WO2020077915A1 - Novel use of hydroxytyrosol and derivative thereof in preparing anti-depressant product - Google Patents

Abstract

Provided in the present invention is a novel use of hydroxytyrosol and a derivative thereof in preparing an anti-depressent product, relating to the field of biomedicine. The hydroxytyrosol derivative is selected from at least one of a hydroxytyrosol salt, a hydroxytyrosol ester, or a hydroxytyrosol ether, and may be obtained by means of chemical synthesis, micro-organism fermentation, or extraction from an animal or a plant. The product for preventing and treating depression may be a drug, a food, a functional food, etc. Anti-depressant products prepared using hydroxytyrosol or a derivative thereof as an active ingredient have depression preventing and treating effects, and depression recurrence preventing effects, and may be used to reduce the incidence of depression and improve the quality of life of depression patients.

Description

New application of hydroxytyrosol and its derivatives in the preparation of antidepressant products Technical field

The invention belongs to the field of biomedicine, and specifically relates to the new use of hydroxytyrosol.

Background technique

With the continuous development of social economy, the acceleration of life rhythm, the increasingly fierce social competition, people's psychological pressure gradually increases, and the incidence of depression increases year by year. According to estimates by the World Health Organization, the prevalence of depression worldwide is about 3% to 5%, totaling about 120 million people. By 2022, depression will become the most serious disease burden in developing countries.

Depression, also known as depressive disorder, is a group of clinical symptom groups or states centered on self-experience in depressed mood, with persistent and significant spontaneous mood depression as the main clinical feature and the main type of mood disorder. The main clinical symptoms are: low mood, out-of-group, out-of-group, physical discomfort, loss of appetite and sleep disturbances; some cases have obvious anxiety and motor agitation; in severe cases, mental symptoms such as hallucinations and delusions may occur. There are the following major types of depression: endogenous depression, reactive depression, occult depression, depression characterized by learning difficulties, secondary depression caused by drugs, secondary depression caused by physical diseases Syndrome, postpartum depression, etc. At present, the drugs for depression in the market include paroxetine, sertraline, fluvoxamine, and venlafaxine. There are many side effects of drugs, such as drowsiness, constipation, gastrointestinal discomfort, respiratory depression, memory loss and Dependence, etc., it is of great significance to find and develop safer antidepressants.

At present, there are many theoretical theories about the pathogenesis of depression: monoamine neurotransmitter theory, neuroendocrine theory, neuroplasticity theory, etc. Among them, the study of monoamine neurotransmitter theory is the most extensive and in-depth. It believes that the onset of depression is related to the decrease of neurotransmitter levels of monoamine neurotransmitters such as serotonin, norepinephrine, and dopamine. Dopamine is an important catecholamine neurotransmitter used to help cells transmit pulsed chemicals. This kind of brain endocrine matter is related to people's passions and feelings, and can transmit messages of excitement and happiness. Dopamine can be damaged in the nucleus accumbens and striatum, and when the level of dopamine decreases, depression symptoms such as lack of pleasure, anxiety, and loss of appetite will appear. The level of dopamine in the brain may be one of the indicators of the severity of depression and the effect of treatment, so regulating the level of dopamine in the brain is of great significance for the treatment of depression.

Hydroxytyrosol (CtyHrosio, C ₈ H ₁₀ O ₃ , CAS: 10597-60-1), also known as 3,4-dihydroxy phenylethanol (3,4-dihydroxy phenylethanol), is a hydrophilic and lipophilic natural Polyphenol compounds. At room temperature and low purity, it is yellow oily substance, and at high concentration, it is colorless oily substance. They are all fluid; they are mainly present in the form of oleuropein in the fruits and leaves of the olive family of the olive family. Due to the structure of catechol and phenethyl alcohol, hydroxytyrosol has strong antioxidant properties and has received extensive attention in recent years. Studies have shown that hydroxytyrosol has a strong antioxidant effect, has a good inhibitory and preventive effect on the occurrence and development of inflammation; it has a potential neuroprotective effect on dopaminergic cells, and can be used to treat Parkinson's and Alzheimer's synthesis Sign. The prior art has disclosed a preparation method of hydroxytyrosol and its use in the manufacture of cartilage protection, prevention and treatment of osteoarthritis, blood lipid lowering, and treatment of macular eye disease products, but the use of hydroxytyrosol as a prevention and treatment of depression Not yet public.

Summary of the invention

In view of the problems in the prior art, the present invention provides a new use of hydroxytyrosol in the preparation of antidepressant products, which has the functions of preventing and treating depression and its complications, and preventing the recurrence of depression.

To achieve the above objective, the present invention adopts the following technical solutions.

The application of hydroxytyrosol and its derivatives in the preparation of products for the prevention and treatment of depression. It can be used to alleviate and improve the mental illness caused by psychological stress; it can be used to alleviate and improve various symptoms of depression, such as low mood, out of group, physical discomfort, loss of appetite and sleep disorders.

The hydroxytyrosol derivative is at least one selected from the group consisting of hydroxytyrosolate, hydroxytyrosol ester and hydroxytyrosol ether, and its structural formula is part or all of -OH in the compound represented by formula (I) Salts, esters or ethers with bases, acids, alcohols:

The above-mentioned hydroxytyrosol derivatives can be converted into hydroxytyrosol by hydrolysis in the human body or animal body, producing the same or similar physiological activities.

The hydroxytyrosol salt is a physiologically acceptable salt formed by hydroxytyrosol and an inorganic or organic base. These include, but are not limited to, alkali metal salts of hydroxytyrosol, such as sodium and potassium salts; alkaline earth metal salts of hydroxytyrosol, such as calcium and magnesium salts; ammonium salts; and salts of hydroxytyrosol with nitrogen-containing organic bases. The nitrogen-containing organic base includes but is not limited to trimethylamine, triethylamine, tributylamine, pyridine, N, N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, Dicyclohexylamine, dibenzylamine, N-benzyl-β-phenethylamine, N, N′-dibenzylethylenediamine, procaine, amphetamine.

The hydroxytyrosol ester is a physiologically acceptable ester of hydroxytyrosol and an inorganic or organic acid; the inorganic acid is selected from sulfuric acid, phosphoric acid, carbonic acid, and hydrochloric acid; the organic acid is selected from formic acid, acetic acid, propionic acid, Butyric acid, maleic acid, oxalic acid, methanesulfonic acid, succinic acid or fatty acids.

The hydroxytyrosol ether includes hydroxytyrosol alkyl ether, hydroxytyrosol aryl ether and hydroxytyrosol glycoside. The alkyl ether can be methyl, ethyl, n-propyl, n-butyl, n-hexyl, n-octyl, isopropyl or tert-butyl. The aryl ether may be an aromatic ether containing carbon atoms, or a heteroaromatic ring ether containing nitrogen atoms, sulfur atoms, and oxygen atoms; the hydroxytyrosol glycoside may be hydroxytyrosol glucoside, hydroxytyrosine Alcohol fructoside and so on.

The hydroxytyrosol and its derivatives may be derived from chemical synthesis, microbial fermentation, or animal and plant extraction, for example, from olive fruits, olive leaves, or other extracts containing hydroxytyrosol and its derivatives.

The product for preventing and treating depression can be medicine, food, functional food, special medical food or cosmetics.

The antidepressant product may further contain acceptable auxiliary materials for preparing medicines, foods, functional foods, special medical foods, or cosmetics. For example, fillers, carriers, emulsifiers, excipients, etc.

The product for preventing and treating depression can be in the form of oral, injection or external use. The oral dosage form includes, but is not limited to, lozenges, tablets, capsules, and granules; it is preferably a buccal dosage form, such as a sublingual lozenge. The injection dosage form includes, but is not limited to, injection solution and powder injection. The external dosage form includes, but is not limited to, suppositories, aerosols, films, and patches; it is preferably a pharmaceutical form for lumens, such as a pharmaceutical form for nasal passages, which can absorb the medicine through the nasal mucosa.

The antidepressant foods include but are not limited to baked goods, condiments, beverages, alcohols, milk and dairy products, frozen drinks, soups, nutrition bars, spreads, dietary supplements, food or feed additives, functional foods.

The products for preventing and treating depression can be used in combination with other antidepressant products to reduce the dosage of other products and reduce side effects. It can also be a compound preparation composed of hydroxytyrosol and its derivatives and other active ingredients, which has a synergistic effect and can improve the effect of treating depression and reduce side effects.

Other active ingredients are selected from but not limited to ingredients with antidepressant activity, such as sertraline hydrochloride, fluoxetine, paroxetine, citalopram, fluvoxamine, doxepin hydrochloride, selegiline, safinamide , Toloxadone; any sedative and hypnotic active ingredients, such as kwazepam, temazepam, sulazepam, γ-aminobutyric acid; or any combination of ingredients with antioxidant activity, such as salidroside ; Hydroxysalidium glycosides, tyrosol; components with neurasthenia, such as gastrodin.

In the product for preventing and treating depression, the content of hydroxytyrosol and its derivatives is 0.01% -99.99%; preferably 0.05% -95%; more preferably 0.05% -65%; the% is 100% by volume Fractional content (w / v) or mass percentage content (w / w).

Further, the percentage content in the oral product is 0.05% -60%; preferably 0.1% -40%. The percentage content in the product for injection is 0.1% -95%; preferably 0.5% -50%. The percentage content in external products is 0.1% -65%.

The beneficial effects of the present invention are:

The present invention provides a new use of hydroxytyrosol and its derivatives for the preparation of antidepressant products, which can be used to prevent or treat depression and its complications, prevent the recurrence of depression, reduce the incidence of depression and improve patients with depression The quality of life, combined with other antidepressant drugs, can reduce the side effects of other products.

detailed description

The present invention will be further described below with reference to examples, but the present invention is not limited by the following examples.

Example 1 Preparation of hydroxytyrosol sublingual lozenge

The sublingual lozenges are prepared by conventional procedures. The formula (made into 1000 tablets) is as follows:

Using wet granulation and tableting method, weigh the prescribed amount of sucrose and microcrystalline cellulose, and then mix hydroxytyrosol and 10% starch slurry, add it as a binder to the soft material, pass a 20 mesh sieve, wet granules Dry at 70 ℃ for 1h, sift through 20 mesh sieve, add talcum powder and mix well, then press to obtain.

Example 2 Preparation of hydroxytyrosol soft capsules

Soft capsules are prepared by conventional procedures, and the formula (made of 1000 capsules) is as follows:

Take a prescribed amount of hydroxytyrosol, add vegetable oil to dissolve it, and use it as a medical solution; take another glycerin and water to 70 ℃, stir to dissolve, add gelatin, stir for 2h, go out the floating foam, and filter. Turn on the soft capsule machine, turn on the left and right plastic boxes and spray body for heating, and turn on the water pump circulation and refrigeration at the same time. The spray body is set at 43 ° C, and the left and right plastic boxes are set at 60 ° C. After preheating is completed, adjust the thickness of the leather on both sides to 0.70mm. After the rubber has been debugged, add the liquid medicine to the hopper, adjust the filling volume, and press the soft capsule. Wash the soft capsules and dry them.

Example 3 Preparation of hydroxytyrosol tablets

The tablets are prepared through conventional procedures and the formula (made into 1000 tablets) is as follows:

Mix the powdered sugar, microcrystalline cellulose and sodium carboxymethyl starch, and then mix the hydroxytyrosol and starch slurry as a binder, add soft materials, make wet granules through a 14 mesh nylon sieve, and dry at 45 ℃. Add talc powder, mix it, pass a 12-mesh nylon sieve. After passing the content test, the granules can be compressed.

Example 4 Preparation of hydroxytyrosol granules

The granules are prepared by conventional procedures. The formula (1000 bags) is as follows:

Sodium bisulfite, sucrose and microcrystalline cellulose were mixed, and then hydroxytyrosol and starch slurry were mixed and added as a binder to make a soft material and passed through a 12 mesh nylon sieve. The edible flavor that has passed through the 80 mesh sieve is mixed with the whole granules, and packed separately.

Example 5 Preparation of hydroxytyrosol injection

The injection solution (1000 pcs) is prepared through the conventional process, and the formula is as follows:

In the preparation container, add 80% of the prescribed amount of water for injection, saturated with carbon dioxide, after adding hydroxytyrosol to dissolve, add the pre-formulated disodium edetate solution and sodium bisulfite solution, stir well, add carbon dioxide saturated Water is sufficient for injection and filtered to remove impurities. Packing, potting and sterilization under carbon dioxide flow.

Example 6 Preparation of hydroxytyrosol injection

The injections (100 pcs) are prepared by conventional procedures, the formula is as follows:

Dissolve vitamin E in refined soybean oil, then add hydroxytyrosol, homogenize, filter and remove impurities, perfuse, and sterilize.

Example 7 Preparation of health drinks

The health drink is prepared through the conventional process, the formula is as follows:

Weigh the raw material according to the above weight, dissolve it with a small amount of water, and then make up to 1000mL with water, sterilize and sub-pack. Among them, hydroxytyrosol and tyrosine are the main materials. In order to further adjust the flavor and taste of the product, the product formula of the present invention also adds an appropriate amount of citric acid and sucralose flavoring agent, and edible flavors. The specific preparation process is in accordance with the food Routine operations in the field can be carried out.

Example 8 Preparation of compound hydroxytyrosol tablets

The tablets are prepared through conventional procedures and the formula (made into 1000 tablets) is as follows:

Fluoxetine, powdered sugar, microcrystalline cellulose and sodium carboxymethyl starch were mixed, and then hydroxytyrosol and 10% starch slurry were mixed and added to make a soft material. Wet granules were made through a 14 mesh nylon sieve at 45 ° C dry. The dried granules are sized through a 12-mesh nylon sieve, and then the granules are mixed with talc powder, and then passed through a 12-mesh nylon sieve. After passing the content test, the granules are compressed and obtained.

Example 9 Preparation of hydroxytyrosol caproate

The preparation method is as follows:

Dissolve 1 g of hydroxy-p-hydroxyphenyl ethanol in 2.3 mL of pyridine, dilute with 10 mL of toluene and cool in an ice-water bath. 3mL of hexanoyl chloride was added to produce a solid. Overnight, the mixture was allowed to reach room temperature. The product was dissolved in a mixed solution of ethyl acetate and water, the organic phase was retained, and the aqueous phase was removed. The organic phase was washed successively with 1.5mol / L HCl solution and 4% sodium bicarbonate solution, dried and concentrated to obtain hydroxytyrosol caproate, whose hydrogen spectrum is characterized by:

¹ H NMR (CDCl ₃ ) δ 7.0-6.8 (m, 3H); 4.40 (t, 2H, J = 7.1 Hz); 2.90 (t, 2H, J = 6.8 Hz); 2.22 (t, 2H, J = 7.3Hz); 2.23 (t, 2H, J = 7.3Hz); 2.25 (t, 2H, J = 7.4Hz); 1.35-2.32 (m, 6H); 1.56-1.25 (m, 12H), 0.93-0.87 ( m, 9H);

The structure of the compound is as follows:

Example 10 Preparation of hydroxytyrosol caproate sublingual buccal tablets

The sublingual buccal tablets of hydroxytyrosol caproate are prepared by conventional procedures. The formula (made into 1000 tablets) is as follows:

Using wet granulation and tableting method, weigh the prescribed amount of sucrose and microcrystalline cellulose, and then mix hydroxytyrosol and 10% starch slurry, add it as a binder to the soft material, pass a 20 mesh sieve, wet granules Dry at 70 ℃ for 1h, sift through 20 mesh sieve, add talcum powder and mix well, then press to obtain.

Example 11 Model test of acquired despair depression by hanging tail method

Hydroxytyrosol and its derivatives were provided by Shandong College of Pharmaceutical Sciences. 70 ICR mice, weighing 18-22g, provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd., animal room temperature controlled at 20 ℃ -25 ℃, relative humidity 45% -55%, 12h / 12h day and night, free water intake and diet . After adapting to the environment for one week, they were randomly divided into 7 groups: model control group (0mg / kg / d), hydroxytyrosol low-dose group (1mg / kg / d), hydroxytyrosol medium-dose group (4mg / kg / d) , Hydroxytyrosol high-dose group (10mg / kg / d), Example 8 group (each tablet contains hydroxytyrosol 20mg, fluoxetine 5mg), fluoxetine group (3mg / kg / d), hydroxytyrosol Caproate group (4mg / kg / d). Each of the 6 test groups was gavaged with the sample once a day for 30 days with 0.1mL / 10g body weight; the model control group was gavaged with the corresponding solvent, and the gavage volume was 0.1mL / 10g body weight. Two hours after the last administration of the sample, the tail of each group of mice (at 2 cm from the tip of the tail) was glued to the upper bracket of the hanging box (30 cm × 30 cm × 25 cm) with tape to make it upside down, the head About 5cm from the bottom of the box, two mice were hung at a time, separated by a partition. The mouse struggled to overcome the abnormal posture, but the activity was intermittently immobile for a period of time, showing a state of disappointment. The suspension time is 6min, and the cumulative immobility time of the suspended tail within 4min after the statistics (the immobile state means that the mouse stops struggling or has no activity), the results are shown in Table 1:

Table 1 The effect of hydroxytyrosol on tail suspension test in mice (mean ± SD, n = 10)

Group	Dosage (mg // kg / d)	Number of animals (only)	Hanging tail immobile time (s)
Model control group	0	10	101 ± 7.01
Hydroxytyrosol low-dose group	1	10	87 ± 5.45 *
Hydroxytyrosol medium dose group	4	10	74 ± 7.35 *
Hydroxytyrosol high-dose group	10	10	62 ± 4.37 *
Example 8 group	4 hydroxytyrosol + fluoxetine	10	61 ± 5.83 *
Fluoxetine group	3	10	70 ± 6.28 *
Hydroxytyrosol caproate group	4	10	72 ± 5.48 *

Note: Compared with the model control group, * P <0.05.

It can be seen from Table 1 that there is a significant difference between the dosing and immobilization time of the animal-administered group and the model control group. Among them, the more the dose of hydroxytyrosol is administered, the tail-suspending time of mice is shortened in a dose-dependent manner. There was no significant difference between the hydroxytyrosol medium dose group (4mg / kg / d), the hydroxytyrosol caproate group (4mg / kg / d) and the fluoxetine group (3mg / kg / d), indicating that hydroxytyrosol Alcohol and its caproate derivatives have the effect of improving the condition or assisting the treatment of depression. At the same time, in Example 8 group (4mg / kg / d hydroxytyrosol + 1mg / kg / d fluoxetine) compared with the hydroxytyrosol group (10mg / kg / d), there was no significant difference in the dangling time, And significantly better than hydroxytyrosol (4mg / kg / d) and fluoxetine group (3mg / kg / d), indicating that the combined use of the composition has a synergistic effect and helps to relieve the disease.

Example 12 Open field experiment

Hydroxytyrosol and its derivatives were provided by Shandong College of Pharmaceutical Sciences. 70 ICR mice, weighing 18-22g, provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd., animal room temperature controlled at 20 ℃ -25 ℃, relative humidity 45% -55%, 12h / 12h day and night, free water intake and diet . After adapting to the environment for one week, the mouse depression model was injected intraperitoneally with corticosterone for 21 days. The success of the model was judged by observing the changes in animal behavior. After successful modeling, they were randomly divided into 7 groups: model control group (0mg / kg / d), low-dose hydroxytyrosol group (1mg / kg / d), medium-dose hydroxytyrosol group (4mg / kg / d) , Hydroxytyrosol high-dose group (10mg / kg / d), Example 8 group (each tablet contains hydroxytyrosol 20mg, fluoxetine 5mg), fluoxetine group (3mg / kg / d), hydroxytyrosol In the caproate group (4 mg / kg / d), 10 mice per group. Each of the 7 test groups was given a sample by gavage once a day for 30 days, with an intragastric amount of 0.1 mL / 10g body weight; the model control group was gavaged with the corresponding solvent, with an intragastric amount of 0.1 mL / 10g body weight. Two hours after the last administration of the sample, each group of mice was placed in an open box (55cm × 55cm × 40cm) without a lid, and the bottom of the box was divided into small squares of 11cm × 11cm with a white line. The four walls are made of black to make a good contrast with the mouse in the darker room. The camera was fixed above the center of the room, and the spontaneous activity of the mice was measured by video recording. After the model was built for 21 days, the moving distance and the number of upright times of the mice were measured. The mouse was placed in the middle of the bottom of the box and allowed to move freely to adapt to the environment for 5 minutes. Then it was measured for 5 minutes, and recorded and observed with the camera each time. Between the two experiments, each cell at the bottom of the box was wiped clean with 70% ethanol. Each measurement time is 4 o'clock in the afternoon, the room is stable at 25 ℃ -28 ℃, and keep the room quiet. Record the spontaneous activity of the mouse: every time the mouse passes a small grid or spans 10cm, it is counted as a parallel point, and the mouse's forelimbs standing upright or touching the carton wall are recorded as standing upright. The results are shown in Table 2:

Table 2 The effect of hydroxytyrosol on the number of parallel moving lattices and upright times of mice (mean ± SD, n = 10)

Group	Dosage (mg // kg / d)	Number of animals (only)	Parallel shift	Erect times
Model control group	0	10	1 ± 0.41	0 ± 0
Hydroxytyrosol low-dose group	1	10	10 ± 1.28 *	1 ± 0.31 *
Hydroxytyrosol medium dose group	4	10	19 ± 1.23 *	2 ± 0.28 *
Hydroxytyrosol high-dose group	10	10	37 ± 3.19 *	3 ± 0.25 *
Example 8 group	4 hydroxytyrosol + fluoxetine	10	35 ± 2.74 *	3 ± 0.34 *
Fluoxetine group	3	10	18 ± 3.62 *	2 ± 0.49 *
Hydroxytyrosol caproate group	4	10	18 ± 4.21 *	2 ± 0.18 *

Note: Compared with the control group, * P <0.05.

It can be seen from Table 2 that the number of parallel movements and the number of upright times in the animal-administered group are significantly different from the model control group. Among them, the more the dosage of hydroxytyrosol, the dose-dependent increase in the number of mice activities, the exploration behavior increased. There was no significant difference between the hydroxytyrosol medium-dose group (4mg / kg / d) and the hydroxytyrosol caproate group (4mg / kg / d) compared with the fluoxetine group (3mg / kg / d), indicating that the hydroxyl group Tyrosol and its caproate derivatives have the effect of improving the condition and treating depression. At the same time, in the Example 8 group (4 mg / kg / d hydroxytyrosol + 1 mg / kg / d fluoxetine) compared with the hydroxytyrosol group (10 mg / kg / d), the number of parallel shifts and the number of upright times were not significant Difference, and significantly better than hydroxytyrosol (4mg / kg / d) and fluoxetine group (3mg / kg / d), indicating that the combined use of the composition has a synergistic effect and helps to relieve the disease.

Example 13 Rat model of chronic unpredictable mild stress depression

Hydroxytyrosol and its derivatives were provided by Shandong College of Pharmaceutical Sciences. 70 SD rats, weighing 183-224g, provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd., animal room temperature controlled at 20 ℃ -25 ℃, relative humidity 45% -55%, day and night 12h / 12h, free water and food .

After adapting to the environment for one week, they were randomly divided into 7 groups: model control group (0mg / kg / d), hydroxytyrosol low-dose group (0.5mg / kg / d), hydroxytyrosol medium-dose group (2mg / kg / d) ), Hydroxytyrosol high dose group (6mg / kg / d), Example 8 group (each tablet contains hydroxytyrosol 20mg, fluoxetine 5mg), fluoxetine group (2mg / kg / d), hydroxytyrosol Alcohol hexanoate group (4mg / kg / d). Modeling and administration: The rats in the blank control group were fed and drank normally, without any stimulation, and were fed in 5 cages; the remaining rats will be constructed for 6 weeks of chronic unpredictable mild stimulation depression models (single cage feeding). Choose one type of stress every day, different stress sources are randomly distributed, and the same stress source should be applied at least 7 days apart. Except for 24 hours of continuous stress, stress is applied every day from 8:30 to 12:00. Stress methods include: night lighting 12h, squirrel cage tilt 45 °, cold water (4 ℃ -8 ℃) swimming 5min, 45 ℃ hot water swimming 5min, behavior limit 1h, wet litter 24h, tail pinching 1min, water ban 24h, Fasting for 24 hours, overnight in crowded squirrel cages, etc. After 2 weeks of chronic stress, all rats underwent another forced swimming test and sucrose preference test. Then, the administration group started administration, once a day, for 4 consecutive weeks (the model was continued during the administration period), the model control group was orally administered the corresponding vehicle, and the forced swimming test and sucrose preference test were used to evaluate the depression of the drug in mice. Behavioral impact.

Before the sugar water test, two bottles of water were used to adjust the animals to 48h. In the first 24h, both bottles were filled with an equal amount of 1% (w / v) sucrose water. In the second 24h, one bottle was filled with 1% sugar water, and the other bottle was filled with equal amount of purified water, and the positions of the two water bottles were changed after 12h. After that, the animals were banned from water and fasting. After 24 hours, the sugar water / pure water consumption experiment was conducted. Place one rat per cage, and give each rat two pre-weighed two bottles of water at the same time: one bottle of 1% sugar water and another bottle of purified water. The test time is 2h, and the position of the two bottles is switched after 1h. After the test, the double bottles are weighed again. Sugar preference of each mouse = [sugar water consumption / (white water consumption + sugar water consumption)] × 100%, the results are shown in Table 3:

Table 3 The effect of hydroxytyrosol on the sugar preference and forced swimming behavior of rats (mean ± SD, n = 10)

Note: Compared with the model control group, * P <0.05.

The sugar water preference test and forced swimming results showed that compared with the model control group, the sugar water preference and swimming immobility of the administration group were significantly improved, and were positively correlated with the dose. There was no significant difference between the hydroxytyrosol medium-dose group (2mg / kg / d) and the hydroxytyrosol caproate group (2mg / kg / d) compared with the fluoxetine group (2mg / kg / d), indicating that hydroxytyrosol Alcohol and its caproate derivatives have the effect of treating or assisting the treatment of depression. At the same time, in Example 8 group (2mg / kg / d hydroxytyrosol + 0.5mg / kg / d fluoxetine) compared with hydroxytyrosol group (6mg / kg / d), sugar water preference and swimming immobility time were not significant Sexual difference, and significantly better than hydroxytyrosol (2mg / kg / d) and fluoxetine group (2mg / kg / d), indicating that the combination of the composition has a synergistic effect and helps to relieve the disease.

Example 14 Effect of hydroxytyrosol on the content of monoamine neurotransmitters in the brain of depressed rats

Hydroxytyrosol and its derivatives were provided by Shandong College of Pharmaceutical Sciences. 70 SD rats, weighing 183-224g, provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd., animal room temperature controlled at 20 ℃ -25 ℃, relative humidity 45% -55%, day and night 12h / 12h, free water intake and diet .

After adapting to the environment for one week, they were randomly divided into 7 groups: model control group (0mg / kg / d), hydroxytyrosol low-dose group (0.5mg / kg / d), hydroxytyrosol medium-dose group (2mg / kg / d) ), Hydroxytyrosol high dose group (6mg / kg / d), Example 8 group (each tablet contains hydroxytyrosol 20mg, fluoxetine 5mg), fluoxetine group (2mg / kg / d), hydroxytyrosol Alcohol caproate group (2mg / kg / d).

模 模 方法 同 实施 例 13。 The modeling method is the same as in Example 13.

After 2 weeks of chronic stress, the administration group started administration, once a day for 4 weeks (the model was continued during administration), and the model control group was orally administered the corresponding vehicle. After the administration, the rats were anesthetized by intraperitoneal injection of 0.35mL / 100g, and the brain was quickly stripped. On the ice tray, separate the cerebellum and the surrounding tissue of the hematoma. Weighing, taking 400mg tissue into a tissue homogenizer, added 0.1mol / L perchloric acid (containing _{0.05% EDTA-Na 2) 1mL} , homogenized in an ice-water bath. After completion, transfer the homogenate into a 1.5mL centrifuge tube, centrifuge at 10000r / min for 10min at 4 ℃, take the supernatant and store in -80 ℃ refrigerator.

Accurately weigh the appropriate amount of standard norepinephrine (NE), dopamine (DA) and serotonin (5-HT) respectively, mix with 0.1mol / L perchloric acid (containing 0.1% cysteine) solution Into a standard stock solution with a mass concentration of 1 mg / mL, and dilute the stock solution to NE concentrations 0.1, 0.5, 1.5, 3, 4.5 μg / mL, DA concentrations 0.2, 1, 2, 4, 5 μg / mL, 5- The HT concentration is 0.5, 1, 1.5, 2, 3 μg / mL, which is used as the standard control solution of each component.

Detection by HPLC. Chromatographic conditions: CLC-ODS column was selected as the column. The mobile phase of the chromatography was pH = 2.5 ion pair reagent (0.15g sodium dodecyl sulfate, EDTA-Na ₂ 0.028g, pH = 2.5-3 sulfuric acid. Solution 0.2mL, 10% methanol solution 400mL, distilled water volume to 1000mL); flow rate 1.2mL / min; column temperature 40 ℃. Fluorescence excitation wavelength 285nm, emission wavelength 313nm, injection volume 20μL, quantified by peak area external standard method.

Table 4 The effect of hydroxytyrosol on the content of monoamine neurotransmitters in the rat brain (mean ± SD, n = 10)

Group	Dosage (mg / kg / d)	Number of animals (only)	NE (μg / g)	DA (μg / g)	5-HT (μg / g)
Model control group	0	10	223 ± 64.45	273 ± 58.29	389 ± 65.19
Hydroxytyrosol low-dose group	0.5	10	458 ± 60.15 *	398 ± 56.17 *	567 ± 82.41 *
Hydroxytyrosol medium dose group	2	10	658 ± 72.11 *	516 ± 55.02 *	762 ± 75.84 *
Hydroxytyrosol high-dose group	6	10	858 ± 62.75 *	637 ± 54.91 *	1003 ± 84.32 *
Example 8 group	2 hydroxytyrosol + 0.5 fluoxetine	10	869 ± 73.12 *	625 ± 76.90 *	994 ± 85.32 *
Fluoxetine group	2	10	634 ± 61.94 *	523 ± 72.58 *	774 ± 72.65 *
Hydroxytyrosol caproate group	2	10	662 ± 73.52 *	531 ± 73.76 *	754 ± 849.47 *

Note: Compared with the model control group, * P <0.05.

As can be seen from Table 4, compared with the model control group, the monoamine neurotransmitters in the administration group were significantly improved, and were positively correlated with the dose of hydroxytyrosol administered. There was no significant difference between the hydroxytyrosol medium-dose group (2mg / kg / d) and the hydroxytyrosol caproate group (2mg / kg / d) compared with the fluoxetine group (2mg / kg / d), indicating that hydroxytyrosol Alcohol and its caproate derivatives can increase the level of monoamine neurotransmitters in the brains of depressed animals. At the same time, the level of monoamine neurotransmitters in the brain of the Example 8 group (2 mg / kg / d hydroxytyrosol + 0.5 mg / kg / d fluoxetine) compared with the hydroxytyrosol group (6 mg / kg / d) No significant difference was seen, and it was significantly better than hydroxytyrosol (2mg / kg / d) and fluoxetine group (2mg / kg / d), indicating that the combination of the composition has a synergistic effect and helps the depression Of relief.

Example 15 Pharmacokinetic characteristics of hydroxytyrosol sublingual lozenges, hydroxytyrosol tablets and hydroxytyrosol caproate sublingual lozenges

Hydroxytyrosol sublingual buccal tablets (20mg / tablet), hydroxytyrosol tablets (20mg / tablet) and hydroxytyrosol caproate sublingual buccal tablets (20mg / tablet) were provided by Shandong Provincial College of Pharmaceutical Sciences, and the preparation methods are as in the examples One, two and ten are shown. 18 Beagle dogs, half male and half female, weighing 8.0-12kg, provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd. Animal room temperature is controlled at 20 ℃ -25 ℃, relative humidity 45% -55%, day and night 12h / 12h, free water intake and diet.

The doses are all 40mg / piece. Randomly divided into three groups A, B and C. After fasting for 12 hours, group A was given hydroxytyrosol sublingual lozenges, group B was given hydroxytyrosol tablets orally, and group C was given hydroxytyrosol caproate sublingual lozenges. Before and after administration, 0.083, 0.167, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10h, 12 hours of blood were taken from the saphenous vein of the hind limbs, and the blood was collected by heparin anticoagulation , Separate the plasma and store it in the refrigerator at -20 ℃ to be tested. Animals were fed uniformly 4 hours after administration.

The Begle dogs were given the second dose 7 days after the first dose, and were killed immediately after 1 hour and the brain was quickly stripped. On the ice tray, separate the cerebellum and the surrounding tissue of the hematoma, weigh it, take 500mg of brain tissue, put it in a tissue homogenizer, add 2.5mL of physiological saline, and homogenize in an ice water bath. After completion, transfer the homogenate into a 1.5mL centrifuge tube, centrifuge at 10000r / min for 10min at 4 ℃, take the supernatant and store in -80 ℃ refrigerator.

Thaw the plasma and brain tissue homogenate samples at room temperature, take 200 μL in a 1.5 mL EP tube, add 10 μL of 20 μg / mL salicin (internal standard) reference solution, vortex to mix for 3 min, add 1 mL of acetonitrile, vortex Spin-mix for 3min, centrifuge at 14800r / min for 12min, take 1mL of supernatant, and dry at 37 ° C with nitrogen. The residue was reconstituted with 120μL of initial mobile phase. Centrifuge at 14800r / min for 15min. Take the supernatant and measure by injection.

The content of hydroxytyrosol was analyzed by LC-MS / MS. Column: ACQUITY UPLC BEH C ₁₈ column (2.1mm × 50mm, 1.7μm); chromatographic conditions: mobile phase acetonitrile (A) -0.1% formic acid (B), gradient elution (0-1min, 95% B; 1 -3.5min, 95% → 5% B; 3.5-5min, 5% B; 5-6min, 5% → 95% B; 6-7.5min, 95% B); column temperature: 35 ℃; volume flow: 0.3 mL / min; injection volume 5μL; mass spectrometry conditions: MRM multi-reaction monitoring, electrospray negative ion source; capillary voltage 2.5kV; extraction voltage 3V; ion source temperature 120 ° C; desolvent gas temperature 400 ° C; collision voltage 15V; cone hole Voltage 30V; desolvent gas volume flow 800L / h; selection of hydroxytyrosol and salicin to detect ion-to-nucleus ratio m / z of 153.01 → 123.01, 285.28 → 123.04, respectively.

Methodological verification results show that this method is suitable for the determination of hydroxytyrosol in biological samples with a linear range of 5-1000ng / mL. The pharmacokinetic parameters of the obtained hydroxytyrosol sublingual buccal tablets, hydroxytyrosol tablets and hydroxytyrosol caproate in Beagle dogs are shown in Table 5:

Table 5 Pharmacokinetic parameters of hydroxytyrosol in plasma of Beagle dogs (mean ± SD, n = 6)

Table 6 Concentration of hydroxytyrosol in brain tissue of Beagle dogs 1h after administration (mean ± SD, n = 6)

The results show that, compared with oral tablets, hydroxytyrosol can enter the blood circulation to reach the peak faster through sublingual administration, and hydroxytyrosol can be enriched in the brain faster. Hydroxytyrosol caproate can increase the plasma and brain tissue exposure levels of hydroxytyrosol, which is beneficial for hydroxytyrosol to enter the brain tissue through the blood-brain barrier to play the role of prevention and treatment of depression.

Claims (11)

1. The application of hydroxytyrosol and its derivatives in the preparation of products for the prevention and treatment of depression; the structural formula of the hydroxytyrosol derivatives is part or all of the -OH and alkali, acid, Salt, ester or ether formed by alcohol:

   

   Formula (I).
2. The use according to claim 1, characterized in that the hydroxytyrosol salt is selected from alkali metal salts, alkaline earth metal salts, ammonium salts of hydroxytyrosol and salts of hydroxytyrosol with nitrogen-containing organic bases.
3. The use according to claim 2, wherein the nitrogen-containing organic base is selected from trimethylamine, triethylamine, tributylamine, pyridine, N, N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, dibenzylamine, N-benzyl-β-phenethylamine, N, N'-dibenzylethylenediamine, procaine or Amphetamine.
4. The use according to claim 1, characterized in that the hydroxytyrosol ester is a physiologically acceptable ester of hydroxytyrosol and an inorganic or organic acid; the inorganic acid is preferably selected from sulfuric acid, phosphoric acid, carbonic acid or hydrochloric acid; The organic acid is preferably selected from formic acid, acetic acid, propionic acid, butyric acid, maleic acid, oxalic acid, methanesulfonic acid, succinic acid or fatty acid.
5. The use according to claim 1, characterized in that the hydroxytyrosol ether comprises hydroxytyrosol alkyl ether, hydroxytyrosol aryl ether and hydroxytyrosol glycoside.
6. The hydroxytyrosol and its derivatives according to claim 1 can be derived from chemical synthesis, microbial fermentation or animal and plant extraction; the plant can be olive fruit, olive leaf or other plants containing hydroxytyrosol or its derivatives.
7. The use according to claim 1, wherein the product for preventing and treating depression is selected from medicine, food, functional food or special medical food.
8. The use according to claim 7, characterized in that the depression and prevention products are oral, injectable, and external medicines; oral medicines are preferably sublingual dosage forms; injection medicines are preferably injections; and external medicines are preferably nasal Medication.
9. The use according to claim 1, characterized in that hydroxytyrosol and its derivatives can be combined with other active ingredients to form a compound preparation.
10. Use according to claim 9, characterized in that the other active ingredients are selected from those with antidepressant activity; such as sertraline hydrochloride, fluoxetine, paroxetine, citalopram, fluvoxamine, doxe hydrochloride Ping, selegiline, safinamide, toloxone; ingredients with sedative and hypnotic activity; such as any one or combination of quasi-pan, temazepam, sulazepam, γ-aminobutyric acid; with anti- Oxidatively active components; such as salidroside; hydroxysalidium, tyrosol; components with a therapeutic effect on neurasthenia; such as gastrodin.
11. The use according to any one of claims 1-10, characterized in that, in the product for preventing and treating depression, the content of hydroxytyrosol and its derivatives is 0.01% -99.99%; preferably 0.05% -95% ; More preferably, it is 0.05% -65%; the% is the percentage by mass (w / v) or the percentage by mass (w / w).