CN115737787A

CN115737787A - Application of lactoferrin and choline in preparation of medicine for preventing and/or treating Alzheimer's disease

Info

Publication number: CN115737787A
Application number: CN202211620474.7A
Authority: CN
Inventors: 成果; 张�林; 张伶俐
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2022-12-15
Filing date: 2022-12-15
Publication date: 2023-03-07
Anticipated expiration: 2042-12-15
Also published as: CN115737787B

Abstract

The invention provides an application of lactoferrin and choline in combination in preparing a medicine for preventing and/or treating Alzheimer's disease. The lactoferrin and the choline are combined in a specific ratio to serve as drug effect substances, so that the level of an anti-inflammatory factor in a mouse with neurodegenerative diseases can be improved, the level of a proinflammatory factor is reduced, the anti-inflammatory reaction capability of the brain is enhanced, tau protein phosphorylation and serum A beta level can be reduced, the learning capability and cognitive function are improved, the autonomous exploration capability is improved, and a new choice is provided for clinically preventing or treating Alzheimer's disease.

Description

Application of lactoferrin and choline in preparation of medicine for preventing and/or treating Alzheimer's disease

Technical Field

The invention particularly relates to application of lactoferrin and choline in combination in preparation of a medicine for preventing and/or treating Alzheimer's disease.

Background

Alzheimer's Disease (AD) is the most common neurodegenerative disease and the leading cause of senile dementia (about 60% to 80%), and is clinically characterized by global dementia such as memory impairment, aphasia, disuse, agnosia, impairment of visual-spatial skills, executive dysfunction, and personality and behavioral changes. At present, with the aging degree of the social population and the extension of the life expectancy of human beings, the number of people suffering from AD is continuously increased, and serious burden is brought to the society and families. At present, clinically, drugs for treating AD are mainly symptomatic therapy, and the therapeutic effect is not yet ideal, so that researches on prevention and treatment of AD are urgent.

Lactoferrin is an iron-binding glycoprotein with the molecular weight of 80kDa, belongs to the transferrin family, not only regulates the iron metabolism of the organism, but also has important effects on the anti-inflammation and the antioxidation of the organism. The research proves that the lactoferrin receptor has large expression quantity in neurons and capillary endothelial cells of the brain of a Parkinson disease patient, and the lactoferrin is combined with the lactoferrin to inhibit the propagation of prion protein, so that the potential application value of the lactoferrin in preventing and treating neurodegenerative diseases is prompted by the effect of the lactoferrin.

Choline, a positively charged tetravalent base, is a constituent of all biological membranes and a precursor of acetylcholine in cholinergic neurons, and studies have shown that choline protects the brain from alzheimer's disease through two pathways. First, choline helps to reduce homocysteine levels, a potent neurotoxin that can lead to neurodegeneration and amyloid plaque formation, doubling the prevalence of alzheimer's disease. Choline acts as a chemical conversion agent, converting harmful homocysteine into beneficial methionine. Secondly, choline can reduce the activation of microglia, and although the latter can function to clear debris from the brain, activated microglia are susceptible to uncontrolled, resulting in brain inflammation and ultimately neuronal death.

In the research report, lactoferrin and choline have an effect of improving alzheimer disease, but the benefit of patients is limited in the practical use process of the lactoferrin and choline as health food, so that the need for providing a medicament or health food which has a more remarkable effect of treating or preventing alzheimer disease and is more suitable for practical use is needed.

Disclosure of Invention

In order to solve the technical problems, the invention provides application of lactoferrin and choline in combination in preparing a medicament for preventing and/or treating Alzheimer's disease.

The invention also provides application of the lactoferrin and choline combined food in preparation of the food for assisting in improving the memory function.

Further, the mass ratio of the lactoferrin to the choline is 2.5-7.5 mg:1 to 5g.

Further, the mass ratio of lactoferrin to choline is 5mg:2.5g.

The present invention also provides a combination for the prevention and/or treatment of alzheimer's disease comprising lactoferrin and choline for simultaneous or separate administration.

Further, the mass ratio of the lactoferrin to the choline is 2.5-7.5 mg: 1-5 g.

Further, the mass ratio of the lactoferrin to the choline is 5mg:2.5g.

The invention also provides a composition for assisting in improving memory function, which is an oral preparation prepared by taking lactoferrin and choline as active ingredients and adding acceptable auxiliary materials; the oral preparation is granule, powder, pill, capsule or solution.

Further, the mass ratio of the lactoferrin to the choline is 2.5-7.5 mg:1 to 5g, preferably 5mg:2.5g.

The invention finally provides a process for the preparation of the aforesaid composition, which comprises the following steps:

weighing lactoferrin and choline according to the proportion, adding acceptable auxiliary materials or auxiliary components, and mixing uniformly to obtain the lactoferrin choline injection.

Animal experiments prove that compared with the single use of the lactoferrin or the choline, the lactoferrin and the choline which are combined in a specific proportion are used as functional substances, the level of an anti-inflammatory factor in a mouse with neurodegenerative diseases is improved, the level of the pro-inflammatory factor is reduced, the anti-inflammatory response capability of the brain is enhanced, tau protein phosphorylation and serum A beta level can be reduced, the learning capability and the cognitive function are improved, the autonomous exploration capability and the memory capability are improved, and a new choice is provided for clinically preventing or treating the Alzheimer's disease.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 shows the effect of choline in combination with lactoferrin on spatial memory in mice;

figure 2 is a graph of the effect of choline in combination with lactoferrin on the voluntary movements of the mice (5 min exploration performance of the OFT in each group of mice, respectively central zone residence time (a), frequency of entry into the central zone (b), mean rate of movement (c) and total distance of movement (d). ^* P<0.05, compared to model group mice; ^# P<0.05, compared to lactoferrin group mice; ^& P<0.05, compared to the choline group mice);

FIG. 3 shows the effect of choline in combination with lactoferrin on the cerebral inflammatory factors in mice ((a) - (f) IL-1 α, IL-1 β, IL-6, TGF- β 1, MCP-1, and COX-2mRNA expression levels P <0.05 compared to model mice, # P <0.05, & P <0.05 compared to lactoferrin group mice, and & lt 0.05 compared to choline group mice);

FIG. 4 shows choline in combination with lactoferrinEffect of the combination on ERK and JNK phosphorylation levels in mouse brain tissue ((a) ERK and p-ERK expression levels, (b) JNK and p-JNK expression levels. ^* P<0.05, compared to model group mice; ^# P<0.05, compared to lactoferrin group mice);

FIG. 5 is a graph of the effect of choline in combination with lactoferrin on mouse serum A β levels ((a) serum A β _1-40 And (4) horizontal. (b) Serum Abeta _1-42 And (4) horizontal. ^* P<0.05, compared with the model group mice, ^# P<0.05, compared with the lactoferrin group of mice, ^& P<0.05, compared to the choline group mice);

FIG. 6 shows LPR1 protein levels (40 μm scale) for each group of mice. ^* P<0.05, compared to model group mice; ^# P<0.05 compared with choline group, ^& P<0.05, compared with lactoferrin group mice, ^$ P<0.05 compared to blank control);

FIG. 7 shows the tau phosphorylation levels in mice of each group (scale bar 40 μm. Times. P <0.01, compared to model group mice, # P <0.05 compared to lactoferrin group, & P <0.05 compared to choline group mice).

Detailed Description

Example 1 compositions of the invention

The formula is as follows: lactoferrin 5mg, choline 2.5g

The preparation method comprises the following steps: weighing lactoferrin and choline according to the proportion, and adding pharmaceutically or food acceptable auxiliary materials to obtain the product.

Example 2 compositions of the invention

The formula is as follows: lactoferrin 7.5mg, choline 5g

Example 3 compositions of the invention

The formula is as follows: lactoferrin 4.5mg, choline 3g

The advantageous effects of the present invention will be further described by the following test examples

Test example 1 Effect of Choline in combination with Lactoferrin on spatial memory Capacity of SAMP-8 mice

1. Experimental Material

48 SAMP-8 mice, 4 months old, and 12 SAMR-1 mice, 4 months old, were purchased from Wintorithua animal laboratories, beijing.

2. Experimental procedure

After 1 week of adaptive feeding of all mice, SAMP-8 mice were randomly divided into 4 groups, model group, LF group, choline group, LF + choline group, 12 animals per group, and SAMR-1 was used as a blank control group, with no statistical significance for the differences in objective scores of the animals in each group (P > 0.05). LF group was administered with 10mg/kg/d exogenous lactoferrin. Choline group was given 5g/kg/d choline. The LF + Choline group was administered a combination of lactoferrin at 5mg/kg/d and choline at 2.5 g/kg/d. The model group and the blank control group were given the same dose of physiological saline.

3. Method of producing a composite material

The water maze experiment included two items: positioning navigation test and space exploration test. The experiment is used for detecting the space memory function of the mouse; in the space exploration test, animals participating in the test need to spend more time and energy to find out quadrants of the escape platform according to the prompt of space memory. The quadrants are arranged clockwise and are divided into a first quadrant, a second quadrant, a third quadrant and a fourth quadrant in sequence. The region in which the platform is located is a first quadrant, and the third quadrant is on the opposite side of the first quadrant. The experimental period is five days, the positioning navigation experiment is carried out four days before, the space exploration experiment is carried out on the fifth day, and the program is a classic experimental Morris water maze test program.

3.1 positioning sailing test

Before starting the experiment, each group of mice was placed on the platform and allowed to acclimatize for 20s. Then the surface wall is placed in a pool in the third quadrant; the timing was started and the time recorded to stop as model control mice climbed on the platform and stayed for 5s.60s is the longest record of the experiment, if the mouse can not board within the specified time, the mouse is actively guided to board the platform and stay for 10s, finally the mouse is cleaned, and the mouse is put into a mouse cage after the experiment is finished. Then, according to the flow, the first quadrant, the second quadrant, the third quadrant and the fourth quadrant are orderly moved for one circle in the clockwise direction every day for 4 days. The latency time on the platform was recorded for 4 quadrant mice per experiment and the mean value was estimated for several quadrants to assess the spatial learning ability of the mice.

3.2 space exploration experiment

And (3) removing the platform under the water maze in the last day of the experiment, keeping the experiment environment, the water temperature and the positioning navigation experiment of the water maze unchanged, placing the test mouse in the 3 rd quadrant surface wall pool, recording and observing the water swimming route passing by the mouse in 60s, cleaning the water stain on the mouse body, and placing the mouse into the mouse cage. The experiments in the other quadrants need not be performed. The assessment of the spatial memory capacity of the mice was done by recording the number of times the mice crossed the platform, and the ratio of the time required for the mice to reach the third quadrant to the total time, etc.

4. Results of the experiment

The specific results are shown in fig. 1, and it is known that the mobility range of the blank control mice is mainly concentrated in the first quadrant, the number of crossing the platform is large, and the mobility tracks of the model group mice are mostly at the edge of the pool, which is not good. When choline or lactoferrin is used alone, the mouse has increased times of crossing, but the activity of the mouse is still useless, and the activity track is more scattered. The activity tracks of the mice interfered by the combined use of the lactoferrin and the choline are concentrated in one quadrant and four quadrants, and the times of crossing the platform are obviously increased. The LF + choleline can effectively improve the cognitive dysfunction of the mouse with neurodegenerative disease and improve the learning ability of the mouse.

Test example 2 Effect of Choline in combination with Lactoferrin on the autonomous exploration ability of SAMP-8 mice

1. Carrying out the step

Experimental groups and intervention treatments were set up as in experimental example 1. Open Field Test (OFT) is a method for evaluating animal autokinetic exploration behavior, and is widely used in the field of spirits. In the unfamiliar open environment, mice are stringent, i.e. tend to move around the open field rather than in the central area; meanwhile, the device also has the characteristic of spontaneous autonomous activity. The anxiety degree and the autonomous exploration capability of the mouse are measured by placing the mouse in an open-field reaction box and observing the indexes such as the movement track, the movement distance and the like of the mouse in a specified time.

The OFT test chamber is an area with the length of 60cm, the width of 60cm and the height of 40cm, the periphery of the OFT test chamber is dark gray, the bottom of the OFT test chamber can be divided into an edge area and a central area, and the central area accounts for 25% of the total area. The mouse is placed into a certain fixed edge facing the area, the mouse freely moves in the area for 5min, and the residence time, the frequency of entering the central area, the horizontal movement distance and the like of the mouse in the central area and the edge area of the open field are recorded through a camera and a detection and analysis system. Keeping the test environment quiet, constant temperature and humidity and uniform light; the open field area and the inner walls were cleaned with 75% ethanol before and after each mouse experiment to remove the effect of residual odor on the assay.

2. Results

In the OFT assay, the ability of each group of mice to explore autonomously is shown in fig. 2. Compared with the normal control group mice, the mice in the model group have obviously reduced residence time in the central area and times of entering the central area. But the total distance moved and the average rate of activity increased significantly (P < 0.05), suggesting that the mice had significant anxiety. The anxiety state of the individual intervention groups of LF and Choline was improved. While the choline and lactoferrin combination compositions were longer in the central region (P < 0.05), more frequent in the central region (P < 0.05), slower in the mean locomotor speed (P < 0.05), and shorter in the horizontal locomotor distance (P < 0.05) than either lactoferrin or choline alone. The choline and lactoferrin combined composition can be used for improving the autonomous exploration capacity of mice.

Test example 3 Effect of Choline in combination with Lactoferrin on the cerebral inflammatory factor of SAMP-8 mice

1. Carrying out the steps

Experimental groups and intervention treatments were set up as in experimental example 1. 3 mice are selected in each group, total RNA of cells in each group is extracted from brain tissues of the mice in each group by using an RNA extraction kit, the total RNA is reversely transcribed into cDNA after the concentration of the total RNA is measured, the cDNA is diluted by 5 to 10 times and then is subjected to PCR amplification, the mRNA expression levels of IL-1 alpha, IL-1 beta, IL-6, TNF-beta 1, MCP-1 and COX-2 are detected by taking beta-actin as an internal reference, the detection is repeated for three times for each sample, the relative gene expression quantity is calculated by adopting a 2-delta-Ct method, and the primer sequence is shown in the following table 1.

TABLE 1 primer names and sequences

2. Results of the experiment

Specific results as shown in figure 3, lactoferrin or choline alone reduced levels of some inflammatory factors, such as TGF- β 1 and COX-2 (P < 0.05), compared to the placebo group. However, the combination of lactoferrin and choline can significantly reduce the levels of various inflammatory factors such as IL-1 alpha, IL-1 beta, IL-6, TGF-beta 1, MCP-1, COX-2 (P < 0.05), and the like, and the combination of choline and lactoferrin can significantly reduce the levels of IL-1 alpha, IL-1 beta, IL-6, TGF-beta 1, and MCP-1 (P < 0.01) as compared to the use of lactoferrin or choline alone. The results indicate that the choline and lactoferrin combination composition can significantly improve the anti-inflammatory response ability of mice compared to lactoferrin or choline alone.

Test example 4 Effect of Choline in combination with Lactoferrin on phosphorylation of ERK and JNK proteins in SAMP-8 mouse brain tissue

1. Carrying out the step

Experimental groups and intervention treatments were set up as in experimental example 1. After the intervention of the mice, 3 mice in each group are dissected and brain tissues are collected, the brain tissues are crushed by a tissue homogenizer, and the total protein of the brain tissues is extracted. The Bradford method is used for quantifying the protein, and Western Blot is used for detecting the phosphorylation levels of JNK protein and ERK protein.

2. Results of the experiment

Specific results are shown in fig. 4, and compared with the model group, the phosphorylation levels of lactoferrin or choline ERK and JNK proteins alone were slightly reduced, but the difference was not statistically significant (P > 0.05). And the combined use of lactoferrin and choline results in significantly reduced levels of ERK and JNK protein phosphorylation (P < 0.05). Furthermore, the levels of ERK and JNK protein phosphorylation were also significantly reduced with lactoferrin in combination with choline compared to the LF group (P < 0.05). The results indicate that the choline and lactoferrin combination composition is capable of significantly inhibiting ERK and JNK phosphorylation levels compared to lactoferrin or choline alone.

Test example 5 Effect of Choline in combination with Lactoferrin on serum A β levels in mice

The main pathological features of AD are senile plaques formed by abnormal aggregation of extracellular β -amyloid protein (a β) and neurofibrillary tangles formed by abnormal hyperphosphorylation of intracellular Tau protein. Among these, abnormal accumulation of the neurotoxic substance a β has a major impact on the pathological course of AD, as the earliest and most important change in AD pathology. Studies have shown that, on the one hand, abnormal accumulation of extracellular Α β directly induces loss of neuronal synapses, exacerbating the condition of AD; on the other hand, the abnormal aggregation of a β activates microglia, induces a series of downstream events such as neuroinflammation and oxidative stress, and further causes abnormal hyperphosphorylation of Tau protein, and finally leads to the change and even death of neuron functions. Therefore, reducing the production of a β and/or promoting clearance and degradation of a β is of great importance to alleviate the pathological course of AD.

1. Carrying out the steps

Experimental groups and intervention treatments were set up as in experimental example 1. After completion of the mouse intervention, 3 mice (the same batch of mice as in test example 4) were collected from each group, blood was collected from the inner canthus, and serum was extracted by centrifugation. Coating a target antibody in a 48-hole microporous plate by adopting a sandwich enzyme-linked immunosorbent assay (ELISA) to prepare a solid phase carrier, respectively adding a standard substance or a sample into each micropore, wherein the target is connected with the antibody on the solid phase carrier for binding, then adding the horseradish peroxidase-labeled antibody, washing the unbound antibody, and adding the TMB substrate again for color development. TMB is converted to blue by the catalysis of peroxidase and to the final yellow by the action of an acid. The shade of the color is positively correlated with the target in the sample. The absorbance (OD value) was measured at a wavelength of 450nm using a microplate reader, and the sample concentration was calculated.

2. Results of the experiment

The specific results are shown in fig. 5, from which it can be seen that: lactoferrin or choline, serum A β alone, compared to the model group _1-40 The level is obviously reduced (P)<0.05 But a β) _1-42 The level does not change significantly (P)>0.05). And lactoferrin is used in combination with choline, serum A beta _1-42 The level is remarkably reduced (P)<0.05). Mouse serum A beta using lactoferrin in combination with choline compared to lactoferrin or choline alone _1-40 The level is further reduced. The results show that the combination composition of lactoferrin and choline can further reduce the serum A beta concentration and improve the effect of preventing or treating AD compared with the use of lactoferrin or choline alone.

Test example 6 Effect of Choline in combination with Lactoferrin on mouse hippocampus LRP1 expression

1. Experimental procedure

Experimental groups and intervention treatments were set up as in experimental example 1. After the intervention of the mice, 3 mice are taken from each group of mice, the hippocampus tissues are dissected and extracted, and the section is stained after paraffin embedding. Hematoxylin staining cell nucleus is blue, and DAB shows positive expression as brown yellow.

2. Experimental methods

Specifically, as shown in fig. 6, the results show that: there was no significant difference in the expression of choline LRP1 alone compared to the control group (P > 0.05). LRP1 expression was significantly increased with lactoferrin alone (P < 0.05), indicating that lactoferrin achieves clearance and degradation of a β by binding to LRP 1. Compared with other groups, the lactoferrin and choline combined composition can obviously improve the expression of LPR1 (P is less than 0.05), and the mechanism that the prevention and treatment effect of AD is obviously improved by the combined use of lactoferrin and choline is probably that, on one hand, LF improves the expression of LPR1 of a receptor thereof, accelerates the elimination and degradation of A beta, and reduces the inflammatory reaction in the brain of a mouse. On the other hand, choline provides raw materials for synthesis of LPR1 and provides energy for transporting A beta by LPR 1. Therefore, the combined use of the two can further improve the prevention and treatment effect of AD.

Test example 7 Effect of Choline in combination with Lactoferrin on the expression of mouse hippocampus p-tau

1. Carrying out the step

2. Results of the experiment

Specific results are shown in fig. 7, from which it can be seen that: the phosphorylation levels of tau in mouse cerebral hippocampus were significantly reduced compared to the control group using choline or lactoferrin alone (P < 0.01). The phosphorylation level of tau protein in mouse brain hippocampus was further reduced compared to LF or choline groups (P < 0.05) using lactoferrin in combination with choline. The result shows that compared with the single use of lactoferrin or choline, the combined use of lactoferrin and choline can better inhibit tau protein phosphorylation of brain tissues and better play a role in preventing or treating AD.

As is clear from the above test examples 1-7, the combined use of lactoferrin and choline can increase the level of anti-inflammatory factors, decrease the level of pro-inflammatory factors, and enhance the anti-inflammatory response in the brain of mice, as compared to the use of lactoferrin or choline alone. The lactoferrin and choline are used together, so that the phosphorylation level of tau protein in the brain tissue of a SAMP-8 mouse and the level of A beta in the serum of the mouse can be reduced, and the learning ability and the cognitive function of the mouse are improved. Choline can promote lactoferrin to be combined with LRP1, and improve the capacity of clearing and degrading A beta. Therefore, the invention proves the potential application value of the combined use of the lactoferrin and the choline in the Alzheimer's disease, and also provides a theoretical basis for the application of the lactoferrin and the choline as a combination in the prevention and/or treatment of the Alzheimer's disease.

Claims

1. Use of lactoferrin in combination with choline in the manufacture of a medicament for the prevention and/or treatment of alzheimer's disease.

2. Use of lactoferrin in combination with choline in the preparation of a food for assisting in improving memory function.

3. Use according to claim 1 or 2, characterized in that: the mass ratio of the lactoferrin to the choline is 2.5-7.5 mg:1 to 5g.

4. Use according to claim 3, characterized in that: the mass ratio of the lactoferrin to the choline is 5mg:2.5g.

5. A combination for the prevention and/or treatment of alzheimer's disease, characterized in that: it contains lactoferrin and choline for simultaneous or separate administration.

6. The combination according to claim 5, wherein: the mass ratio of the lactoferrin to the choline is 2.5-7.5 mg:1 to 5g.

7. The combination according to claim 5, wherein: the mass ratio of the lactoferrin to the choline is 5mg:2.5g.

8. A composition for aiding in the improvement of memory, comprising: the oral preparation is prepared by taking lactoferrin and choline as active ingredients and adding acceptable auxiliary materials; the oral preparation is granule, powder, pill, capsule or solution.

9. The composition of claim 7, wherein: the mass ratio of the lactoferrin to the choline is 2.5-7.5 mg:1 to 5g, preferably 5mg:2.5g.

10. A process for preparing a composition according to claim 8 or 9, characterized in that: it comprises the following steps: