Preparation method of black soybean hull extract and application of black soybean hull extract in treatment of Alzheimer's disease

Abstract

The invention relates to the field of biological medicines, in particular to a preparation method of a novel black soybean hull extract and application of the black soybean hull extract in preparation of a medicine for treating Alzheimer's disease. The preparation method of the black soybean hull extract does not need toxic organic solvent extraction, is different from only extraction of black soybean hull anthocyanin, can reserve active ingredients of black soybean seed coats to the maximum extent, improves the yield and extraction efficiency of a final compound product, and reduces toxic substances possibly generated in the preparation process. Wild C57BL / 6 mice and disease model APP / Ps1 mice are utilized to research the influence of the black soybean hull extract on the Alzheimer's disease. Compared with disease model mice fed with standard mixed grain feed, the black soybean hull extract is added into diet, so that the learning and memory ability and the movement ability of the disease model mice can be remarkably improved, and the anxiety degree of the disease model mice is reduced. Therefore, the black bean peel extract has a remarkable treatment effect on the Alzheimer's disease. The black bean peel extract has the following advantages in the aspect of treating the Alzheimer's disease: the natural plant extract is non-toxic and has no side effect; raw materials are easily available and the preparation cost is low; the diet intake is convenient and practical. The black soybean hull extract has important scientific significance and application value for treating the Alzheimer's disease.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a method for preparing a black bean peel extract and its application in the treatment of Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is characterized by cognitive impairment, decreased learning and memory abilities, and visuospatial skill impairment. Histopathological features of AD include abnormal deposition of extraneuronal amyloid plaques, hyperphosphorylation of tau proteins associated with the cytoskeleton leading to the formation of intracellular neurofibrillary tangles, and ultimately, irreversible neuronal death. Currently, the efficacy of drugs developed for AD is unstable, and many carry risks of toxic side effects.

[0003] Black beans, derived from the black, dried seeds of the soybean plant (Soybean family), are a traditional food and medicinal crop. Black beans are rich in protein, unsaturated fatty acids, B vitamins, and vitamin E. The seed coat also contains abundant oils, pigments, and dietary fiber. Initially, black bean seed coats were widely studied as a substitute for synthetic pigments. The main components of anthocyanins extracted from black bean seed coats are anthocyanins, primarily cyanidin-3-O-glucoside. Subsequent studies have found that black bean seed coat anthocyanins also possess anti-inflammatory, antioxidant, and blood sugar and lipid-lowering functions. Based on these properties, black bean seed coat extract has great potential as a treatment for Alzheimer's disease. However, current methods for preparing black bean seed coat extract often focus on extracting high-purity anthocyanins, neglecting the importance of other nutrients. As a complex extract, there are no reports on how to maximize the preservation of its nutritional components, avoid the generation of toxic substances during the extraction process, and improve the yield of the final product during the extraction process.

[0004] The C57BL / 6 mouse is a widely used laboratory mouse strain in biomedical research. It is an inbred strain with a highly consistent genetic background and stable phenotypic characteristics. Its nervous system is similar to that of humans, and its brain structure and function share high homology with humans. Therefore, the APP / Ps1 disease model mouse based on the C57BL / 6 mouse can better simulate the pathological features of human Alzheimer's disease (AD) patients, such as β-amyloid (Aβ) deposition and tau protein hyperphosphorylation. The experimental conditions of mouse models are relatively easy to control and highly standardized, providing more reliable experimental data. Mouse models offer advantages in AD drug development, including high physiological and pathological similarity, precise gene manipulation capabilities, comprehensive drug efficacy evaluation methods, and higher experimental reproducibility, making them a superior choice for simulating complex human diseases and conducting in-depth research on drug mechanisms. Summary of the Invention

[0005] The purpose of this invention is to provide a novel and efficient method for preparing black soybean peel extract and its application in the treatment of Alzheimer's disease.

[0006] According to the application of the present invention, the black bean husk extract is prepared by the following method:

[0007] (1) Grinding: Grind the raw material black bean skin into powder.

[0008] (2) Extraction: Take an appropriate amount of black bean skin grinding material and put it into 22-28 times the volume of acidic ethanol extract. Adjust the pH to between 1 and 2 and extract at a constant temperature of 50-60℃ for 2 hours. During this period, perform two ultrasonic-assisted extractions, each for 15 minutes.

[0009] (3) Centrifugation: Centrifuge at 4℃, 5000rpm for 10min, and take the supernatant for later use.

[0010] (4) Concentration: Centrifuge the supernatant obtained by centrifugation at low temperature under vacuum until there is no alcohol odor, and concentrate the solution to a Baume degree close to 15.

[0011] (5) Drying: Black bean skin extract was obtained by vacuum spray drying.

[0012] (6) Yield: 25-35%.

[0013] This invention also provides the application of black bean husks in the preparation of formulations that enhance the learning and memory abilities of disease model mice.

[0014] This invention also provides the application of black bean husks in the preparation of formulations that enhance the motor function of disease model mice.

[0015] The present invention also provides the application of black bean husks in the preparation of formulations that reduce anxiety levels in disease model mice.

[0016] The method for preparing black soybean husk extract provided by this invention significantly increases the yield of black soybean husk extract, and the preparation process does not involve or generate toxic organic substances, and the extraction process is simple and efficient. Unlike methods that only extract anthocyanins from black soybean husks, this method retains a variety of active nutrients from the black soybean seed coat to the greatest extent, ultimately yielding a complex product, black soybean husk extract.

[0017] To verify the therapeutic effect of black soybean husk extract on Alzheimer's disease symptoms, this invention utilized wild-type C57BL / 6 mice and APP / Ps1 disease model mice for research. Mice, as commonly used model organisms in the biomedical field, possess advantages in AD drug development, including high physiological and pathological similarity, precise gene manipulation capabilities, comprehensive efficacy evaluation methods, and higher experimental reproducibility—advantages not found in other non-mammalian animal models. Starting at 3 months of age, C57BL / 6 mice and APP / Ps1 mice were fed a standard mixed grain diet and a grain diet supplemented with black soybean husk extract, respectively. Mice with different genotypes under different dietary conditions were cultured to 9 months of age and then subjected to water maze, open field, and elevated cruciate maze tests. Experiments demonstrated that the black soybean husk extract provided by this invention significantly improved the learning and memory abilities and motor skills of the disease model mice, and significantly reduced their anxiety levels. The above experiments confirm that the black bean peel extract provided by this invention can significantly improve the symptoms of Alzheimer's disease, which is of great scientific significance for research in the field of neurodegenerative diseases, and is expected to play a key role in the treatment of Alzheimer's disease symptoms.

[0018] This invention provides a highly efficient and non-toxic preparation process for black soybean husk extract, preserving the original active ingredients of the black soybean seed coat to the greatest extent. The therapeutic effect of black soybean husk extract on Alzheimer's disease was demonstrated using mouse model organisms. Researching and utilizing black soybean husk extract to treat Alzheimer's disease can reduce the risk of toxic side effects from chemical drugs. Furthermore, the raw material for black soybean husk extract is readily available, which can reduce treatment costs. Therefore, this invention has significant scientific and practical value for the treatment of Alzheimer's disease. Attached Figure Description

[0019] Figure 1 This study demonstrates the effects of feeding standard mixed grain diets and grain diets supplemented with black soybean hull extract on the learning and memory abilities of wild-type and disease model mice.

[0020] Figure 2 This study demonstrates the effects of feeding standard mixed grain diets and grain diets supplemented with black soybean hull extract on the locomotor function of wild-type and disease model mice.

[0021] Figure 3 This study demonstrates the effects of feeding standard mixed grain diets and grain diets supplemented with black soybean hull extract on anxiety levels in wild-type and disease model mice. Detailed Implementation

[0022] Example 1: Effects of standard mixed grain diet and grain diet supplemented with black soybean hull extract on learning and memory abilities in wild-type and disease model mice.

[0023] 1. Preparation of black soybean peel extract

[0024] (1) Grinding: Grind the raw material black bean skin into powder.

[0025] (2) Extraction: Take an appropriate amount of black bean skin grinding material and put it into 22-28 times the volume of acidic ethanol extract. Adjust the pH to between 1 and 2 and extract at a constant temperature of 50-60℃ for 2 hours. During this period, perform two ultrasonic-assisted extractions, each for 15 minutes.

[0026] (3) Centrifugation: Centrifuge at 4℃, 5000rpm for 10min, and take the supernatant for later use.

[0027] (4) Concentration: Centrifuge the supernatant obtained by centrifugation at low temperature under vacuum until there is no alcohol odor, and concentrate the solution to a Baume degree close to 15.

[0028] (5) Drying: Black bean skin extract was obtained by vacuum spray drying.

[0029] (6) Yield: 50-70%

[0030] 2. Formulation of cereal feed with added black soybean hull extract

[0031] Mix the black bean husk extract evenly into the grain feed according to the specified ratio.

[0032] 3. Testing of the water maze in mice with different genotypes under different dietary conditions.

[0033] Adaptation period (day 1): Three days in advance, transfer mice that have been cultured to 9 months of age to the test room. At the beginning of day 1, mice with different genotypes and dietary conditions are placed in a water maze device and allowed to explore freely to adapt to the environment. Each mouse explores for 2 minutes, and the exploration is repeated 3 times.

[0034] Learning period 1 (days 2-3): The test platform is 1 cm above the water surface. Each mouse is placed in the quadrant opposite to the platform and tested again. If a mouse does not find the platform within 60 seconds, it is guided to the target platform to rest for 20 seconds. Each mouse undergoes three learning cycles.

[0035] Learning Period 2 (Days 4-6): The test platform was submerged in water, and titanium dioxide was added to blur the surface of the water. Each mouse was placed in the quadrant opposite to the platform and tested again. If a mouse could not find the platform within 60 seconds, it was guided to the target platform to rest for 20 seconds. Each mouse underwent three learning cycles.

[0036] Testing period (day 7): The testing platform was removed, and each mouse was placed in the quadrant opposite to the quadrant where the learning platform was located for testing. The testing time was 60 seconds. Each mouse was tested three times.

[0037] like Figure 1As shown in the figure, A represents the latency time for mice to enter the target quadrant during the testing period. A shorter latency time indicates that the mouse enters the target quadrant earlier and has better spatial memory. B represents the duration of time mice stay in the target quadrant during the testing period. The longer the mouse stays in the target quadrant, the stronger its spatial memory. C represents the latency time for mice to enter the platform area during the testing period. A shorter latency time indicates that the mouse can find the platform location during the learning period faster and has better learning and memory abilities. D represents the number of times mice cross the platform area during the testing period. The more times a mouse crosses the platform, the stronger its learning and memory abilities.

[0038] Compared to disease model mice fed a standard mixed grain diet, mice fed a grain diet supplemented with black soybean hull extract significantly shortened latency in the target quadrant and plateau region, significantly prolonged residence time in the target quadrant, and significantly increased the number of times they crossed the plateau region. Furthermore, wild-type mice fed a grain diet supplemented with black soybean hull extract showed a significantly shorter latency in the target quadrant compared to wild-type mice fed only a standard mixed grain diet. The addition of black soybean hull extract to the diet significantly restored the lost learning and memory abilities in disease model mice and also showed some improvement in the learning and memory abilities of wild-type mice.

[0039] Note: Each group should be repeated at least 6 times. The method for analyzing significant differences is: mean ± SD (n≥6, two-tailed t test, *p<0.05, **p<0.01, ***p<0.001). "Normal" refers to feeding standard mixed grain feed; "BSCE" refers to feeding grain feed supplemented with black soybean hull extract.

[0040] Example 2: Effects of standard mixed grain diet and grain diet supplemented with black soybean hull extract on the motor function of disease model mice.

[0041] Black soybean hull extract was prepared as described in Example 1, and a grain feed containing black soybean hull extract was added.

[0042] Open field tests were conducted on mice of different genotypes under varying dietary conditions.

[0043] Three days prior to the experiment, mice aged 9 months were transferred to the test room to acclimatize to the environment. At the start of the experiment, the mice were placed near the boundary between the central and peripheral areas and allowed to move freely in the test chamber for 5 minutes to acclimatize. The mice's activity in the test chamber was then recorded for 2 minutes.

[0044] like Figure 2As shown in the figure, A represents the total distance the mouse moved in the experimental box. The longer the distance, the more active the mouse and the stronger its motor ability. B represents the number of times the mouse traversed the area laterally in the experimental box. The more times it traversed, the more active and capable the mouse was. Compared with disease model mice fed a standard mixed grain diet, feeding them a grain diet supplemented with black soybean hull extract significantly increased the distance the disease model mice moved in the experimental box and the number of times they traversed the area laterally. However, feeding them a grain diet supplemented with black soybean hull extract had no significant effect on the motor ability of wild-type mice. Adding black soybean hull extract to the diet significantly restored the decreased motor ability of disease model mice.

[0045] Note: Each group had 8 replicates. The method for analyzing significant differences was mean ± SD (n = 8, two-tailed t-test, *p < 0.05, **p < 0.01, ***p < 0.001). "Normal" referred to feeding with a standard mixed grain diet; "BSCE" referred to feeding with a grain diet supplemented with black soybean hull extract.

[0046] Example 3: Effects of standard mixed grain diet and grain diet supplemented with black soybean hull extract on anxiety levels in disease model mice.

[0047] Black soybean hull extract was prepared as described in Example 1, and a grain feed containing black soybean hull extract was added.

[0048] Open field tests were conducted on mice of different genotypes under different dietary conditions, as described in Example 2.

[0049] Conducting elevated cross maze tests on different genotypes grown from different foods:

[0050] Three days prior to the experiment, mice aged 9 months were transferred to the testing room to acclimatize to the environment. At the start of the experiment, the mice were placed in the central area and allowed to move freely in the maze for 5 minutes to adapt to the environment. Their activity in the maze was then recorded for 2 minutes.

[0051] like Figure 3As shown in the figure, A represents the proportion of the distance the mouse traveled in the central area of ​​the experimental box during the open field test. A higher proportion means the mouse had more time to move in the central area, had a stronger desire to explore, was more relaxed, and had a lower level of anxiety. B represents the number of times the mouse entered the open arm in the elevated cross maze test. The more times the mouse entered the open arm, the lower its level of anxiety. C represents the anxiety index of the mouse in the elevated cross maze test, which is calculated from the proportion of the number of times the mouse entered the closed arm to the total traversed area. A higher anxiety index means a higher level of anxiety. D represents the proportion of the time the mouse spent in the open arm in the elevated cross maze test to the total test time, reflecting the mouse's comfort and sense of security in the open arm. The longer the time spent in the open arm, the lower the level of anxiety.

[0052] Compared with disease model mice fed a standard mixed grain diet, feeding a grain diet supplemented with black soybean hull extract significantly increased the proportion of movement distance in the central area of ​​the test chamber in the open field test, significantly increased the number of times the disease model mice entered the open arm in the elevated cross maze test, significantly reduced the anxiety index exhibited by the mice in the elevated cross maze test, and significantly prolonged the time the mice spent in the open arm area in the elevated cross maze test. However, feeding a grain diet supplemented with black soybean hull extract had no significant effect on the anxiety level of wild-type mice. Adding black soybean hull extract to the diet significantly reduced the increased anxiety level in disease model mice.

[0053] Note: Each group had at least 6 replicates. The method for analyzing significant differences was: mean ± SD (n ≥ 6, two-tailed t-test, *p < 0.05, **p < 0.01, ***p < 0.001). "Normal" referred to feeding standard mixed grain diets; "BSCE" referred to feeding grain diets supplemented with black soybean hull extract.

Claims

1. The application of black soybean husk extract in the preparation of drugs for treating Alzheimer's disease, characterized in that, The black bean peel extract was prepared by the following method. (1) Grinding: Grind the raw material black bean skin into powder. (2) Extraction: Take an appropriate amount of black bean skin grinding material and put it into 22-28 times the volume of acidic ethanol extract. Adjust the pH to between 1 and 2 and extract at a constant temperature of 50-60℃ for 2 hours. During this period, perform two ultrasonic-assisted extractions, each for 15 minutes. (3) Centrifugation: Centrifuge at 4℃, 5000rpm for 10min, and take the supernatant for later use. (4) Concentration: Centrifuge the supernatant obtained by centrifugation at low temperature under vacuum until there is no alcohol odor, and concentrate the solution to a Baume degree close to 15. (5) Drying: Black bean skin extract was obtained by vacuum spray drying. (6) Yield: 25-35%.

2. Application of black bean husks in the preparation of formulations that enhance the learning and memory abilities of disease model mice.

3. Application of black bean husks in the preparation of formulations that enhance the motor function of disease model mice.

4. Application of black bean husks in the preparation of formulations that reduce anxiety levels in disease model mice.

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