Use of lactoferrin in combination with ergothioneine in preparation of drug for preventing and / or treating alzheimer's disease

Abstract

The present invention provides a use of lactoferrin in combination with ergothioneine in the preparation of a drug for preventing and / or treating Alzheimer's disease. Compared with the use of lactoferrin or ergothioneine alone, in the present invention, the combined use of lactoferrin and ergothioneine at a specific ratio as an active pharmaceutical ingredient can reduce cell damage caused by Aβ25-35, reduce the expression of a p-Tau protein, lower the oxidative stress level and regulate apoptosis, alleviate memory impairment and cognitive dysfunction, and can reduce Aβ deposition in mouse plasma.

Description

Use of lactoferrin combined with ergothioneine in preparation of drugs for preventing and / or treating Alzheimer's disease TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to use of lactoferrin combined with ergothioneine in preparation of drugs for preventing and / or treating Alzheimer's disease. BACKGROUND

[0002] Alzheimer's disease (AD) is the most common neurodegenerative disease and the leading cause of dementia. Currently, there are more than 40 million AD patients worldwide, and this number is expected to exceed 130 million by 2050. In the later stages of AD, patients will lose cognitive ability and self-care ability, and cannot live independently, which seriously damages the quality of life of patients and their families, and brings a huge burden to families and society. The pathogenesis of AD is very complex, and has not been fully elucidated. The pathological changes involved are complex and interrelated, which hinders the development of new drugs. So far, there is no drug that can cure and stop the progression of the disease. Therefore, the prevention and improvement of AD is imminent.

[0003] Lactoferrin (LF) is an iron-binding glycoprotein with a molecular weight of 80 kDa, which not only regulates iron metabolism in the body, but also plays an important role in anti-inflammation and anti-oxidation. Lactoferrin plays an important role in neural development, cognition and neuroprotection by increasing the expression of brain-derived neurotrophic factor in experimental animals, reducing neuronal loss and neuroinflammation. A randomized controlled trial showed that compared with standard treatment, lactoferrin capsules (250 mg / day for three months) improved cognitive symptoms, oxidative stress, inflammation, apoptosis, and biomarkers related to beta-amyloid and tau protein pathology in patients with mild to moderate AD.

[0004] Ergothioneine (EGT) is a sulfur-containing histidine derivative that is abundant in mushrooms and has significant antioxidant and neuroprotective effects. It is widely used in food, health products, cosmetics, and pharmaceutical industries. In the food industry, ergothioneine is widely used as a natural antioxidant in various functional foods. In the cosmetics industry, it is often added to anti-aging, whitening and spot-removing products to help improve skin condition and delay aging. In the pharmaceutical field, ergothioneine is used to treat oxidative stress-related diseases due to its strong antioxidant properties. It can also protect the brain from oxidative damage and neuroinflammation, and has potential application value in the prevention and treatment of neurodegenerative diseases.

[0005] There is no combination of lactoferrin and ergothioneine for preventing and treating Alzheimer's disease. SUMMARY

[0006] The present application provides the use of lactoferrin combined with ergothioneine in the preparation of a medicine for preventing and / or treating Alzheimer's disease.

[0007] The present application also provides the use of lactoferrin combined with ergothioneine in the preparation of a food for assisting in improving memory function.

[0008] Further, the mass ratio of the lactoferrin to the ergothioneine is 1:2-8.

[0009] Further, the mass ratio of the lactoferrin to the ergothioneine is 1:2 or 1:8.

[0010] The present application also provides a combined medicine for preventing and / or treating Alzheimer's disease, which contains lactoferrin and ergothioneine for simultaneous or separate administration.

[0011] Further, the mass ratio of the lactoferrin to the ergothioneine is 1:2-8.

[0012] Further, the mass ratio of the lactoferrin to the ergothioneine is 1:2 or 1:8.

[0013] The present application also provides a composition for assisting in improving memory function, which is an oral preparation prepared by adding acceptable adjuvants to lactoferrin and ergothioneine as active ingredients; the oral preparation is granules, powder, pills, capsules or solution.

[0014] Further, the mass ratio of the lactoferrin to the ergothioneine is 1:2-8, preferably 1:2 or 1:8.

[0015] The present application also provides a method for preparing the above-mentioned composition, which comprises the following steps:

[0016] The lactoferrin and ergothioneine are weighed according to the proportion, and then acceptable adjuvants or auxiliary ingredients are added and mixed, to obtain the product.

[0017] The use of lactoferrin combined with ergothioneine in the preparation of a medicine for preventing and / or treating Alzheimer's disease, or in the preparation of a food for assisting in improving memory function, according to the present application, shows that, compared with the use of lactoferrin or ergothioneine alone, the combination of lactoferrin and ergothioneine as a pharmacodynamic substance in a specific ratio can more significantly reduce Aβ 25-35The cell damage caused reduces p-Tau protein expression, decreases oxidative stress levels, regulates apoptosis, and reduces Aβ deposition in plasma, thus playing a synergistic role in improving memory function and alleviating cognitive impairment, providing a new option for the clinical prevention and treatment of Alzheimer's disease.

[0018] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0019] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0020] Figure 1. Effects of different intervention methods on Aβ 25-35 A graph showing the induced nerve cell survival rate.

[0021] Figure 2. Effects of different interventions on AD markers (A: Protein bands of p-Tau and Tau proteins in cells after different experimental group treatments; B: Expression of p-Tau relative to Tau in cells after different experimental group treatments).

[0022] Figure 3. Effects of different interventions on oxidative stress (ROS) (A: Intracellular fluorescence expression after different experimental group treatments; B: Relative expression of intracellular fluorescence after different experimental group treatments).

[0023] Figure 4. Effects of different intervention methods on changes in MDA activity in cells.

[0024] Figure 5. Effects of different intervention methods on changes in SOD activity in cells.

[0025] Figure 6. Effects of different interventions on apoptosis (A: Protein bands of apoptosis-related proteins Cleaved-Caspase-3, Bax, and Bcl-2 after different interventions; B: Expression of Cleaved-Caspase-3 relative to β-actin after different interventions; C: Expression of Bax relative to β-actin after different interventions; D: Expression of Bcl-2 relative to β-actin after different interventions).

[0026] Figure 7. Effects of different interventions on mitochondrial membrane potential (A: Schematic diagram of JC-1 staining fluorescence in cells; B: Results of mitochondrial membrane potential).

[0027] Figure 8. Effects of different interventions on the ability of mice to spatially short-term working memory (A: total number of times of entering the arm of the mice; B: spontaneous alternation rate).

[0028] Figure 9. Effects of different interventions on the ability of mice to spatial memory (A: schematic diagram of the location navigation of the water maze; B: experimental results of escape latency; C: number of times of crossing the platform of the mice in the target quadrant; D: residence time of the mice in the target quadrant).

[0029] Figure 10. Effects of different interventions on the content of Aβ in the plasma of mice (A: content of Aβ in the plasma of mice under different treatments; B: content of Aβ in the plasma of mice under different treatments). 1-40 1-42 DETAILED DESCRIPTION

[0030] The raw materials and equipment used in the present application are known products, which are obtained by purchasing commercially available products.

[0031] Example 1, the composition of the present application

[0032] Formulation: lactoferrin 50 mg, ergothioneine 100 mg

[0033] Preparation method: lactoferrin and ergothioneine are weighed according to the proportion, and then pharmaceutical or food acceptable excipients are added, to obtain the product.

[0034] Example 2, the composition of the present application

[0035] Formulation: lactoferrin 50 mg, ergothioneine 400 mg

[0036] Preparation method: lactoferrin and ergothioneine are weighed according to the proportion, and then pharmaceutical or food acceptable excipients are added, to obtain the product.

[0037] The beneficial effects of the present application are demonstrated by the following experimental examples.

[0038] Experimental Example 1, Effect of lactoferrin combined with ergothioneine on the survival rate of nerve cells induced by Aβ 25-35

[0039] 1. Experimental method

[0040] In order to detect the effect of lactoferrin and ergothioneine and their combined use on the survival rate of nerve cells (N2a), cells in exponential growth phase were selected, and after trypsin digestion of the cells, a single cell suspension was prepared with MEM complete culture medium, and the cell suspension was inoculated into a 96-well plate at a concentration of 1.0 x 10 4 ​​​The N2a cells were seeded at a density of 1 x 104 / well in 96-well plates and incubated overnight in a cell incubator (37°C, 5% CO2). After 24 h of treatment with 100 μL of ergothioneine (160 μg / mL), 100 μL of lactoferrin (80 μg / mL), and 100 μL of a combination of the two (2:1 group: ergothioneine at 160 μg / mL and lactoferrin at 80 μg / mL; 8:1 group: ergothioneine at 640 μg / mL and lactoferrin at 80 μg / mL), the medium was discarded, and 100 μL of 20 μmol / L Aβ 25-35 The cells were stimulated for 24 h. Then the medium was discarded, and 1 / 10 volume of Cell Counting Kit-8 (CCK-8) was added directly to the cell culture medium, mixed thoroughly, and 100 μL of the mixed medium was added to each well. The cells were incubated for another 1-4 h in the cell incubator, and the optical absorbance at 450 nm was measured using a microplate reader to calculate the cell viability.

[0041] 2. Experimental results

[0042] The experimental results are shown in Figure 1. The Aβ 25-35 The cell viability was about 70% after 24 h of treatment. Compared with the model group treated with Aβ 25-35 Compared with the model group treated with Aβ, the treatment with ergothioneine and lactoferrin both improved the cell viability (P < 0.005), but the combined treatment with ergothioneine and lactoferrin significantly improved the cell viability of the AD model cells (P < 0.05) and reduced cell death by about 20%, showing a good synergistic effect.

[0043] Example 2: Effect of lactoferrin combined with ergothioneine on AD markers

[0044] 1. Experimental method

[0045] Neurofibrillary tangles formed by the abnormal hyperphosphorylation of Tau protein in cells are one of the main pathological features of AD. The effect of ergothioneine and lactoferrin intervention on the level of phosphorylated Tau (p-Tau) protein in model cells (i.e., N2a cells treated with Aβ 25-35 According to the operation procedure of Example 1, the N2a cells were seeded and dosed. After drug intervention, the cell culture plate was placed on ice, and IP lysis buffer containing PMSF and phosphatase inhibitor was added, mixed thoroughly, and then the cells were collected into a centrifuge tube using a cell scraper and incubated on ice for 20 min to allow the cells to be fully lysed. The cells were centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was collected as the total protein. The protein concentration was quantified using the BCA (bicinchoninic acid) method, and the expression of p-Tau and Tau protein was detected by Western blot.

[0046] 2. Experimental results

[0047] The experimental results are shown in Figure 2. Compared with the blank control group, the intracellular p-Tau protein level was significantly increased (P < 0.001) after treatment with 20 μmol / L of Aβ 25-35 After 24 h of induction, the intracellular p-Tau protein level was significantly increased (P < 0.001), while the p-Tau protein level was reduced (P < 0.01) after 24 h of pretreatment with ergothioneine and lactoferrin. Compared with the lactoferrin and ergothioneine single treatment groups, the protein expression was significantly reduced (P < 0.01) in the lactoferrin and ergothioneine combination group, indicating that the combination can synergistically reduce the pathological markers of AD model cells.

[0048] Example 3, Effect of Lactoferrin Combined with Ergothioneine on Oxidative Stress (ROS)

[0049] 1. Experimental method

[0050] N2a cells were inoculated and dosed according to the operation procedure of Example 1. The 2,7-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe was diluted with serum-free culture solution at a ratio of 1:1000 to obtain a final concentration of 10 μmol / L. The cell culture solution was removed, and an appropriate volume of diluted DCFH-DA fluorescent probe was added to fully cover the cells. Incubation was performed at 37°C in a cell culture box for 20 minutes, and the cells were washed three times with PBS buffer to sufficiently remove the DCFH-DA fluorescent probe that did not enter the cells. Finally, a fluorescence microscope was used for observation and photography.

[0051] 2. Experimental results

[0052] The experimental results are shown in Figure 3. ROS is an important marker of cellular oxidative damage. Aβ 25-35 After treatment, the intracellular ROS level was significantly increased (P < 0.001) compared with the blank control group, indicating that excessive ROS has been produced in the cells, the body's antioxidant defense system cannot effectively remove ROS, and the oxidation and antioxidant imbalance causes cellular oxidative damage. Ergothioneine and lactoferrin treatment significantly reduced the ROS level in the cells (P < 0.01), and the combination of ergothioneine and lactoferrin reduced more than 40% of the ROS in the cells compared with the Aβ model group, and was significantly lower than the single treatment group (P < 0.01), indicating that the combination of lactoferrin and ergothioneine can synergistically improve the oxidative damage of cells.

[0053] Example 4, Effect of Lactoferrin Combined with Ergothioneine on Oxidative Stress Products (MDA and SOD)

[0054] 1. Experimental method

[0055] Malondialdehyde (MDA) content is an important parameter reflecting cellular damage and the body's antioxidant potential. Superoxide dismutase (SOD) is a key enzyme in preventing cellular oxidative stress damage. By measuring the effects of ergothioneine and lactoferrin on cellular MDA and SOD activity, intracellular antioxidant capacity can be determined. Following the MDA and SOD kit instructions, the optical density (OD) value of the samples was measured using a microplate reader. The samples were prepared according to the inoculation and administration methods described in Example 1. Intracellular MDA and SOD content was calculated according to the instructions.

[0056] 2. Experimental Results

[0057] The results (Figure 4) showed that, compared with the blank control group, Aβ 25-35 Treatment significantly increased intracellular MDA levels (P<0.001). The MDA levels in the ergothioneine and lactoferrin groups decreased by 0.36 and 0.4 nmol / mg, respectively (P<0.01), while the MDA levels in the ergothioneine and lactoferrin combination decreased by more than 0.6 nmol / mg, which was significantly lower than that in the single treatment groups (P<0.05).

[0058] The changes in SOD activity in cells are shown in Figure 5. In Aβ... 25-35 Under the induction of ergothioneine, the activity of SOD was significantly lower than that of the control group (P<0.001). Compared with the Aβ treatment group, the SOD activity of the ergothioneine and lactoferrin group increased by more than 0.2 mU / mg, which was statistically significant (P<0.05). The SOD activity of the combination of ergothioneine and lactoferrin increased by more than 0.3 mU / mg, which was significantly higher than that of the single treatment group (P<0.05), indicating that the combination of ergothioneine and lactoferrin can synergistically improve the antioxidant capacity of cells.

[0059] Experiment Example 5: Effects of lactoferrin combined with ergothioneine on cell apoptosis

[0060] 1. Experimental Methods

[0061] Aβ can promote apoptosis by regulating the expression of apoptosis-related proteins. Western blot was used to examine the effects of different intervention methods on the expression levels of apoptosis-related proteins Cleaved-Caspase-3, Bax, and Bcl-2. Specific samples were prepared according to the inoculation and drug administration methods described in Example 1.

[0062] 2. Experimental Results

[0063] The results (Figure 6) showed that, compared with the blank control group, after treatment with 20 μmol / L Aβ... 25-35After induction for 24 h, the expression of pro-apoptotic proteins Cleaved-Caspase-3 (P<0.005) and Bax (P<0.001) increased, while the expression of anti-apoptotic protein Bcl-2 decreased (P<0.001). Compared with the model group, after intervention for 24 h by ergothioneine and lactoferrin and their combination, the expression of Cleaved-Caspase-3 (P<0.01) and Bax (P<0.05) proteins decreased, and the expression of Bcl-2 increased significantly (P<0.05). Compared with the ergothioneine group, the expression of pro-apoptotic proteins decreased and the expression of anti-apoptotic proteins increased in the lactoferrin group (P<0.05), indicating that lactoferrin and ergothioneine can synergistically inhibit cell apoptosis.

[0064] Example 6, Effect of Lactoferrin Combined with Ergothioneine on Mitochondrial Membrane Potential

[0065] 1. Experimental method

[0066] In the early stage of apoptosis, the mitochondrial transmembrane potential decreases, causing mitochondrial dysfunction, ATP synthesis disorder, and ROS accumulation, and further producing oxidative stress. Therefore, detecting the changes in mitochondrial membrane potential is one of the effective means to determine whether early apoptosis occurs. An appropriate amount of mitochondrial membrane potential detection reagent (JC-1, 200X) is diluted according to the ratio of 8 ml ultrapure water per 50 μl JC-1 (200X). Vortex to fully dissolve and mix the JC-1. Then add 2 ml of JC-1 staining buffer (5X) and mix well to obtain the JC-1 staining working solution. Add 0.5 ml of cell culture medium to a 12-well plate, then add 0.5 ml of JC-1 staining working solution and mix well. Incubate in a cell culture incubator at 37°C for 20 minutes. During the incubation period, prepare an appropriate amount of JC-1 staining buffer (1X) according to the ratio of 4 ml distilled water per 1 ml JC-1 staining buffer (5X) and place it in an ice bath. After 37°C incubation, remove the supernatant, wash twice with JC-1 staining buffer (1X), and then add 1 ml of cell culture medium. Observe and take pictures under a fluorescence microscope.

[0067] 2. Experimental results

[0068] The staining results (Figure 7) show that compared with the blank control group, after treatment with 20 μmol / L of Aβ 25-35 After induction for 24 h, a large amount of JC-1 in the matrix exists in monomer form, producing a large amount of green fluorescence (P<0.001). Compared with the model group, after pre-intervention for 24 h by ergothioneine and lactoferrin, the green fluorescence intensity of the cells decreased and the red fluorescence intensity increased (P<0.05), indicating that ergothioneine and lactoferrin can effectively alleviate the mitochondrial membrane potential decrease caused by Aβ 25-35The mitochondrial membrane potential of the cells was reduced, and after the combination of ergothioneine and lactoferrin, the membrane potential was further reduced (P<0.05), indicating that the combination of ergothioneine and lactoferrin can further alleviate the early apoptosis of cells.

[0069] Example 7, Effect of Lactoferrin Combined with Ergothioneine on the Spatial Short-term Working Memory Ability of APP / PS1 Mice

[0070] 1. Experimental method

[0071] (1) Experimental animal grouping and treatment method: 40 five-month-old APP / PS1 mice and 8 wild-type mice of the same litter were purchased from Beijing Zishan Health Medical Research Institute Co., Ltd. After all the mice were adaptively fed for one week, the APP / PS1 mice were randomly divided into five groups, namely the model group (Saline), the EGT group, the LF group, the EGT+LF (2:1) group, and the EGT+LF (8:1) group, with 8 mice in each group, and the wild-type mice were used as the blank control group (Ctrl). The LF group was given 50 mg / kg / d of exogenous lactoferrin, the EGT group was given 100 mg / kg / d of ergothioneine, the EGT+LF (2:1) group was given 50 mg / kg / d of exogenous lactoferrin and 100 mg / kg / d of ergothioneine, and the EGT+LF (8:1) group was given 50 mg / kg / d of exogenous lactoferrin and 400 mg / kg / d of ergothioneine. The model group and the blank control group were given the same dose of normal saline. Each group was treated by gavage for 90 days.

[0072] (2) Detection method: Y maze was used to detect the spatial short-term working memory ability of animals. The Y maze box was composed of three branches and a connecting area, with an angle of 120° between the three arms, and each arm was 30 cm long. After the test mice were placed at the end of one arm, the mice were allowed to freely explore the three arms of the Y maze for 5 minutes. The number of times and total distance of the mice in each arm were detected, and when the mouse's center point was positioned in the branch, it was considered that the mouse had entered the arm. After each test, the instrument was cleaned with 75% ethanol and thoroughly dried. Index: spontaneous alternation percentage (%) = [alternation times / (total number of times entering each arm - 2)] x 100%.

[0073] 2. Experimental results

[0074] The results are shown in Table 1 and Figure 8. As shown in Figure 8, there was no significant difference in the total number of arm entries among the mice in each group (P>0.05) in the spontaneous alternation test in the Y maze (Figure 8A), indicating that there was no difference in the motor ability of the mice in each group. However, compared with the wild-type mice (see Table 1), the spontaneous alternation accuracy of the mice in the APP / PS1+Saline group was reduced by 46% (P<0.001), and the spontaneous alternation accuracy of the model mice treated with lactoferrin or ergothioneine was increased by more than 15% (P<0.05). The spontaneous alternation accuracy of the mice treated with lactoferrin and ergothioneine combined was increased by at least 21% compared with the accuracy of the mice treated with lactoferrin or ergothioneine alone, and the difference was statistically significant (P<0.05) (Figure 8B), indicating that the combination of lactoferrin and ergothioneine can better alleviate the impairment of working memory in AD model mice.

[0075] Table 1. Spontaneous alternation rate after treatment in different experimental groups

[0076] Further comparison of the increase in the alternation rate of the mice in the lactoferrin, ergothioneine, and lactoferrin and ergothioneine combined groups relative to the Saline group in Table 1 showed that the increase in the alternation rate after the combination of lactoferrin and ergothioneine (2:1 group: 41%, 8:1 group: 39%) was higher than the sum of the increase in the alternation rate after treatment with lactoferrin alone and ergothioneine alone (33%), indicating that the combination of lactoferrin and ergothioneine has a synergistic effect on improving the spontaneous alternation rate.

[0077] Example 8, Effect of Lactoferrin Combined with Ergothioneine on the Spatial Memory Ability of APP / PS1 Mice

[0078] 1. Experimental method

[0079] The Morris water maze test was used to evaluate the spatial learning and reference memory ability of animals. The water maze body is a circular barrel structure with a diameter of 120 cm and a height of 50 cm. Before the experiment, the body was filled with an appropriate amount of water, and an appropriate amount of titanium white powder was added and mixed to make the water milky white, so that the camera and recording system can record and analyze the movement trajectory of the mice. The water temperature was maintained at 22-26°C during the entire experiment.

[0080] The body was divided into four quadrants by computer software, and the escape platform was hidden about 1 cm below the water surface in the target quadrant. Four black marks of different shapes were made on the inner wall of the body above the water surface to facilitate the mice to find the platform. The experiment mainly included positioning navigation experiment and spatial exploration experiment, which were used to test the spatial learning ability and spatial reference memory ability of mice, respectively.

[0081] In the positioning navigation experiment, the mice were randomly placed into the water from one of the quadrants each time, facing the pool wall. When the mice found the platform, they were allowed to stay on the platform for 10 s. If the mice did not find the platform within the specified 60 s, they were guided to the platform and stayed on the platform for 10 s. The mice were trained 4 times a day, once in each quadrant, with an interval of 20 min each time. The experiment lasted for 4 days, during which the escape latency of the mice in finding the underwater platform was mainly observed.

[0082] On the 5th day, the spatial exploration experiment was performed, during which the platform was removed, and the mice were placed into the pool in one quadrant other than the target quadrant to swim freely for 1 min. The percentage of the swimming time of the mice in the target quadrant in the total swimming time and the number of times of crossing the platform were recorded to evaluate the memory ability of the mice.

[0083] The specific grouping of experimental animals and the treatment method were the same as in Experimental Example 7.

[0084] 2、Experimental results

[0085] The results of the positioning navigation experiment of the water maze (Figure 9) showed that, compared with the wild-type control group of mice, the escape latency of the APP / PS1+Saline group of mice was prolonged by 26.6 s on the 4th day (P<0.001), indicating that the APP / PS1 mice of this age had shown a spatial learning ability disorder; after the intervention of lactoferrin and ergothioneine, the escape latency of the model mice was shortened by more than 9.8 s on the 4th day (P<0.001). Compared with the single treatment group, the lactoferrin and ergothioneine composition group could significantly shorten the latency time (P<0.05) (Figures 9A and 9B), indicating that the lactoferrin and ergothioneine composition could synergistically alleviate the spatial learning ability disorder of the APP / PS1 mice. In the spatial exploration experiment, the residence time and the number of times of crossing the platform of the APP / PS1+Saline group of mice, i.e., the model group of mice, were significantly less than those of the wild-type control group of mice (P<0.001), but after the combined use of lactoferrin and ergothioneine, the residence time and the number of times of crossing the platform of the mice were significantly more than those of the model group of mice (P<0.001), and were stronger than those of the single use of lactoferrin and ergothioneine groups (P<0.05) (Figures 9C and 9D). This result indicated that the lactoferrin and ergothioneine composition group could improve the spatial reference memory ability of the APP / PS1 mice and alleviate the cognitive dysfunction.

[0086] Experimental Example 9, Effect of Lactoferrin Combined with Ergothioneine on the Content of Aβ in the Plasma of APP / PS1 Mice

[0087] 1、Experimental method

[0088] The blood of the mice treated in Experimental Example 7 was collected, centrifuged at 4°C and 1000xg for 15 min, and the supernatant was taken to detect the Aβ in the plasma using an ELISA kit 1-40and Aβ 1-42 levels.

[0089] 2、Experimental results

[0090] Results (Figure 10) show that compared with wild type control group mice, the Aβ 1-40 and Aβ 1-42 contents in the plasma of APP / PS1+Saline group mice increased by 313 pg / mL and 188 pg / mL (P<0.001), respectively, and the difference was statistically significant; the Aβ 1-40 and Aβ 1-42 contents of lactoferrin and ergothioneine fed APP / PS1 mice were reduced by 169 pg / mL and 191 pg / mL (P<0.05) compared with the Saline group. The Aβ 1-40 content of lactoferrin combined with ergothioneine was significantly reduced (P<0.05) compared with the single use group, and the Aβ 1-42 content of the 8:1 combined use ratio group was slightly reduced, but the difference was not statistically significant (P>0.05). It is shown that lactoferrin combined with ergothioneine significantly reduces the Aβ content in the plasma of AD model mice.

[0091] From the above experimental results, it can be seen that the combined use of lactoferrin and ergothioneine can reduce the cell damage caused by Aβ 25-35 , reduce the expression of p-Tau protein, reduce the level of oxidative stress and regulate apoptosis, relieve memory impairment and cognitive dysfunction, and reduce Aβ deposition in the plasma of mice.

[0092] In summary, the present application provides the use of lactoferrin combined with ergothioneine in the preparation of a drug for preventing and / or treating Alzheimer's disease. Experimental results show that compared with the use of lactoferrin or ergothioneine alone, lactoferrin and ergothioneine combined as a drug efficacy substance in a specific ratio play a synergistic effect in improving memory function and relieving cognitive dysfunction, providing a new choice for the clinical prevention or treatment of Alzheimer's disease.

Claims

1. Use of lactoferrin in combination with ergothioneine for the preparation of a medicament for the prevention and / or treatment of Alzheimer's disease, characterized in that: The mass ratio of the lactoferrin to the ergothioneine is 1:2-8.

2. Use of lactoferrin in combination with ergothioneine for the preparation of a foodstuff for the auxiliary improvement of memory function, characterized in that: The mass ratio of the lactoferrin to the ergothioneine is 1:2-8.

3. Use according to claim 1 or 2, characterized in that: The mass ratio of the lactoferrin to the ergothioneine is 1:2 or 1:

8.

4. A combination medicament for preventing and / or treating Alzheimer's disease, comprising: (a) a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof: ###0000014### (b) donepezil hydrochloride. It contains lactoferrin and ergothioneine for simultaneous or separate administration, characterized in that the mass ratio of the lactoferrin to the ergothioneine is 1:2-8.

5. The combination of claim 4, wherein: The mass ratio of the lactoferrin to the ergothioneine is 1:2 or 1:

8.

6. A composition for assisting improvement of memory function, characterized by: It is a preparation prepared with lactoferrin and ergothioneine as active ingredients and acceptable adjuvants, characterized in that the mass ratio of the lactoferrin to the ergothioneine is 1:2-8.

7. The composition of claim 6, wherein: The mass ratio of the lactoferrin to the ergothioneine is 1:2 or 1:

8. And / or, the preparation is an oral preparation; the oral preparation is granules, powder, pills, capsules or solution.

8. Process for the preparation of a composition according to claim 6 or 7, characterized in that: It comprises the following steps: The lactoferrin and the ergothioneine are weighed according to the proportion, and then acceptable adjuvants or auxiliary ingredients are added and mixed, to obtain the preparation.

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