CN115737787B

CN115737787B - Application of lactoferrin in combination with choline in preparation of medicines for preventing and/or treating Alzheimer's disease

Info

Publication number: CN115737787B
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: 2024-04-19
Anticipated expiration: 2042-12-15
Also published as: CN115737787A

Abstract

The invention provides application of lactoferrin in combination with choline in preparing a medicament for preventing and/or treating Alzheimer's disease. The lactoferrin and choline are combined in a specific proportion to serve as drug effect substances, so that the level of anti-inflammatory factors in mice with neurodegenerative diseases can be improved, the level of pro-inflammatory factors can be reduced, the brain anti-inflammatory response capacity can be improved, tau protein phosphorylation and serum Abeta level can be reduced, learning ability and cognitive function can be improved, autonomous exploration ability can be improved, and a new choice is provided for clinical prevention or treatment of Alzheimer's disease.

Description

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

Technical Field

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

Background

Alzheimer's Disease (AD) is the most common neurodegenerative disease, and is the most common cause (about 60% -80%) of Alzheimer's disease, and is characterized clinically by memory impairment, aphasia, disuse, disbelief, impairment of visual space skills, executive dysfunction, and global dementia such as personality and behavior modification. At present, along with the increasing of the aging degree of the social population and the prolonging of the life expectancy of human beings, the number of the patients suffering from AD is continuously increased, and a serious burden is brought to the society and families. At present, medicines for treating the AD mainly adopt symptomatic treatment, and the treatment effect is not ideal, so that researches on prevention and treatment of the AD are urgent.

Lactoferrin is an iron-binding glycoprotein with a molecular weight of 80kDa, belongs to transferrin families, not only regulates iron metabolism of organisms, but also plays an important role in anti-inflammatory and antioxidant of organisms. The research proves that the lactoferrin receptor has large expression quantity in neurons and capillary endothelial cells of the brain of the parkinsonism patient, the lactoferrin is combined with the lactoferrin receptor to inhibit the transmission of prion, and the effect of the lactoferrin suggests the potential application value of the lactoferrin receptor to the prevention and the treatment of neurodegenerative diseases.

Choline is a tetravalent base with positive charges, 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 channels. First, choline helps reduce homocysteine levels, which is a potent neurotoxin that can lead to neurodegeneration and amyloid plaque formation, doubling the prevalence of Alzheimer's disease. Choline plays a role as a chemical conversion agent, converting harmful homocysteine into beneficial methionine. Secondly, choline reduces activation of microglia, which, although the latter may act to clear debris from the brain, is susceptible to runaway resulting in brain inflammation and ultimately neuronal death.

Lactoferrin and choline have been reported to have an improving effect on Alzheimer's disease, but there is a limit in patient's benefits in the practical use as a health food, and therefore, it is necessary to provide a medicament or health food which has a more remarkable effect of treating or preventing Alzheimer's disease and is more suitable for practical use.

Disclosure of Invention

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

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

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

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

The invention also provides a combination for preventing and/or treating Alzheimer's disease, which comprises 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 lactoferrin to 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 preparation method of the composition, which comprises the following steps:

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

The application of the lactoferrin and the choline in preparing medicaments for preventing and/or treating Alzheimer's disease or foods for assisting in improving memory function is proved by animal experiments, compared with the single use of the lactoferrin or the choline, the lactoferrin and the choline are combined in a specific proportion to serve as functional substances, so that the level of anti-inflammatory factors in mice with neurodegenerative diseases is improved, the level of pro-inflammatory factors is reduced, the brain anti-inflammatory response capacity is improved, tau protein phosphorylation and serum Abeta level can be reduced, learning ability and cognitive function are improved, autonomous exploring ability and memory ability are improved, and a new choice is provided for clinically preventing or treating Alzheimer's disease.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

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

FIG. 2 is the effect of choline in combination with lactoferrin on the autonomous movement of mice (each group of mice explored performance at 5min of OFT for central zone residence time (a), frequency of entry into central zone (b), average movement rate (c) and total movement distance (d) ^* P <0.05, compared to model group mice; ^# P <0.05, compared to lactoferrin group mice; ^& P <0.05, compared to choline group mice);

FIG. 3 is the effect of choline in combination with lactoferrin on the rat encephalitis factor ((a) - (f) IL-1 alpha, IL-1 beta, IL-6, TGF-beta 1, MCP-1 and COX-2mRNA expression levels, <0.05, # P <0.05 compared to model mice, # P <0.05 compared to lactoferrin mice, & P <0.05 compared to choline mice,);

FIG. 4 is the effect of choline in combination with lactoferrin on the levels of ERK and JNK phosphorylation in mouse brain tissues ((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 the effect of choline in combination with lactoferrin on serum Abeta levels in mice ((a) serum Abeta _1-40 levels, (b) serum Abeta _1-42 levels ^* P <0.05, ^# P <0.05 compared to model group mice, ^& P <0.05 compared to lactoferrin group mice, compared to choline group mice);

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

fig. 7 shows tau protein phosphorylation levels (40 μm scale.) for mice in each group, 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 inventive composition

The formula comprises the following components: lactoferrin 5mg, choline 2.5g

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

EXAMPLE 2 compositions of the invention

The formula comprises the following components: lactoferrin 7.5mg, choline 5g

EXAMPLE 3 compositions of the invention

The formula comprises the following components: lactoferrin 4.5mg, choline 3g

The beneficial effects of the present invention are further illustrated 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 materials

48 SAMP-8 mice at 4 months of age and 12 SAMR-1 mice at 4 months of age were purchased from beijing vitelli Hua animal laboratory company.

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 no statistical significance (P > 0.05) of the difference in objective scores of animals in each group, SAMR-1 as a blank group. LF group was given 10mg/kg/d exogenous lactoferrin. Choline group was given 5g/kg/d choline. The LF+choline group was given a combination of lactoferrin at 5mg/kg/d and choline at 2.5 g/kg/d. The model group and the blank group were given the same dose of physiological saline.

3. Method of

The water maze test includes two items: positioning navigation test and space exploration test. The positioning navigation test is used for repeatedly training the mice, enhancing the memory of the mice to the surrounding environment, finding an underwater escape platform in a short time, and detecting the spatial memory function of the mice; the space exploration test, the animal participating in the test spends more time and energy to find the quadrant of the escape platform according to the prompt of the space memory. The quadrants are arranged clockwise and are sequentially divided into a first quadrant, a second quadrant, a third quadrant and a fourth quadrant. The area where the platform is located is the first quadrant, and the third quadrant is on the opposite side of the first quadrant. The experimental period is five days, four days before the positioning navigation experiment, and the space exploration experiment is carried out on the fifth day, wherein the program is a classical experimental Morris water maze test program.

3.1 Positioning navigation test

Before starting the test, each group of mice was placed on a platform, and the mice were first acclimatized for 20s. Then placing the wall of the third quadrant surface in the pool; starting timing, the time to stop recording was 5s while the mice of the model control group climbed the platform. 60s is the longest record of the experiment, if the mice cannot land within the specified time, the mice are actively guided to land on the platform and stay for 10s, the mice are finally cleaned, and the mice are placed in a mouse cage after the experiment is finished. Following this procedure, the first, second, third and fourth quadrants were sequentially rotated clockwise one revolution per day for 4 days. The latency of 4 quadrant mice per experiment on the platform was recorded, and the average of several quadrants was estimated to assess the spatial learning ability of the mice.

3.2 Space exploration experiments

The last day of the experiment, the platform under the water maze is removed, the experimental environment, the water temperature and the positioning navigation experiment of the water maze are all kept unchanged, a test mouse is placed in a wall pool of a3 rd quadrant, then a swimming route passed by the mouse in 60s is recorded and observed, the water stain on the mouse is cleaned, and the mouse is placed in a mouse cage. Experiments in other quadrants need not be performed. Assessment of the spatial memory of the mice was accomplished by recording the number of mice crossing the platform, and the ratio of time the mice reached the third quadrant to total time.

4. Experimental results

As shown in fig. 1, the movement range of the blank mice is mainly concentrated in the first quadrant, the number of times of passing the mice is large, and the movement track of the mice in the model group is mostly the edge of the pool, so that the mice have no purpose. The choline or lactoferrin is used alone, so that the number of times of mouse passing through the platform is increased, but the activity of the mouse is still without purposefulness, and the activity track is relatively dispersed. The mouse activity track of the combined use intervention of lactoferrin and choline is concentrated in one quadrant and four quadrants, and the number of times of channel penetration is obviously increased. The result shows that LF+choline can effectively improve cognitive dysfunction of mice with neurodegenerative diseases and improve learning ability of the mice.

Test example 2 Effect of Choline in combination with lactoferrin on autonomous exploratory ability of SAMP-8 mice

1. Implementation steps

Experimental grouping and intervention treatments were set up as in experimental example 1. Open field test (open FILED TEST, OFT) is a method for evaluating animal voluntary motor exploration behaviors, and is widely used in the mental field. In an open environment, mice have a tendency to touch, i.e., to move around the open field rather than in the central region; and simultaneously, the device also has the characteristic of spontaneously and autonomously carrying out activities. The anxiety degree and the autonomous exploration ability of the mice are measured by placing the mice in an open field reaction box and observing indexes such as movement track, movement distance and the like of the mice in a designated time.

The OFT test box is an area with the length of 60cm, the width of 60cm and the height of 40cm, dark gray is formed around, the bottom can be divided into an edge area and a central area, and the central area accounts for 25% of the total area. The method comprises the steps of placing a mouse facing to an area into a certain fixed edge, enabling the mouse to freely move in the area for 5min, and recording the stay time of the mouse in the central area and the edge area of an open field, the frequency of entering the central area, the horizontal movement distance and the like through a camera and a detection analysis system. The test environment is kept quiet, the temperature and humidity are constant, and the light is uniform; the open field area and the inner walls were cleaned with 75% ethanol before and after each mouse test to remove the effect of residual odors on the test.

2. Results

In the OFT test, the autonomous exploratory capacity of each group of mice is shown in fig. 2. The time spent in and the number of entries into the central zone were significantly reduced in the model group compared to the normal control group. But total distance of locomotion and average rate of locomotion increased significantly (P < 0.05), suggesting that the mice were significantly anxious. The anxiety states of the LF and choline alone intervention groups were improved. While the choline and lactoferrin combination composition had longer residence time in the central zone (P < 0.05), higher frequency of entry into the central zone (P < 0.05), slower average locomotor speed (P < 0.05), and shorter horizontal locomotor distance (P < 0.05) than lactoferrin or choline mice alone. Indicating that the choline and lactoferrin combination composition can alter the ability of mice to explore autonomously.

Test example 3 Effect of Choline in combination with lactoferrin on the Japanese encephalitis factor in SAMP-8 mice

1. Implementation steps

Experimental grouping and intervention treatments were set up as in experimental example 1.3 mice are selected in each group, the total RNA of each group of cells is extracted from brain tissues of each group of mice by using an RNA extraction kit, the concentration of the total RNA is measured, the total RNA is reversely transcribed into cDNA, the cDNA is diluted 5 to 10 times and then is subjected to PCR amplification, the expression level of mRNA of IL-1 alpha, IL-1 beta, IL-6, TNF-beta 1, MCP-1 and COX-2 is detected by taking beta-actin as an internal reference, the detection is repeated three times for each sample, the relative expression quantity of genes 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. Experimental results

Specific results as shown in figure 3, lactoferrin or choline alone reduced partial inflammatory factor levels, such as TGF- β1 and COX-2 (P < 0.05), compared to the blank model group. However, the use of lactoferrin in combination with choline significantly reduced the levels of a variety of inflammatory factors such as IL-1α, IL-1β, IL-6, TGF- β1, MCP-1, COX-2 (P < 0.05), and the use of choline in combination with lactoferrin significantly reduced the levels of IL-1α, IL-1β, IL-6, TGF- β1, and MCP-1 (P < 0.01) compared to lactoferrin or choline alone. The results indicate that the choline and lactoferrin combination composition is able to more significantly increase the ability of the mice to resist inflammatory responses than lactoferrin or choline alone.

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

1. Implementation steps

Experimental grouping and intervention treatments were set up as in experimental example 1. After the mouse intervention is finished, 3 mice are dissected and brain tissues are collected from each group of mice, the brain tissues are crushed by using a tissue homogenizer, and the total protein of the brain tissues is extracted. The Bradford method quantifies the protein and Western Blot detects the phosphorylation level of JNK protein and ERK protein.

2. Experimental results

The specific results are shown in fig. 4, where lactoferrin or choline ERK and JNK proteins alone have slightly reduced phosphorylation compared to the model group, but the differences were not statistically significant (P > 0.05). Whereas the combined use of lactoferrin with choline significantly reduced the level of ERK and JNK protein phosphorylation (P < 0.05). Furthermore, the level of ERK and JNK protein phosphorylation was also significantly reduced (P < 0.05) in combination with lactoferrin with choline compared to LF group. The results indicate that the choline and lactoferrin combination composition is able to inhibit ERK and JNK phosphorylation levels more significantly than lactoferrin or choline alone.

Test example 5 Effect of Choline in combination with lactoferrin on serum Aβ levels in mice

The main pathological features of AD are senile plaques formed by abnormal aggregation of extracellular β -amyloid (β -amyliod protein, aβ) and neurofibrillary tangles formed by abnormal hyperphosphorylation of intracellular Tau protein. Among them, abnormal aggregation of the neurotoxic substance aβ is the earliest and most predominant change in AD pathology, and has an important influence on the pathological course of AD. Research shows that on one hand, abnormal aggregation of extracellular Abeta can directly induce loss of neuronal synapses and aggravate the condition of AD; on the other hand, abnormal aggregation of aβ activates microglial cells, induces a series of downstream events such as neuroinflammation and oxidative stress, and further causes abnormal hyperphosphorylation of Tau protein, and finally leads to alteration of neuronal function and even death. Thus, reducing aβ production and/or promoting aβ clearance and degradation is of great importance for alleviating the pathological processes of AD.

1. Implementation steps

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

2. Experimental results

The specific results are shown in fig. 5, from which it can be seen: serum aβ _1-40 levels were significantly reduced (P < 0.05) but no significant changes in aβ _1-42 levels (P > 0.05) with lactoferrin or choline alone compared to the model group. Whereas serum aβ _1-42 levels were significantly reduced (P < 0.05) with lactoferrin in combination with choline. Serum aβ _1-40 levels were further reduced in mice with lactoferrin in combination with choline, as compared to lactoferrin alone or choline groups. The results show that the lactoferrin and choline combined composition can further reduce serum aβ concentration and improve the effect of preventing or treating AD compared to lactoferrin or choline alone.

Test example 6 Effect of Choline in combination with lactoferrin on mouse hippocampal LRP1 expression

1. Experimental procedure

Experimental grouping and intervention treatments were set up as in experimental example 1. After the mouse intervention is finished, 3 mice are taken from each group of mice, the sea horse tissue is dissected and extracted, and the slices are stained after paraffin embedding. Hematoxylin-stained nuclei were blue and DAB showed positive expression as brown yellow.

2. Experimental method

As shown in fig. 6, it is clear from the results that: there was no significant difference in choline LRP1 expression (P > 0.05) when used alone compared to the control group. Lactoferrin LRP1 expression was significantly increased (P < 0.05) alone, indicating that lactoferrin achieves aβ clearance and degradation by binding to LRP 1. Compared with other groups, the lactoferrin and choline combined composition can obviously improve the expression of LPR1 (P < 0.05), which indicates that the mechanism of obviously improving the AD control effect by combining the lactoferrin and the choline is possible, on one hand, LF improves the expression of LPR1 receptor, accelerates the clearance and degradation of Abeta and reduces the inflammatory reaction in the brain of mice. On the other hand, choline provides a raw material for the synthesis of LPR1, and provides energy for the transportation of aβ by LPR 1. Therefore, the combined use of the two can further improve the AD prevention and treatment effect.

Experimental example 7 influence of Choline in combination with lactoferrin on the expression of p-tau in the hippocampus of mice

1. Implementation steps

2. Experimental results

The specific results are shown in fig. 7, from which it can be seen that: the rat brain hippocampal tau phosphorylation levels were significantly reduced (P < 0.01) compared to the control group using choline or lactoferrin alone. The mice had a further reduced brain hippocampal tau phosphorylation level (P < 0.05) compared to LF or choline groups when lactoferrin was used in combination. The results show that compared with the single use of lactoferrin or choline, the combined use of lactoferrin and choline can better inhibit brain tissue tau protein phosphorylation and better prevent or treat AD.

As is evident from test examples 1 to 7 above, the use of lactoferrin in combination with choline increases the level of anti-inflammatory factor, decreases the level of pro-inflammatory factor, and enhances the ability of the brain of mice to resist inflammatory responses, as compared to lactoferrin or choline alone. Lactoferrin and choline can also reduce the phosphorylation level of tau protein in brain tissue of a SAMP-8 mouse, and improve the learning ability and cognitive function of the mouse by virtue of the serum Abeta level of the mouse. Choline can promote the binding of lactoferrin to LRP1, enhancing its ability to clear and degrade aβ. Therefore, the invention proves the potential application value of the combined use of the lactoferrin and the choline in the Alzheimer's disease, and provides a theoretical basis for the application of the lactoferrin and the choline as the combination in the prevention and/or treatment of the Alzheimer's disease.

Claims

1. The use of lactoferrin in combination with choline in the manufacture of a medicament for the treatment of alzheimer's disease, characterized in that: the mass ratio of the lactoferrin to the choline is 2.5-7.5 mg: 1-5 g.

2. Use of lactoferrin in combination with choline in the preparation of a food product for aiding in improving memory function, characterized in that: the mass ratio of the lactoferrin to the choline is 2.5-7.5 mg: 1-5 g.

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

4. A combination medicament for treating alzheimer's disease, which is characterized in that: it contains lactoferrin and choline for simultaneous or separate administration;

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

5. The combination according to claim 4, wherein: the mass ratio of the lactoferrin to the choline is 5mg:2.5 g.

6. A composition for aiding in improving memory, characterized in that: 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;

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

7. The composition of claim 6, wherein: the mass ratio of the lactoferrin to the choline is 5mg:2.5 g.

8. A process for the preparation of a composition as claimed in claim 6 or 7, characterized in that: it comprises the following steps: