CN116421589A

CN116421589A - Application of Mito Q and curcumin in treating AD

Info

Publication number: CN116421589A
Application number: CN202211534185.5A
Authority: CN
Inventors: 都秀波; 乐霖枫; 李雪霞
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2022-12-02
Filing date: 2022-12-02
Publication date: 2023-07-14

Abstract

The invention provides an application of Mito Q and curcumin in treating AD. We have found that Mito Q has a synergistic effect with curcumin for the treatment of Alzheimer's disease.

Description

Application of Mito Q and curcumin in treating AD

Technical Field

The invention relates to the field of medicines, in particular to application of Mito Q and curcumin in treating AD.

Background

Alzheimer's Disease (AD) was first discovered and named by Alois Alzheimer in 1906, an age-related neurodegenerative disease that is clinically manifested as memory decline, cognitive decline, and the like. As a major type of senile dementia, AD has extremely high morbidity and mortality in the elderly, and the morbidity increases with age.

The causes of AD are complex, senile plaques formed by aggregation of Abeta protein, nerve fiber tangles formed by hyperphosphorylation aggregation of tau protein, reduced synapse number, neuron deficiency, oxidative stress, mitochondrial injury, autophagy disorder and the like are formed, and the course of disease is long, so that no medicine capable of fundamentally treating AD is available at home and abroad at present, and therefore, the search of specific medicine for AD is urgent. The pathogenesis of AD is very complex and the mechanisms interact with each other, and the effect of intervention from a single target is often poor.

Curcumin is polyphenol compound extracted from rhizome of Curcuma longa, and has chemical formula of C ₂₁ H ₂₀ O ₆ . Mito Q, chemical formula C ₃₇ H ₄₆ O ₄ P.CH ₃ O ₃ S is an analog of coenzyme Q. Coenzyme Q is present in the mitochondria of all living cells and is an important carrier of the respiratory chain of the inner mitochondrial membrane (protons h+ and electrons). C compared with common coenzyme Q ₃₇ H ₄₆ O ₄ P.CH ₃ O ₃ S has positive charge and can directly enter mitochondria with negative charge, C ₃₇ H ₄₆ O ₄ P.CH ₃ O ₃ S crosses mitochondria hundreds of times more efficiently than common coenzyme Q. At present, the New Zealand company has developed a main drug effect component of C ₃₇ H ₄₆ O ₄ P.CH ₃ O ₃ Drug Mito Q of S has been successfully marketed.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide an application of Mito Q-compatible curcumin in the treatment of alzheimer's disease, for solving the problem that the treatment of alzheimer's disease in the prior art lacks an effective means.

One aspect of the invention provides C ₃₇ H ₄₆ O ₄ P.CH ₃ O ₃ S or pharmaceutically acceptable salt thereof is matched with curcumin or pharmaceutically acceptable salt thereof in preparation of medicines for treating Alzheimer disease.

Further, the medicament may be a tablet, a sugar-coated tablet, a film-coated tablet, an enteric-coated tablet, a capsule, a hard capsule, a soft capsule, an oral liquid, a buccal agent, a granule, a pill, a powder, a paste, a pellet, a suspension, a powder, a solution, an injection, a suppository, an ointment, a plaster, a cream, a spray, a drop, a patch, or the like; preferred are oral dosage forms, such as: capsule, tablet, oral liquid, granule, pill, powder, pellet, or unguent.

Further, the pharmaceutically acceptable salt is an inorganic salt or an organic salt.

Further, the pharmaceutically acceptable salt is C ₃₇ H ₄₆ O ₄ P.CH ₃ O ₃ S or salts of curcumin with acids.

Further, the acid is any one of inorganic acid such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, aspartic acid, and glutamic acid.

Further, the medicament has one or more of the following effects:

a) Relieving anxiety;

b) Improving exploration and cognitive ability;

c) Enhancing memory;

d) Lowering blood glucose levels;

e) Lowering oligomer levels, sappβ, and BACE1 levels;

f) Reducing hyperphosphorylation and neurofibrillary tangles of Tau protein;

g) Protecting tissue synaptoproteins and neuronal activity.

In another aspect, the invention provides a medicament for treating Alzheimer's disease, comprising a therapeutically effective amount of C ₃₇ H ₄₆ O ₄ P.CH ₃ O ₃ S or a pharmaceutically acceptable salt thereof, and curcumin or a pharmaceutically acceptable salt thereof.

Further, the medicament has one or more of the following effects:

a) Relieving anxiety;

b) Improving exploration and cognitive ability;

c) Enhancing memory;

d) Lowering blood glucose levels;

e) Lowering oligomer levels, sappβ, and BACE1 levels;

f) Reducing hyperphosphorylation and neurofibrillary tangles of Tau protein;

g) Protecting tissue synaptoproteins and neuronal activity.

Further, the medicine also contains a carrier acceptable to human bodies.

Further, the carrier comprises: saline, buffers, dextrose, water, glycerol, ethanol, powders, and combinations thereof.

The compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions.

The medicament, formulation or pharmaceutical composition of the invention may be administered to a subject in need thereof (e.g., human and non-human mammals) by conventional means. Representative modes of administration include (but are not limited to): one or more routes of administration, such as oral or injectable (including one or more of intravenous, intravenous drip, intramuscular or subcutaneous injection, etc.). When used, the pharmaceutical composition is administered to a mammal in a safe and effective amount. Of course, the particular dosage and method will also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

As described above, the Mito Q compatible curcumin has the following beneficial effects:

our study found C ₃₇ H ₄₆ O ₄ P.CH ₃ O ₃ The S-compatibility curcumin has better treatment effect on Alzheimer disease and can play a synergistic effect.

Drawings

FIG. 1 open field experiment. A. The number of lattices passed by the mice; B. number of standing times of mice; c number of times the mice urinate. (n=10, < p <0.05, < p <0.01, < p <0.001,: vs AD)

Fig. 2 overhead plus maze experiment. A. E, the time when the mice enter the open arms for the first time and close the arms; B. f, the total path of the movement of the mice on the open arms and the closed arms; C. g, total time that the mice stay in the open arms and the closed arms; D. h, number of times mice entered open arm, closed arm. (n=10, < p <0.05, < p <0.001,: vs AD)

Fig. 3 new object recognition experiment. A. Total number of searches for each group of mice during familiarity; B. the resolution of the old and new objects by the mice during the test period. (n=10, < p <0.05, < p <0.01, < p <0.001,: vs AD)

Fig. 4Y maze experiment examined spontaneous alternation rate of mice behavior (n=10, < p <0.05, < p <0.01, < p <0.001,: vs AD). The scenario fear experiments of fig. 5 detect the ability to correlate between learning, memory unpleasant experiences and environment. A. Time of congeal of mice during the association test; B. the congealing time of the mice during the change-associated test; C. the time of congeal in mice during auditory cue test. (n=10, < p <0.05, < p <0.01,: vs AD)

Figure 6Morris water maze examined the spatial learning and memory ability of mice. A. Positioning the escape latency of the mice during voyage; B. average swimming speed of each group of mice; C. swimming time of each group of mice in the quadrant where the original platform is located during short-term and long-term memory test; D. the number of platform crossings during short-term and long-term memory testing in each group of mice. (n=10, < p <0.05, < p <0.01, < p <0.001, & p <0.05, @ p <0.05, #p < 0.01)

FIG. 7 detection of biochemical indicators in mice. A. The level of blood lipid-related biochemical indicators TG, apoB and TC/CHO; B. levels of liver-related biochemical indices ALT and AST; C. levels of kidney-related biochemical indicators UREA and Cre-P; D. levels of pancreas-related biochemical index AMY; E. blood glucose level. (n=10, < P <0.05, < P <0.01,: vs AD)

FIG. 8 detection of A beta pathology-related proteins in mouse brain tissue. AWestern Blot detects the expression level of mouse hippocampal Abeta oligomer, APP-beta and BACE1 protein; gray value analysis of the B mouse hippocampal Abeta oligomer, APP-beta and BACE1 protein; c Western Blot detects the expression level of Abeta oligomer, APP-beta and BACE1 protein in the mouse cortex area; d, analyzing gray values of Abeta oligomers, APP-beta and BACE1 proteins in the cortex area of the mice; e immunofluorescence the expression levels of aβ in the cortex and hippocampal region of each group of mice were examined. (n=4, < P <0.05, < P <0.01, < P <0.001, < P <0.0001, bar:200 μm)

FIG. 9 detection of tau pathology-related proteins in mouse brain tissue. Detection of mouse hippocampal phosphorylated tau protein p-S202 by A Western Blot

Expression levels of p-T231 and p-S404; gray value analysis of B mouse hippocampal phosphorylated tau proteins p-S202, p-T231 and p-S404; c Western Blot detects expression levels of phosphorylated tau proteins p-S202, p-T231 and p-S404 in the cortical region of the mouse; gray scale analysis of phosphorylated tau proteins p-S202, p-T231 and p-S404 in cortical areas of D mice; e silver glycine immersion plating nerve staining kit detects the content of the NFT in the cortex and hippocampus of each group of mice. (n=4, < P <0.05, < P <0.01, < P <0.001, < P <0.0001, bar:50 μm)

FIG. 10 mouse brain tissue synaptoprotein levels and Nib body detection. A Western Blot detects the expression levels of mouse hippocampal synapse-related proteins Syna, syn1 and PSD 95; gray value analysis of proteins of hippocampal Syna, syn1 and PSD95 of B mice; c Western Blot detects expression levels of synapse-related proteins Syna, syn1 and PSD95 in a mouse cortex region; gray value analysis of Syna, syn1 and PSD95 proteins in the cortical areas of the D mice; e, detecting nikohlrabi of the brain tissue of the mouse by nikohlrabi staining; statistical analysis of mice brain tissue nikovia. (n=4, < P <0.05, < P <0.01, < P <0.001, < P <0.0001, bar:100 μm)

Detailed Description

The subject selects B6.129-PS1M146V/APPSwe/Tau P301L triple transgenic AD model mice (3 xTg AD mice) cultivated in Jackson laboratories in the United states and non-transgenic mice (Wli Type mice, WT) with the same genetic background as experimental subjects, and carries out pharmacodynamics study on the anti-AD effect of Mito Q-compatible curcumin.

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention. It should be understood that the process equipment or devices not specifically identified in the examples below are all conventional in the art. Furthermore, it is to be understood that the reference to one or more method steps in this disclosure does not exclude the presence of other method steps before or after the combination step or the insertion of other method steps between these explicitly mentioned steps, unless otherwise indicated; it should also be understood that the combined connection between one or more devices/means mentioned in the present invention does not exclude that other devices/means may also be present before and after the combined device/means or that other devices/means may also be interposed between these two explicitly mentioned devices/means, unless otherwise indicated. Moreover, unless otherwise indicated, the numbering of the method steps is merely a convenient tool for identifying the method steps and is not intended to limit the order of arrangement of the method steps or to limit the scope of the invention in which the invention may be practiced, as such changes or modifications in their relative relationships may be regarded as within the scope of the invention without substantial modification to the technical matter.

MitoQ is given to New Zealand Biotechnology, CAS:845959-55-9,Product Code:71700. We performed experiments with pure compounds.

We used curcumin at a dose of 100 mg/kg/d and Mito Q at 25 mg/kg/d. The mice groups were set up as in table 1.

TABLE 1

Curcumin is mixed into the feed of mice, mito Q is dissolved into drinking water of the mice, and the mice are treated by adopting a free feeding and drinking mode. Each group of mice was dosed starting at july age and was tested behaviourally five months after dosing.

Example 1 open field experiment

Also known as open-box experiments, a method for evaluating the autonomous behavior and exploring the behavior and the tension of experimental animals in a new and different environment is widely used: the animals were placed in the center of the bottom in the box and simultaneously imaged and timed. After 3min of observation, the imaging was stopped. And cleaning the inner wall and the bottom surface of the square box to prevent the information (such as the urine, the urine and the smell of the animal) remained by the last animal from affecting the next test result. The animals were changed and the experiment continued. And (3) observing the indexes: number of checks crossed by animals per unit time, number of rearing limbs, number of urination and defecation.

As shown in fig. 1, AD group mice showed a decrease in the number of passes through the lattice within 180s compared to WT group mice, indicating that AD group mice had weaker spontaneous locomotion than WT group mice; the standing times of the AD mice are reduced, which indicates that the exploration of the outside things by the AD group mice is reduced; increased stool frequency indicated an increased anxiety in AD mice. Mito Q increases the number of crossing lattices of AD mice, and curcumin reduces the number of defecation times of AD mice. The Mito Q compatibility curcumin greatly increases the crossing grid number and standing times of the AD mice and reduces the defecation times of the AD mice.

Example 2 overhead maze test

The elevated plus maze has a pair of open arms and a pair of closed arms. Mice were allowed to move in the open arms for curiosity and exploratory in fresh environment; however, the elevated plus maze is relatively high from the ground, which is equivalent to a person standing on a cliff, and mice tend to move in the closed arms due to fear and darkness of the highly suspended open arms. Before the experiment, in order to increase the total number of times of putting the mice into arms and avoid the mice to be hidden in closed arms, the mice are firstly put in an open field (open field) for adaptation for 3min and then put into the vagus palace. The mice were placed into the maze from the central grid towards the closed arms at the beginning of the experiment and activity was recorded within 3 minutes. After the experiment is completed, the mice are taken out, the two arms are cleaned up, and alcohol is sprayed to remove smell. The number of times of entering the open arms and the residence time are inversely related to the anxiety emotion of the mice, and the smaller the number of times of entering the open arms, the shorter the residence time, which indicates that the anxiety emotion of the mice is more serious. Notice that: the mice are fully touched before the experiment, and animals have to be completely adapted to experimenters and experimental environments without fear; if the animal falls to the ground, the animal is preferably removed; the experimenter was 1 meter from the maze.

As shown in fig. 2, the experimental results show that, compared with the WT mice, the AD mice have longer time to enter the open arms for the first time, less times to enter the open arms, and longer residence time in the closed arms, which indicates that the exploratory performance of the AD mice in the new and abnormal environment is obviously reduced and the anxiety emotion is serious. Mito Q-compatible curcumin reduced the time for AD group mice to first enter the open arm and increased the number of times to enter the open arm.

Example 3 New object identification experiment

New object identification experiments utilize the exploratory nature of mice on fresh objects to test the memory capacity of mice. The new object recognition experiment was less irritating to mice than the water maze.

Mainly consists of 3 stages: adaptation period, familiarity period, and testing period. There are three objects a, B, C, where the a, a 'object is identical and the B object is completely different from the a, a' object.

1) The adaptation period is as follows: the mice are placed in the experimental device without any object for 3min in sequence, so that the mice can be freely explored to adapt to the experimental environment, and the stress of the animals in the experimental process is reduced as much as possible.

2) Familiarity period: two objects A and A' are placed at the left end and the right end of one side wall, the mouse is placed in the field back to the two objects, and the lengths of the nasal tips of the mouse from the two objects are consistent. The mice were placed for 3min, the video recording device was turned on immediately after placement, the experimenter left the test room immediately, and the contact of the mice with the two objects was recorded, including the number of times the nose or mouth touched the object and the number of times the object was explored in the range of 2-3cm away (the front paws were strapped on the object, the nose sniffed the object, licked the object, etc. all belonged to the explored object, and the swinging of the frame or climbing onto the object could not be counted as an explore of a new object).

3) The test period is similar to the familiarity period except that a' in two identical objects is replaced with another, different object B, and the objects during the two object test periods relative to the familiarity period are referred to as the familiarity object a and the novelty object B, respectively. In order to prevent experimental errors caused by special preference of experimental animals on a certain object or a certain position during the experiment in the test period, the positions of a familiar object A and a novel object B are exchanged when the mice are sequentially subjected to the experiment. In addition, in order to exclude the influence of odors, the objects and the laboratory boxes should be cleaned in time. The most basic index in the object recognition experiment is the total exploring times of each group of mice in the familiarity period and the resolution ratio of new and old objects in the test period, wherein the "resolution ratio" is generally expressed by DR, and a specific calculation formula is as follows: dr=n/(n+f), "N" represents the number of times the animal explores the novelty object during the test period, and "F" represents the number of times the animal explores the familial object during the test period.

As shown in fig. 3, the experimental results showed that AD group mice had reduced exploration numbers for both subjects during familiarity (as in fig. 3 a) compared to WT group mice, indicating reduced curiosity and desire to explore for fresh things. Meanwhile, the resolution ratio of the AD group mice to the new and old objects is reduced (as shown as B in fig. 3) in the test period, which shows that the memory capacity of the AD group mice is obviously reduced. The Mito Q and Mito Q compatible curcumin respectively and remarkably and extremely remarkably increase the exploring times of AD group mice on two objects in the familiarity period and the distinguishing ratio of the AD group mice on new and old objects in the testing period.

Example 4Y maze experiment

The Y maze test utilizes the natural exploration habit of mice and curiosity of the novel environment to detect the autonomous exploration and memory capacity of the mice. The Y labyrinth device consists of three identical and communicated arms (1), (2) and (3), wherein the size of each arm is 30cm multiplied by 8cm multiplied by 15cm (length multiplied by width multiplied by height), and the included angle of each arm is 120 degrees. The experimental mice were placed at the end of either arm before the start of the experiment, the order of entry of each mouse into the three arms (1), (2), (3) was observed and recorded for 3min, and finally the number of alternations, such as (1) (2) (3), (3) (1) (2), (2) (3) (1) and so on, was counted. To reduce the operating errors, after each mouse experiment was tested, the experimental device was wiped with alcohol to eliminate the odor from interfering with the experiment.

The working memory capacity of mice was examined by the Y maze and spontaneous alternation behavior was defined as continuous entry into three different arms. Spontaneous alternation rate = number of spontaneous alternation actions/(total number of arm advances-2) ".

As seen from the results of fig. 4, AD mice had a reduced spontaneous alternation rate compared to WT mice, and either curcumin alone or Mito Q alone significantly increased spontaneous alternation rate in AD mice. Mito Q-compatible curcumin significantly increased spontaneous alternation rate in AD mice.

Example 5 scene fear experiments

The scenario fear test is the ability to determine the association between animal learning, memory unpleasant experiences and the environment. Pairing with sound or aversive stimuli (e.g. foot shock) occurs, mice learn that there is a link between sound and shock (auditory cue condition fear), and that there is some link between shock and surrounding environment (association condition fear). When the mouse animals experience fear, a phenomenon of immobility (stagnation) occurs to defend. Stagnation is considered a reliable indicator for evaluation of rodent fear. Normally, the mice have much more congealing behavior in auditory cues than in association tests, and in modification of association tests, the congealing behavior is minimal because the association condition box has been modified to be "completely non-planar" and the association of the mice to the surrounding environment is almost absent. If the mice were to exhibit congealing in the association test and auditory cue test the next day, but not in the change association test, it could be assumed that: the sense and the motor function of animals are normal; can remember the implication signal that the previous day appears paired with the aversive stimulus; the implication signal that the previous day did not appear in pairs with aversive stimuli can be resolved, indicating that the animal has normal memory. Any of these tests increases or decreases suggesting that the neuroanatomy, neurotransmitters and genes that regulate the affective portion of memory may be altered.

1) Training phase (first day)

The instrument was commissioned to ensure that the grid floor had current stimulus and the microphone had sound stimulus, and current intensity and sound intensity (db) were recorded, respectively. Mice were placed in a conditional fear box for 2min, and the time to arrest of the animals was recorded as baseline over the first 2min. Next, a click sound, 80D b,30s, was added. Immediately after the shock was delivered, 0.35mA,2s. No stimulation was followed for 20s. The animal lag time(s) was recorded throughout the training phase to measure the unconditional fear of the animals. The mice were removed from the control box and the control box was thoroughly cleaned with 75% alcohol in preparation for the training of the next animal.

2) The test phase is performed the next day after training, including association testing, altering associations, and conditional stimulus testing. There was no shock stimulus during the test phase. There was also no auditory condition stimulus in the correlation test and the change correlation test, but the same control box was used for each animal as for the training phase.

a. And (3) association test: the animals were placed in the box for 2min and the computer automatically recorded the animal's stagnation behavior. The fear of the relevant condition of the animal was measured using the time of stagnation of the animal in the same operating box recorded at this stage. This is the association test. After the observation is finished, the animals are put back into the cage, the operation box is cleaned by 75% alcohol, and the next animal is observed.

b. Altering the association test and the auditory condition stimulus test: the association test was performed after 1 h. The operation is as follows:

the computer is tuned to the desired program. The operation box is modified as follows: replacing the grid floor in the associated condition box with a smooth plastic plate; a colored plastic plate is added on the diagonal line of the box, so that the cuboid operation box is changed into a triangle; change olfactory cues (olfactor cues), clean the control box thoroughly with 4% acetic acid solution. The change association and auditory condition stimulation experiments are then initiated. Animals were placed in the modified association box (starting from the first animal in the association experiment). First, no stimulus was applied for 2min. Auditory condition stimulation was then added for 2min. And then no stimulation for 60s. Animals were returned to the cage and the control box was cleaned with 4% acetic acid solution for testing of the next animal.

It was found (see fig. 5) that mice in AD group showed reduced clotting behavior (as in a, fig. 5C) in the association test and auditory cue test and increased clotting behavior (as in B, fig. 5) in the change association test compared to mice in WT group, indicating that mice in AD group had some impairment in sensation, motor and memory. Curcumin increased the stagnating behavior of AD mice in the association test, mito Q increased the stagnating behavior of AD mice in the association test and auditory cue test. Mito Q-compatible curcumin significantly increased the clotting behavior of AD group mice in both the association test and the auditory cue test, and reduced the clotting behavior of AD group mice in the altered association test.

EXAMPLE 6Morris Water maze experiment

The Morris water maze (Morris water maze, MWM) device is a cylindrical pool of 160cm in diameter and 50cm in height. Before the experiment, water was added to the tank to about 26cm, and the water temperature was maintained at 20.+ -. 1 ℃. A video device is arranged above the water tank, and the periphery of the water tank is shielded by a curtain to prevent the influence of the surrounding environment on the mice. The pool is artificially divided into four quadrants, and a circular platform with a height of 1-2cm lower than the water surface is placed in one quadrant at a position 30cm away from the pool wall, wherein the diameter of the platform is 12cm.

1) Positioning navigation: positioning and sailing for 4 days, carrying out experiments at the same time every day, storing the mice on a platform for 10s before the experiments, then putting the mice into water from the opposite quadrant to the pool wall, and recording the time for the mice to find the platform. At time limit 60s, if the mouse cannot find the platform within 60s, the mouse is placed on the platform again for memorizing for 10s, and finally the mouse is wiped dry and placed back into the mouse cage.

2) Space exploration: space exploration experiments were performed the next and fourth days after the end of the positioning voyage. The platform was removed before the experiment, and the number of times the mouse passed through the position of the original platform and the time of stay in the quadrant of the platform within 120s were recorded as the short time (24 h) and long time memory (72 h) of the mouse to the position of the platform. The movement of the mice was recorded using a SMART v3.0 video tracking system and data analysis was performed using Graphpad Prism 7.0 software.

As shown in fig. 6, in the positioning voyage test, the escape latency of AD group mice was significantly longer than WT mice from the third day, indicating a decrease in spatial learning ability of AD mice. Mito Q and curcumin reduced the escape latency of AD mice from day three and day four, respectively. Whereas Mito Q-compatible curcumin reduced escape latency in AD mice from the next day. The swimming speeds of the mice in each group are not different, which indicates that the escape latency is caused by the learning and memory ability of the mice and is irrelevant to the exercise ability.

The short-term memory test and the long-term memory test of the mice are respectively carried out 24 hours and 72 hours after the positioning navigation experiment is finished. Short term (24 h) memory findings for each group of mice were examined: within 60s, the swimming time of the mice in the curcumin-compatible Mito Q treatment group in the effective quadrant and the frequency of crossing the position of the original platform are obviously more than those of AD mice, and the effect is better than that of independent curcumin and Mito Q.

Example 7 detection of blood lipid and blood glucose levels

Taking blood from eyeballs of mice, and measuring blood sugar by using a kefu glucometer; the supernatant was centrifuged at 3000r for 15min and the blood lipid level was measured using an Imagic-M7 biochemical analyzer.

As shown in fig. 7, the level of Triglyceride (TG) was elevated in AD mice compared to WT mice. Mito Q and Mito Q compatible curcumin respectively remarkably and extremely remarkably reduce the TG content of AD mice. There was no significant difference in the levels of apolipoprotein B (ApoB) and total cholesterol (Total Cholesterol, TC/CHO) between the groups of mice. In each group of mice, there was no significant difference in the levels of glutamic pyruvic transaminase (Alanine transaminase, ALT) and glutamic oxaloacetic transaminase (Aspartate transaminase, AST), the levels of UREA (UREA) and Creatinine (cretinine, cre-P), the biochemical indicators associated with kidney metabolism, and the levels of Amylase (AMY) that reacted with pancreatic function. Three groups of drugs were suggested to be non-toxic to the liver, kidney and pancreas of mice. Interestingly, both curcumin and Mito Q significantly reduced blood glucose levels in AD mice, whereas Mito Q-compatible curcumin can significantly reduce blood glucose levels in AD mice.

Example 8A beta oligomer and fiber aggregate levels

western blot experiment: preparing a separating gel, and gelling for 30min; preparing concentrated glue, quickly inserting a comb after the concentrated glue is poured into a glue plate, and gelling for 30min. Adding 3-5 mu L of protein marker or 10-20 mu g of protein sample into each hole, and carrying out 150V voltage electrophoresis to the bottom of the gel; electrotransport transfer of proteins to PVDF membranes; sealing the transferred PVDF film by using 5% skimmed milk; adding primary antibody, standing overnight at 4 ℃, and washing 3 times by TBST; adding a secondary antibody, incubating for 2 hours at room temperature, and washing 3 times by using TBST; and (5) developing.

Immunofluorescence experiments on frozen tissue sections: firstly, washing brain slices for 2 times by using PBS, fixing 4% PFA for 20-30min, and washing the brain slices for 2 times by using PBS; punching with 0.2% Triton X-100 (PBS) for 10min; adding a sealing liquid to seal for 1.5 hours at room temperature; adding a primary antibody, incubating at 4 ℃ overnight, and washing 3 times with PBS; adding a secondary antibody, and incubating for 1.5h at room temperature; PBS was washed 3 times; adding DAPI for dyeing for 4min, and washing with PBS for 3 times; and sealing the anti-fluorescence quenching agent into a piece and preventing light. And observing by a confocal microscope.

The massive production and aggregation of aβ to form amyloid plaques is one of the main pathological features of AD, and among the various aggregated forms of aβ, soluble oligomers are considered as the most toxic form. Aβ is produced by cleavage of APP by BACE1 and gamma secretase, while also producing the soluble cleavage fragment sAPPβ; the expression levels of BACE1 and sAPPβ reflect the pathological course of Aβ production.

As shown in fig. 8, the AD group mice had significantly elevated levels of aβ oligomers, sappβ, and BACE1 in the hippocampus and cortex of the brain as compared to the WT group. Curcumin alone reduced the levels of AD murine hippocampal 4.5×,6× aβ oligomers and BACE1 and reduced the levels of cortical 6×,18× APP- β, but had no significant effect on hippocampal 4×,10×,13×,23× APP- β, and cortical BACE1 levels. Mito Q alone reduced levels of hippocampal 4.5×,6×,23× Abeta oligomers and BACE1 and reduced levels of cortical 6×,18× APP-beta, but had no significant effect on hippocampal 4×,10×,13× APP-beta, and cortical BACE1 levels. Whereas Mito Q is compatible with curcumin, the levels of Abeta oligomers, sAPP beta and BACE1 of mice treated with curcumin are significantly reduced. And compared to curcumin alone, mito Q-compatible curcumin-treated AD mice had significantly reduced levels of 6 x Abeta oligomers and BACE1, and 6 x, 18 x Abeta oligomers, APP-beta and BACE1 in the cerebral cortex region. Compared to mitoq alone, the level of the 6 x aβ oligomers, and APP- β and BACE1 levels in the cortical areas were significantly reduced in the mitoq-compatible curcumin-treated AD mice brainhaima. In addition, AD groups have a greater amount of aβ plaque deposition than WT groups; the cortical astrocytes are activated to some extent and are not tightly surrounded by plaque; curcumin group and Mito Q group Abeta plaque reduction; the extent of astrocyte activation in the cortical areas was reduced and most plaques in the Mito Q group were tightly surrounded by astrocytes; the aβ plaques in the curcumin + Mito Q group almost completely disappeared, approaching the WT group.

Example 9 mouse brain Tau pathological index

Hyperphosphorylation of Tau protein and formation of neurofibrillary tangles (NFT) are another important pathological feature of AD.

As shown in FIG. 9, the levels of p-S202, p-T231 and p-S404 were significantly elevated in the hippocampal and cortical areas of mice in the AD group compared to the WT group, and curcumin or Mito Q alone reduced the levels of p-S202 and p-S404 and p-S202, p-T231 and p-S404 in the hippocampal and cortical areas of mice, but had no significant effect on the levels of p-T231 in the hippocampal areas. Whereas the levels of p-S202, p-T231 and p-S404 were significantly reduced in Mito Q in combination with curcumin in brain hippocampus and cortex of mice. And compared with curcumin alone, the levels of brain hippocampus p-S202 and p-S404, and the levels of p-S202, p-T231 and p-S404 in the cerebral cortex region of the AD mice treated with Mito Q compatible curcumin are significantly reduced. The levels of p-S202, p-T231 and p-S404 were significantly reduced in both brain hippocampus and cortical areas of the Mito Q-compatible curcumin-treated AD mice compared to Mito Q alone. In addition, the levels of NFT in the cerebral cortex region and in the brain hippocampus CA1, CA3 and DG regions of mice in AD groups were significantly increased, and the levels of NFT in each brain region of mice in AD groups were significantly reduced by both three groups of drugs. Mito Q-compatible curcumin reduced NFT better than curcumin alone or Mito Q, and treated mice had similar levels of NFT in each brain region to WT group mice.

EXAMPLE 10 protection of Compound against synaptoprotein and neuronal Activity in mouse hippocampal tissue

Neurofibrillary tangle staining: the experimental steps are strictly carried out according to the specification of a Servicebio glycine silver staining kit.

Nib staining: the experimental procedure was strictly followed by the Solarbio Niy staining kit instructions.

As shown in fig. 10, the postsynaptic compact protein PSD95 level of the brain hippocampus, and the levels of the cortical synaptosin Syna, syn1 and PSD95 were significantly reduced in AD mice compared to WT mice. While the expression levels of hippocampal Syna and Syn1 did not change significantly. Curcumin alone or Mito Q increased levels of Syn1 and PSD95 in the cortical areas of AD mice, but had no significant effect on levels of hippocampal Syna, syn1 and PSD95, as well as Syna in the cortical areas. Mito Q-compatible curcumin increased levels of PSD95 in the brain hippocampus region of AD mice and levels of Syna, syn1 and PSD95 in the cerebral cortex region. The levels of brain hippocampus PSD95, as well as the levels of Syna, syn1 and PSD95 in the cortical areas were significantly elevated in Mito Q-compatible curcumin-treated AD mice compared to curcumin alone. Both Syna and PSD95 levels were significantly elevated in brain cortex areas of the Mito Q-compatible curcumin-treated AD mice compared to Mito Q alone. The number of Nib bodies in neurons is positively correlated with the state and viability of neurons. As shown, the number of nikovia bodies in the cortical and hippocampal CA3 and DG regions of the AD group was significantly reduced. Curcumin alone or Mito Q increases the number of Nib bodies in the cortical and hippocampal DG regions, whereas Mito Q in combination with curcumin increases the number of Nib bodies in the cortical and hippocampal CA3 and DG regions.

Through the experiments, the compound medicine has obvious influence on the key indexes for treating AD, and the compound medicine can produce synergistic effect when being used simultaneously.

The above examples are provided to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, many modifications and variations of the methods and compositions of the invention set forth herein will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the present invention.

Claims

1.C ₃₇ H ₄₆ O ₄ P.CH ₃ O ₃ S or pharmaceutically acceptable salt thereof is matched with curcumin or pharmaceutically acceptable salt thereof in preparation of medicines for treating Alzheimer disease.

2. Use according to claim 1, characterized in that: the medicine is any one of tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquid, buccal agents, granules, medicinal granules, pills, powder, ointment, pellets, suspension, powder, solution, injection, suppositories, ointments, plaster, cream, spray, drops and patches.

3. Use according to claim 1, characterized in that: the pharmaceutically acceptable salt is an inorganic salt or an organic salt.

4. Use according to claim 3, characterized in that: the pharmaceutically acceptable salt is C ₃₇ H ₄₆ O ₄ P.CH ₃ O ₃ S or salts of curcumin with acids.

5. Use according to claim 4, characterized in that: the acid is any one of inorganic acid such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid and the like, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, aspartic acid and glutamic acid.

6. Use according to claim 1, characterized in that: the medicament has one or more of the following effects:

a) Relieving anxiety;

b) Improving exploration and cognitive ability;

c) Enhancing memory;

d) Lowering blood glucose levels;

e) Lowering oligomer levels, sappβ, and BACE1 levels;

f) Reducing hyperphosphorylation and neurofibrillary tangles of Tau protein;

g) Protecting tissue synaptoproteins and neuronal activity.

7. A medicament for treating alzheimer's disease, characterized in that: the medicine isThe composition contains a therapeutically effective amount of C ₃₇ H ₄₆ O ₄ P.CH ₃ O ₃ S or a pharmaceutically acceptable salt thereof, and curcumin or a pharmaceutically acceptable salt thereof.

8. A medicament according to claim 7, characterized in that: the medicament has one or more of the following effects:

a) Relieving anxiety;

b) Improving exploration and cognitive ability;

c) Enhancing memory;

d) Lowering blood glucose levels;

e) Lowering oligomer levels, sappβ, and BACE1 levels;

f) Reducing hyperphosphorylation and neurofibrillary tangles of Tau protein;

g) Protecting tissue synaptoproteins and neuronal activity.

9. A medicament according to claim 7, characterized in that: the medicine also contains a carrier acceptable to human bodies.

10. A medicament according to claim 9, characterized in that: the carrier is selected from any one or more of saline, buffer solution, glucose, water, glycerol, ethanol, powder and combination thereof.