Preparation method and application of red kidney bean anti-inflammatory active peptide
The method for preparing anti-inflammatory active peptides from red kidney beans has solved the problem of insufficient research on anti-inflammatory active peptides from red kidney beans. The obtained peptides showed significant anti-inflammatory effects in in vitro experiments and are suitable for the preparation of anti-inflammatory drugs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANXI UNIV
- Filing Date
- 2023-03-21
- Publication Date
- 2026-06-12
Smart Images
Figure CN116121325B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of preparation and application of plant polypeptides, specifically to a method for preparing and applying an anti-inflammatory active peptide from red kidney beans. Background Technology
[0002] Red kidney beans (Phaseolus vulgaris), a herbaceous plant belonging to the genus *Phaseolus* of the legume family, are scientifically known as *Phaseolus vulgaris*. They are rich in potassium, amino acids, and dietary fiber, making them highly nutritious. In traditional Chinese medicine, red kidney beans are considered sweet and neutral in nature, warming the middle and lower body, benefiting the intestines and stomach, stopping hiccups, and tonifying the kidneys and replenishing vital energy. The shell, seeds, and roots of red kidney beans can all be used medicinally, possessing significant medicinal value. Modern medical research indicates that red kidney beans are a high-potassium, high-magnesium, and low-sodium food, making them particularly suitable for patients with heart disease, arteriosclerosis, hyperlipidemia, and hypokalemia. Red kidney beans contain urease, various globulins, and albumin, which can enhance immunity, improve skin metabolism, and also have weight-loss, lipid-lowering, and digestive benefits. The saponins in red kidney beans can relieve joint pain and neuralgia, and have significant anti-inflammatory effects.
[0003] Inflammation is the body's defensive response to external stimuli. When inflammation is activated regularly over a certain period of time, it is beneficial to the body. However, when excessive inflammation occurs, it can cause various serious diseases such as cardiovascular disease, cancer, and diabetes.
[0004] With the development of society and the economy, people's living standards have also improved, gradually shifting from simply having enough to eat to eating well, and healthy eating has become the mainstream of the times. Food-derived anti-inflammatory peptides have become a research hotspot due to their high safety and easy absorption. To date, the chemical and pharmacological research on red kidney bean anti-inflammatory peptides both domestically and internationally is not systematic or in-depth, and there are relatively few research reports on anti-inflammatory bioactive peptides from red kidney beans. Summary of the Invention
[0005] To address the above-mentioned problems, the present invention aims to provide a method for preparing an anti-inflammatory active peptide from red kidney beans, and the application of this active peptide in the preparation of anti-inflammatory drugs.
[0006] To achieve the above objectives, the present invention employs the following technical solutions:
[0007] This invention provides a method for preparing anti-inflammatory active peptides from red kidney beans, comprising the following steps:
[0008] Step 1: Take red kidney beans, grind and crush them, add distilled water and stir for 30 minutes, then heat to 60℃ and soak for 2-4 hours. After centrifugation, take the supernatant. The filter residue can be extracted 1-2 times. Freeze-dry the obtained supernatant to obtain red kidney bean protein.
[0009] Step 2: Dissolve the red kidney bean protein powder from Step 1 in distilled water to obtain a mixture. Adjust the protein concentration of the mixture, add pepsin, and perform enzymatic hydrolysis. After the reaction is complete, place the hydrolysate in a boiling water bath and cool to room temperature. Centrifuge at 4℃ and 4500r / min for 15min, collect the supernatant, freeze-dry, and obtain the red kidney bean enzymatic hydrolysate.
[0010] Step 3: Dissolve the red kidney bean enzymatic hydrolysis product powder from Step 2 in distilled water, centrifuge using an ultrafiltration tube to separate the different liquid components, and freeze-dry them.
[0011] Furthermore, in step 1, the mass-to-volume ratio of red kidney beans to distilled water is 1:20-35.
[0012] Preferably, in step 1, the mass-to-volume ratio of red kidney beans to distilled water is 1:30, and then the mixture is heated to 60°C and soaked for 3 hours.
[0013] Furthermore, in step 2, pepsin 1000 U / g to 4000 U / g is added, and enzymatic hydrolysis is carried out at a pH of 2 to 3 and a temperature of 35 to 45°C.
[0014] Preferably, in step 2, 2000 U / g of pepsin is added, and enzymatic hydrolysis is carried out at pH 2 and temperature 40°C.
[0015] Furthermore, the enzymatic hydrolysis time in step 2 is 20–30 min.
[0016] Furthermore, in step 2, the enzymatic hydrolysate is placed in a boiling water bath for 10–30 minutes.
[0017] Preferably, the enzymatic hydrolysis time in step 2 is 30 min, and the enzymatic hydrolysate in step 2 is placed in a boiling water bath for 15 min.
[0018] Furthermore, in step 3, the ultrafiltration tube has a molecular weight cutoff of 10KD and 3KD.
[0019] Preferably, in step 3, the ultrafiltration tube has a molecular weight cutoff of 3 kDa.
[0020] This invention also provides the application of the red kidney bean anti-inflammatory active peptides prepared by the above method in the preparation of anti-inflammatory drugs.
[0021] Compared with the prior art, the present invention has the following advantages:
[0022] The red kidney bean anti-inflammatory active peptide obtained by this invention was tested for its anti-inflammatory activity in vitro. The components of the active peptide had no obvious toxic side effects on RAW264.7 cells, and could significantly inhibit the release of NO from LPS-induced RAW264.7 cells, thus exhibiting good anti-inflammatory activity. It can be used in the preparation of anti-inflammatory drugs. Attached Figure Description
[0023] Figure 1 Effects of various components of red kidney bean anti-inflammatory active peptides on the viability of RAW264.7 cells;
[0024] Figure 2 Effects of red kidney bean anti-inflammatory peptides on LPS-induced NO release in RAW264.7 cells. * in the figure represents P<0.05, ** represents P<0.01. Detailed Implementation
[0025] The technical solutions of the present invention will be described in detail below with reference to the embodiments and accompanying drawings. It should be noted that those skilled in the art can make several modifications and improvements without departing from the principle of the present invention, and these should also be considered to fall within the protection scope of the present invention.
[0026] Example 1
[0027] The preparation of anti-inflammatory active peptides from red kidney beans includes the following steps:
[0028] Step 1: Take 4g of red kidney beans, grind them in a grinder, add 120mL of distilled water, stir for 30min, soak at 60℃ for 3h, centrifuge at 4℃ and 4500rpm for 20min, and take the supernatant. The filter residue can be extracted 1-2 times. Freeze-dry the obtained supernatant to obtain red kidney bean protein.
[0029] Step 2: Take 0.5g of the red kidney bean protein powder obtained in Step 1 and add it to 500mL of distilled water while stirring. Place it in a 70℃ water bath and heat and stir. Adjust the pH of the solution to 2 and the temperature to 40℃. Add 2000U / g pepsin and enzymatically hydrolyze for 30min. Heat in a boiling water bath for 10min. Centrifuge at 4℃ and 4500rpm for 15min. Collect the supernatant and freeze-dry to obtain the red kidney bean enzymatic hydrolysate.
[0030] Step 3: Dissolve the red kidney bean enzymatic hydrolysate obtained in step 2 in distilled water, separate it using 10KD and 3KD ultrafiltration centrifuge tubes, and collect and freeze-dry each component.
[0031] Example 2
[0032] The preparation of anti-inflammatory active peptides from red kidney beans includes the following steps:
[0033] Step 1: Take 4g of red kidney beans, grind them in a grinder, add 110mL of distilled water, stir for 30min, soak at 60℃ for 3h, centrifuge at 4℃ and 4500rpm for 20min, and take the supernatant. The filter residue can be extracted 1-2 times. Freeze-dry the obtained supernatant to obtain red kidney bean protein.
[0034] Step 2: Take 0.5g of the red kidney bean protein powder obtained in Step 1 and add it to 500mL of distilled water while stirring. Place it in a 70℃ water bath and heat and stir. Adjust the pH of the solution to 2 and the temperature to 45℃. Add 2500U / g pepsin and enzymatically hydrolyze for 25min. Heat in a boiling water bath for 15min. Centrifuge at 4℃ and 4500rpm for 15min. Collect the supernatant and freeze-dry to obtain the red kidney bean enzymatic hydrolysate.
[0035] Step 3: Dissolve the red kidney bean enzymatic hydrolysate obtained in step 2 in distilled water, separate it using 10KD and 3KD ultrafiltration centrifuge tubes, and collect and freeze-dry each component.
[0036] Example 3
[0037] The preparation of anti-inflammatory active peptides from red kidney beans includes the following steps:
[0038] Step 1: Take 4g of red kidney beans, grind them in a grinder, add 120mL of distilled water, stir for 30min, soak at 60℃ for 2h, centrifuge at 4℃ and 4500rpm for 20min, and take the supernatant. The filter residue can be extracted 1-2 times. Freeze-dry the obtained supernatant to obtain red kidney bean protein.
[0039] Step 2: Take 0.5g of the red kidney bean protein powder obtained in Step 1 and add it to 500mL of distilled water while stirring. Place it in a 70℃ water bath and heat and stir. Adjust the pH of the solution to 3 and the temperature to 35℃. Add 3000U / g pepsin and enzymatically hydrolyze for 20min. Heat in a boiling water bath for 20min. Centrifuge at 4℃ and 4500rpm for 15min. Collect the supernatant and freeze-dry to obtain the red kidney bean enzymatic hydrolysate.
[0040] Step 3: Dissolve the red kidney bean enzymatic hydrolysate obtained in step 2 in distilled water, separate it using 10KD and 3KD ultrafiltration centrifuge tubes, and collect and freeze-dry each component.
[0041] Example 4
[0042] Effects of Red Kidney Bean Anti-inflammatory Active Peptides on RAW264.7 Cell Survival
[0043] RAW264.7 cells in logarithmic growth phase were subjected to a 1×10⁻⁶ thiocyanate incubation. 4Each sample was transferred to a 96-well plate. After incubation at 37°C in a 5% CO2 incubator for 24 hours, the red kidney bean anti-inflammatory active peptide from Example 1 was added to each well to a final concentration of 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, with six replicates for each concentration. A medium containing the same volume of Tris-HCl buffer was used as a control. After 24 hours of incubation, 20 μL of MTT solution (5 mg / mL) was added to each well, and incubation continued for another 4 hours before termination. The culture supernatant was carefully aspirated from each well, and 150 μL of LDMSO was added to each well. The plates were shaken for 10 minutes to fully dissolve any crystals. The absorbance of each well was measured at 570 nm using a microplate reader.
[0044] Figure 1 The results showed that the anti-inflammatory active peptides of red kidney beans had no significant effect on the growth of RAW264.7 cells.
[0045] Example 5
[0046] Analysis of the anti-inflammatory activity of red kidney bean anti-inflammatory peptides
[0047] RAW264.7 cells in logarithmic growth phase were subjected to a 1×10⁻⁶ thiocyanate incubation. 5 Each well was transferred to a 30 mm dish. After incubation at 37°C in a 5% CO2 incubator for 24 h, the red kidney bean anti-inflammatory active peptide from Example 1 was added to final concentrations of 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively. After 4 h, LPS was added to a final concentration of 0.5 μg / mL. Each concentration was tested in triplicate, and culture medium containing the same volume of Tris-HCl buffer was used as a control. After 18 h of incubation, the supernatant was collected, and the NO content was detected using the Griess method. Specifically, 25 μL of supernatant was added to each well with 25 μL of Griess I and 25 μL of Griess II, and the absorbance of each well was measured at 540 nm using a microplate reader. The NO concentration was calculated based on the standard curve.
[0048] Figure 2 The results showed that the anti-inflammatory active peptides of red kidney beans of different components could inhibit the NO production induced by LPS from RAW264.7, and the peptides with a molecular weight <3KD had the strongest inhibitory ability, that is, the peptides with small molecular weight have the strongest anti-inflammatory activity.
[0049] Contents not described in detail in this specification are prior art known to those skilled in the art. Although illustrative specific embodiments of the invention have been described above to facilitate understanding by those skilled in the art, it should be understood that the invention is not limited to the scope of the specific embodiments. Various modifications are readily apparent to those skilled in the art as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims, and all inventions utilizing the concept of this invention are protected.
Claims
1. A method for preparing an anti-inflammatory active peptide from red kidney bean, characterized in that, Includes the following steps: Step 1: Take red kidney beans, grind and crush them, add distilled water and stir for 30 minutes, then heat to 60°C and soak for 2-4 hours. After centrifugation, take the supernatant. The filter residue can be extracted 1-2 times. Freeze-dry the obtained supernatant to obtain red kidney bean protein. Step 2: Dissolve the red kidney bean protein powder from Step 1 in distilled water to obtain a mixture. Adjust the protein concentration of the mixture, add pepsin, and perform enzymatic hydrolysis. After the reaction is complete, place the hydrolysate in a boiling water bath and cool to room temperature. Centrifuge at 4°C and 4500 r / min for 15 min, collect the supernatant, freeze-dry, and obtain the red kidney bean enzymatic hydrolysate. Step 3: Dissolve the red kidney bean enzymatic hydrolysis product powder from Step 2 in distilled water, centrifuge using an ultrafiltration tube to separate the different liquid components, and freeze-dry them. In step 1, the mass-to-volume ratio of red kidney beans to distilled water is 1:30, and then the mixture is heated to 60°C and soaked for 3 hours. In step 2, pepsin 1000 U / g to 4000 U / g is added, and enzymatic hydrolysis is carried out at a pH of 2 to 3 and a temperature of 35 to 45°C. The enzymatic hydrolysis time in step 2 is 20–30 min; In step 2, the enzymatic hydrolysate is placed in a boiling water bath and reacted for 15 minutes. In step 3, the ultrafiltration tube has a molecular weight cutoff of 10KD and 3KD.
2. The preparation method of the anti-inflammatory active peptide of red kidney bean according to claim 1, characterized in that, In step 2, 2000 U / g of pepsin is added, and enzymatic hydrolysis is carried out at pH 2 and temperature 40°C.
3. The method for preparing an anti-inflammatory active peptide from red kidney beans according to claim 1, characterized in that, The enzymatic hydrolysis time in step 2 is 30 minutes.
4. The application of the red kidney bean anti-inflammatory active peptide prepared by the method described in claim 1 in the preparation of anti-inflammatory drugs.