Preparation method of broad bean leaf extract as well as product and application of broad bean leaf extract

Abstract

The invention discloses a preparation method of a broad bean leaf extract and a product and application thereof, and the preparation method comprises the following steps: S1, freeze-drying fresh broad bean leaves, crushing and grinding to obtain broad bean leaf powder; s2, adding the broad bean leaf powder into a hydrochloric acid aqueous solution for ultrasonic extraction to obtain a mixture; s3, centrifuging the mixture, and collecting supernate, namely the broad bean leaf extract. The content of levodopa in the broad bean leaf extract is detected through high performance liquid chromatography, it is found that the content of levodopa in the broad bean leaf extract is higher than that of other broad bean part extracts, and the broad bean leaf extract can be used for preparing the medicine for treating and improving the Alzheimer's disease. The technical support is provided for extending the broad bean industry chain and promoting the rapid development of the broad bean deep processing industry.

Description

Technical Field

[0001] This invention belongs to the field of plant extract technology, specifically relating to a method for preparing broad bean leaf extract, its products, and applications. Background Technology

[0002] Levodopa (L-DOPA, L-3,4-dihydroxyphenylalanine) is a precursor to dopamine and plays a crucial role in the central nervous system. It is a core drug component in the treatment of Parkinson's disease, as the main pathological feature of Parkinson's disease is the degeneration of dopaminergic neurons in the substantia nigra; supplementing with levodopa can effectively alleviate symptoms. Therefore, the efficient and high-purity extraction of levodopa is of great significance to the pharmaceutical industry.

[0003] The seeds of legumes such as broad beans contain high concentrations of levodopa, making it a natural and renewable plant source with development potential. However, current traditional methods for extracting levodopa from broad bean flour have several major problems:

[0004] (1) Low extraction efficiency

[0005] Traditional extraction methods mainly include water extraction and alcohol extraction. Although these methods are simple to operate, their extraction efficiency is relatively low, primarily because:

[0006] Solubility limitation: L-DOPA has limited solubility in water or ethanol, making it difficult to completely dissolve the target component during extraction.

[0007] Cell wall barrier: The dense structure of plant cell walls prevents levodopa from being released from the cell into the extract.

[0008] Long extraction time and low temperature: Traditional methods often use room temperature or low temperature extraction, which makes it difficult to effectively destroy cell structure, resulting in insufficient extraction.

[0009] (2) Insufficient purity

[0010] During the extraction process, in addition to levodopa, a large number of other plant components are also extracted, such as proteins, polysaccharides, flavonoids, phenols, and pigments. These impurities not only affect the purity of the final product but may also interfere with subsequent separation and purification steps. The main problems include:

[0011] Lack of selectivity: Traditional solvents have the ability to dissolve a variety of components, making it difficult to selectively extract levodopa.

[0012] Co-extraction is severe: many compounds with similar polarity are extracted along with levodopa, increasing the difficulty of subsequent purification.

[0013] The purification process is complex, requiring multiple column chromatography and recrystallization steps to obtain a high-purity product, which increases operating costs and time.

[0014] (3) High cost

[0015] The large amounts of chemical reagents and complex purification steps used in traditional extraction processes significantly increase production costs.

[0016] High reagent consumption: The large amount of reagents used, such as ethanol, acids and bases, increases the cost of raw materials.

[0017] High energy consumption: Steps such as heating, concentration, and drying require a large amount of energy.

[0018] High waste disposal costs: The use of chemical reagents generates a large amount of waste liquid, which requires environmentally friendly treatment, further increasing costs.

[0019] Large equipment investment: To achieve high-purity products, complex separation and purification equipment (such as high-performance liquid chromatography, membrane separation, etc.) is required.

[0020] (4) The process is complex and difficult to industrialize.

[0021] While traditional extraction methods can achieve certain results in the laboratory stage, they face many challenges in industrial production:

[0022] The operation is complicated: it involves multiple steps, including extraction, filtration, concentration, purification, crystallization, and drying. The process is lengthy and has a low degree of automation.

[0023] Poor batch repeatability: Due to the complexity of the operation, the product quality varies greatly between different batches, affecting the consistency of the drug.

[0024] High equipment requirements: Industrialized production requires continuous and automated equipment support, which traditional methods cannot meet.

[0025] Significant environmental pressure: The use of large amounts of organic solvents and chemical reagents puts pressure on the environment and is inconsistent with the development trend of green chemistry.

[0026] In summary, traditional methods for extracting L-DOPA from broad bean flour suffer from problems such as low efficiency, poor purity, high cost, and complex processes, which limit its application in industrial production.

[0027] Therefore, there is an urgent need for an efficient, environmentally friendly, and industrially viable method for extracting levodopa. Summary of the Invention

[0028] The purpose of this invention is to provide a method for preparing broad bean leaf extract, its products, and applications, in order to solve the problems of low efficiency, poor purity, high cost, and complex process in the extraction of L-DOPA from broad bean powder.

[0029] To achieve the above objectives, the first aspect of the present invention provides a method for preparing broad bean leaf extract, comprising the following steps:

[0030] S1: Freeze-dry fresh broad bean leaves and grind them to obtain broad bean leaf powder;

[0031] S2: Broad bean leaf powder was added to hydrochloric acid aqueous solution and subjected to ultrasonic extraction to obtain a mixture;

[0032] S3: Centrifuge the mixture and collect the supernatant, which is the broad bean leaf extract.

[0033] Preferably, in step S1, after crushing and grinding, the broad bean leaf powder is obtained by passing it through a 200-mesh sieve.

[0034] Preferably, in step S2, the concentration of the hydrochloric acid aqueous solution is 1–3 wt%.

[0035] Preferably, in step S2, the mass-to-volume ratio of broad bean leaf powder to hydrochloric acid aqueous solution is 1g:40-60mL.

[0036] Preferably, in step S2, the ultrasonic power is 200W and the time is 20-40min.

[0037] The second aspect of the present invention provides a method for detecting levodopa in broad bean leaf extract. The broad bean leaf extract obtained by the above preparation method is used as the test solution, and high performance liquid chromatography is used for analysis and detection. Before each injection, the sample is filtered through a 0.22 μm microporous membrane and stored in a -4°C refrigerator for later use.

[0038] Preferred analytical conditions for high performance liquid chromatography are as follows:

[0039] Chromatographic column: CAPCELL PAK CR (4.6 mm l.D. × 150 mm, 5 μm) column;

[0040] Mobile phase: 0.1% trifluoroacetic acid buffer solution and methanol were used as the mobile phase;

[0041] Flow rate: 0.8 mL / min;

[0042] Column temperature: 30℃;

[0043] Detection wavelength: 280nm;

[0044] Injection volume: 15 μL.

[0045] Preferably, the content of levodopa in the broad bean leaf extract is 1.21%.

[0046] The third aspect of this invention provides the use of the broad bean leaf extract prepared by the aforementioned method in the preparation of drugs for the treatment and prevention of Alzheimer's disease.

[0047] The fourth aspect of this invention provides the application of the broad bean leaf extract prepared by the aforementioned method in foods or health products that help improve memory.

[0048] Preferably, the dosage form of the pharmaceutical, food, or health product is freeze-dried powder, capsules, oral liquid, pills, granules, or tablets. The broad bean leaf extract of this invention can be prepared by adding commonly used excipients in the art according to the dosage form.

[0049] Therefore, the present invention provides a method for preparing a broad bean leaf extract with the above-described structure, as well as the product and its application, which have the following beneficial effects:

[0050] (1) This invention uses levodopa from broad beans as the main active ingredient. It is a natural plant extract with high safety and biocompatibility. Compared with traditional chemical drugs, broad bean extract has no obvious toxic side effects and is suitable for long-term use, especially for early intervention and adjuvant improvement of Alzheimer's disease (AD) patients.

[0051] (2) In addition to being a potential drug ingredient, the levodopa in the broad beans of this invention can also be developed into functional foods or health products, such as capsules and oral liquids, to further expand its application scope. This multi-purpose development not only meets market demand but also provides more choices for AD patients.

[0052] (3) Broad beans are a widely cultivated crop with abundant resources and low cost. This invention utilizes levodopa in broad beans, which not only increases the added value of broad beans, but also provides a new direction for the development of agricultural by-products, and has significant economic and social benefits.

[0053] (4) This invention optimizes the extraction rate and purity of levodopa, reduces resource waste, meets the requirements of sustainable development, and the development of natural plant extracts reduces dependence on chemically synthesized drugs, which is beneficial to environmental protection and ecological balance.

[0054] (5) This invention optimizes the sample extraction and processing method by using hydrochloric acid aqueous solution and ultrasound-assisted extraction, achieving high-efficiency and high-purity levodopa extraction. In addition, through single-factor and orthogonal experimental design, parameters such as liquid-to-solid ratio, solvent pH and extraction time are optimized to improve the extraction yield of levodopa, making the entire extraction process more environmentally friendly and suitable for industrial production.

[0055] The technical solution of the present invention will be further described in detail below with reference to embodiments and accompanying drawings. Attached Figure Description

[0056] Figure 1 This is a process flow diagram of the present invention;

[0057] Figure 2 The high-performance liquid chromatograms of the levodopa reference standard and the test solution are shown.

[0058] Figure 3 This is the standard curve for levodopa;

[0059] Figure 4 The effects of different extraction methods (a), ultrasonic time (b), ultrasonic power (c), and ultrasonic extraction temperature (d) on the L-DOPA content in broad beans.

[0060] Figure 5 The expression of APP protein in WT group cells and APP695 group cells is shown in (a) Western blot results and (b) grayscale analysis.

[0061] Figure 6 The results show the expression of APP after intervention with broad bean leaves. (a) shows the results of Western blot, from left to right: control group, low-dose group of broad bean leaf freeze-dried powder (50 mg / mL), medium-dose group of broad bean leaf freeze-dried powder (100 mg / mL), and high-dose group of broad bean leaf freeze-dried powder (200 mg / mL). (b) shows the statistical analysis results of APP expression.

[0062] Figure 7 The expression of Aβ42 after intervention with freeze-dried broad bean leaf powder is shown in (a) Western blot results and (b) statistical analysis results of Aβ42 expression.

[0063] Figure 8 The images show the expression of Tau and p-tau after intervention with freeze-dried broad bean leaf powder. (a) shows the results of Western blot, (b) shows the statistical analysis results of Tau expression, and (c) shows the statistical analysis results of p-tau expression. Detailed Implementation

[0064] The present invention will be further described below. It should be noted that this embodiment is based on the present technical solution and provides detailed implementation methods and specific operation processes, but the present invention is not limited to this embodiment.

[0065] Example 1

[0066] This embodiment provides a method for preparing broad bean leaf extract, characterized by comprising the following steps:

[0067] S1: Freeze-dry fresh broad bean leaves and grind them into powder. After grinding, pass the powder through a 200-mesh sieve to obtain broad bean leaf powder.

[0068] S2: Accurately weigh 0.500g of broad bean leaf powder and place it in a 25mL Erlenmeyer flask with a stopper. Add 25mL of 2% hydrochloric acid aqueous solution and extract it under ultrasonic treatment at 200W for 30min. Then, add water to the mark to obtain a mixture.

[0069] S3: Centrifuge the mixture at 4500 r / min for 15 min and collect the supernatant, which is the broad bean leaf extract.

[0070] Comparative Example 1

[0071] The difference between this comparative example and Example 1 is that the raw materials used for extraction are different. In this comparative example, the fresh broad bean leaves in Example 1 are replaced with fresh broad bean stems.

[0072] Comparative Example 2

[0073] The difference between this comparative example and Example 1 is that the raw materials used for extraction are different. In this comparative example, the fresh broad bean leaves in Example 1 are replaced with fresh broad bean seeds.

[0074] Example 2

[0075] This embodiment provides a method for detecting levodopa in broad bean extract. The extracts prepared in Example 1 and Comparative Examples 1-2 are used as test solutions. The high performance liquid chromatography is used for analysis and detection. Before each injection, the sample is filtered through a 0.22 μm microporous membrane and stored in a -4℃ refrigerator for later use.

[0076] The chromatographic conditions for high performance liquid chromatography are as follows:

[0077] Chromatographic column: CAPCELL PAK CR (4.6 mm l.D. × 150 mm, 5 μm) column;

[0078] Mobile phase: 0.1% trifluoroacetic acid buffer solution (phase A) and methanol (phase B) were used as the mobile phase;

[0079] Flow rate: 0.8 mL / min;

[0080] Column temperature: 30℃;

[0081] Detection wavelength: 280nm;

[0082] Injection volume: 15 μL.

[0083] Detailed analysis process:

[0084] (1) Inject the filtered test solution into the HPLC system, perform qualitative analysis based on the retention time, and record the peak area.

[0085] (2) Linear relationship: A standard curve was plotted with peak area as the ordinate (Y) and levodopa reference concentration as the abscissa (X). The regression equation was calculated, and the linear relationship equation was: Y = 5.093X - 29.393 (r = 0.9998). The results showed that there was a good linear relationship between the peak area and mass concentration of levodopa.

[0086] from Figure 1 As can be seen from the data, the retention time of the levodopa reference standard was 8.447 min. Figure 3 The linear relationship curve of levodopa can be seen in the graph.

[0087] (3) Sample content determination

[0088] The broad bean leaf extract of Example 1, the broad bean stem extract of Comparative Example 1, and the fresh broad bean seed extract of Comparative Example 2 were each tested in triplicate, and the content of the samples was determined according to the above chromatographic conditions. Figure 2 The peak areas of the extracts from Examples 1 and Comparative Examples 1-2 show that the content order is broad bean leaves > broad bean stems > fresh broad bean seeds. Calculations show that the L-DOPA content in the broad bean leaf extract of Example 1 is 1.21 ± 0.0134%, the L-DOPA content in the broad bean stem extract of Comparative Example 1 is 0.25 ± 0.0005%, and the L-DOPA content in the broad bean seed extract of Comparative Example 2 is 0.07 ± 0.0015%. The L-DOPA content in the broad bean stems and fresh beans is 20.66% and 5.79% of that in the fresh broad bean leaves, respectively, indicating that the L-DOPA content in the broad bean leaves is significantly higher than in other parts.

[0089] (4) Precision, repeatability, stability and recovery of samples

[0090] Precision test: The prepared sample solution was measured repeatedly 6 times. The RSD value of levodopa was 0.14%, indicating that the detection method has good precision.

[0091] Repeatability test: Six portions each of broad bean leaves, broad bean stems and fresh broad bean seeds were accurately pipetted from the test solution, and the content of levodopa was detected under the above chromatographic conditions. The results showed that the RSD values ​​of broad bean leaves, broad bean stems and fresh broad bean seeds were 1.25%, 0.18% and 2.16%, respectively, indicating that the detection method had good repeatability.

[0092] Stability test: The same batch of test solution was injected at 0, 2, 4, 6, 8, 12 and 24 h according to the chromatographic conditions. The RSD value of levodopa was 0.48%, indicating that the test solution was stable within 24 h.

[0093] Recovery experiment: Based on the repeatability test results, the average levodopa content was used as the calculated background value for different parts of broad bean. Six portions of broad bean leaves, stems, and fresh beans with known content were precisely measured for each group. Different volumes of levodopa reference solution were precisely added to each group, and the content was determined according to the chromatographic conditions described above. The recovery rate was calculated. The results showed that the average spiked rates were 90.76%, 95.13%, and 100.80%, respectively, and the RSD values ​​were 0.96%, 0.66%, and 0.54%, respectively, indicating good recovery performance.

[0094] Therefore, the high performance liquid chromatography method of this invention is used to test the content of levodopa in the extract, and the test results are relatively accurate.

[0095] Comparative Example 3

[0096] The difference between this comparative example and Example 1 is that the solvent used for extraction is different; in this comparative example, the 2% hydrochloric acid aqueous solution in Example 1 is replaced with a 0.1 mol / L acetic acid solution. The content of levodopa in the obtained extract was detected according to the method in Example 2, and the content of levodopa in the extract was 0.97 ± 0.0423%.

[0097] Comparative Example 4

[0098] The difference between this comparative example and Example 1 is that the solvent used for extraction is different; in this comparative example, the 2% hydrochloric acid aqueous solution in Example 1 is replaced with a 0.1 mol / L phosphoric acid solution. The content of levodopa in the obtained extract was detected according to the method in Example 2, and the content of levodopa in the extract was 0.82 ± 0.0204%.

[0099] Comparative Example 5

[0100] The difference between this comparative example and Example 1 lies in the extraction method. In this comparative example, the ultrasonic extraction in Example 1 is replaced with hot reflux extraction. Specifically, 0.5g of broad bean leaf powder is weighed and added to a 2% hydrochloric acid aqueous solution, brought to a final volume of 25ml, refluxed at 90℃ for 1 hour, centrifuged for 15 minutes, and filtered through a 0.22μm filter membrane. The content of levodopa in the extract is 1.00±0.0261%.

[0101] Comparative Example 6

[0102] The difference between this comparative example and Example 1 is that the extraction method is different. In this comparative example, the ultrasonic extraction in Example 1 is replaced by cold soaking. The specific process is as follows: 0.5g of broad bean leaf powder is weighed and added to a 2% hydrochloric acid aqueous solution, the volume is adjusted to 25ml, and it is refrigerated at 4°C for 24 hours. After centrifugation for 15 minutes, it is filtered through a 0.22μm filter membrane. The content of levodopa in the extract is 1.14±0.0496%.

[0103] Comparative Example 7

[0104] The difference between this comparative example and Example 1 is that the extraction method is different. In this comparative example, the ultrasonic extraction in Example 1 is replaced by a combined ultrasonic and microwave extraction. The specific process is as follows: 0.5g of broad bean leaf powder is weighed and added to a 2% hydrochloric acid aqueous solution, and the volume is adjusted to 25ml. The temperature is 60℃, the power is 200W, the time is 30min, the centrifugation time is 15min, and the mixture is filtered through a 0.22μm filter membrane. The content of levodopa in the extract is 1.02±0.0826%.

[0105] Example 3

[0106] After concentrating the broad bean leaf extract prepared in Example 1, it was frozen at -80℃ for 3 days, dried in a freeze dryer, and then pulverized to obtain broad bean leaf freeze-dried powder. The broad bean leaf freeze-dried powder obtained above was used in Alzheimer's disease. The test process was as follows: (1) Prepare broad bean mother liquor (weigh 7.4mg of broad bean leaf freeze-dried powder and dissolve it in 1ml of DMSO to prepare a 20mM mother liquor, and store it at -20℃ in the dark), SH-SY5Y / WT medium (performed in a clean bench. For every 100ml of medium prepared, 94ml of DMEM, 5ml of fetal bovine serum, and 1ml of penicillin and streptomycin are required. Store at 4℃.), APP695 medium (performed in a clean bench. For every 100ml of medium prepared, 93.9ml of DMEM, 5ml of fetal bovine serum, 1ml of penicillin and streptomycin are required. zeocin. Store at 4℃. ), Low-dose group culture medium of broad bean leaf freeze-dried powder (in a clean bench, mix broad bean mother liquor with APP (SH-SY5Y / APP695swe) culture medium at a ratio of 1:20000. Prepare fresh and use immediately.), Medium-dose group culture medium of broad bean leaf freeze-dried powder (medium-dose group culture medium of broad bean: in a clean bench, mix broad bean mother liquor with APP (SH-SY5Y / APP695swe) culture medium at a ratio of 1:8000. Prepare fresh and use immediately.), High-dose group culture medium of broad bean leaf freeze-dried powder (in a clean bench, mix broad bean mother liquor with APP (SH-SY5Y / APP695swe) culture medium at a ratio of 1:4000. Prepare fresh and use immediately.); (2) Divide the cells into normal group (SH- SY5Y / WT cells), model group (SH-SY5Y / APP695swe cells), solvent group (APP695 cells treated with DMSO only without broad bean leaf freeze-dried powder) and low, medium and high dose groups of broad bean leaf freeze-dried powder (SH-SY5Y / APP695swe cells treated with different broad bean culture media for 24h); (3) The corresponding cells were revived, changed and passaged. The corresponding cells with good growth status were taken, the corresponding culture medium was changed, and the cells were cultured for 24h. The collected cell proteins were lysed on ice for 20min with RIPA cell lysis buffer. The quantification was performed according to the requirements of BCA kit and instructions. The mixed sample was placed in boiling water for 5min for denaturation. The protein samples were separated on 8% to 10% SDS-PAGE and transferred to a membrane. The membrane was blocked at room temperature for 2h with 5% skim milk blocking buffer. The corresponding antibody was diluted with blocking buffer to an appropriate concentration (the above proteins are all about 1:1000). The membrane was contacted with the antibody and incubated at 4℃ for at least 24h. The following day, the membrane was diluted with the matched secondary antibody and then incubated at room temperature for 1 hour. Protein images were obtained using a high-sensitivity chemiluminescence detection reagent on a chemiluminescence imaging system. ImagJ was used to analyze the band density and calculate the optical density ratio of the target band to the internal control band.

[0107] SH-SY5Y cells are human neuroblastoma cells with a functionally mature neuronal phenotype. Their cell morphology and physiological and biochemical functions are similar to normal nerve cells, making them suitable as a substitute for primary neuronal cultures. They are widely used to establish in vitro models of neurodegenerative diseases, especially Parkinson's and Alzheimer's diseases. In the SH-SY5Y / APP695 cell line, APP expression is significantly higher than in the SH-SY5Y / WT cell line, which may mimic the pathological state of APP overexpression in Alzheimer's disease. Therefore, it can serve as a good AD model.

[0108] The test results are as follows: Analysis of the effect of freeze-dried broad bean leaf powder on Alzheimer's disease (AD) (cellular level).

[0109] (1) Verify the expression of APP in AD model cells APP695.

[0110] from Figure 5 As can be seen, the protein expression level of APP in APP695 cells was significantly higher than that in SH-SY5Y / WT cells. Gray-scale analysis and statistical analysis of the two groups showed a statistically significant difference (P<0.001). The experimental results indicate that APP695 cells can stably and highly express APP protein and can be used as an AD model cell.

[0111] (2) Effect of freeze-dried broad bean leaf powder on APP protein expression

[0112] from Figure 6 As can be seen, compared with the internal control, the expression levels of APP in the low, medium, and high dose treatment groups of broad bean leaf freeze-dried powder were significantly lower than those in the control group, and the comparisons were statistically significant (P < 0.01). The experimental results indicate that broad bean leaf freeze-dried powder can reduce the expression of APP in APP695 cells.

[0113] (3) Effects of broad bean Aβ42 and BACE1 expression

[0114] from Figure 7 As can be seen, compared with the control group, the expression level of Aβ42 in the medium and high dose groups of broad bean leaf freeze-dried powder (experimental group) was significantly reduced, and the difference was statistically significant (P<0.001). Furthermore, in terms of BACE1 expression, the expression level was higher in the control group, increased in the low dose group after intervention with broad bean leaf freeze-dried powder, but decreased in the medium dose group, and the difference was statistically significant (P<0.05). The experimental results indicate that broad bean leaf freeze-dried powder can reduce the expression of Aβ42 in APP695 cells, and at appropriate concentrations, it can also reduce the expression of BACE1 in APP695 cells.

[0115] (4) Effects of broad beans on the expression of Tau and p-tau

[0116] from Figure 8 As can be seen, the change in tau expression after intervention with freeze-dried broad bean leaf powder was not significant, therefore no further analysis was performed. In P-tau, compared with the control group, the expression levels in the medium and high dose groups of freeze-dried broad bean leaf powder were significantly reduced, with statistically significant differences (P < 0.001). The experimental results indicate that the phosphorylation level of P-tau protein in cells after intervention with freeze-dried broad bean leaf powder was significantly lower than that in the control group.

[0117] Therefore, in cell experiments, APP expression was significantly reduced at all three concentrations compared to the control group after treatment with freeze-dried broad bean leaf powder. APP is a precursor protein of Aβ, and Aβ accumulation is one of the key pathological features of AD. The reduction in APP expression may indicate a decrease in Aβ production, which helps in understanding the pathological mechanism of AD. Aβ42 expression decreased in the medium- and high-dose groups, suggesting that high-dose treatment may effectively reduce Aβ42 expression. Aβ42 plaque accumulation may lead to neurotoxicity, inducing neuronal death and synaptic dysfunction. Reducing Aβ42 plaques can alleviate this neurotoxicity, thereby delaying the neurodegenerative process. The reduction of Aβ42 plaques may help restore synaptic function and improve signal transmission between neurons, thus having a positive impact on cognitive function.

[0118] BACE1 (β-site APP lyase 1) is a key enzyme in the conversion of APP to Aβ, and increased BACE1 activity promotes Aβ production. In the low-dose group, BACE1 expression was significantly higher than in the control group, but significantly lower in the medium- and high-dose groups. This indicates that freeze-dried broad bean leaf powder can inhibit BACE1 expression or activity at higher doses, thereby reducing Aβ production.

[0119] Finally, P-tau protein is a core component of neurofibrillary tangles. Compared with the control group, the expression of P-tau protein decreased in the medium- and high-dose groups, indicating that broad beans can effectively reduce the phosphorylation level of P-tau. Since the decrease in P-tau protein expression can alleviate the damage of neurofibrillary tangles to neurons and improve neuronal function, it may help alleviate the cognitive decline in AD patients as a whole.

[0120] In conclusion, the above results indicate that broad bean leaf extract has a certain effect on improving Alzheimer's disease (AD), which may be mainly attributed to the particularly high content of levodopa among its various extracts.

[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a broad bean leaf extract, characterized in that, Includes the following steps: S1: Freeze-dry fresh broad bean leaves and grind them to obtain broad bean leaf powder; S2: Broad bean leaf powder was added to hydrochloric acid aqueous solution and subjected to ultrasonic extraction to obtain a mixture; S3: Centrifuge the mixture and collect the supernatant, which is the broad bean leaf extract.

2. The method for preparing broad bean leaf extract according to claim 1, characterized in that, In step S1, after crushing and grinding, the powder is passed through a 200-mesh sieve to obtain broad bean leaf powder.

3. The method for preparing broad bean leaf extract according to claim 1, characterized in that, In step S2, the concentration of the hydrochloric acid aqueous solution is 1–3 wt%.

4. The method for preparing broad bean leaf extract according to claim 1, characterized in that, In step S2, the mass-to-volume ratio of broad bean leaf powder to hydrochloric acid aqueous solution is 1 g: 40-60 mL.

5. The method for preparing broad bean leaf extract according to claim 1, characterized in that, In step S2, the ultrasonic power is 200W and the time is 20-40 minutes.

6. A method for detecting levodopa in broad bean leaf extract, characterized in that, The broad bean leaf extract obtained by the preparation method according to any one of claims 1 to 5 was used as the test solution and analyzed by high performance liquid chromatography. Before each injection, the sample was filtered through a 0.22 μm microporous membrane and stored in a -4℃ refrigerator for later use.

7. The method for detecting L-DOPA in broad bean leaf extract according to claim 6, characterized in that, The analytical conditions for high performance liquid chromatography are as follows: Chromatographic column: CAPCELL PAK CR (4.6 mm l.D. × 150 mm, 5 μm) column; Mobile phase: 0.1% trifluoroacetic acid buffer solution and methanol were used as the mobile phase; Flow rate: 0.8 mL / min; Column temperature: 30℃; Detection wavelength: 280nm; Injection volume: 15 μL.

8. The use of the broad bean leaf extract prepared by any one of claims 1 to 5 in the preparation of drugs for the treatment and prevention of Alzheimer's disease.

9. The application of the broad bean leaf extract prepared by any one of claims 1 to 5 in foods or health products that help improve memory.

10. The application of the broad bean leaf extract according to any one of claims 8 or 9, characterized in that: The dosage forms of pharmaceutical, food, or health product products are freeze-dried powder, capsules, oral liquids, pills, granules, or tablets.

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