CN115990146A - Application of BTY in medicines for treating or preventing dementia caused by brain diseases - Google Patents

Abstract

The invention discloses an application of BTY in a medicine for treating or preventing dementia caused by brain diseases, and relates to the technical field of chemical medicines. BTY is known as Benzene,1,2,4-trimethoxy-5- (2-methyl-1-propen-1-yl), and pharmacological activity of BTY was primarily studied in the present invention. In vitro studies prove that BTY can act on sodium ion and chloride ion channels simultaneously to regulate action potential; in vivo studies have demonstrated that BTY can reduce neuronal excitability and inhibit the onset of mouse seizures caused by tetrazolium. On this basis, the inventors have found that BTY is useful for a model mouse with Alzheimer's disease induced by d-galactose in combination with aluminum trichloride, and has a therapeutic or prophylactic effect on Alzheimer's disease.

Description

Application of BTY in medicines for treating or preventing dementia caused by brain diseases

Technical Field

The invention relates to the technical field of chemical medicines, in particular to application of BTY in medicines for treating or preventing dementia caused by brain diseases.

Background

Dementia is usually caused by brain diseases, for example senile dementia, as a nervous system degenerative disease, and its cause is mainly Alzheimer's disease and cerebrovascular disease. At present, the number of patients suffering from Chinese dementia exceeds 900 ten thousand, and the first place in the world. Senile dementia caused by Alzheimer's disease is the most concerned problem on the current dementia problem, and with the aggravation of Chinese aging, the research on senile dementia is related to the future of the whole country and society. Therefore, we mainly look at the current situation of the scientific research of Alzheimer's disease.

Alzheimer's Disease (AD) is a central nervous system degenerative disease with hidden progressive progression. The world Alzheimer's 2018 report shows that every 3 seconds a dementia patient develops worldwide. At least 5000 million dementia patients worldwide are currently estimated to be 1.52 million by 2050, with about 60% -70% of these being Alzheimer's disease patients. AD is highly developed in people over 65 years old and is the most common form of dementia. The main clinical manifestations of AD are memory impairment, cognitive dysfunction, which is characterized by predominantly extracellular β -amyloid (aβ) plaque deposition, hyperphosphorylation of intracellular Tau protein to form neurofibrillary tangles (neurofibrillary tangles, NFTs) with concomitant neuronal and synaptic loss. The number of senile dementia patients is huge worldwide, and long-term treatment is required, which not only seriously affects the life quality of the patients, but also brings heavy burden to families and society, so searching for drugs for effectively treating AD has become an urgent need for human health.

The pathogenesis of AD is complex, the exact causative factor is still unknown, and four major hypotheses exist in the pathogenesis of AD at present: aβ hypothesis based on amyloid plaques, tau hypothesis based on neuronal fiber tangles, inflammatory hypothesis based on brain damage caused by long-term inflammatory reactions, and neuroprotective hypothesis of synapses and neuronal loss. In fact, however, there may be many forms of interactions that complicate the pathogenesis of AD even more. Drugs currently approved by the U.S. food and drug administration are based on neuroprotective hypotheses including tacrine, donepezil, galantamine, rivastigmine, and memantine acting on the glutamatergic system. Unfortunately, these drugs have a general adverse effect and only partially alleviate AD symptoms and do not prevent the disease from further progression. The medicine taking Abeta and Tau proteins as targets is difficult to break through, and the action mechanism of the novel medicine Ab Du Kani monoclonal antibody approved by the FDA for treating AD is to remove Abeta proteins, and then the clinical effect is proved to be poor, so that the medicine has no convincing effect.

The excitation/inhibition balance of the central nervous system (Central nervous system, CNS) has an important role in maintaining its homeostasis. There are studies showing that there is abnormal hyperexcitability of neurons in the hippocampus at early stages of AD, causing imbalance in CNS excitation/inhibition, and ultimately resulting in memory and cognitive dysfunction. Thus, reducing neuronal abnormal excitation, restoring CNS homeostasis, may be a potential treatment for AD. Glutamate is the most prominent excitatory neurotransmitter in the central nervous system, gamma-aminobutyric acid (gamma-aminobutyric acid, GABA) is the most prominent inhibitory neurotransmitter, and imbalance in the regulation of both causes neuronal excitatory toxicity, thereby accelerating the AD process. Perhaps, we can develop drugs that are effective in treating AD.

Disclosure of Invention

Aiming at the problems in the prior art, the invention discovers the application of BTY in the medicines for treating or preventing the dementia caused by the brain diseases, and provides an effective and safe medicine for treating or preventing the dementia caused by the Alzheimer disease and even other brain diseases.

Wherein BTY is fully called Benzene,1,2,4-trimethoxy-5- (2-methyl-1-propen-1-yl), and the structural formula of the compound is shown in formula I.

The technical scheme adopted by the invention is as follows:

the application of BTY in the preparation of medicines for treating or preventing dementia caused by brain diseases is characterized in that the BTY is Benzene,1,2,4-trimethoxy-5- (2-methyl-1-propen-1-yl) and the structural formula is shown in the formula I:

wherein the compound of the formula I is insoluble in water and soluble in organic solvents such as ethyl acetate, is light yellow transparent liquid at normal temperature, and becomes solid at 4 ℃.

When pharmacological activity screening is carried out on a series of compounds prepared by synthesis, the invention unexpectedly discovers that the compounds shown in the formula I can obviously prolong the seizure incubation period of mice with the model of the pentatetrazole epilepsy and can obviously reduce the times of large seizures; the patch clamp experimental result proves that the compound shown in the formula I can simultaneously act on sodium ion and chloride ion channels to regulate action potential. Based on the good pharmacological activity, the invention establishes an Alzheimer disease mouse animal model through long-term administration of aluminum trichloride combined with d-galactose, and carries out treatment by administration of BTY and positive medicine donepezil hydrochloride. The results show that BTY can improve the number of hippocampal nikohlrabi in model mice; down-regulating the expression of inflammatory factors; improving the performance of the model mice in various behavioral tests and improving the learning and memory capacity of the model mice.

Preferably, the brain diseases are Alzheimer's disease and cerebrovascular diseases.

Preferably, the ratio of the effective amount of BTY as an effective component in the medicine to the unit mass of human body is 1.0 mg-10.0 mg/kg. The ratio is obtained by dose conversion relation among animals of different species, the effective dose of mice for treating or preventing Alzheimer's disease in experiments is 10 mg-40 mg/kg, and when the mice are used as medicines for treating or preventing Alzheimer's disease for human, the ratio of the effective dose of BTY obtained by conversion to the unit mass of human body is 1.0 mg-10.0 mg/kg in consideration of individual difference of the dosage.

More preferably, the ratio of the effective dosage of the effective component BTY in the medicine to the unit mass of human body is 1.5 mg-4.0 mg/kg.

Further, the medicament is an injection or an oral preparation. The administration route of the BTY medicine in the invention can be injection, oral administration, subcutaneous implantation, inhalation, transdermal, mucous membrane and the like, and the administration dosage form can be any dosage form which is suitable for different administration routes, provided that the dosage form can lead the BTY to enter the brain to reach effective treatment concentration.

Further, the injection is an emulsion.

Further, the emulsion comprises the following components in percentage by weight: BTY 0.5-5%, oil phase 5-20%, emulsifier 0.6-1.8%, glycerin 0-2.5%, and the balance being purified water or water for injection. Wherein the oil phase is at least one of soybean oil, medium chain oil, olive oil and fish oil. The emulsifier is at least one of egg yolk lecithin, soybean lecithin, pluronic F-68 and polyethylene glycol stearic acid-15 (Solutol HS 15). Oleic acid, sodium oleate or a mixture of the oleic acid and the sodium oleate in any proportion can be added according to the requirement of the emulsifying property. When in preparation, oleic acid is dissolved in an oil phase, sodium oleate is dissolved in a water phase, and the mixture of the oleic acid and the sodium oleate is respectively dissolved in an oil phase and a water phase. The effective amount will vary from subject to subject (e.g., human or animal). The administration volume of the emulsion should be appropriate for the subject to be administered. The concentration of BTY in the emulsion may vary within a range. The concentration range depends on the amount administered, the volume administered, and the solubility of BTY in the oil phase. For example, the concentration range is 0.5% to 5.0%, i.e., 5mg/mL to 50mg/mL. The emulsion may also contain antioxidants, which may include sodium bisulphite, vitamin E, pyrogallic acid esters, and the like. Other suitable additives such as preservatives and flavouring agents may also be used when administered orally.

Further, the preparation process of the emulsion comprises the following steps:

(1) Respectively weighing the BTY and auxiliary materials in the prescription amount;

(2) Under the protection of nitrogen or other inert gases, dissolving BTY in an oil phase preheated to 60-80 ℃, dissolving or dispersing glycerol in an aqueous phase preheated to 60-80 ℃, and dissolving an emulsifier in the oil phase or the aqueous phase;

(3) Mixing the oil phase and the water phase by high-speed shearing to uniformly disperse the oil phase in the water phase, thus preparing the colostrum;

(4) Homogenizing the colostrum for 2-4 times under high pressure to ensure that the average particle size of emulsion drops is not more than 0.5um, and filtering to obtain the drug-carrying emulsion containing BTY; under the protection of nitrogen, the emulsion is obtained by filling the emulsion in a container suitable for medical use and hot-pressing and sterilizing in a proper mode according to the requirement of the administration route.

In summary, compared with the prior art, the invention has the following advantages and beneficial effects:

the invention discloses that BTY has the effect of treating or preventing Alzheimer's disease for the first time, and the research result of pharmacodynamics mechanism shows that BTY has the following effects:

(1) Increasing the number of Nib bodies in the hippocampus of a model mouse, thereby antagonizing the decline of learning and memory capacity caused by a modeling agent

(2) Reduces the expression of inflammatory factors IL-6, IL-1beta and TNF-alpha, and plays an anti-neuroinflammation role;

(3) Antagonizing glutamate-induced neuroexcitatory toxicity, maintaining the excitation-inhibition balance of the central nervous system;

(4) BTY is effective and safe, and has no obvious toxic or side effect in the whole experimental process of the invention.

Drawings

FIG. 1 is a hydrogen spectrum of BTY prepared in example 1: ¹ H NMR(400MHz,Chloroform-d)δ＝6.76(s,1H)，6.51(s,1H)，6.27-6.22(m,1H)，3.89(s,3H)，3.83(s,3H)，3.80(s,3H)，1.91(d,J＝1.5,3H)，1.81(d,J＝1.4,3H)；

FIG. 2 is a carbon spectrum of BTY prepared in example 1: ¹³ C NMR(101MHz,Chloroform-d)δ＝151.37,148.09，142.42，134.74,120.12，119.27，114.54，97.50，56.64，56.43，56.13，26.55，19.59；

FIG. 3 is a high performance liquid chromatogram of BTY prepared in example 1;

fig. 4: effect of BTY on model mouse behavioural experiments, wherein a: swim trace diagrams of mice in each group on the same day of Morris water maze behavioral testing; b: the number of platform crossing times for each group of mice; c: escape latency in each group of mice. The circle indicates the platform position, and the quadrant in which it is located is the target quadrant. P <0.05, P <0.01, ns indicates no significant difference compared to the model group.

Fig. 5: effect of BTY on the number of hippocampal nikohlrabi in model mice, wherein a: representative pictures of each group of mice were nisetum stained (×400); b: statistical analysis of the number of Nib bodies in each group of mice. P <0.05, P <0.01, ns indicates no significant difference compared to the model group.

Fig. 6: effect of BTY on IL-6, IL-1 β, TNF- α expression in mouse brain tissue, wherein a: the content of IL-6 in brain tissue of each group of mice; b: content of IL-1β in brain tissue of each group of mice; c: TNF- α content in brain tissue of each group of mice. P <0.05, < P <0.01 compared to model group.

Fig. 7: effect of BTY on glutamate causing excitotoxicity of HT22 cells, wherein a: representative cell morphology images of each group after 24h co-incubation of glutamate and BTY; b: screening the optimal damage concentration of glutamic acid; c: BTY increases cell viability in the glutamate injury model. P <0.05, < P <0.01 compared to model group.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings and embodiments. It should be understood that the particular embodiments described herein are illustrative only and are not intended to limit the invention, i.e., the embodiments described are merely some, but not all, of the embodiments of the invention.

The following terms and phrases used herein are intended to have the following meanings unless otherwise indicated. A particular term or phrase, unless otherwise specifically defined, should not be construed as being ambiguous or otherwise clear, but rather should be construed in a generic sense. When trade names are presented herein, it is intended to refer to their corresponding commercial products or active ingredients thereof.

Unless otherwise indicated, the term "pharmaceutically acceptable" in the present invention means those compounds, materials, compositions and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Unless otherwise indicated, the term "pharmaceutical excipients" refers to excipients and additives used in the manufacture of medicaments and formulation of prescriptions, are substances which, apart from the active ingredient, have been reasonably evaluated in terms of safety and are included in pharmaceutical preparations. The pharmaceutical excipients not only form, serve as carriers and improve stability, but also have important functions of solubilization, dissolution assistance, sustained and controlled release and the like, and are important components which can influence the quality, safety and effectiveness of the medicine.

Unless otherwise indicated, the term "subject" is any animal that is subject to the experiment, and is the carrier that passively undertakes research. Researchers need to treat subjects humanly and humanly, which is a basic scientific and ethical requirement. "subject" includes any mammal, wherein examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rabbits, guinea pigs, monkeys, humans, etc., preferably mice, and most preferably humans.

The term "treatment" means drug treatment unless otherwise indicated. The medicine treatment refers to using all substances with therapeutic or preventive effects for treating and curing diseases, and keeping the health. In particular, when conditions are involved, treatments are divided into: (1) alleviating one or more biological manifestations of a disease; (2) Interference with one or more biological manifestations of the factors responsible for the occurrence of the disease; (3) Improving one or more biological manifestations of symptoms, effects, or side effects associated with the disorder; (4) One or more biological manifestations that slow the progression of the condition.

The term "prevention" means, unless otherwise indicated, drug prophylaxis, i.e., the taking of a drug in advance to prevent or reduce the occurrence of a certain type of disease.

The term "therapeutically effective amount" refers to a range of drug concentrations or dosages that produce a significant efficacy in the body without causing significant severe adverse side effects, unless otherwise indicated. Wherein, the effective amounts of the treatment of different subjects are different and can be adjusted by the related scientific researchers related to the patent according to the experimental results.

The term "neuroprotectant" is, unless otherwise indicated, a neuromodulatory agent that protects vulnerable neurons, reduces or prevents disease progression, reduces neuronal death.

Unless otherwise indicated, for convenience of description, the compounds of formula I are abbreviated as BTY, i.e., BTY refers to the drug Benzene,1,2,4-trimethoxy-5- (2-methyl-1-propen-1-yl).

Unless otherwise indicated, the reaction temperatures are room temperature, typically 20 to 35 ℃.

The above preferred conditions can be arbitrarily combined on the basis of not deviating from the common knowledge in the art, and thus, each preferred embodiment of the present invention can be obtained.

The reagents and materials used in the present invention are commercially available.

Example 1

This example prepares BTY for use in experiments.

The raw materials are as follows: 2,4, 5-trimethoxybenzaldehyde (T819646, shanghai Meilin Biochemical Co., ltd.), isopropyl magnesium bromide (1822702, shanghai Meilin Biochemical Co., ltd.), tetrahydrofuran (T818767, shanghai Meilin Biochemical Co., ltd.), anhydrous copper sulfate (Q12 HB3731-2017, tianjin, miou chemical Co., ltd.), anhydrous magnesium sulfate (Q/12 KM3936-2019, tianjin, miou chemical Co., ltd.), silica gel plate (10052521046809, qingdao ocean chemical Co., ltd.).

The preparation process comprises the following steps: 10.0 to 30.0g of 2,4, 5-trimethoxybenzaldehyde is added into a three-port bottle, 100 to 300mL of anhydrous tetrahydrofuran is injected under the protection of nitrogen, and the solution is clarified by stirring. Slowly injecting 130-390 mL of isopropyl magnesium bromide, and controlling the temperature within 30 ℃. Heating to 60 ℃, enabling the reaction solution to be yellow-green transparent, reacting for 3 hours, enabling the reaction solution to be dark green, and enabling the reaction solution to be petroleum ether: ethyl acetate=2:1 to check if the reaction is complete. After the reaction is finished, cooling in ice bath, adding water to quench unreacted isopropyl magnesium bromide until no bubbles are generated. Tetrahydrofuran was removed by rotary evaporation to give a yellowish green solid attached to the bottle wall, 400mL of water and 400mL of ethyl acetate were added for extraction, the organic layer was taken, the aqueous layer was extracted twice with 400mL of ethyl acetate, and the organic layers were combined. The organic layer was dried by adding an appropriate amount of anhydrous magnesium sulfate, suction-filtered, and spin-dried. 15.6g of greenish black intermediate are obtained. 15.6g of intermediate, 10.4g of anhydrous copper sulfate and 280mL of toluene were added to a three-necked flask. Heating to 120 ℃, dripping reflux liquid on the bottle wall, reacting for 4 hours, petroleum ether: ethyl acetate=2:1 to check if the reaction is complete. After the reaction, toluene is removed by rotary evaporation, and the purified product is subjected to column chromatography (petroleum ether: ethyl acetate=40:1) to obtain a BTY pure product, wherein a hydrogen spectrum, a carbon spectrum and an HPLC (high performance liquid chromatography) spectrum of the BTY pure product are shown in figures 1-3.

Example 2

This example uses the pure BTY product prepared in example 1 to prepare an emulsion for BTY injection, which is prepared as follows:

weighing 15.0g of BTY, 50.0g of soybean oil for injection and 50.0g of Medium Chain Triglyceride (MCT) for injection, placing into a proper container, heating to 60-80 ℃ under the protection of nitrogen, and stirring to dissolve; 12.0g of egg yolk lecithin and 0.3g of sodium oleate are weighed, added into the mixture and stirred to be dissolved, and an oil phase is prepared for later use. Weighing 22.0g of glycerol, weighing about 800mL of water, heating to 60-80 ℃ under the protection of nitrogen, and stirring to dissolve; an aqueous phase is obtained. Adding the oil phase into the water phase, shearing at high speed for 5-15 minutes, and adding water to 1000mL to prepare the colostrum. Homogenizing the primary emulsion for 1-3 times by a high-pressure homogenizer to ensure that the average particle size of the homogenized emulsion drops is not more than 0.5 mu m, filtering by a filter membrane, filling the filtrate into a 5mL or 10mL glass ampoule under the protection of nitrogen filling, and performing rotary heat pressing sterilization at 121 ℃ for 8min to obtain the BTY injection emulsion, wherein the concentration of the BTY is 15mg/mL. A blank injectable emulsion without BTY was prepared in the same manner.

Study 1: influence of BTY on mouse behaviours of AD model

Experimental materials: SPF KM mice, male, weighing 18-22 g, purchased from Mitsui laboratory animal Co., ltd., eligibility: SCXK 2020-030.

Donepezil hydrochloride was purchased from Shanghai Micin Biochemical technologies Co., ltd (specification: 200mg lot number: D859456).

Experimental grouping: the mice to be tested are divided into two groups, the first group is a model building group, and 65 mice are all tested; the second group is a non-manufactured module, 15 in total. Modular mice were co-dosed once daily: the aluminum trichloride is administrated by stomach infusion, 20 mg/kg/d+180 mg/kg/d of d-galactose solution is injected intraperitoneally; non-model mice were dosed once daily: physiological saline was injected intraperitoneally at 5.0mL/kg/d. The two groups are continuously dosed for 6 days and then 1 day, the continuous dosing is repeated for 3 months, then Morris water maze experiments are carried out, 56 mice which are successfully molded are screened according to experimental evaluation results, the mice are divided into 4 groups, and 14 mice in each group are set as a model group, a low-dose group, a high-dose group and a positive drug group. And selecting 14 mice as normal groups according to the evaluation result of the spatial learning and memory capacity. The dosing regimen was as follows, normal (Control) group: physiological saline 5.0mL/kg/d; model group (Model group): blank emulsion prepared in example 2 5.0mL/kg/d; low dose group (BTY-L group): the BTY emulsion prepared in example 2, the BTY dose corresponds to a unit volume ratio of 20mg/kg/d in mice; high dose group (BTY-H group): the BTY emulsion prepared in example 2, the BTY dose corresponds to a unit volume ratio of 40mg/kg/d in mice; positive drug group (Donepezil group): donepezil hydrochloride Ji Rongye mg/kg/d. The administration mode is as follows: intraperitoneal injection. During the administration period, the mice of each group, except the normal group, continued to be given the modeling agent. The administration process was continued for 3 months.

The experimental steps are as follows:

1.1 open field experiments

After 30min adaptation of the test chamber, the mice were placed in the center of a square open field test box (50×50×40 cm) and allowed to move freely, and the total distance of movement of each mouse over 5min WAs recorded and analyzed using a computer tracking system (Noldus Ethovision, tacoma, WA, USA). Before the next rat experiments, the feces were cleaned and sprayed with 70% alcohol to eliminate odors.

1.2 Morris water maze experiment

The water maze is a circular pool with the diameter of 100cm and the depth of 60cm. Warm water (24±2 ℃) was filled into the inside to a water depth of 30cm before the start of the experiment, and then the water was dyed black using a pigment. The pool is equally divided into 4 quadrants, and different marks are attached to the pool walls of different quadrants to show the distinction. A colorless transparent platform with a diameter of 6cm and a height of 25cm was placed in the center of one of the quadrants (the quadrant is set as the third quadrant), and the platform was submerged 1cm. Mice were released in the indicated quadrants following the experimental guidelines after the start of the experiment. On days 1-4, mice were tested 1 time each with water released from 4 different quadrants, each round of test being 30 minutes apart. If the mouse finds the platform within 60 seconds, the mouse stands on the platform for 5 seconds; if not found, it is guided onto the platform with a wooden stick and allowed to stand for 10 seconds. On day 5, the platform WAs removed and the mice were allowed to swim freely for 60 seconds and data such as escape latency, swimming speed and target quadrant search time were recorded using a computer tracking system (Noldus Ethovision, tacoma, WA, USA).

Experimental results:

1.1 open field experiments

Open field experiments can be used to evaluate the voluntary activity and motor function of mice. The experimental results are shown in table 1, and there was no statistical difference in total distance traveled by each group of mice at the same time, indicating that administration did not significantly affect spontaneous locomotor activity and locomotor function.

TABLE 1 Total distance traveled by mice of each group over 5min

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Note that: ns indicates no statistical difference compared to the Control group (normal group). Data are presented as x±sem and multiple sets of comparative analysis are performed with ANOVA.

1.2 Morris water maze experiment

The Morris water maze can be used to evaluate mice for spatial perception and learning memory. As shown in fig. 4A, compared with the normal group, the model group mice have a disturbed swimming track and poor purposiveness, and take longer time to approach the platform, so that the mice can approach the platform more quickly after the drug treatment. As shown in fig. 4B and 4C, the model mice have long escape latency and less crossing of the target platform, indicating poor learning and memory ability, and improved after drug treatment, wherein the BTY-L effect is most remarkable.

Study 2: influence of the number of BTY on the Nib bodies of the hippocampus of model mice

Experimental materials:

chloral hydrate (lot number: C894539, shanghai Michlin Biochemical technologies Co., ltd.);

paraformaldehyde (lot number: C104188, shanghai Ala Biochemical technologies Co., ltd.);

absolute ethanol (lot number: 2201121, specification: 2.5L/barrel, sichuan Xie Co., ltd.);

xylene (lot number: 2201111, specification: 500 mL/bottle, sichuan Shang Sci Co., ltd.);

hematoxylin dye (lot number: CR2109146, specification: 500 mL/bottle, wohan Seville Biotechnology Co., ltd.);

toluidine blue (lot number: CR2104004, specification: 25g, wohan Seville Biotechnology Co., ltd.).

The experimental steps are as follows:

after the behavioural experiment is finished, the mice are anesthetized by 10% chloral hydrate, dissected, fully exposed, perfused with 15mL of physiological saline and 15mL of 4% paraformaldehyde fixing solution through the heart, and rapidly broken to take brains. The fixed tissue is dehydrated by a full-automatic dehydrator (dehydration time length: 75% alcohol 30h,85% alcohol 4h,95% alcohol 8h,100% alcohol 2h,100% alcohol 1h, xylene 45min, paraffin 5 h), embedded, and sliced as follows:

(1) Slide anti-slip processing: soaking with APES, taking out slices, and placing in an oven at 60deg.C for 60min to adhere slices tightly;

(2) Slice dewaxing to water, specific operation: xylene I5-10min, xylene II5-10min, absolute ethanol I5min, absolute ethanol II5min,95% ethanol 5min,85% ethanol 5min,75% ethanol 5min, and UP water soaking for 5min;

(3) Putting into 1% toluidine blue water solution preheated to 50 ℃, and dyeing for 20min in a 56 ℃ incubator;

(4) Washing with distilled water;

(5) Soaking in 70% alcohol for 1min;

(6) Differentiation of 95% alcohol, control under a mirror, and display definition in positive expression;

(7) Rapidly dehydrating absolute alcohol;

(8) Xylene is transparent and neutral gum is blocked. The above specimens were all processed according to the pathology test SOP procedure. Dehydrating, trimming, embedding, slicing, dyeing, sealing and the like, and finally performing microscopic examination and image acquisition.

Experimental results:

toluidine blue can dye Nib's bodies to dark blue, other tissues to light blue, and the background is colorless. As shown in the experimental results in FIG. 5, compared with the normal group, the model group has obviously reduced hippocampal neuronal Nib bodies, the low and high dose drug combination positive drug groups can increase the number of Nib bodies, and the low and high dose drug administration group is superior to the positive drug group.

Study 3: effect of BTY on expression of inflammatory factors IL-6, IL-1. Beta. And TNF-alpha

Experimental materials:

chloral hydrate (lot number: C894539, shanghai Michlin Biochemical technologies Co., ltd.);

PBS,1× (GP 21090010951 Whansai Weibull Biotech Co., ltd.);

mouse IL-6 ELISA Kit (Wuhan Eboltag Biotechnology Co., ltd.; cat# RK 00008);

mouse IL-1beta ELISA Kit (Wuhan Eboltag Biotechnology Co., ltd.; cat# RK 00006);

mouse TNF-alpha ELISA Kit (Wuhan Eboltake Biotechnology Co., ltd., cat# RK 00027)

The experimental steps are as follows:

after the behavioural experiment is finished, the mice are anesthetized with 10% chloral hydrate, dissected, fully exposed to the heart, perfused with about 20mL of physiological saline through the heart, and rapidly broken to take the brain. Taking left and right half brain hippocampus tissues for standby. Fresh brain tissue was weighed at about 60mg, the tissue was rinsed with pre-chilled PBS (0.01 m, ph=7.4), residual blood was removed (to avoid the effect of lysed erythrocytes in the homogenate on the measurement), and the tissue was minced after weighing. The sheared tissue is combined with a corresponding volume of PBS (typically 9mL of PBS for a 1:9 weight to volume ratio, such as 1g tissue sample, the specific volume can be adjusted appropriately as required by the experiment and recorded). Homogenizing (50 Hz,30s, repeating for 3 times) at-4deg.C, centrifuging at 5000r/min for 5-10min, and collecting supernatant.

Reagents and standards of different concentrations are prepared strictly according to the instruction of the kit, absorbance is detected at 450nm by using an enzyme-labeled instrument, a standard curve is drawn, and the concentration of the target protein is measured.

Experimental results:

the experimental results are shown in FIG. 6, in which the amounts of IL-6, IL-1. Beta. And TNF-. Alpha.in the model group were all significantly increased as compared to the normal group, while the amounts of inflammatory factors in the brains of mice in the low, high dose administration group and the positive drug group were significantly decreased as compared to the model group. It is shown that BTY can play a role in neuroprotection by down-regulating mouse brain tissue inflammatory factors, and the effect is equivalent to that of donepezil as a positive drug.

Study 4: protection of glutamate-injured HT22 cells by BTY

Experimental materials:

HT22 cells were from Sichuan university student's biological treatment national emphasis laboratory;

l-glutamic acid (Soy pal, #G0010);

CCK-8 (solebao, CA 1210);

DMEM high sugar medium (AG 29301810, hyclone, usa);

fetal bovine serum (20010401, gibco, usa);

penicillin-streptomycin solution (diabody) (20201220, hyclone company, usa);

PBS powder (WK 173618-1, beijing Zhongshan mountain bridge Biotechnology Co., ltd.);

DMSO (20201220, beijing Soy Bao technology Co., ltd.).

The experimental steps are as follows:

complete medium: DMEM high sugar medium, fetal bovine serum and penicillin-streptomycin solution (diabody) were mixed at 90:9:1, and uniformly mixing the components according to the volume ratio, and storing the components in a refrigerator at the temperature of 4 ℃.

Serum-free medium: DMEM high sugar medium and penicillin-streptomycin solution (diabody) were mixed at 99:1, and uniformly mixing the components according to the volume ratio, and storing the components in a refrigerator at the temperature of 4 ℃.

Glutamic acid stock solution: 0.147g of glutamic acid powder is weighed into a 50mL centrifuge tube, 20mL of serum-free culture medium is added, the mixture is dissolved by ultrasonic treatment, and a 0.22 mu m filter membrane is used for filtration, so that 60mM glutamic acid stock solution is obtained.

BTY stock: 111mgBTY powder was weighed and dissolved in 5mL of cell grade DMSO and filtered through a 0.22 μm filter to give a 100mM BTY stock solution.

HT22 cells in logarithmic phase cultured in complete medium were inoculated into 96-well plates at a density of 5X 103/100. Mu.L/well, and the peripheral wells were filled with sterile PBS, incubated at 37℃for 24 hours with 5% CO2 until the cells were completely adherent; the supernatant was discarded, and glutamic acid solutions of 0mM, 10mM, 20mM, 30mM and 40mM were added to the supernatant, and the mixture was cultured at 37℃for 24 hours under 5% CO 2; then 110. Mu.L of 10% CCK-8 solution was added to each well, and the mixture was incubated at 37℃for 2 hours under 5% CO 2; the OD value at 450nm is detected by an enzyme-labeled instrument, the activity of HT22 cells is calculated, and the concentration of glutamic acid corresponding to the cell viability of about 60% under the experimental condition is screened out according to the formula of 'cell viability= [ (average absorbance value of experimental group-average absorbance value of zeroing hole)/(average absorbance value of normal control group-average absorbance value of zeroing hole ]', so that the concentration is selected as the damage concentration in the subsequent cell experiment and used for the following cell pharmacodynamics experiment.

HT22 cells in logarithmic phase cultured in complete medium were inoculated into 96-well plates at 5X 103/100. Mu.L/well, and the peripheral wells were filled with sterile PBS and cultured at 37deg.C under 5% CO2 for 24 hr until the cells were completely adherent; the supernatant was discarded, and 80. Mu.L of BTY stock diluted with serum-free medium at different concentrations (to give final concentrations of 2.5. Mu.M, 5. Mu.M, 12.5. Mu.M, 25. Mu.M) was added to each of the other dosing groups except the control group and model group. After 2 hours of incubation, glutamic acid solution diluted with serum-free medium was added to each of the above-mentioned administration groups at a final concentration of 20mM, and incubation was continued at 37℃for 24 hours under 5% CO2 at 40. Mu.L per well. The control group was only added with the same volume of serum-free medium; the model group was sequentially added with 80. Mu.L of serum-free medium and 40. Mu.L of glutamic acid solution diluted with serum-free medium to a final concentration of 20 mM; the control and model groups were otherwise identical to the dosing group. After 24h, a picture of the cells was taken under an inverted microscope, followed by addition of 110. Mu.L of 10% CCK-8 solution per well, and incubation at 37℃for 2 hours under 5% CO 2; OD values at 450nm were measured using an ELISA reader and the results are shown in FIG. 7.

Experimental results: as shown in fig. 7, after the model group cells are incubated with glutamic acid, the cells shrink, the adherence is not firm, and the cell viability is obviously reduced; the cell morphology and survival rate were significantly improved after treatment with different concentrations of BTY. The BTY has good neuroprotection.

The foregoing examples merely represent specific embodiments of the present application, which are described in more detail and are not to be construed as limiting the scope of the present application. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present application, which fall within the protection scope of the present application.

Claims (8)

1. The application of BTY in the preparation of medicines for treating or preventing dementia caused by brain diseases is characterized in that the BTY is Benzene,1,2, 4-tri-methoxy-5- (2-methyl-1-propen-1-yl) and the structural formula is shown in the formula I:

   
2. 2. the use according to claim 1, wherein the brain diseases are alzheimer's disease and cerebrovascular diseases.
3. 3. The use according to claim 1, wherein the ratio of the effective amount of BTY as an active ingredient in the medicament to the unit mass of the human body is 1.0 mg-10.0 mg/kg.
4. 4. The use according to claim 3, wherein the ratio of the effective amount of BTY as an active ingredient in the medicament to the unit mass of the human body is 1.5mg to 4.0mg/kg.
5. 5. The use according to claim 1 or 3 or 4, wherein the medicament is an injection or an oral formulation.
6. 6. The use according to claim 5, wherein the injection is an emulsion.
7. 7. The use according to claim 6, wherein the emulsion comprises the following components in percentage by weight: BTY 0.5-5%, oil phase 5-20%, emulsifier 0.6-1.8%, glycerin 0-2.5%, and the balance being purified water or water for injection.
8. 8. The use according to claim 7, wherein the emulsion is prepared by:

   (1) Respectively weighing the BTY and auxiliary materials in the prescription amount;

   (2) Under the protection of nitrogen or other inert gases, dissolving BTY in an oil phase preheated to 60-80 ℃, dissolving or dispersing glycerol in an aqueous phase preheated to 60-80 ℃, and dissolving an emulsifier in the oil phase or the aqueous phase;

   (3) Mixing the oil phase and the water phase by high-speed shearing to uniformly disperse the oil phase in the water phase, thus preparing the colostrum;

   (4) Homogenizing the colostrum for 2-4 times under high pressure to ensure that the average particle size of emulsion drops is not more than 0.5um, and filtering to obtain the drug-carrying emulsion containing BTY; under the protection of nitrogen, the emulsion is obtained by filling the emulsion in a container suitable for medical use and hot-pressing and sterilizing in a proper mode according to the requirement of the administration route.

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