Nostoc sphaeroids kutz peptide composition with anti-inflammatory activity, anti-inflammatory active peptide as well as preparation method and application of nostoc sphaeroids kutz peptide composition
By preparing gesina peptides with specific amino acid sequences, the shortcomings of traditional antibiotics and existing anti-inflammatory peptides have been overcome, achieving highly efficient anti-inflammatory effects and industrial applications, and making them suitable for the preparation of anti-inflammatory drugs and functional foods.
Patent Information
- Application Number
- CN202511209496.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-27
- Publication Date
- 2025-12-12
AI Technical Summary
In existing technologies, traditional antibiotics have problems such as intestinal flora disruption, drug resistance, drug residues, and environmental pollution when treating animal inflammation. Meanwhile, naturally derived anti-inflammatory peptides have drawbacks such as low activity and complex preparation processes. The anti-inflammatory activity and large-scale application of Ge Xian Mi small peptides have not been fully developed.
By preparing small molecule kudzu peptides with specific amino acid sequences, including PAG, LVG, LGV and PVL, highly efficient anti-inflammatory active peptides were screened using multi-step enzymatic hydrolysis and Caco-2 cell transmembrane transport experiments. The preparation process is green and efficient, and suitable for industrial production.
Ge Xianmi peptide can effectively inhibit the secretion of inflammatory factors in macrophages, improve intestinal health and growth efficiency in animals, and has good anti-inflammatory effects. It is suitable for the preparation of anti-inflammatory drugs, functional foods and feed additives.
Smart Images
Figure CN121102434A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biotechnology, in particular to a nostoc commune peptide composition with anti-inflammatory activity, an anti-inflammatory active peptide and a preparation method and application thereof. BACKGROUND
[0002] In current aquaculture, animal inflammation problems (such as intestinal inflammation, respiratory tract infection, etc.) are one of the main challenges affecting production efficiency and animal health. Inflammation is an immune stress response of the body to external stimulation, infection and endogenous tissue damage. Its occurrence is usually accompanied by excessive secretion and release of cytokines (such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1, IL-6, IL-8, etc.), which in turn triggers an inflammatory cascade reaction of tissues or organs, affecting the health and production performance of animals.
[0003] In aquaculture, antibiotics are usually used to combat inflammation problems in farmed animals. However, traditional antibiotics such as amoxicillin and clarithromycin can destroy the normal intestinal flora and may cause antibiotic-associated diarrhea or clostridium difficile infection (pseudomembranous enteritis). Gentamicin and vancomycin can damage the kidneys, and macrolides (such as erythromycin) or tetracyclines can affect liver function. The abuse of antibiotics can lead to bacterial resistance, making future infections more difficult to treat (such as MRSA methicillin-resistant Staphylococcus aureus), and also causes drug residues and environmental pollution.
[0004] Natural anti-inflammatory active substances (such as plant or microbial source anti-inflammatory peptides) have become a research hotspot due to their high efficiency, low toxicity and multiple mechanisms of action. Compared with traditional anti-inflammatory drugs (such as NSAIDs or glucocorticoids), they have the following significant advantages: many anti-inflammatory peptides can specifically act on inflammation-related signaling molecules (such as NF-κB, MAPK, COX-2, etc.), reducing damage to non-target tissues and avoiding gastrointestinal damage or immunosuppression of traditional anti-inflammatory drugs; peptides are easily metabolized by the body, with low residual toxicity and reduced liver and kidney burden. Plant or microbial peptides often regulate multiple inflammation links (such as antioxidant, inhibition of inflammatory cytokines, promotion of anti-inflammatory factor secretion), with better comprehensive effect than single target drugs. Some peptides regulate Th1 / Th2 balance or Treg cell function, suitable for autoimmune diseases. Plant or microbial source anti-inflammatory peptides are widely available and their structures can be optimized.
[0005] Natural anti-inflammatory active substances have become a research hotspot, but existing plant or microbial source anti-inflammatory peptides have the defects of low activity, complex preparation process or high cost. Nostoc commune is a high-protein cyanobacterium rich in various bioactive substances, but the anti-inflammatory activity of its small peptides and large-scale application have not been fully developed. SUMMARY
[0006] In order to solve the problems in the prior art, the present application provides a Nostoc sphaeroides peptide composition with anti-inflammatory activity, an anti-inflammatory activity peptide, and a preparation method and application thereof, the Nostoc sphaeroides small peptide has high anti-inflammatory activity, has good anti-inflammatory effect, can inhibit the secretion of inflammatory factors in macrophages, improve the intestinal health and growth efficiency of animals, and the preparation process is green and efficient, and is suitable for industrial production.
[0007] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows:
[0008] A Nostoc sphaeroides peptide with anti-inflammatory activity, the amino acid sequence of the Nostoc sphaeroides peptide with anti-inflammatory activity comprises one or more of PAG, LVG, LGV and PVL.
[0009] Further, the molecular weight of the Nostoc sphaeroides peptide with anti-inflammatory activity is less than 500 Da.
[0010] Further, the preparation method of the Nostoc sphaeroides peptide with anti-inflammatory activity comprises the following steps:
[0011] (1) crushing the Nostoc sphaeroides to break the wall, adding phosphate buffer solution for homogenization, taking the supernatant and adding ethanol solution, separating to obtain polypeptide supernatant, rotary evaporation and freeze-drying to obtain polypeptide crude extraction powder;
[0012] (2) dissolving the polypeptide crude extraction powder in phosphate buffer solution, sequentially hydrolyzing by pepsin and trypsin, collecting the supernatant, and freeze-drying to obtain the digested polypeptide;
[0013] (3) inoculating Caco-2 cells into a Transwell well plate, and culturing until the transmembrane resistance reaches 700-900 Ω·cm 2 After the above, the digested polypeptide obtained in step (2) is dissolved in Hank's balanced salt solution to obtain a polypeptide solution, the polypeptide solution is added to the upper chamber, Hank's balanced salt solution is added to the lower chamber, the lower chamber culture medium is cultured and collected, and freeze-drying is performed to obtain the polypeptide after transmembrane transport.
[0014] Further, the following steps are further included: desalting the polypeptide after transmembrane transport obtained in step (3) and performing LC-MS / MS identification, screening the Nostoc sphaeroides peptide with anti-inflammatory activity by using PeptideRanker and denovo model, determining the amino acid sequence of the Nostoc sphaeroides peptide with anti-inflammatory activity, and synthesizing the Nostoc sphaeroides peptide with anti-inflammatory activity.
[0015] Further, the step (1) is crushed and broken wall of the Nostoc commune after 100-500 mesh screen; the pH value of the phosphate buffer is 7.2-7.4; the mass ratio of the phosphate buffer and Nostoc commune is 1:1-1.5; the volume fraction of the ethanol solution is 65-75%, and the added amount of the ethanol solution is 2-4 times of the volume of the supernatant.
[0016] Further, the concentration of the polypeptide crude powder in the step (2) is 10 mg / mL in the phosphate buffer; the hydrolysis conditions of the pepsin are that the pH is 1.5-2.5; the hydrolysis temperature is 36.5-37.5 DEG C, and the hydrolysis time is 1.5-2.5 h; after the end, the inactivation is boiled for 8-12 min or the pH is adjusted to 7.1-8.5 to terminate the hydrolysis reaction.
[0017] Further, the hydrolysis conditions of the trypsin in the step (2) are that the pH is 7.0-8.0; the temperature is 36.5-37.5 DEG C, and the time is 1.5-2.5 h; after the end, the inactivation is boiled for 8-12 min.
[0018] Further, the added amount of the pepsin in the step (2) is 3-5% E / S of the polypeptide crude powder, and the added amount of the trypsin is 3-5% E / S of the polypeptide crude powder.
[0019] Further, the seeding density of the Caco-2 cells in the step (3) is (1.8-2.2) x 10 5 cells / mL, the culture temperature is 36-38 DEG C, the volume fraction of CO2 in the incubator is 5%, the concentration of the polypeptide solution is 0.5-1.5 mg / mL, and the culture time is 110-130 min.
[0020] Further, the anti-inflammatory active Nostoc commune peptide is applied to the preparation of anti-inflammatory drugs, functional foods or feed additives.
[0021] Further, the inflammatory factors in the anti-inflammatory include NO and TNF-α.
[0022] The beneficial effects of the application are as follows:
[0023] 1, the application provides a Nostoc commune peptide composition, an anti-inflammatory active peptide and a preparation method thereof, the method takes Nostoc commune as raw material, and the anti-inflammatory active peptide is screened out through in vitro simulation of gastrointestinal digestion and Caco-2 cell transmembrane transport experiment, the method is simple and has strong operability. A new way is provided for high-value utilization of Nostoc commune, and good economic benefits and social benefits are obtained.
[0024] 2. The amino acid sequence of the anti-inflammatory active peptide provided by the present invention is one or more of PAG, LVG, LGV or PVL; the anti-inflammatory active peptide prepared by the present invention has good gastrointestinal stability during gastrointestinal digestion and can maintain good in vivo activity after transmembrane transport.
[0025] 3. The Ge Xian Mi peptide composition provided by the present invention has good anti-inflammatory effects and can inhibit the secretion of inflammatory factors NO and TNF-α by macrophages. It can be used to prepare anti-inflammatory drugs, food, health products or functional feed additives. Attached Figure Description
[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0027] Figure 1 The effect of the application of the 'Ge Xian Mi' polypeptide in Example 1 on NO secretion in RAW264.7 cells is shown in the figure.
[0028] Figure 2 The effect of the application of the 'Ge Xian Mi' polypeptide in Example 1 on the secretion of TNF-α by RAW264.7 cells is shown in the figure.
[0029] NC was the negative control group; PC was the positive control group; GP contained undigested kudzu root peptides; SGP contained kudzu root peptides digested. Detailed Implementation
[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0031] Example 1
[0032] This embodiment provides a method for preparing a Nostoc commune polypeptide composition with anti-inflammatory activity, comprising the following steps:
[0033] (1) Extraction of crude peptides
[0034] 100g of *Ge Xian Mi* (a type of medicinal herb) was pulverized to 100 mesh and placed in a 500mL centrifuge bottle. 100mL of 0.02mol / L, pH 7.0 phosphate buffer was added, and the mixture was homogenized using a homogenizer (18000r / min, 12s, with 15s intervals) until the homogenate was uniform and viscous. The centrifuge bottle was then incubated at 4℃ for 2 hours, centrifuged at 12000×g for 20 minutes at 4℃, and the supernatant was collected. Three volumes of 70% ethanol solution were added to the supernatant, and the mixture was incubated at 4℃ for 8 hours. Finally, it was centrifuged at 12000×g for 20 minutes at 4℃ to obtain the peptide supernatant. The supernatant was then subjected to rotary evaporation and freeze-drying to obtain a crude peptide powder sample, which was stored at -20℃ for later use.
[0035] (2) In vitro simulation of gastrointestinal digestion
[0036] The crude peptide powder was dissolved in phosphate buffer to prepare a 10 mg / mL solution, and the pH was adjusted to 2.0 by adding 1 mol / L hydrochloric acid. The solution was then incubated in a 37°C water bath for 10 min, while pepsin (4% of the peptide content) was added for hydrolysis for 2 h. After hydrolysis, the mixture was boiled for 10 min to inactivate the pepsin, and centrifuged at 5000×g for 15 min at 4°C. The pH of the centrifuged solution was adjusted to 7.5 by adding 1 mol / L NaOH solution, and trypsin (4% of the peptide content) was added for 2 h of incubation in 37°C water. After digestion, the solution was boiled for 10 min, centrifuged at 5000×g for 15 min at 4°C, and the supernatant was collected and labeled DXP. After freeze-drying, the digested peptides were obtained, which is the *Ge Xian Mi* peptide composition with anti-inflammatory activity.
[0037] Example 2
[0038] This embodiment provides a method for preparing anti-inflammatory active peptides, which, based on Example 1, further includes the following steps:
[0039] Caco-2 cell transmembrane transport experiment
[0040] Caco-2 cells were seeded in culture medium (DMEM + 10% fetal bovine serum + 1% penicillin-streptomycin) and cultured in an incubator at 37°C and 5% CO2. Caco-2 cells were then cultured at a rate of 2 × 10⁶ cells / year. 5 Cells / mL were seeded in Transwell plates (0.4 μm pore size, 12 mm diameter, 1.12 cm² surface area). 2Add 0.5 mL of cell suspension and 1.5 mL of cell culture medium to the upper and lower chambers, respectively. After 24 hours of culture, change the culture medium. Change the culture medium every other day for the first 7 days, and then daily thereafter until 21 days of culture. Measure the transmembrane resistance (TEER) of the cell monolayer using a resistance meter. When the TEER reaches 800 Ω·cm... 2 The above meets the experimental requirements. Prepare a 1 mg / mL digested peptide solution (dissolved in Hank's balanced salt solution), add 0.5 mL of peptide sample solution to the upper chamber, add 1.5 mL of HBSS to the lower chamber, place in a cell culture incubator for 120 min, and finally remove all the culture medium from the lower chamber. After freeze-drying, obtain the transmembrane transported kudzu peptide with anti-inflammatory activity.
[0041] Identification and synthesis of anti-inflammatory active peptide sequences
[0042] The transmembrane transported peptides were pretreated using MonoSpinC. 18 Desalting was performed using a desalting column. The liquid chromatography eluent consisted of 0.1% formic acid (buffer A) and acetonitrile (buffer B), with the following elution gradient: 0-2 min, 2% buffer B; 2-19 min, 2%-30% buffer B; 19-20 min, 30%-95% buffer B; 20-25.5 min, 95% buffer B; 25.5-26 min, 95%-2% buffer B; 26-30 min, 2% buffer B. After peptide loading, gradient elution was performed, followed by primary and secondary mass spectrometry scanning, with a full scan range of m / z 100-1200. The results were analyzed using MassLyna 4.1 software. The mass spectrometry data were analyzed for peptide composition using the PeaksStudio database and the denovo model, identifying anti-inflammatory active peptides including PAG, LVG, LGV, and PVL. The above peptides were prepared using chemical synthesis methods, with the purity of the synthesized peptides all exceeding 95%.
[0043] Example 3
[0044] This embodiment provides a method for preparing an anti-inflammatory peptide composition of *Ge Xian Mi* (a type of herb) and an anti-inflammatory peptide, including the following steps:
[0045] (1) Extraction of crude peptides
[0046] 100g of *Ge Xian Mi* (a type of medicinal herb) was pulverized to 500 mesh and placed in a 500mL centrifuge bottle. 150mL of 0.015mol / L phosphate buffer (pH 6.8) was added, and the mixture was homogenized using a homogenizer (18000r / min, 12s, with 15s intervals) until the homogenate was uniform and viscous. The centrifuge bottle was then incubated at 3℃ for 2 hours, centrifuged at 3℃ and 12000×g for 20 minutes, and the supernatant was collected. Three volumes of 70% ethanol solution were added to the supernatant, and the mixture was incubated at 3℃ for 6 hours. Finally, it was centrifuged at 3℃ and 12000×g for 20 minutes to obtain the peptide supernatant. The supernatant was concentrated by rotary evaporation and freeze-dried to obtain the crude peptide powder sample, which was stored at -20℃ for later use.
[0047] (2) In vitro simulation of gastrointestinal digestion
[0048] The crude peptide powder was dissolved in phosphate buffer to prepare an 8 mg / mL solution, and the pH was adjusted to 1.5 by adding 1 mol / L hydrochloric acid. The solution was then incubated in a 37.5°C water bath for 10 min, while pepsin (3.5 wt% of peptide content) was added for hydrolysis for 1.5 h. After hydrolysis, the mixture was boiled for 12 min to inactivate the pepsin, and centrifuged at 5000×g for 15 min at 4°C. The pH of the centrifuged solution was adjusted to 7.0 by adding 1 mol / L NaOH solution, and trypsin (4.5% ES of peptide content) was added for 2.5 h of incubation in 36.5°C water. After digestion, the solution was boiled for 12 min, centrifuged at 5000×g for 15 min at 4°C, and the supernatant was collected and labeled DXP. The digested peptides were obtained after freeze-drying.
[0049] (3) Caco-2 cell transmembrane transport experiment
[0050] Caco-2 cells were seeded in culture medium (DMEM + 10% fetal bovine serum + 1% penicillin-streptomycin) and cultured in an incubator at 37°C and 5% CO2. Caco-2 cells were cultured at a rate of 2.2 × 10⁻⁶ cells / year. 5 Cells / mL were seeded in Transwell plates (0.4 μm pore size, 12 mm diameter, 1.12 cm² surface area). 2 Add 0.5 mL of cell suspension and 1.5 mL of cell culture medium to the upper and lower chambers, respectively. After 24 hours of culture, change the culture medium. Change the culture medium every other day for the first 7 days, and then daily thereafter until 21 days of culture. Measure the transmembrane resistance (TEER) of the cell monolayer using a resistance meter. When the TEER reaches 700 Ω·cm... 2The above conditions were met. A 0.5 mg / mL digested peptide solution (dissolved in Hank's balanced salt solution) was prepared. 0.5 mL of the peptide sample solution was added to the upper chamber, and 1.5 mL of HBSS was added to the lower chamber. The mixture was placed in a cell culture incubator for 110 min. Finally, all the culture medium in the lower chamber was removed, and the mixture was freeze-dried to obtain the transmembrane transported, anti-inflammatory *Gnaphalium affine* peptide. The sequence identification and synthesis steps of the anti-inflammatory peptide were the same as in Example 2.
[0051] Example 4
[0052] This embodiment provides a method for preparing an anti-inflammatory peptide composition of *Ge Xian Mi* (a type of herb) and an anti-inflammatory peptide, including the following steps:
[0053] (1) Extraction of crude peptides
[0054] 100g of *Ge Xian Mi* (a type of medicinal herb) was pulverized to 600 mesh and placed in a 500mL centrifuge bottle. 150mL of 0.015mol / L phosphate buffer (pH 7.4) was added, and the mixture was homogenized using a homogenizer (18000r / min, 12s, 15s interval) until the homogenate was uniform and viscous. The centrifuge bottle was then incubated at 6℃ for 2 hours, centrifuged at 3℃ and 12000×g for 20 minutes, and the supernatant was collected. Three volumes of 70% ethanol solution were added to the supernatant, and the mixture was incubated at 3℃ for 10 hours. Finally, it was centrifuged at 6℃ and 15000×g for 10 minutes to obtain the peptide supernatant. The supernatant was concentrated by rotary evaporation and freeze-dried to obtain a crude peptide powder sample, which was stored at -20℃ for later use.
[0055] (2) In vitro simulation of gastrointestinal digestion
[0056] The crude peptide powder was dissolved in phosphate buffer to prepare a 12 mg / mL solution, and the pH was adjusted to 2.5 by adding 1 mol / L hydrochloric acid. The solution was then incubated in a 36.5°C water bath for 10 min, while pepsin (4.5% ES of peptide content) was added for hydrolysis for 2.5 h. After hydrolysis, the mixture was boiled for 8 min to inactivate the pepsin, and centrifuged at 5000×g for 15 min at 4°C. The pH of the centrifuged solution was adjusted to 8.0 by adding 1 mol / L NaOH solution, and trypsin (3.5% of peptide content) was added for 1.5 h of incubation in 37.5°C water. After digestion, the solution was boiled for 12 min, centrifuged at 5000×g for 15 min at 4°C, and the supernatant was collected and labeled DXP. The digested peptides were obtained after freeze-drying.
[0057] (3) Caco-2 cell transmembrane transport experiment
[0058] Caco-2 cells were seeded in culture medium (DMEM + 10% fetal bovine serum + 1% penicillin-streptomycin) and cultured in an incubator at 37°C and 5% CO2. Caco-2 cells were incubated at a growth rate of 1.8 × 10⁻⁶ cells / year. 5 Cells / mL were seeded in Transwell plates (0.4 μm pore size, 12 mm diameter, 1.12 cm² surface area). 2 Add 0.5 mL of cell suspension and 1.5 mL of cell culture medium to the upper and lower chambers, respectively. After 24 hours of culture, change the culture medium. Change the culture medium every other day for the first 7 days, and then daily thereafter until 21 days of culture. Measure the transmembrane resistance (TEER) of the cell monolayer using a resistance meter. When the TEER reaches 900 Ω·cm... 2 The above meets the experimental requirements. Prepare a 1.5 mg / mL digested peptide solution (dissolved in Hank's balanced salt solution), add 0.5 mL of peptide sample solution to the upper chamber, add 1.5 mL of HBSS to the lower chamber, place in a cell culture incubator for 130 min, and finally remove all the culture medium from the lower chamber. After freeze-drying, obtain the transmembrane transported kudzu peptide with anti-inflammatory activity.
[0059] The identification and synthesis steps of the anti-inflammatory active peptide sequence are the same as in Example 2.
[0060] Example 5
[0061] Inflammation induction experiment
[0062] Sample preparation: RAW264.7 cells (mouse mononuclear macrophage cell line) were seeded in 12-well plates at a density of 1×10⁻⁶ cells / well. 5 cells / cm 2 Cell plates were incubated at 37°C and 5% CO2 for 24 hours. The corresponding peptides were dissolved in DMEM medium to prepare a 1 mg / mL peptide solution, which was then filtered through a 0.22 μm filter. When RAW264.7 cells reached approximately 80% confluence, the original culture medium was removed, and the cells were washed with PBS. 500 μL of the peptide solution was added for pretreatment for 1 hour. Then, 50 μL of 10 μg / mL of 50 μL LPS (a Gram-negative inflammation inducer that activates the TLR4 pathway in macrophages, triggering an inflammatory response and releasing NO, TNF-α, etc.) was added to DMEM medium to induce an inflammatory response. After co-incubation for 12 hours, the cell supernatant was collected, and the levels of NO and TNF-α were measured.
[0063] Application Example 1
[0064] RAW264.7 cells were cultured according to the above-described inflammatory induction experiment. NC was set as a blank control group, and no inflammatory induction experiment was performed. PC was the positive control group, and the inflammatory induction experiment was performed according to the above method without adding polypeptide solution. GP undigested *Ge Xian Mi* polypeptide was the crude polypeptide powder prepared in step (1) of Example 1 of this invention. SGP digested *Ge Xian Mi* polypeptide was the digested *Ge Xian Mi* polypeptide prepared by the method in Example 1 of this invention. The above-described inflammatory induction experiment was performed, and the NO content results are as follows: Figure 1 As shown, compared with the NC group, the NO content in the PC group was significantly increased. Both undigested and digested *Gynostemma pentaphyllum* polypeptides could reduce the NO content in macrophages. Specifically, the NO content in the digested *Gynostemma pentaphyllum* polypeptide-treated group (SGP) was 7.45 μmol / L, significantly lower than the 11.256 μmol / L in the undigested polypeptide (GP) group.
[0065] TNF-α content as Figure 2 As shown, compared with the NC group, the TNF-α content in the PC group was significantly increased, and both undigested and digested *Gynostemma pentaphyllum* peptides could reduce the TNF-α content in macrophages. Specifically, the TNF-α content in the digested *Gynostemma pentaphyllum* peptide-treated group (SGP) was 467.3 pg / mL, significantly lower than the 763.8 pg / mL in the undigested peptide (GP) group. This indicates that the digested *Gynostemma pentaphyllum* peptides have a more significant anti-inflammatory effect.
[0066] Application Example 2
[0067] Experimental design: NC (no inflammation induction), PC (treatment method same as in Application Example 1), and peptide fragments PAG, LVG, LGV, and PVL were set up. The results are shown in Table 1. Compared with the inflammation-positive control group, the NO and TNF-α contents of the equal amounts of PAG, LVG, LGV, and PVL treatment groups were significantly reduced (P<0.05). Among them, the NO content in the LVG treatment group was the lowest, at 7.92 μmol / L, and the TNF-α content was the lowest, at 648.32 pg / mL, indicating that these peptides have good anti-inflammatory activity.
[0068] Table 1:
[0069] RAW264.7 cell treatment samples TNF-α content pg / L NO content μmol / L NC (no inflammation induction) 412.28 6.23 PC (Gram-negative inflammation inducer) 1638.37 14.74 PAG 982.45 12.32 LVG 648.32 7.92 LGV 912.54 11.68 PVL 1067.25 10.13
[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A composition of kudzu root peptides with anti-inflammatory activity, characterized in that, The Ge Xian Mi peptide composition is prepared using Ge Xian Mi as raw material. The Ge Xian Mi peptide composition contains anti-inflammatory active peptides, and the molecular weight of the anti-inflammatory active peptides is less than 500 Da.
2. The Ge Xian Mi peptide composition with anti-inflammatory activity according to claim 1, characterized in that, The amino acid sequence of the anti-inflammatory active peptide is one or more of PAG, LVG, LGV and PVL.
3. The method for preparing the kudzu root peptide composition with anti-inflammatory activity according to claim 1 or 2, characterized in that, The preparation method includes the following steps: (1) Pulverize and break the cell wall of Nostoc commune, add phosphate buffer solution to homogenize, take the supernatant, add ethanol solution to it, let stand and centrifuge to obtain polypeptide supernatant, concentrate the polypeptide supernatant and freeze dry to obtain crude polypeptide powder. (2) Dissolve the crude polypeptide powder in phosphate buffer, and then hydrolyze it with pepsin and trypsin in sequence. Collect the supernatant and freeze-dry it to obtain the digested polypeptide, which is the Ge Xian Mi peptide composition with anti-inflammatory activity.
4. A method for preparing an anti-inflammatory active peptide, characterized in that, The method includes the following steps: seeding Caco-2 cells in Transwell plates and culturing them until the transmembrane resistance of a monolayer of cells reaches 700-900 Ω·cm. 2 The above describes the process of dissolving the digested polypeptide of claim 3 in Hank's balanced salt solution to obtain a polypeptide solution. The obtained polypeptide solution is added to the upper chamber, and Hank's balanced salt solution is added to the lower chamber. The culture medium from the lower chamber is cultured and collected. After freeze-drying, the polypeptide after transmembrane transport is obtained. The transmembrane transported polypeptide is desalted and identified by LC-MS / MS. Anti-inflammatory active peptides are screened using the Peptide Ranker and de novo models to determine the amino acid sequence of the anti-inflammatory active peptides. The anti-inflammatory active peptides are then synthesized.
5. The method for preparing the kudzu root peptide composition with anti-inflammatory activity according to claim 3, characterized in that, In step (1), the pH value of the phosphate buffer is 6.8-7.4; the temperature for centrifugation is 3-6℃ and the standing time is 2-10h; the mass ratio of phosphate buffer to gesian rice is 1:1-1.5; the volume fraction of the ethanol solution is 65-75%, and the amount of ethanol solution added is 2-4 times the volume of the supernatant.
6. The method for preparing the kudzu root peptide composition with anti-inflammatory activity according to claim 3, characterized in that, In step (2), the concentration of the crude polypeptide powder in phosphate buffer is 8-12 mg / mL; the amount of pepsin added is 3-5% E / S of the crude polypeptide powder; the pH of hydrolysis is 1.5-2.5; the temperature is 36.5-37.5℃; and the time is 1.5-2.5 h. After the hydrolysis is completed, the peptide is boiled for 8-12 min to inactivate it or the pH is adjusted to 7.0-8.0 to terminate the hydrolysis reaction.
7. The method for preparing the kudzu root peptide composition with anti-inflammatory activity according to claim 3, characterized in that, In step (2), after the pepsin is hydrolyzed, the pH is adjusted to 7.0-8.0, and then trypsin is hydrolyzed at a temperature of 36.5-37.5℃ for 1.5-2.5 hours. After the hydrolysis is completed, the mixture is boiled for 8-12 minutes to inactivate the trypsin. The amount of trypsin added is 3-5% E / S of the crude polypeptide extract powder.
8. The method for preparing the anti-inflammatory active peptide according to claim 4, characterized in that, The concentration of the polypeptide solution in the upper chamber is 0.5-1.5 mg / mL, and the incubation time is 110-130 min.
9. The use of the kudzu root peptide composition with anti-inflammatory activity as described in claim 1 or 2 in the preparation of anti-inflammatory drugs, functional foods or feed additives.
10. The application according to claim 9, characterized in that: The gesnipeptide composition can inhibit the secretion of inflammatory factors in macrophages, including NO and TNF-α.