3 times, concentrating the obtained solution, mixing with sample, and performing column chromatography to obtain 3-benzyloxy-3 ', 4' -dibenzyloxy-phenethylbutyrate;

synthesis I of S4, 3-hydroxy-3 ', 4' -dihydroxy-butyric acid phenethyl ester

Weighing the 3-benzyloxy-3 ', 4' -dibenzyloxy-phenethyl butyrate prepared by the S3, putting the 3-benzyloxy-3 ', 4' -dibenzyloxy-phenethyl butyrate into a reaction container, adding anhydrous methanol for dissolving, adding 10% Pd/C, introducing hydrogen at room temperature, stirring for reacting for 16 hours, filtering, concentrating, and carrying out column chromatography to obtain the target product, namely 3-hydroxy-3 ', 4' -dihydroxy-phenethyl butyrate.

3. The method according to claim 2, wherein the crotonic acid, benzyl alcohol, mercury acetate, 3N sodium hydroxide, 0.5M sodium borohydride, and 3N sodium hydroxide aqueous solution are added in a ratio of 1 to 30 mmol: 1-300 ml: 1-300 mmol: 1-300 ml: 1-300 ml;

the adding amount ratio of the 3, 4-dihydroxy phenethyl alcohol, the potassium carbonate, the anhydrous acetone and the benzyl bromide in the S2 is 1-20 mmol: 1-100 mmol: 1-200 ml: 1-80 mmol;

the addition proportion of the beta-benzyloxy butyric acid, THF, 3, 4-dibenzyloxyphenyl ethanol, EDCI and DMAP in S3 is 1-15 mmol: 1-300 ml: 1-20 mmol: 1-60 mmol: 1-500 mg;

in the S4, the adding amount ratio of the 3-benzyloxy-3 ', 4' -dibenzyloxy-butyric acid phenethyl ester to the anhydrous methanol to the 10% Pd/C is 1-20 mmol: 1-400 ml: 1-1000mg, said hydrogen gas pressure being one atmosphere.

4. The method according to claim 3, wherein the crotonic acid, benzyl alcohol, mercury acetate, 3N sodium hydroxide, and a 3N aqueous solution of 0.5M sodium borohydride are added in a ratio of 29.7 mmol: 30 ml: 30 mmol: 30 ml: 30 ml;

the adding amount ratio of the 3, 4-dihydroxy phenethyl alcohol, the potassium carbonate, the anhydrous acetone and the benzyl bromide in the S2 is 6.49 mmol: 25.9 mmol: 20 ml: 13.62 mmol;

the addition ratio of the beta-benzyloxy butyric acid, THF, 3, 4-dibenzyloxyphenyl ethanol, EDCI and DMAP in S3 is 6.4 mmol: 45 ml: 4 mmol: 8 mmol: 50 mg;

in S4, the adding amount ratio of the 3-benzyloxy-3 ', 4' -dibenzyloxy-butyric acid phenethyl ester to the anhydrous methanol to the 10% Pd/C is 3.92 mmol: 40 ml: 200mg, the pressure of the hydrogen gas being one atmosphere.

5. A process for the preparation of a compound of formula i as claimed in claim 1, comprising the steps of:

synthesis of S1 and 3, 4-dibenzyloxy phenethyl alcohol

Weighing 3, 4-dihydroxy phenethyl alcohol and potassium carbonate in a reaction container, adding anhydrous acetone for dissolving, adding benzyl bromide, stirring and refluxing for 4-5h at 70 ℃, detecting by TLC (thin layer chromatography) for complete reaction, filtering out potassium carbonate, concentrating, mixing samples, and carrying out column chromatography to obtain 3, 4-dibenzyloxy phenethyl alcohol;

preparation of S2, 3-oxobutanoic acid

Measuring ethyl acetoacetate in a reaction container, adding a newly prepared 1N NaOH aqueous solution, carrying out oil bath reaction at 60 ℃, carrying out plate counting detection reaction after 3 hours, completely reacting, cooling the reaction liquid to room temperature, placing the reaction container in an ice bath environment at 0 ℃, slowly dropwise adding 10% dilute hydrochloric acid into the reaction liquid, acidifying to pH 3, adding NaCl solid to saturate the reaction liquid after the temperature of the reaction liquid is raised back to the room temperature, extracting the reaction liquid for three times by using ethyl acetate, combining organic layers, concentrating, carrying out dry-process sample mixing, and carrying out column chromatography to obtain a transparent liquid 3-oxobutyric acid;

synthesis of S3, 3-oxo-3 ', 4' -dibenzyloxy-phenethylbutyrate

Weighing 3, 4-dibenzyloxy phenethyl alcohol prepared in S1 and 3-oxobutyric acid prepared in S2 in a reaction container, adding DCM, ultrasonically dissolving, sequentially adding EDCI and DMAP into the mixed solution, reacting at room temperature, performing dot plate detection reaction after 1h, completely reacting, concentrating the reaction solution, adding saturated sodium chloride aqueous solution, extracting with ethyl acetate, concentrating an organic layer, mixing samples by a dry method, performing column chromatography, drying, and freezing to obtain 3-oxo-3 ', 4' -dibenzyloxy-phenethyl butyrate;

synthesis of S4, 3-oxo-3 ', 4' -hydroxy-butyric acid phenethyl ester

Weighing 3-oxo-3 ', 4' -dibenzyloxy-phenethyl butyrate prepared by S3 in a reaction container, adding methanol, ultrasonically dissolving, adding Pd/C under the protection of argon, introducing hydrogen into the reaction container, replacing for three times under a vacuum state, reacting at room temperature, performing spot plate detection reaction after 2 hours, completely reacting, performing suction filtration on reaction liquid, taking filtrate, concentrating, drying and freezing to obtain 3-oxo-butyric acid 3, 4-dihydroxy phenethyl ester;

synthesis of S5, 3-hydroxy-3 ', 4' -dihydroxy-butyric acid phenethyl ester II

Weighing 3-oxo-3 ', 4' -hydroxy-phenethyl butyrate prepared in S4 in a reaction container, adding absolute ethyl alcohol, ultrasonically dissolving, placing the reaction container in an ice bath at 0 ℃, slowly adding NaBH4 in batches after the temperature of the reaction liquid is stable, performing spot plate detection reaction after 15min, completely reacting, then dropwise adding anhydrous acetone into the reaction liquid, quenching the residual NaBH4, dropwise adding a saturated hydrochloric acid ethanol solution into the reaction liquid, acidifying to a pH value of 5, concentrating the reaction liquid, performing dry-method sample mixing, and performing column chromatography to obtain a target product, namely 3, 4-dihydroxy phenethyl 3-hydroxybutyrate.

6. The preparation method according to claim 5, wherein the 3, 4-dihydroxyphenylethanol, the potassium carbonate, the anhydrous acetone and the benzyl bromide in S1 are added in an amount of 1-20 mmol: 1-100 mmol: 1-200 ml: 1-80 mmol;

the addition ratio of the ethyl acetoacetate to the 1N NaOH aqueous solution in S2 was 1 ml: 3-10 ml;

the adding amount ratio of the 3, 4-dibenzyloxy phenethyl alcohol, the 3-oxobutyric acid, the DCM, the EDCI and the DMAP in the S3 is 1-15 mmol: 1-60 mmol: 1-200 ml: 1-10 g: 1-1000 mg;

the adding amount ratio of the 3-oxo-3 ', 4' -dibenzyloxy-phenethylbutyrate, the methanol and the Pd/C in the S4 is 1-10 g: 1-300 ml: 1-1000mg, said hydrogen gas pressure being one atmosphere;

the adding amount ratio of the 3-oxo-3 ', 4' -hydroxy-butyric acid phenethyl ester, the absolute ethyl alcohol and the NaBH4 in the S5 is 0.5g-5 g: 20-300 ml: 50-1000 mg.

7. The preparation method according to claim 6, wherein the addition amount ratio of the 3, 4-dihydroxyphenylethanol, the potassium carbonate, the anhydrous acetone and the benzyl bromide in S1 is 6.49 mmol: 25.9 mmol: 20 ml: 13.62 mmol;

the addition ratio of the ethyl acetoacetate to the 1N NaOH aqueous solution in S2 was 26 ml: 100 ml;

the adding amount ratio of the 3, 4-dibenzyloxy phenethyl alcohol, the 3-oxobutyric acid, the DCM, the EDCI and the DMAP in the S3 is 5 mmol: 10 mmol: 30 ml: 1.85 g: 100 mg;

the adding amount ratio of 3-oxo-3 ', 4' -dibenzyloxy-phenethylbutyrate, methanol and Pd/C in S4 is 1.00 g: 25 ml: 100mg, the pressure of the hydrogen gas is one atmosphere;

the adding amount proportion of the 3-oxo-3 ', 4' -hydroxy-butyric acid phenethyl ester, the absolute ethyl alcohol and the NaBH4 in the S5 is 500 mg: 20 ml: 95.78 mg.

8. The compound shown in the formula I, optically pure isomers of the compound, mixtures of enantiomers in any proportion or pharmaceutically acceptable salts thereof are applied to the preparation of health-care foods and medicines for relieving brain fatigue, improving learning and memory abilities and manic symptoms.

9. A health food or a pharmaceutical composition for relieving brain fatigue, improving learning and memory ability and mania symptoms, which comprises a compound shown as a formula I, an optically pure isomer of the compound, a mixture of enantiomers in any ratio or a pharmaceutically acceptable salt thereof.

10. The health food or pharmaceutical composition for alleviating brain fatigue and improving learning and memory ability and manic symptoms according to claim 9, wherein the effective intake concentration for an adult is in the range of 8.8 mg per kg body weight per day.

Technical Field

The invention relates to the fields of biology and medicine, in particular to a new compound 3-hydroxy-3 ', 4' -dihydroxy-butyric acid phenethyl ester, a preparation method thereof and application thereof in preparing health-care food and medicines for improving 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. Various degrees of 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 can also activate the biosynthesis of mitochondria, reduce the proportion of damaged mitochondria by increasing the number of healthy mitochondria and ensure 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.

The applicant utilizes esterification reaction of two mitochondrial nutrients Hydroxytyrosol (HT) and beta-hydroxybutyric acid (beta-HB) to form a novel compound, the compound has a novel structure, does not have the possibility of giving any revelation due to other compounds, has outstanding substantive characteristics, simultaneously has remarkable progress for improving the brain fatigue, and provides possibility for developing novel health-care food and medicine for improving the brain fatigue. The invention relates to an application of a novel compound in preparing health-care food and medicines for improving brain fatigue, which is disclosed for the first time. The new compound molecule can improve brain fatigue, particularly improve learning and memory ability and mania.

Disclosure of Invention

The invention aims to provide a novel compound 3-hydroxy-3 ', 4' -dihydroxy-butyric acid phenethyl ester (hydroxytyrosol hydroxybutyrate (HT-HB)), a preparation method thereof and application thereof in preparing health-care food and medicines for relieving brain fatigue, improving learning and memory ability and improving mania related to brain fatigue.

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

a compound of formula i having the formula:

a process for the preparation of a compound of formula I comprising the steps of:

s1 synthesis of beta-benzyloxy butyric acid

Weighing crotonic acid in a reaction vessel, adding benzyl alcohol, adding mercuric acetate, and stirring at normal temperature overnight; cooling the reaction container in a low-temperature condensation tank to 0 deg.C, adding 3N sodium hydroxide within 5-10min, adding 0.5M sodium borohydride 3N sodium hydroxide aqueous solution, and maintaining the solution at 0 deg.C for 3-10 min; taking out the reaction vessel, stirring at room temperature for 1-2h, filtering to obtain filtrate, and extracting with diethyl ether for 3-4 times to remove excessive benzyl alcohol; acidifying the water layer with 10 wt% hydrochloric acid to pH2 to separate out great amount of white solid, and filtering to obtain beta-benzyloxy butyric acid;

synthesis of S2 and 3, 4-dibenzyloxy phenethyl alcohol

Weighing 3, 4-dihydroxy phenethyl alcohol and potassium carbonate in a reaction container, adding anhydrous acetone for dissolving, adding benzyl bromide, stirring and refluxing for 4-5h at 70 ℃, detecting by TLC (thin layer chromatography) for complete reaction, filtering out potassium carbonate, concentrating, mixing samples, and carrying out column chromatography to obtain 3, 4-dibenzyloxy phenethyl alcohol;

synthesis of S3, 3-benzyloxy-3 ', 4' -dibenzyloxy-phenethylbutyrate

Weighing beta-benzyloxy butyric acid prepared by S1, adding THF (tetrahydrofuran) for dissolving, adding 3, 4-dibenzyloxy phenethyl alcohol prepared by S2, EDCI and DMAP (dimethyl formamide) and stirring in an oil bath at 30 ℃ for reacting for 3-4h, detecting by TLC (thin layer chromatography), and stopping the reaction when the 3-benzyloxy butyric acid disappears; concentrating, dissolving and washing with EA for 2-3 times, concentrating the obtained solution, mixing with sample, and performing column chromatography to obtain 3-benzyloxy-3 ', 4' -dibenzyloxy-phenethylbutyrate;

synthesis I of S4, 3-hydroxy-3 ', 4' -dihydroxy-butyric acid phenethyl ester

Weighing the 3-benzyloxy-3 ', 4' -dibenzyloxy-phenethyl butyrate prepared by the S3, putting the 3-benzyloxy-3 ', 4' -dibenzyloxy-phenethyl butyrate into a reaction container, adding anhydrous methanol for dissolving, adding 10% Pd/C, introducing hydrogen at room temperature, stirring for reacting for 16 hours, filtering, concentrating, and carrying out column chromatography to obtain the 3-hydroxy-3 ', 4' -dihydroxy-phenethyl butyrate.

Further, in S1, the addition ratio of crotonic acid, benzyl alcohol, mercury acetate, 3N sodium hydroxide, and a 3N sodium hydroxide aqueous solution of 0.5M sodium borohydride is 1 to 30 mmol: 1-300 ml: 1-300 mmol: 1-300 ml: 1-300ml, preferably 29.7 mmol: 30 ml: 30 mmol: 30 ml: 30 ml;

the adding amount ratio of the 3, 4-dihydroxy phenethyl alcohol, the potassium carbonate, the anhydrous acetone and the benzyl bromide in the S2 is 1-20 mmol: 1-100 mmol: 1-200 ml: 1-80mmol, preferably 6.49 mmol: 25.9 mmol: 20 ml: 13.62 mmol;

the addition proportion of the beta-benzyloxy butyric acid, THF, 3, 4-dibenzyloxyphenyl ethanol, EDCI and DMAP in S3 is 1-15 mmol: 1-300 ml: 1-20 mmol: 1-60 mmol: 1-500mg, preferably 6.4 mmol: 45 ml: 4 mmol: 8 mmol: 50 mg;

in the S4, the adding amount ratio of the 3-benzyloxy-3 ', 4' -dibenzyloxy-butyric acid phenethyl ester to the anhydrous methanol to the 10% Pd/C is 1-20 mmol: 1-400 ml: 1-1000mg, preferably 3.92 mmol: 40 ml: 200mg, the pressure of the hydrogen gas being one atmosphere.

Or a process for the preparation of a compound of formula I, comprising the steps of:

synthesis of S1 and 3, 4-dibenzyloxy phenethyl alcohol

Weighing 3, 4-dihydroxy phenethyl alcohol and potassium carbonate in a reaction container, adding anhydrous acetone for dissolving, adding benzyl bromide, stirring and refluxing for 4-5h at 70 ℃, detecting by TLC (thin layer chromatography) for complete reaction, filtering out potassium carbonate, concentrating, mixing samples, and carrying out column chromatography to obtain 3, 4-dibenzyloxy phenethyl alcohol;

preparation of S2, 3-oxobutanoic acid

Measuring ethyl acetoacetate into a reaction container, adding a newly prepared 1N NaOH aqueous solution, carrying out oil bath reaction at 60 ℃, carrying out plate counting detection reaction after 3 hours, completely reacting, cooling the reaction liquid to room temperature, placing the reaction container into an ice bath environment at 0 ℃, slowly dropwise adding 10% (mass percent) of dilute hydrochloric acid into the reaction liquid, acidifying to pH 3, adding NaCl solid to saturate the reaction liquid after the temperature of the reaction liquid is raised back to room temperature, extracting the reaction liquid for three times by using ethyl acetate, combining organic layers, concentrating, carrying out dry-process sample mixing and carrying out column chromatography to obtain a transparent liquid 3-oxobutyric acid;

synthesis of S3, 3-oxo-3 ', 4' -dibenzyloxy-phenethylbutyrate

Weighing 3, 4-dibenzyloxy phenethyl alcohol prepared in S1 and 3-oxobutyric acid prepared in S2 in a reaction container, adding DCM, ultrasonically dissolving, sequentially adding EDCI and DMAP into the mixed solution, reacting at room temperature, performing dot plate detection reaction after 1h, completely reacting, concentrating the reaction solution, adding saturated sodium chloride aqueous solution, extracting with ethyl acetate, concentrating an organic layer, mixing samples by a dry method, performing column chromatography, drying, and freezing to obtain 3-oxo-3 ', 4' -dibenzyloxy-phenethyl butyrate;

synthesis of S4, 3-oxo-3 ', 4' -hydroxy-butyric acid phenethyl ester

Weighing 3-oxo-3 ', 4' -dibenzyloxy-phenethyl butyrate prepared by S3 in a reaction container, adding methanol, ultrasonically dissolving, adding Pd/C under the protection of argon, introducing hydrogen into the reaction container, replacing for three times under a vacuum state, reacting at room temperature, performing spot plate detection reaction after 2 hours, completely reacting, performing suction filtration on reaction liquid, taking filtrate, concentrating, drying and freezing to obtain 3-oxo-butyric acid 3, 4-dihydroxy phenethyl ester;

synthesis of S5, 3-hydroxy-3 ', 4' -dihydroxy-butyric acid phenethyl ester II

Weighing 3-oxo-3 ', 4' -hydroxy-butyric acid phenethyl ester prepared by S4, adding absolute ethyl alcohol into a reaction container, ultrasonically dissolving, placing the reaction container in an ice bath at 0 ℃, slowly adding NaBH4 in batches after the temperature of the reaction liquid is stable, performing spot plate detection reaction after 15min, completely reacting, then dropwise adding anhydrous acetone into the reaction liquid, quenching the residual NaBH4, dropwise adding saturated hydrochloric acid ethanol solution into the reaction liquid, acidifying to the PH value of 5, concentrating the reaction liquid, performing dry-method sample mixing, and performing column chromatography to obtain the 3, 4-dihydroxy phenethyl 3-hydroxybutyrate.

Further, the adding amount ratio of the 3, 4-dihydroxy phenethyl alcohol, the potassium carbonate, the anhydrous acetone and the benzyl bromide in the S1 is 1-20 mmol: 1-100 mmol: 1-200 ml: 1-80mmol, preferably 6.49 mmol: 25.9 mmol: 20 ml: 13.62 mmol;

the addition ratio of the ethyl acetoacetate to the 1N NaOH aqueous solution in S2 was 1 ml: 3-10ml, preferably 26 ml: 100 ml;

the adding amount ratio of the 3, 4-dibenzyloxy phenethyl alcohol, the 3-oxobutyric acid, the DCM, the EDCI and the DMAP in the S3 is 1-15 mmol: 1-60 mmol: 1-200 ml: 1-10 g: 1-1000mg, preferably 5 mmol: 10 mmol: 30 ml: 1.85 g: 100 mg;

the adding amount ratio of the 3-oxo-3 ', 4' -dibenzyloxy-phenethylbutyrate, the methanol and the Pd/C in the S4 is 1-10 g: 1-300 ml: 1-1000mg, preferably 1.00 g: 25 ml: 100mg, the pressure of the hydrogen gas is one atmosphere;

the adding amount ratio of the 3-oxo-3 ', 4' -hydroxy-butyric acid phenethyl ester, the absolute ethyl alcohol and the NaBH4 in the S5 is 0.5g-5 g: 20-300 ml: 50-1000mg, preferably 500 mg: 20 ml: 95.78 mg.

The compound shown in the formula I, optically pure isomers of the compound, mixtures of enantiomers in any proportion or pharmaceutically acceptable salts thereof are applied to the preparation of health-care foods and medicines for relieving brain fatigue, improving learning and memory abilities and manic symptoms. The beta hydroxybutyric acid (and various ester compounds thereof) produced by the metabolism of the beta hydroxybutyric acid and the hydroxytyrosol (and various ester compounds derived therefrom) jointly play a role in improving the brain fatigue remarkably.

A health food or a pharmaceutical composition for relieving brain fatigue, improving learning and memory ability and mania symptoms, which comprises a compound shown as a formula I, an optically pure isomer of the compound, a mixture of enantiomers in any proportion or a pharmaceutically acceptable salt thereof.

Further, an effective intake concentration range for adults is 8.8 mg per kg 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 new compound hydroxytyrosol hydroxybutyrate is found for the first time to be capable of effectively improving the learning and memory capacity reduction and the manic mood brought by brain fatigue;

(2) the novel compound of hydroxytyrosol hydroxybutyrate is similar to the effect of hydroxytyrosol acetate or beta-ethyl hydroxybutyrate on improving manic mood, but is better than the effect of hydroxytyrosol acetate or beta-ethyl hydroxybutyrate on improving the learning and memory capacity reduction caused by brain fatigue.

Drawings

FIG. 1 is a graph showing the effect of hydroxytyrosol acetate, ethyl beta-hydroxybutyrate and hydroxybutyrate on the improvement of learning and memory abilities, respectively, wherein the abscissa is the grouping of ingested drugs and the ordinate is the ratio of the time spent in the quadrant of the rat water maze inspection test platform;

FIG. 2 is a graph showing the effect of hydroxytyrosol acetate, ethyl hydroxybutyrate and hydroxybutyrate on the improvement of learning and memory ability when used respectively, wherein the abscissa is a group of ingested drugs and the ordinate is the moving distance ratio of the quadrant of a rat water maze inspection test platform;

FIG. 3 is a graph of the improvement in manic mood using hydroxytyrosol acetate, ethyl hydroxybutyrate and hydroxybutyrate, respectively, with the abscissa being the group of ingested drugs and the ordinate being the rate of movement of rats;

FIG. 4 is a graph of the improvement in manic mood using hydroxytyrosol acetate, ethyl hydroxybutyrate and hydroxybutyrate, respectively, with a grouping of ingested drugs on the abscissa and the number of movements by the rat on the ordinate;

FIG. 5 is a graph of the improvement in manic mood using hydroxytyrosol acetate, ethyl hydroxybutyrate and hydroxybutyrate, respectively, with a grouping of ingested drugs on the abscissa and a ratio of the internal region movement path of the rat on the ordinate;

FIG. 6 is a scheme of beta-benzyloxybutyric acid¹H NMR spectrum;

FIG. 7 is a schematic representation of 3, 4-dibenzyloxyphenyl ethanol¹H NMR spectrum;

FIG. 8 is a schematic representation of 3-benzyloxy-3 ', 4' -dibenzyloxy-butyric acid phenethyl ester¹H NMR spectrum;

FIG. 9 is a drawing of 3-oxo-3 ', 4' -dibenzyloxy-butyric acid phenethyl ester¹H NMR spectrum;

FIG. 10 is a scheme of 3-oxo-3 ', 4' -hydroxy-butyric acid phenethyl ester¹H NMR spectrum;

FIG. 11 is a schematic representation of phenethyl 3-hydroxy-3 ', 4' -dihydroxy-butanoate¹H NMR spectrum;

FIG. 12 is a 13C NMR spectrum of phenethyl 3-hydroxy-3 ', 4' -dihydroxy-butyrate;

FIG. 13 is a HRMS (ESI) spectrum of 3-hydroxy-3 ', 4' -dihydroxy-butyric acid phenethyl ester.

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.

Example 1

3-hydroxy-3 ', 4' -dihydroxy-butyric acid phenethyl ester (hydroxytyrosol hydroxybutyrate), the structure of which is shown as formula I:

the preparation method comprises the following steps:

s1, Synthesis of beta-benzyloxybutyric acid (5)

Crotonic acid (1) (2.55g,29.7mmol) was weighed into a 150ml round bottom flask, benzyl alcohol (3) (30ml) was added, mercury acetate (9.63g,30mmol) was added, stirring was carried out at normal temperature overnight, the flask was placed in a low temperature condensation tank to be cooled to 0 ℃ and 30ml of 3N sodium hydroxide was added within 5-10min, then 30ml of 0.5M (0.57g) sodium borohydride in 3N sodium hydroxide aqueous solution was added, and the solution was maintained at 0 ℃ for 3-10 min. Taking out, stirring at room temperature for 1-2h, filtering to obtain filtrate, and extracting with 75ml diethyl ether for 3-4 times to remove excessive benzyl alcohol. The aqueous layer was taken out and acidified with 10% hydrochloric acid to pH2 to precipitate a large amount of white solid, which was filtered to give β -benzyloxybutyric acid (5) as a white solid in a yield of 75% in 4.32 g. 1H NMR (400MHz, CDCl3) δ 7.40-7.28 (m,5H),4.58(dd, J ═ 33.0,11.6Hz,2H), 4.30-4.19 (m,1H), 3.93-3.18 (m,1H),3.55(d, J ═ 5.5Hz,1H),1.44(d, J ═ 5.9Hz,3H), β -benzyloxybutyric acid¹The H NMR spectrum is shown in FIG. 6.

Synthesis of S2, 3, 4-dibenzyloxyphenyl ethanol (6)

3, 4-dihydroxyphenethyl alcohol (2) (1g,6.49mmol) and potassium carbonate (3.59g,25.9mmol) were weighed into a 50ml round-bottom flask, and anhydrous acetone (20ml) was added to dissolve, then benzyl bromide (4) (1.62ml,13.62mmol) was added, and the mixture was stirred at 70 ℃ under reflux for 4-5h, checked by TLC for completion, filtered off the potassium carbonate, concentrated, and sample stirred, and column chromatography (DCM: EA ═ 20:1) gave 3, 4-dibenzyloxyphenethanol (6) as a white solid, 1.86g, 86% yield. 1H NMR (400MHz, CDCl3) delta 7.45-7.43 (m,4H), 7.39-7.26 (m,6H),6.88d, J ═ 8.1Hz,1H),6.81(d, J ═ 2.0Hz,1H),6.73(dd, J ═ 8.1,2.0Hz,1H),5.15(s,2H),5.13(s,2H),3.77(q, J ═ 6.3Hz,2H),2.75(t, J ═ 6.4Hz,2H), 3, 4-dibenzyloxyphenethanol¹The H NMR spectrum is shown in FIG. 7.

Synthesis of S3, 3-benzyloxy-3 ', 4' -dibenzyloxy-phenethylbutyrate (7)

Beta-benzyloxybutyric acid (5) (1.24g,6.4mmol) was weighed into a 100ml round-bottomed flask, THF (45ml) was added and dissolved, and 3, 4-dibenzyloxyphenylethanol (6) (1.34g,4mmol), EDCI (1.53g,8mmol), DMAP (50mg) were added in an oil bath at 30 ℃ and stirred for 3-4h, TLC detection was carried out, and 3-benzyloxybutyric acid disappeared to stop the reaction. Concentrating, dissolving and washing with EA for 2-3 times, concentrating the obtained solution, mixing,column chromatography (PE: DCM ═ 1:1) afforded 3-benzyloxy-3 ', 4' -dibenzyloxy-butyric acid phenethyl ester (7) in the form of a pink oil 823mg, 40% yield. 1H NMR (400MHz, CDCl3) δ 7.47-7.39 (m,4H), 7.38-7.26 (m,10H), 7.25-7.23 (m,1H),6.85(d, J ═ 8.2Hz,1H),6.80(d, J ═ 1.9Hz,1H),6.71(dd, J ═ 8.2,2.0Hz,1H),5.12(s,2H),5.11(s,2H),4.49(dd, J ═ 33.0,11.6Hz,2H), 4.28-4.17 (m,2H), 4.02-3.92 (m,1H),2.81(t, J ═ 7.1Hz,2H),2.61(dd, J ═ 15.0,7.3, 1H),2.39, 7.7, 7, 3, 7, 5, 7.7, 5, 3, 5, 7, 5, 3, 5, 3, 7, 2H), benzyl-benzyloxy (dd, 3, 2H), benzyl-1, 2H, 1, 2H), benzyl-1, 2,1, 2¹The H NMR spectrum is shown in FIG. 8.

S4 Synthesis of 3-hydroxy-3 ', 4' -dihydroxy-butyric acid phenethyl ester I (8)

3-benzyloxy-3 ', 4' -dibenzyloxy-butyric acid phenethyl ester (7) (2g,3.92mmol) was weighed into a 100ml round-bottom flask, and was dissolved by adding anhydrous methanol (40ml), followed by addition of 10% Pd/C (200mg), stirring at room temperature with hydrogen (pressure of hydrogen is one atmosphere) for 16h, filtration, concentration, and column chromatography (DCM: MeOH: 80:1) to give 3-hydroxy-3 ', 4' -dihydroxy-butyric acid phenethyl ester (8) as a colorless to pale yellow oil 762mg, 80% yield. 1H NMR (400MHz, CDCl3) δ 6.76(d, J ═ 8.0Hz,1H),6.70(d, J ═ 1.8Hz,1H), 6.69-6.51 (M,2H),6.33(s,1H), 4.32-4.23 (M,2H), 4.23-4.14 (M,1H),3.42(s,1H),2.79(t, J ═ 6.8Hz,2H), 2.48-2.37 (M,2H),1.20(d, J ═ 6.3Hz,3H), 13C NMR (101MHz, CDCl3) δ 172.88,143.92,142.69,130.17,121.04,115.95,115.52,65.51,64.71,42.89,34.25,22.31. hrems (calcd for C12H 16H 5+ [ M + Na + ] ++ ]]+, 263.0890; process for preparing found 263.0891.3-hydroxy-3 ', 4' -dihydroxy-butyric acid phenethyl ester¹The H NMR spectrum is shown in FIG. 11. The 13C NMR spectrum of 3-hydroxy-3 ', 4' -dihydroxy-butyric acid phenethyl ester is shown in FIG. 12. The HRMS (ESI) spectrum of 3-hydroxy-3 ', 4' -dihydroxy-butyric acid phenethyl ester is shown in FIG. 13.

The second preparation method comprises the following steps:

synthesis of S1, 3, 4-dibenzyloxyphenyl ethanol (6)

Weighing 3, 4-dihydroxy phenethyl alcohol (2)) (1g,6.49mmol) and potassium carbonate (3.59g,25.9mmol) were dissolved in anhydrous acetone (20ml) in a 50ml round bottom flask, and benzyl bromide (4) (1.62ml,13.62mmol) was added, and the mixture was refluxed at 70 ℃ for 4-5h, and the reaction was checked by TLC for completion, after potassium carbonate was filtered off, concentrated, and sample-stirred, column chromatography (DCM: EA ═ 20:1) gave 3, 4-dibenzyloxyphenyl ethanol (6) as a white solid, 1.86g, 86% yield. 1H NMR (400MHz, CDCl3) delta 7.45-7.43 (m,4H), 7.39-7.26 (m,6H),6.88d, J ═ 8.1Hz,1H),6.81(d, J ═ 2.0Hz,1H),6.73(dd, J ═ 8.1,2.0Hz,1H),5.15(s,2H),5.13(s,2H),3.77(q, J ═ 6.3Hz,2H),2.75(t, J ═ 6.4Hz,2H), 3, 4-dibenzyloxyphenethanol¹The H NMR spectrum is shown in FIG. 7.

S2, preparation of 3-oxobutanoic acid (10)

Weighing and placing 26ml of ethyl acetoacetate (9) into a round-bottom flask, adding 100ml of newly prepared 1N NaOH aqueous solution, carrying out oil bath reaction at 60 ℃, carrying out plate counting detection after 3 hours, completely reacting, placing a reaction bottle in an ice bath environment at 0 ℃ after the reaction liquid is cooled to room temperature, slowly dropwise adding 10% dilute hydrochloric acid into the reaction liquid, acidifying to pH 3, adding NaCl solid when the temperature of the reaction liquid is increased back to the room temperature to saturate the reaction liquid, extracting the reaction liquid for three times by using ethyl acetate, combining organic layers, concentrating, carrying out dry-process sample mixing and column chromatography to obtain 2.80g of transparent liquid 3-oxobutyric acid (frozen waxy solid) with the yield of 80%.

Synthesis of S3, 3-oxo-3 ', 4' -dibenzyloxy-phenethylbutyrate (11)

3, 4-dibenzyloxyphenyl ethanol (1.70g, 5mmol) and 3-oxobutyric acid (1.02g, 10mmol) are weighed into a round-bottom flask, 30ml of DCM is added, ultrasonic dissolution is carried out, EDCI1.85 g and DMAP100 mg are sequentially added into a mixed solution, room temperature reaction is carried out, after 1h, the reaction is detected by a dot plate, the reaction is complete, and a developing agent PE: EA ═ 3:1 and Rf ═ 0.7, the reaction mixture was concentrated, 25ml of a saturated aqueous solution of sodium chloride was added, extraction was performed with ethyl acetate, the organic layer was concentrated, and dry-method sample mixing and column chromatography were performed to obtain an off-white waxy solid (obtained by drying and freezing) 3-oxo-3 ', 4' -dibenzyloxy-phenethylbutyrate 1.88g, with a yield of 88%. 1H NMR (400MHz, CDCl3) δ 7.49-7.26 (m,10H),6.87(d, J ═ 8.2Hz,1H),6.81(d, J ═ 2.0Hz,1H),6.71(dd, J ═ 8.2,2.0Hz,1H),5.13(d, J ═ 7.0Hz,1H),4.28(t, J ═ 7.0Hz,2H),3.38(s,2H),2.85(t, J ═ 7.0Hz,2H),2.17(s,3H), 3-oxo-3 ', 4' -dibenzyloxy-butyric acid phenethyl ester¹The H NMR spectrum is shown in FIG. 9.

Synthesis of S4, 3-oxo-3 ', 4' -hydroxy-butyric acid phenethyl ester (12)

Weighing 1.00g of 3-oxo-3 ', 4' -dibenzyloxy-phenethyl butyrate in a double-neck bottle, adding 25ml of methanol, ultrasonically dissolving, adding 100mg of Pd/C under the protection of argon, introducing hydrogen into a reaction bottle, replacing for three times under a vacuum state, reacting at room temperature, performing dot-plate detection reaction after 2 hours, completely reacting, performing suction filtration on a reaction solution, taking a filtrate, concentrating to obtain a white wax-like solid (prepared by drying and freezing) 0.54g of 3-oxobutyric acid 3, 4-dihydroxy phenethyl ester, wherein the yield is 95%. 1H NMR (400MHz, DMSO) δ 8.71(d, J ═ 23.2Hz,2H),6.63(d, J ═ 8.0Hz,1H),6.60(d, J ═ 1.7Hz,1H),6.47(dd, J ═ 7.9,1.7Hz,1H),4.17(t, J ═ 7.1Hz,2H),3.56(s,2H),2.70(t, J ═ 7.0Hz,2H),2.14(s,3H), 3-oxo-3 ', 4' -hydroxy-butyric acid phenethyl ester¹The H NMR spectrum is shown in FIG. 10.

S5 Synthesis of 3-hydroxy-3 ', 4' -dihydroxy-butyric acid phenethyl ester II (8)

Weighing 500mg of 3-oxo-3 ', 4' -hydroxy-butyric acid phenethyl ester into a 50ml round-bottom flask, adding 20ml of absolute ethyl alcohol, ultrasonically dissolving, placing a reaction bottle into an ice bath at 0 ℃, slowly adding NaBH495.78mg in batches after the temperature of the reaction solution is stable, performing spot plate detection reaction after 15min, completely reacting, then dropwise adding absolute acetone into the reaction solution, quenching the residual NaBH4, dropwise adding a saturated hydrochloric acid ethanol solution into the reaction solution, acidifying to the PH value of 5, concentrating the reaction solution, performing dry sample mixing and column chromatography to obtain 430mg of colorless to light yellow oily substance 3, 4-dihydroxy phenethyl 3-hydroxybutyrate, wherein the yield is 85%. 1H NMR (400MHz, CDCl3) δ 6.76(d, J ═ 8.0Hz,1H),6.70(d, J ═ 1.8Hz,1H), 6.69-6.51 (M,2H),6.33(s,1H), 4.32-4.23 (M,2H), 4.23-4.14 (M,1H),3.42(s,1H),2.79(t, J ═ 6.8Hz,2H), 2.48-2.37 (M,2H),1.20(d, J ═ 6.3Hz,3H), 13C NMR (101MHz, CDCl3) δ 172.88,143.92,142.69,130.17,121.04,115.95,115.52,65.51,64.71,42.89,34.25,22.31. hrems (calcd for C12H 16H 5+ [ M + Na + ] ++ ]]+, 263.0890; process for preparing found 263.0891.3-hydroxy-3 ', 4' -dihydroxy-butyric acid phenethyl ester¹The H NMR spectrum is shown in FIG. 11. 3-hydroxy groupThe 13C NMR spectrum of phenethyl-3 ', 4' -dihydroxy-butyrate is shown in FIG. 12. The HRMS (ESI) spectrum of 3-hydroxy-3 ', 4' -dihydroxy-butyric acid phenethyl ester is shown in FIG. 13.

Example 2

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 (22-28 degrees) and humidity (60%) and light 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) simultaneously performing intragastric administration of normal saline (hereinafter referred to as brain fatigue group) every day by using a brain fatigue model established by a sleep deprivation method (3) simultaneously performing intragastric administration of 35mg/kg of hydroxytyrosol acetate every day during brain fatigue; (hereinafter abbreviated as HTac group, shown in the following table) (4) cerebral fatigue while intragastric administering 23.6mg/kg of beta-hydroxybutyrate ethyl ester per day (hereinafter abbreviated as HBET group, shown in the following table); (5) 42.9mg/kg of hydroxytyrosol hydroxybutyrate (hereinafter referred to as HTHB group, shown in the following table) was gavaged daily while treating brain fatigue.

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, and in order to avoid falling into the water, the animal must keep slightly grasping the edge of the platform, so that the aim of causing the brain fatigue of the rat due to sleep depriv.

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 experiment 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. The training of the other two quadrants is completed. 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. Swimming situations. 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. In the software tracking system, the inside of an open field box is divided into 16 squares of 4 × 4, wherein 12 squares defining the periphery are the peripheral area, and 4 squares inside are the central area.

The open field experiment operation method comprises the following steps: after the water maze examination test is finished, the rat is put back into the cage for rest for 10min, and then the animal is put into the center of the bottom surface in the box under a quiet environment, and shooting and timing are carried out simultaneously. And stopping shooting after 5min of observation. The inner wall and the bottom surface of the square box are cleaned, so that the influence of the information smell and the excrement left by the animal at the last time on the next test result is avoided. Animals were replaced and the experiment continued. And calculating parameters such as the number of observable movements, the movement speed, the movement distance of each square grid and the like according to computer software.

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

3) Statistical analysis

The results are expressed in Mean ± s.e.m form, where blank group data is statistical results of 15 rats, other 5 group data are statistical results of 10 rats, and a few unreasonable data are removed according to 99.9% confidence intervals; data analysis using One Way-ANOVA analysis method, significant statistical significance was p <0.05, p <0.01, p <0.001, p < 0.0001.

4. Function of hydroxytyrosol hydroxybutyrate in improving learning and memory of rats with brain fatigue

The improvement of the learning and memory ability of the hydroxytyrosol hydroxybutyrate 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 control group, brain fatigue group, HTac group, HBET group, and HTHB group, respectively, in the water maze examination test in the quadrant (i.e., the fourth quadrant) in which the platform was located at the time of training. The results show that in the fourth quadrant residence time index, the brain fatigue groups have significant difference compared with the control group; there was a significant difference between the HTHB group and the brain fatigue group. On the fourth quadrant swimming distance index, the brain fatigue group has significant difference compared with the control group; there was a significant difference between the HTHB group and the brain fatigue group. Compared with a control group, the brain fatigue model group has obviously reduced learning and memory abilities, and compared with two medicaments of hydroxytyrosol acetate and beta-ethyl hydroxybutyrate, the use of a new medicament of hydroxytyrosol hydroxybutyrate has more obvious tendency of improving the learning and memory abilities of a brain fatigue model rat.

5. Improvement effect of hydroxytyrosol hydroxybutyrate on manic mood of rats with brain fatigue

The improvement of the manic emotion of the rats with the brain fatigue model by the hydroxytyrosol hydroxybutyrate is detected by adopting a field-open 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 novel compound 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, fig. 4 and fig. 5 show the mean locomotor speed, the number of movements and the internal region movement path ratio in the open field experiment of the rats of the control group, brain fatigue group, HTac group, HBET group and HTHB group. The faster a manic rat will move in the open field over time, the less it will tend to stop moving, so the total number of movements is correspondingly reduced, while there is a greater tendency to move in the interior regions of the mine rather than at the edges of the open field. . 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 HTHB group all had significant differences compared to 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 HTHB group were significantly different from the brain fatigue group. On the internal region moving distance ratio index, the brain fatigue group has significant difference compared with the control group; the HTac group and the HTHB group were significantly different from the brain fatigue group. In the case of brain fatigue, the manic mood of the rats is remarkably stronger than that of the control group, and the three drugs of hydroxytyrosol acetate, beta-ethyl hydroxybutyrate and hydroxytyrosol hydroxybutyrate are respectively used, so that the manic mood of the rats is remarkably improved to different degrees. The effect of the synthesized novel molecular hydroxytyrosol hydroxybutyrate on improving emotion is proved to extend the effectiveness of hydroxytyrosol acetate and beta-ethyl hydroxybutyrate, and the performance of partial indexes is superior to that of the hydroxytyrosol acetate or the beta-ethyl hydroxybutyrate.