Anti-Alzheimer's disease composition containing human albumin as well as preparation method and application of anti-Alzheimer's disease composition

Abstract

The invention discloses an anti-Alzheimer's disease composition containing human albumin as well as a preparation method and application of the anti-Alzheimer's disease composition, and relates to the field of medicines, and the anti-Alzheimer's disease composition comprises 5-40 parts by mass of human albumin; 1-20 parts of polyunsaturated fatty acid or ester thereof, and the balance of deionized water. The anti-Alzheimer's disease composition is simple in components and preparation method, good in anti-Alzheimer's disease effect and wide in application prospect in the field of medicine.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an anti-Alzheimer's disease composition containing human albumin, and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD) is a disease that occurs in the brain. It has an insidious onset and no typical symptoms during the onset, so it is very easy to be ignored. After cardiovascular and cerebrovascular diseases and malignant tumors, it has become the third largest disease causing disability and death among the elderly. Alzheimer's disease has become a global health crisis. According to data released by the 2020 World Alzheimer's Report, approximately 50 million people in the world currently suffer from Alzheimer's disease. Due to the increase in the overall average life expectancy of people and the aggravation of the development of an aging population, the prevalence of Alzheimer's disease is also continuing to increase. It is estimated that by 2050, the number of Alzheimer's patients will increase to more than 150 million. Unfortunately, to date, there is still a lack of effective treatments for Alzheimer's disease in the world. Summary of the invention

[0003] In view of the problems and shortcomings of the prior art, the present invention provides an anti-Alzheimer's disease composition containing human albumin, and a preparation method and application thereof. The composition of the present invention comprises human albumin and polyunsaturated fatty acids.

[0004] Human serum albumin (HSA) is a monomeric protein produced by the liver. Mature human serum albumin contains 585 amino acid residues and has a molecular weight of 66KD. Blood-derived human serum albumin products are a therapeutic drug extracted from human plasma and are currently widely used in the treatment of liver ascites and related diseases such as burns and scalds.

[0005] Polyunsaturated fatty acids (PUFAs) are mainly derived from marine organisms or deep-sea fish, etc. The content of PUFAs in red blood cells can improve central nervous system inflammation and oxidative damage, reduce the release of pro-inflammatory cytokines, increase the synthesis of neurotrophic factors, inhibit cell apoptosis, promote nerve development and regeneration, and thus improve memory ability.

[0006] The scheme of the present invention is as follows: In a first aspect, the present invention provides an anti-Alzheimer's disease composition containing human albumin, characterized in that the composition comprises the following components in parts by mass: 5 to 40 parts by mass of human albumin; 1 to 20 parts by mass of polyunsaturated fatty acids (PUFAs), and preferably, the balance of the composition is deionized water.

[0007] Furthermore, the human albumin is selected from at least one of recombinant human albumin and hematogenous human albumin.

[0008] Further, the polyunsaturated fatty acid is selected from two of docosahexaenoic acid (22:6; DHA) or its ester, docosapentaenoic acid (22:5; DPA) or its ester, and eicosapentaenoic acid (20:5; EPA) or its ester. Preferably, the ester is selected from at least one of polyunsaturated fatty acid methyl esters or polyunsaturated fatty acid ethyl esters. More preferably, the polyunsaturated fatty acid methyl esters include at least one of docosapentaenoic acid methyl ester, docosahexaenoic acid methyl ester, and eicosapentaenoic acid methyl ester; the polyunsaturated fatty acid ethyl esters include at least one of docosapentaenoic acid ethyl ester, docosahexaenoic acid ethyl ester, and eicosapentaenoic acid ethyl ester.

[0009] Further, the mass ratio of two polyunsaturated fatty acids or their esters in docosahexaenoic acid (22:6; DHA) or its ester, docosapentaenoic acid (22:5; DPA) or its ester, and eicosapentaenoic acid (20:5; EPA) or its ester is 1:(0.05-20). For example, it can be 1:0.05, 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20 and any range therebetween.

[0010] Preferably, the mass ratio of two polyunsaturated fatty acids or their esters among docosahexaenoic acid (22:6; DHA) or its ester, docosapentaenoic acid (22:5; DPA) or its ester, and eicosapentaenoic acid (20:5; EPA) or its ester is 1:(0.1~10).

[0011] Furthermore, the composition also includes additives acceptable in the fields of medicines, foods, and health products.

[0012] Preferably, the additive comprises at least one of polysorbates, polyoxyethylene fatty acid esters, parabens, gum arabic, gum tragacanth, and stearate.

[0013] Furthermore, the composition can also be used in combination with other drugs for treating Alzheimer's disease. Preferably, the drug for treating Alzheimer's disease includes at least one of galantamine, donepezil, and huperzine A.

[0014] The second aspect of the present invention provides the use of the composition in the preparation of anti-Alzheimer's disease drugs, foods, and health products.

[0015] Furthermore, the medicines, foods and health products can be administered orally or by injection.

[0016] Furthermore, the dosage form of the medicine, food, and health product includes at least one of oral solution, soft capsule, emulsion, and injection.

[0017] Furthermore, the administration by injection includes at least one of intramuscular injection or intravenous injection.

[0018] Furthermore, when the medicine, food, and health care product are administered orally, they need to be released through a sustained-release route, including at least one of capsules, nano-, microcapsules, and microspheres.

[0019] It should be noted that the present invention does not specifically limit the type of product. Those skilled in the art can understand that other products that add the composition described in this application to play the same role as the composition in the present invention should also be within the scope of protection of the present invention.

[0020] The third aspect of the present invention provides a method for preparing the composition, the method comprising the following steps: S1: mixing different types of polyunsaturated fatty acid solutions by weight; S2: adding the polyunsaturated fatty acid solution obtained in step S1 to a human albumin aqueous solution or human albumin salt buffer by mass, and making up the balance with deionized water to obtain a mixed solution of the composition. Preferably, the human albumin salt buffer comprises at least one of human albumin phosphate buffer, human albumin citrate buffer, and human albumin acetate buffer, and preferably, the human albumin buffered saline solution is human albumin PBS buffer.

[0021] Furthermore, the mixing conditions in step S1 are: 40-60°C, 50-100 rpm, and stirring for 10-30 min.

[0022] Furthermore, step S2 also includes a high-pressure homogenization step. Preferably, the high-pressure homogenization condition is to use a high-pressure homogenizer at 400-1200 N for 10-30 min.

[0023] Furthermore, the method further comprises the step of aseptically filtering the mixed solution of the composition prepared in step S2, wherein the filter diameter is 0.1 μm.

[0024] The beneficial effects of the present invention include but are not limited to: 1. The present invention provides an anti-Alzheimer's disease composition containing human albumin, which uses human albumin as the main raw material and has a good anti-Alzheimer's disease effect through the synergistic effect of human albumin and two polyunsaturated fatty acids. The composition is simple and the biocompatibility is better.

[0025] 2. The present invention also provides a method for preparing an anti-Alzheimer's disease composition containing human albumin, which has a simple production process and high application prospects. DETAILED DESCRIPTION

[0026] The present invention is described in detail below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of the present invention are purchased through commercial channels.

[0027] Definition: Recombinant human albumin refers to human albumin prepared by recombinant DNA technology for treatment and prevention.

[0028] Hematogenous human albumin refers to human albumin separated and purified from healthy human blood, plasma or specific immune plasma.

[0029] Source of materials: Recombinant human albumin: Tonghua Anrui Special Biopharmaceutical Co., Ltd., batch number: ART101S; Docosahexaenoic acid (DHA) (Batch number: D100925-100 mg, Shanghai Aladdin Biochemical Technology Co., Ltd.); Eicosapentaenoic acid (EPA) (Batch number: C472120-5mg, Shanghai Aladdin Biochemical Technology Co., Ltd.); Docosapentaenoic acid (DPA) (Batch No.: CCFD200505-25MG, Sinopharm Chemical Reagent Co., Ltd.); Amyloid protein (Aβ 1-42 )Mw: 4514.04, (Batch No.: A494432-2.5mg, Shanghai Aladdin Biochemical Technology Co., Ltd.); Dimethyl sulfoxide (DMSO) (Batch number: D103277-250ml, Shanghai Aladdin Biochemical Technology Co., Ltd.); Hexafluoroisopropanol (Batch No. 213-059-4, Shanghai MacLean Biochemical Technology Co., Ltd.); Physiological sodium chloride solution (batch number: 20241019, Sichuan Kelun Pharmaceutical Co., Ltd.); Isoflurane for animals (Batch No.: R510-22-16, Shenzhen Reward Life Science Co., Ltd.); MTT assay kit (Batch No.: C0009S, Shanghai Bio-Tech Biotechnology Co., Ltd.); SPF grade mice (Batch No.: CS-008, Liaoning Changsheng Biotechnology Co., Ltd.); SH-SY5Y (human neuroblastoma cells) (Lot No.: SNL-092, Wuhan Shanen Biotechnology Co., Ltd.); equipment: Water bath (HH.S21-8, Shanghai Boxun Medical Biological Instrument Co., Ltd.); Multifunctional fluorescence microplate reader (SPMax 3500FL, Shanghai Shanpu Biotechnology Co., Ltd.); Analytical balance (BSA224S, Sartorius Scientific Instruments Beijing Co., Ltd.); Brain stereotaxic instrument (model DB053, Beijing Zhishu Duobao Biotechnology Co., Ltd.); Morris water maze video analysis system (model DB001X, Beijing Zhishu Duobao Biotechnology Co., Ltd.); Y-maze stimulator (model DB003, Beijing Zhishu Duobao Biotechnology Co., Ltd.); Small animal activity recorder (model DB-YLS-1C, Beijing Zhishu Duobao Biotechnology Co., Ltd.); Digital dispersing machine (model IKAT-25, IKA (Guangzhou) Instrument Equipment Co., Ltd.); Digital display electric constant temperature water bath (model HH-4, Shanghai Yiheng Instrument Co., Ltd.); Unless otherwise specified, other reagents and instruments used were commercially available.

[0030] Example 1 The preparation of the anti-Alzheimer's disease composition containing recombinant human albumin comprises the following components in parts by weight: the effective ingredients are shown in Table 1, Table 1 Components of various anti-Alzheimer's disease compositions containing recombinant human albumin

[0031] According to the component information in Table 1, an anti-Alzheimer's disease composition is prepared, and the specific method is as follows: (1) Take the polyunsaturated fatty acids by weight and stir them at 40-60 °C and 50-100 rpm for 10-30 min to mix them evenly; (2) After being mixed evenly, the temperature of the unsaturated fatty acid solution is lowered to 30-35°C, and the solution is added to the human albumin by weight, and homogenized by high pressure at 400-1200 N for 10-30 min using a high pressure homogenizer to obtain a mixed solution of an anti-Alzheimer's disease composition.

[0032] (3) The mixed solution of the anti-Alzheimer's disease composition is aseptically filtered through a 0.1 μm filter to obtain an anti-Alzheimer's disease composition containing human albumin.

[0033] Test Example 1: Cell Viability Experiment 1 μg / μl Aβ 1-42 Solution preparation: Accurately weigh Aβ 1-42 1 mg of powder was dissolved in 20 μl of anhydrous DMSO. After fully dissolved, 980 μl of saline was added, mixed evenly, and placed in a 4°C refrigerator to obtain 1 μg / μl Aβ 1-42 Solution.

[0034] SH-SY5Y cells in good logarithmic growth phase were cultured at 5×10 3 The cells were seeded at a density of 100 μl / 100 μl in a 96-well plate and cultured at 37 °C for 24 hours. Then the cells were treated in the following groups, each for 24 hours: blank control group: SH-SY5Y cells only (group 38); positive control group (Aβ 1-42 Treatment, group 39): SH-SY5Y cells + 1 μg / μl Aβ 1-42 ; Sample group: SH-SY5Y cells + 1 μg / μl Aβ 1-42 +10 μg / μl sample (experimental groups: 1-37). After treatment, SH-SY5Y cells in each group were incubated with MTT (final concentration in the incubation system was 0.5 mg / mL) for 4 h, and then incubated with 10% SDS solution at 37 °C for another 15 min. The absorbance was measured at 560 nm using a microplate reader. Cell viability was calculated, and the OD value of the experimental group was 560nm Value, recorded as As, control group and blank group OD 560nm The values ​​were recorded as Ac and Ab, and all samples were measured three times. Cell activity was calculated according to the following formula.

[0035] The experimental data are shown in Table 2.

[0036] Cell viability (%) = [(As-Ab) / (Ac-Ab)] × 100% As: absorbance of the experimental group (containing cells, culture medium, MTT solution, and anti-Alzheimer's disease composition); Ac: absorbance of the control group (containing cells, culture medium, and MTT solution, but not containing the anti-Alzheimer's disease composition); Ab: absorbance of blank group (containing culture medium and MTT solution, but not containing cells or anti-Alzheimer's disease composition).

[0037] Table 2 Cell activity test results

[0038] Note: * represents the difference between the recombinant human protein group and the blood-derived human albumin group, P ≤ 0.05, # Represents the difference between the positive control group and the blank group, P ≤ 0.05.

[0039] As shown in Table 2, the effect of a recombinant human albumin anti-Alzheimer's disease composition on the viability of SH-SY5Y cells: compared with the data of the bovine serum albumin group (37), the results of the blood-derived human albumin group (7-12) and the recombinant human albumin group (1-6) were much higher than those of the bovine serum albumin group, indicating that human albumin is more conducive to the growth of SH-SY5Y cells; the recombinant human albumin group (1-6) was better than the blood-derived human albumin group (7-12), with a cell survival rate of more than 129%. The combination of DPA, EPA and albumin (including blood-derived albumin) added at the same ratio (experimental group 3, group 9) was better than the combination of DPA, DHA, albumin (including blood-derived albumin) added at the same ratio (experimental group 27, group 29) and the combination of EPA, DHA, albumin (including blood-derived albumin) (experimental group 28, group 30). The experimental results of adding DHA, EPA and DPA alone showed that in terms of cell survival rate, DPA and EPA were more significant in cell proliferation ability than DHA. Adding three fatty acid compositions (35-36) at the same time did not improve the survival rate of SH-SY5Y cells compared with adding two fatty acid compositions (1-12). Therefore, the combination of two unsaturated fatty acids DPA and EPA is preferred. The cell survival rate of the blank control group (containing only cells, culture medium, MTT solution, group 38) was 100%, and the cell survival rate of the positive control group was only 70%, indicating that the anti-Alzheimer's disease composition prepared by this method was significantly different from the blank control and the positive control, indicating that the recombinant human albumin anti-Alzheimer's disease composition has the effect of promoting cell proliferation on SH-SY5Y.

[0040] At the same time, the experimental data of the experimental groups (groups 13-17) to which only recombinant human albumin, DHA, EPA, and DPA were added showed the lowest cell survival rate compared with the experimental groups (groups 1-12 and groups 18-36), indicating that the single component had the weakest activity on cell proliferation; at the same time, when only two of the three functional ingredients were added, the data results of the experimental groups (18-26) were 82%-108%, which was far lower than the data of the experimental groups (groups 1-12) of 119%, indicating that the anti-Alzheimer's disease composition can maintain a high cell activity under the premise of the coexistence of the three groups of active ingredients; at the optimal formula ratio, the experimental groups (groups 1-6) were much higher than other groups (7-30), and the cell activity value was >129%, indicating that the anti-Alzheimer's disease composition containing recombinant human albumin prepared by this method has a strong activity on the growth of human neuroblastoma cells.

[0041] Test Example 2: Alzheimer's disease animal behavior experiment Animals: 190 SPF mice, half male and half female, weighing 25-30 g, were purchased and kept in a clean environment, fed with special feed, and had unlimited food and water. Animal surgeries were performed in accordance with the Guide for the Care and Use of Laboratory Animals. Based on the results of the cell activity test, the experimental groups (1-12) and groups (27-30) were selected as sample groups for animal experiments.

[0042] After 7 days of adaptive feeding, 190 mice were randomly divided into blank group, sham operation group, model group and sample group (group: 1-19), a total of 19 groups, 10 mice in each group, anesthetized with isoflurane, fixed on a mouse stereotaxic instrument, and injected with 1 μg / μl Aβ in the lateral ventricle of mice in all groups except the blank group and the sham operation group. 1-42 Solution 3 μl, 2 min (constant speed), the needle was retained for 5 min to ensure sufficient diffusion of the solution. Penicillin sodium powder was sprinkled on the surgical site to prevent infection, the scalp was sutured, and 0.1 ml 40000U penicillin sodium solution was injected intramuscularly for 3 consecutive days after modeling. The sham operation group mice were injected with 3 μl of normal saline in the lateral ventricle at the same location, and the postoperative treatment method was the same as above.

[0043] Mice in the blank group, sham operation group, and model group were gavaged with 0.5% CMA-Na solution, and mice in the sample group (groups: 1-16) were gavaged with 80 mg / kg of the corresponding combination for 14 consecutive days. The experimental mice were subjected to Y maze and Morris water maze tests to verify the changes in their cognitive abilities.

[0044] (1) Y-maze experiment: The Y-maze experiment was conducted with reference to the method of Kim DH et al. (Neurobiol Learn Mem, 2007, 87 (4): 536-546). The Y-maze consists of 120° arms A, B, and C. The material is an iron plate with black paint on both the inner and outer surfaces. The arm is 47 cm long, 16 cm high, and 3 cm wide. The intersection of the three arms is an equilateral triangle. During the experiment, a layer of fresh bedding was laid on the bottom, and it was replaced after each mouse experiment. The smell left by the previous mouse was eliminated with 70% ethanol. Before the experiment began, the mouse was placed facing the A arm and allowed to explore the maze freely for 10 min. The total number of arm entries (entry into an arm is counted as one entry if the entire body except the tail enters the arm) and the order of arm entry were recorded, and the spontaneous alternation reaction rate was calculated. The formula is spontaneous alternation reaction rate = [number of correct alternation reactions / (total number of alternation reactions - 2)] × 100%. The data results are shown in Table 3.

[0045] Table 3 Y-maze test results

[0046] Note: # represents the difference between the model group and the blank group, P≤0.05, * represents the difference between the recombinant human protein group and the hematogenous human albumin group, P≤0.05.

[0047] According to the data in Table 3, the spontaneous alternation reaction rate of mice in the model group was reduced (52.2%) compared with the blank group (81.5%), indicating that intracerebroventricular injection of Aβ 1-42 The short-term memory of mice was damaged; compared with the model group, the spontaneous alternation reaction rate of mice in groups (1-16) increased, indicating that albumin can alleviate the damage caused by intracerebroventricular injection of Aβ 1-42 The short-term memory damage caused by Aβ injection in mice was better than that in groups (1-6) and (7-12). 1-42 The short-term memory damage caused is better than that of blood-derived human albumin.

[0048] (2) Morris water maze test: The Morris water maze test was conducted according to the method of Mutlu O (Drug Target Insights, 2015, 9 (1): 1-8). The Morris water maze consists of a circular pool with a diameter of 120 cm and a height of 40 cm, an automatic video recording and analysis system, and black ink is added after water is added to prevent interference. The water temperature is maintained at around 25 °C. The pool is covered with black curtains around it, and different decorations are hung on the curtains as reference objects. The water maze is divided into four quadrants: southeast, northeast, southwest, and northwest. A black circular platform 1 cm below the horizontal plane is placed in the center of a quadrant. During the test, the mouse is placed in the water facing the pool wall of a quadrant. The time required for the mouse to find and climb onto the platform is recorded according to the water maze video tracking system, that is, the escape latency. Each mouse is trained twice a day, and the above-mentioned positioning and navigation test is performed for 5 consecutive days. If the time exceeds 60 s in the first few training sessions, the mouse is guided to stay on the platform for 10 s. In this experiment, the maximum escape latency was set to 120 s, that is, if the mouse did not find the platform within 120 s, the escape latency was recorded as 120 s. The data results are shown in Table 4.

[0049] Table 4 Morris water maze test results

[0050] Note: # represents the difference between the model group and the blank group after the fifth day of administration and the first day of administration, P≤0.05, * represents the difference between the recombinant human protein group and the hematogenous human albumin group after the fifth day of administration and the first day of administration, P≤0.05.

[0051] Compared with the blank group, the escape latency of mice in the model group was prolonged from the second day, indicating that intracerebroventricular injection of Aβ1-42 The mice suffered from memory impairment of orientation and navigation, while the escape latency of the mice in the sham operation group (group 18) was not significantly different from that in the blank group, indicating that the surgery did not cause any impairment to the mice's orientation and navigation cognitive function; compared with the model group, the escape latency of the mice gavaged with the albumin combination (groups 1-16) was significantly shortened from the 4th day; the escape latency of the mice gavaged with the recombinant albumin combination (groups 1-6) was significantly shortened from the 5th day, and was lower than that of the mice gavaged with the blood-derived human albumin combination (groups 7-12), indicating that the recombinant human albumin group (1-6) had a better effect than the blood-derived human albumin group (7-12).

[0052] The above description is only an embodiment of the present invention. The protection scope of the present invention is not limited by these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent substitution, improvement, etc. made within the technical ideas and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An anti-Alzheimer's disease composition containing human albumin, characterized in that: The composition comprises the following components in parts by weight: 5 to 40 parts by weight of human albumin; and 1 to 20 parts by weight of polyunsaturated fatty acids (PUFAs) or their esters.

2. The composition according to claim 1, characterized in that The human albumin is selected from at least one of recombinant human albumin and hematogenous human albumin.

3. The composition according to claim 1, characterized in that The polyunsaturated fatty acids are selected from two of docosahexaenoic acid (22:6; DHA) or its esters, docosapentaenoic acid (22:5; DPA) or its esters, and eicosapentaenoic acid (20:5; EPA) or its esters.

4. The composition according to claim 1, characterized in that The composition also includes additives acceptable in the fields of medicines, foods and health products.

5. Use of the composition according to any one of claims 1 to 4 in the preparation of anti-Alzheimer's disease drugs, foods, and health products.

6. A method for preparing the composition according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: S1: mixing different types of polyunsaturated fatty acid solutions by weight; S2: adding the polyunsaturated fatty acid solution obtained in step S1 to a human albumin aqueous solution or a human albumin salt buffer solution in parts by mass, and making up the balance with deionized water to obtain a mixed solution of the composition.

7. The method according to claim 6, characterized in that The conditions for uniform mixing in step S1 are: 40-60°C, 50-100 rpm, and stirring for 10-30 min.

8. The method according to claim 6, characterized in that Step S2 also includes a high-pressure homogenization step.

9. The method according to claim 8, characterized in that The high-pressure homogenization conditions are: using a high-pressure homogenizer at 400-1200 N and high-pressure homogenization for 10-30 min.

10. The method according to claim 6, characterized in that The method further comprises the step of aseptically filtering the mixed solution of the composition prepared in step S2, wherein the filter diameter is 0.1 μm.

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