Preparation method and application of pericarpium citri reticulatae and radix puerariae polypeptide composition

Abstract

The invention provides a preparation method and application of a pericarpium citri reticulatae and radix puerariae polypeptide composition, the method combines traditional Chinese medicine pericarpium citri reticulatae and radix puerariae with bioactive polypeptide I9-27, and a novel lipid-lowering preparation is prepared through the steps of microwave extraction, compounding, drying and the like. According to the invention, the traditional Chinese medicines of pericarpium citri reticulatae and radix puerariae are combined with a modern biological polypeptide technology for the first time, a microwave-assisted extraction technology is combined, the pericarpium citri reticulatae and radix puerariae polypeptide compound preparation is developed, the optimal compatibility ratio of pericarpium citri reticulatae and radix puerariae extracts to I9-27 polypeptide is determined through systematic research, and the remarkable effects of the combination in the aspects of lipid lowering and blood vessel protection are proved; the activity of vascular endothelial cells after ox-LDL damage is improved, the ROS and MDA levels are reduced, the oxidation resistance is enhanced, and the oxidative stress state is improved; furthermore, expression of inflammatory factors TNF-alpha and IL-1beta can be remarkably reduced, cell viability is remarkably improved through a single polypeptide group, and the composition has the advantages that metabolite of the composition is amino acid, toxicity is low, and stability and activity are enhanced through structural modification.

Description

Technical Field

[0001] The present invention relates to the technical field of bioactive polypeptides, and in particular to a preparation method and application of a tangerine peel and kudzu root polypeptide composition. Background Art

[0002] Hyperlipidemia is a complex lipid metabolism disorder characterized by abnormally elevated plasma levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C), and / or decreased high-density lipoprotein cholesterol (HDL-C). According to the World Health Organization, approximately 40% of adults worldwide suffer from hyperlipidemia, making it a major modifiable risk factor for atherosclerotic cardiovascular disease. In particular, elevated LDL-C levels are strongly associated with the risk of coronary heart disease and ischemic stroke. Currently, statins (HMG-CoA reductase inhibitors) are the primary first-line treatment in clinical practice. While they offer significant lipid-lowering effects, long-term use is associated with numerous limitations, including muscle toxicity, liver damage, and varying tolerance. Furthermore, drugs such as fibrates and bile acid sequestrants can also cause gastrointestinal discomfort and interfere with vitamin absorption. Therefore, the development of safe and effective lipid-lowering agents is of great clinical significance and application value.

[0003] According to Traditional Chinese Medicine (TCM), the pathogenesis of hyperlipidemia primarily involves phlegm, blood stasis, and liver depression, with dysfunction of the internal organs, particularly the liver, spleen, and kidneys, being the key. The Suwen Zhizhen Yao Da Lun states, "All swellings and fullness due to dampness are attributed to the spleen." Splenic dysfunction is a key factor in the endogenous generation of phlegm. Tangerine peel (Chenpi) is warm in nature, pungent and bitter in flavor, and enters the lung and spleen meridians. Its primary effects are regulating qi and strengthening the spleen, drying dampness and resolving phlegm, and regulating the stomach and stimulating appetite. Summary of the Invention

[0004] In order to solve the problems in the prior art, the first aspect of the present invention provides a method for preparing a tangerine peel and kudzu root polypeptide composition, the method comprising: Step 1: drying, crushing, and sieving dried tangerine peel to obtain a first powder; drying, crushing, and sieving kudzu root to obtain a second powder; mixing the first powder and the second powder, adding the mixture into water, heating and extracting, filtering, and collecting the filtrate; Step 2: Add I9-27 polypeptide to the filtrate, control the pH and temperature to obtain a composite solution; Step 3: Concentrate the composite liquid, add maltodextrin, and dry into powder to obtain the tangerine peel and kudzu root polypeptide composition.

[0005] In some specific embodiments of the method for preparing the tangerine peel and kudzu vine polypeptide composition of the first aspect, the tangerine peel in step 1 is 2-4 years old tangerine peel, and in some embodiments, is 3 years old tangerine peel.

[0006] In some specific embodiments of the method for preparing the tangerine peel and kudzu vine polypeptide composition of the first aspect, in step 1, the tangerine peel is Guangdong Xinhui tangerine peel.

[0007] In some specific embodiments of the method for preparing the tangerine peel and kudzu vine polypeptide composition of the first aspect, in step 1, the mesh number of the tangerine peel after being crushed and sieved is 60-100 mesh. In some embodiments, in step 1, the mesh number of the tangerine peel after being crushed and sieved is optionally 70 mesh, 80 mesh, or 90 mesh.

[0008] In some specific embodiments of the method for preparing the tangerine peel and kudzu root polypeptide composition of the first aspect, in step 1, the kudzu root is peeled before being dried and crushed.

[0009] In some specific embodiments of the method for preparing the tangerine peel and kudzu root polypeptide composition of the first aspect, the mesh number of the kudzu root sieved after being crushed is 60-100 mesh. In some specific embodiments of the method for preparing the tangerine peel and kudzu root polypeptide composition of the first aspect, the mesh number of the kudzu root sieved after being crushed is optionally 70 mesh, 80 mesh, or 90 mesh.

[0010] In some specific embodiments of the method for preparing the tangerine peel and kudzu vine polypeptide composition of the first aspect, in step 1, the heating extraction method is microwave heating, and the microwave heating power is 500~700W. In some specific embodiments of the method for preparing the tangerine peel and kudzu vine polypeptide composition of the first aspect, in step 1, the heating extraction method is microwave heating, and the microwave heating power is optionally 550W, 600W, or 650W.

[0011] In some specific embodiments of the method for preparing the tangerine peel and kudzu vine polypeptide composition of the first aspect, in step 1, the heating extraction time is 10 to 20 minutes. In some specific embodiments of the method for preparing the tangerine peel and kudzu vine polypeptide composition of the first aspect, in step 1, the heating extraction time is optionally 12 minutes, 14 minutes, 16 minutes, or 18 minutes.

[0012] In some specific embodiments of the method for preparing the tangerine peel and kudzu vine polypeptide composition of the first aspect, in step 1, after heating and extraction, the mesh size of the sieve used for filtration is 90-120 mesh. In some specific embodiments of the method for preparing the tangerine peel and kudzu vine polypeptide composition of the first aspect, in step 1, after heating and extraction, the mesh size of the sieve used for filtration is optionally 95 mesh, 100 mesh, 105 mesh, 110 mesh, or 115 mesh.

[0013] In some specific embodiments of the method for preparing the tangerine peel and kudzu root polypeptide composition of the first aspect, the mass ratio of tangerine peel to kudzu root is 1:(0.5~1.5); in some specific embodiments of the method for preparing the tangerine peel and kudzu root polypeptide composition of the first aspect, the mass ratio of tangerine peel to kudzu root is optionally 1:0.7, 1:0.9, 1:1.1, or 1:1.3.

[0014] In some specific embodiments of the method for preparing the tangerine peel and kudzu root polypeptide composition of the first aspect, in step 2, the molar amount of the I9-27 polypeptide added per 1 L of filtrate is 5~15 μmol. In some specific embodiments of the method for preparing the tangerine peel and kudzu root polypeptide composition of the first aspect, in step 2, the molar amount of the I9-27 polypeptide added per 1 L of filtrate is optionally 7 μmol, 9 μmol, 11 μmol, or 13 μmol.

[0015] In some specific embodiments of the method for preparing the tangerine peel and kudzu vine polypeptide composition of the first aspect, in step 2, the pH is 6-7. In some specific embodiments of the method for preparing the tangerine peel and kudzu vine polypeptide composition of the first aspect, in step 2, the pH is 6.5.

[0016] In some specific embodiments of the method for preparing the tangerine peel and kudzu vine polypeptide composition of the first aspect, the temperature of the temperature control is 20~30°C. In some specific embodiments of the method for preparing the tangerine peel and kudzu vine polypeptide composition of the first aspect, the temperature of the temperature control is optionally 22°C, 24°C, 26°C, or 28°C.

[0017] In some specific embodiments of the method for preparing the Citrus Reticulatae Pellets polypeptide composition of the first aspect, in step 3, the composite liquid is concentrated by reduced pressure concentration.

[0018] In some specific embodiments of the method for preparing the tangerine peel and kudzu vine polypeptide composition of the first aspect, the concentrated composite liquid is concentrated to a relative density of 1.1 to 1.25. In some specific embodiments of the method for preparing the tangerine peel and kudzu vine polypeptide composition of the first aspect, the concentrated composite liquid is concentrated to a relative density of 1.12, 1.14, 1.16, 1.18, 1.20, 1.22, or 1.24.

[0019] In some specific embodiments of the method for preparing the tangerine peel and kudzu vine polypeptide composition of the first aspect, the amount of maltodextrin added is 20-40% by weight of the tangerine peel and kudzu vine polypeptide composition. In some specific embodiments of the method for preparing the tangerine peel and kudzu vine polypeptide composition of the first aspect, the amount of maltodextrin added is optionally 24%, 28%, 32%, or 36% by weight of the tangerine peel and kudzu vine polypeptide composition.

[0020] In some specific embodiments of the method for preparing the Citrus Reticulatae Pellets polypeptide composition of the first aspect, the drying to powder method is spray drying.

[0021] In some specific embodiments of the method for preparing the tangerine peel and kudzu root polypeptide composition of the first aspect, the inlet temperature of the spray drying is 170~190℃. In some specific embodiments of the method for preparing the tangerine peel and kudzu root polypeptide composition of the first aspect, the inlet temperature of the spray drying is optionally 174℃, 178℃, 182℃, or 186℃.

[0022] In some specific embodiments of the method for preparing the tangerine peel and kudzu vine polypeptide composition of the first aspect, the outlet temperature of the spray drying is 70~100℃. In some specific embodiments of the method for preparing the tangerine peel and kudzu vine polypeptide composition of the first aspect, the outlet temperature of the spray drying is optionally 75℃, 80℃, 85℃, 90℃, or 95℃.

[0023] In some specific embodiments of the method for preparing the tangerine peel and kudzu vine polypeptide composition of the first aspect, after drying to powder, the sieve is further sieved, and the mesh number of the sieve is 40-70 mesh. In some specific embodiments of the method for preparing the tangerine peel and kudzu vine polypeptide composition of the first aspect, after drying to powder, the sieve is further sieved, and the mesh number of the sieve is optionally 44 mesh, 48 mesh, 52 mesh, 56 mesh, 60 mesh, 64 mesh, or 68 mesh.

[0024] In some specific embodiments of the method for preparing the Citrus Reticulatae Pellets and Puerariae Radix polypeptide composition of the first aspect, the sequence of the I9-27 polypeptide is: MPDGPAISALEASPHVENMEMDQEVTTQ.

[0025] In some specific embodiments of the method for preparing the Citrus aurantium and Pueraria polypeptide composition of the first aspect, the I9-27 polypeptide is obtained by fermenting the Morchella conicus CCBAS932 strain in one or more steps.

[0026] The second aspect of the present invention provides a Citrus Reticulatae Pellets polypeptide composition prepared by any of the methods described in the first aspect.

[0027] The third aspect of the present invention provides a food comprising the composition according to the second aspect.

[0028] The fourth aspect of the present invention provides an application of the Tangerine Peel and Pueraria lobata polypeptide composition described in the second aspect and the food described in the third aspect in anti-inflammation, lowering blood lipids, and increasing cell vitality.

[0029] The fifth aspect of the present invention provides a polypeptide with the sequence: MPDGPAISALEASPHVENMEMDQEVTTQ.

[0030] The beneficial effects of the present invention are: In a first aspect of the present invention, a polypeptide I9-27 is obtained by fermenting Morchella conicola CCBAS932 strain, and its amino acid sequence is: MPDGPAISALEASPHVENMEMDQEVTTQ. When used alone, it has a good effect of increasing cell viability.

[0031] The second aspect of the present invention combines the traditional Chinese medicine tangerine peel and kudzu root with modern biological polypeptide technology to innovatively develop a tangerine peel and kudzu root polypeptide composite preparation, which has the ability to significantly lower lipids, improve the vitality of vascular endothelial cells after ox-LDL damage, reduce ROS and MDA levels, and improve oxidative stress. Through systematic research, the optimal compatibility ratio of tangerine peel, kudzu root extract and I9-27 polypeptide was determined, and the effect of this combination in reducing the expression of inflammatory factors TNF-α and IL-1β and protecting blood vessels was demonstrated. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The effect of the Citrus aurantium peel and Pueraria root polypeptide of the present invention on lipid deposition in rat aorta tissue; Figure 2 The effect of Citrus aurantium peel and Pueraria root polypeptide on lipid deposition in EA.hy926 cells; Figure 3 Effects of Citrus aurantium and Puerariae Radix polypeptides on oxidative stress indicators in EA.hy926 cells; Figure 4 It is a molecular interaction model between I9-27 peptide and PCSK9 protein; Figure 5 Molecular dynamics energy conformational analysis of the complex between I9-27 peptide and PCSK9; Figure 6 This is the hydrogen bond network topology diagram of the binding interface between I9-27 peptide and PCSK9. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the technical solutions of the present invention, some non-limiting embodiments are further disclosed below to further illustrate the present invention in detail.

[0034] Example 1: Preparation of I9-27 polypeptide Solid medium: malt extract (maltextract): 20.0 g / L; glucose (glucose): 10.0 g / L; peptone (peptone): 5.0 g / L; potassium dihydrogen phosphate (KH2PO4): 1.0 g / L; magnesium sulfate heptahydrate (MgSO4·7H2O): 0.5 g / L; agar: 15-20 g / L; pH: 6.0-6.5; Seed culture medium formula: malt extract: 20.0 g / L; glucose: 12.0 g / L; peptone: 2.0 g / L; KH2PO4: 1.0 g / L; MgSO4·7H2O: 0.5 g / L; pH: 6.0-6.5; Fermentation medium formula: glucose: 30.0 g / L; corn steep liquor: 15.0 g / L; KH2PO4: 1.0 g / L; MgSO4·7H2O: 0.5 g / L; L-cysteine: 0.5 g / L; pH: 6.0; Under aseptic operation, take the frozen glycerol test tube of CCBAS932 strain and inoculate the strain into the above solid culture medium. Incubate at 28°C for 5-7 days to obtain activated bacterial blocks. 2 ) was inoculated into a liquid basal culture medium; cultured in a shaker at 150 rpm and 28°C for 5-7 days to prepare a seed solution of the CCBAS932 strain, and the seed solution was inoculated into 10 L of fermentation medium at a ratio of 10% (v / v) for fermentation culture. The fermentation culture conditions were: temperature 24±1°C, stirring speed 150 rpm, ventilation volume: 0.4 vvm. After 14-16 days of fermentation, the I9-27 polypeptide was extracted; The I9-27 peptide extraction process: The fermentation broth was centrifuged at 1000 × g for 20 minutes at 4°C to remove bacterial impurities. The supernatant was filtered through a 0.45 μm microporous filter to obtain a clear peptide-containing solution. Ammonium sulfate was slowly added to the supernatant to 60% saturation. The peptide was precipitated by incubation at 4°C overnight to precipitate the peptide. The precipitate was recovered by centrifugation and then dissolved in buffer. The peptide was dialyzed against 20 mM Tris-HCl buffer (pH 8.0) at 4°C for 48 hours to remove salts, with the buffer changed every 8 hours. After desalting, the peptide was purified by chromatography. The dialyzed solution was eluted with a DEAE-Sepharose FF anion exchange column using a linear gradient of 0–1 M NaCl to collect the target peptide fraction. The eluate was concentrated and further purified by Sephadex G-50 molecular sieve chromatography. Finally, the peptide was purified by C18 reverse-phase high-performance liquid chromatography (HPLC). The purified I9-27 peptide was verified for purity and molecular weight by high-resolution mass spectrometry and SDS-PAGE. The final product was lyophilized and stored at -20°C. This method yielded approximately 50–80 mg of highly pure (≥98%) I9-27 peptide per 10 L of fermentation broth.

[0035] Example 2: Preparation of Tangerine Peel and Pueraria lobata Polypeptide Prepare the tangerine peel and kudzu root extract as follows: Step (1): 100 g of tangerine peel from Xinhui, Guangdong Province aged for 3 years is selected, impurities are removed from the tangerine peel, the tangerine peel is rinsed with purified water for 2 to 3 times, and the tangerine peel is ground through an 80-mesh sieve after being dried to obtain tangerine peel powder; 200 g of kudzu root grown for 3 years is selected, the kudzu root is peeled, washed, dried, cut into thin slices of about 4 mm, and ground through an 80-mesh sieve to obtain kudzu root powder; the tangerine peel powder and the kudzu root powder are mixed, added into purified water at a solid-liquid ratio of 1:10, soaked for 30 minutes, extracted under a microwave power of 600 W for 15 minutes, filtered through a 100-mesh sieve, and the filtrate is collected.

[0036] Step (2): Add I9-27 polypeptide to the collected filtrate to prepare a mixture so that the concentration of I9-27 polypeptide in the filtrate is 10 μmol / L, adjust the pH to 6.5, and stir at a constant temperature of 25° C. for 1.5 hours to obtain a composite solution.

[0037] Step (3): The obtained composite liquid was concentrated under reduced pressure at 60°C to a relative density of 1.25 (25°C), maltodextrin (calculated as dry powder, i.e., 30% of total solids) was added as an auxiliary material, mixed evenly, and then spray-dried. The spray-drying parameters were an inlet temperature of 180°C and an outlet temperature of 85°C. The spray-dried powder was sieved through a 60-mesh sieve to obtain a finished product.

[0038] Comparative Example 1: Tangerine Peel and Puerariae Radix Group The difference between Comparative Example 1 and Example 2 is that after collecting the filtrate, no I9-27 polypeptide is added, and the rest is the same. Comparative Example 1 is: Step (1): 100 g of tangerine peel from Xinhui, Guangdong Province aged for 3 years is selected, impurities are removed from the tangerine peel, the tangerine peel is rinsed with purified water for 2 to 3 times, the tangerine peel is dried and then crushed through an 80-mesh sieve to obtain tangerine peel powder; 200 g of kudzu root grown for 3 years is selected, the kudzu root is peeled, washed, dried and then cut into thin slices of about 4 mm, the kudzu root is crushed through an 80-mesh sieve to obtain kudzu root powder; the tangerine peel powder and the kudzu root powder are mixed, added into purified water at a solid-liquid ratio of 1:10, soaked for 30 minutes, extracted under a microwave power of 600 W for 15 minutes, filtered through a 100-mesh sieve, and the filtrate is collected.

[0039] Step (2): The obtained composite liquid was concentrated under reduced pressure at 60°C to a relative density of 1.25 (25°C), maltodextrin (based on 30% by weight of the total solid content of the finished dry powder) was added as an auxiliary material, mixed evenly, and then spray-dried. The spray-drying parameters were an inlet temperature of 180°C and an outlet temperature of 85°C. The spray-dried powder was sieved through a 60-mesh sieve to obtain a finished product.

[0040] Comparative Example 2: Peptide Group The I9-27 polypeptide was added to deionized water to obtain a polypeptide solution. The concentration of the I9-27 polypeptide in the polypeptide solution was prepared to be 10 μmol / L. The pH was adjusted to 6.5. The solution was stirred at a constant temperature of 25° C. for 1.5 hours to obtain a composite solution.

[0041] The obtained composite liquid was concentrated under reduced pressure at 60°C to a relative density of 1.25 (25°C), maltodextrin (calculated as dry powder, i.e., 30% of total solids) was added as an auxiliary material, mixed evenly, and then spray-dried. The spray-drying parameters were an inlet temperature of 180°C and an outlet temperature of 85°C. The spray-dried powder was sieved through a 60-mesh sieve to obtain a finished product.

[0042] Example 3: Study on the lipid-lowering effect of Citrus reticulatae and Puerariae root polypeptides in animals According to the preparation method of Example 2, a compound preparation of dried tangerine peel and kudzu root, and a compound preparation of dried tangerine peel and kudzu root and a polypeptide were prepared, and their lipid-lowering effects were compared and studied.

[0043] Sixty healthy SD rats were randomly divided into six groups: a blank control group, a model group, a lovastatin (positive control group), a tangerine peel and kudzu root group, a peptide group, and a tangerine peel and kudzu root peptide group, with 10 rats in each group. Except for the blank control group, all rats in the remaining groups were fed a high-fat diet for 4 weeks to establish a hyperlipidemia model. After successful model establishment, each group received the following treatments: Blank control group and model group: administered with equal volume of normal saline; Positive control group: lovastatin 0.2 mg / kg / d was administered orally; Tangerine peel and Pueraria root group: 0.2 g / kg / d of the extract prepared in Comparative Example 1 was administered orally; Peptide group: Oral administration of the extract prepared in Comparative Example 2 at 0.2 g / kg / d; Tangerine peel and Pueraria root polypeptide group: the extract prepared in Example 2 was administered orally at a rate of 0.2 g / kg / d.

[0044] After 4 weeks of continuous administration, the serum of rats in each group was collected to detect blood lipid indicators: total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C). The results are shown in Table 1: Table 1

[0045] LDL immunohistochemical staining was performed on the aorta tissue. The results showed that LDL deposition in endothelial cells was clearly observed in the model group. Compared with the model group, LDL deposition in the aorta wall of the rats in the positive control group, Tangerine Peel and Pueraria Root group, and Tangerine Peel and Pueraria Root polypeptide group was significantly reduced, while no significant change was observed in the polypeptide group. The reduction in LDL deposition in the aorta wall of the rats in the Tangerine Peel and Pueraria Root polypeptide group was comparable to that in the positive control group (see Figure 1), the polypeptide group did not show obvious lipid-lowering function when used alone, but after microwave extraction with tangerine peel and kudzu root, a tangerine peel and kudzu root polypeptide composition was obtained. The tangerine peel and kudzu root polypeptide group showed good effect in lowering blood lipids. The results of intracellular LDL deposition showed that the tangerine peel and kudzu root polypeptide group could more significantly reduce intracellular lipid deposition than the tangerine peel and kudzu root group, or compared with the polypeptide group used alone.

[0046] Example 4: Study on the mechanism of action of Citrus aurantium and Pueraria polypeptide According to the preparation method of Example 2, a compound preparation of dried tangerine peel and kudzu root, and a compound preparation of dried tangerine peel and kudzu root and a polypeptide were prepared, and their lipid-lowering mechanisms were compared and studied.

[0047] 1. Experimental Design EA.hy926 human vascular endothelial cells were used to establish a vascular endothelial cell injury model induced by oxidized low-density lipoprotein (ox-LDL); Blank control group: normal culture; Model group: ox-LDL (100 μg / ml) treated for 24 h; Tangerine peel and kudzu root polypeptide group: Tangerine peel and kudzu root polypeptide (25 μg / mL) was administered for 24 hours after ox-LDL treatment.

[0048] Tangerine peel and Pueraria root group: Tangerine peel and Pueraria root (25 μg / mL) were administered for 24 hours after ox-LDL treatment.

[0049] Peptide group: Peptide (25 μg / mL) was administered for 24 h after ox-LDL treatment.

[0050] 2. Detection indicators: Cell viability (MTT assay): EA.hy926 cells were counted at 1×10 5 Cells were seeded at 100 μL / well in a 96-well plate and incubated at 37°C in a 5% CO2 incubator for 4 hours. Drug treatment was administered according to grouping and the cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. The culture medium was aspirated and 100 μL of MTT solution (0.5 mg / mL) was added. After incubation at 37°C for 4 hours, the culture medium was aspirated and 150 μL of DMSO was added to each well. The cells were shaken in a microplate reader for 10 minutes to completely dissolve the crystals. OD values ​​were measured at 490 nm and 630 nm using a microplate reader. Cell viability was calculated according to the formula.

[0051] Cell survival rate (%) = cells in the treatment group (OD value at 490 nm - OD value at 630 nm) / cells in the control group (OD value at 490 nm - OD value at 630 nm) × 100%.

[0052] Intracellular LDL deposition (laser confocal microscopy): Prepare 6 mg / mL FITC solution with sterile PBS, extract 20 μL and place it in an EP tube, add 1 mL LDL (2 mg / mL), and mix evenly with a pipette. Place in a 37°C constant temperature incubator, incubate in the dark for 2 hours, transfer to a treated dialysis bag, seal with a 4 cm dialysis bag clamp, and place in a beaker containing 400 μL PBS. Dialyze at 4°C in the dark, change PBS every 24 hours, and dialyze for 3 consecutive days. Transfer the dialyzed FITC-LDL to a brown EP tube and store in a 4°C refrigerator. Take well-grown EA.hy926 cells, wait until the cells grow to about 90% fusion, trypsinize the cells, and use 1×10 5 Cells were seeded into 24-well plates at a density of approximately 70%. Culture medium was then switched to 1% fetal bovine serum (FBS) for 24 hours for cell synchronization. Each group was treated with the corresponding drug and FITC-LDL. 2.5 μL of sterile PBS was dispensed into the center of a sterile 24-well plate, and a 14 mm sterile cell slide was attached to the plate. All steps were performed in a dark environment. After the cell incubation period, the 24-well plate was removed and washed three times with PBS. The cells were trypsinized and resuspended in 200 μL of DMEM medium containing 10% FBS. The cells were then re-plated onto the freshly treated cell slide and incubated in a CO2 incubator for an additional 30 minutes to allow the cells to adhere. After 30 minutes, 300 μL of DMEM medium containing 10% FBS was added to the 24-well plate and incubated for an additional 3 hours at 37°C in a 5% CO2 incubator. After 3 hours, the 24-well plate was removed and washed three times with PBS for 3 minutes each. Add 500 μL of 4% PFA to each well and fix for 15 minutes. Wash three times with PBS for 3 minutes each. Remove the cell slides from the 24-well plate and gently place the cell-side down on a glass slide with an appropriate amount of anti-fluorescence quenching mounting medium. Examine the slides under a laser confocal microscope, selecting random fields at 10×10 magnification. FITC-LDL taken up into the cytoplasm of cells emits green fluorescence.

[0053] Chemiluminescence assay for changes in ROS levels: After drug incubation, cells were removed from the six-well plates, washed twice with PBS, and 1 mL of DCFH-DA probe diluted in 10 μM DMEM was added. A negative control tube was treated with DMEM alone. The cells were incubated at 37°C in a 5% CO2 incubator for 30 min. The culture medium was aspirated, and the cells were washed twice with PBS. The slides were then examined under a fluorescence microscope. Cells not examined under a fluorescence microscope were collected by scraping and suspended in PBS. Fluorescence was measured using a fluorescence microplate reader with an excitation wavelength of 500 ± 15 nm and an emission wavelength of 530 ± 20 nm. The suspension was then homogenized, and protein concentration was determined using the BCA assay. Intergroup comparisons were calculated by dividing the fluorescence value by protein concentration.

[0054] TBA method to detect changes in MDA levels: After incubation, remove the cell culture dish, wash twice with PBS, add 300 μL of the extraction solution in the kit, and use a homogenizer to make a suspension. Proceed as shown in Table 2 below: Table 2

[0055] After mixing the centrifuge tubes, incubate at 95°C for 80 minutes, remove the tubes and cool them in running water. Centrifuge at 4000 rpm for 10 minutes. Measure the absorbance of each sample tube at 530 nm to determine the MDA concentration of each sample.

[0056] TNF-α and IL-1β Content Assay: After drug incubation, cellular RNA was extracted and cDNA synthesized using a reverse transcription kit. Primers were designed with reference to PrimerBank, and qRT-PCR reactions were performed according to the instructions for the GoTaq® qPCR MasterMix PCR Kit (Promega). Melting curves were used to determine the specificity of the target fragments. Amplification curves were used to determine the Ct value and the amplification efficiency of the reaction system. Data were analyzed for relative quantification using the 2-∆∆Ct method. Statistical analysis was performed using SPSS 23.0 software. Data differences between the two groups were analyzed using the independent sample T-test.

[0057] The test results of Example 3 are shown in Table 3: Table 3

[0058] Table 3 shows that compared with the model group, both the peptide group alone and the tangerine peel and kudzu root peptide group could significantly improve the viability of vascular endothelial cells damaged by ox-LDL. The tangerine peel and kudzu root group had a weaker effect on promoting cell viability. The peptide group alone had no obvious effect on MDA, TNF-α, and IL-1β. However, the tangerine peel and kudzu root peptide group could significantly reduce the levels of ROS and MDA compared with the peptide group alone or the tangerine peel and kudzu root feeding group alone, greatly improving the oxidative stress state; and significantly reducing the expression of inflammatory factors TNF-α and IL-1β (P<0.05).

[0059] The results showed that the I9-27 peptide had a strong docking ability with PCSK9, mainly binding through hydrogen bonds and hydrophobic interactions to form a stable molecular complex. Figure 4This is a molecular interaction model between the I9-27 peptide and the PCSK9 protein. This study demonstrated the structural interaction between the I9-27 peptide (presented as a protein backbone) and PCSK9 using molecular docking technology. The crystal structure of PCSK9 is derived from the PDB database (PDB ID: 4NE9). The structural model of the I9-27 peptide was constructed using homology modeling based on the I9 structure in 4NE9. The dark region indicates the interaction interface between the two molecules. Figure 5 This is a molecular dynamics energy conformational analysis of the I9-27 peptide-PCSK9 complex. The dark blue region in the lower right corner of the figure represents the lowest energy conformational state when I9-27 binds to PCSK9.

[0060] 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 in the scope of protection of the present invention.

Claims

1. A method for preparing a tangerine peel and kudzu root polypeptide composition, comprising: Step 1: drying, crushing, and sieving dried tangerine peel to obtain a first powder; drying, crushing, and sieving kudzu root to obtain a second powder; mixing the first powder and the second powder, adding the mixture into water, heating and extracting, filtering, and collecting the filtrate; Step 2: Add I9-27 polypeptide to the filtrate, control the pH and temperature to obtain a composite solution; Step 3: Concentrate the composite liquid, add maltodextrin, and dry into powder to obtain the tangerine peel and kudzu root polypeptide composition.

2. The method for preparing the tangerine peel and kudzu root polypeptide composition according to claim 1, wherein In the step 1, the dried tangerine peel is 2-4 years old dried tangerine peel; and / or, in the step 1, the dried tangerine peel is Guangdong Xinhui dried tangerine peel; and / or, in the step 1, the tangerine peel is sieved through a mesh size of 60-100 mesh after being crushed.

3. The method for preparing the Citrus Reticulatae Puerariae polypeptide composition according to any one of claims 1 and 2, wherein: In the step 1, the kudzu root is peeled before drying and crushing; and / or, the mesh size of the sieve after the kudzu root is crushed is 60-100 mesh; and / or, in the step 1, the heating extraction method is microwave heating, and the microwave heating power is 500-700W; and / or, in the step 1, the heating extraction time is 10-20 minutes; and / or, in the step 1, after the heating extraction, the mesh size of the sieve for filtration is 90-120 mesh; and / or, the mass ratio of tangerine peel to kudzu root is 1:(0.5-1.5).

4. The method for preparing the Citrus Reticulatae Pellets polypeptide composition according to any one of claims 1 to 3, characterized in that: In the step 2, the molar amount of the I9-27 polypeptide added to each 1L of the filtrate is 5-15 μmol; and / or, in the step 2, the pH is 6-7; and / or, the temperature of the temperature control is 20-30°C; and / or, in the step 3, the method of concentrating the composite liquid in the step 3 is reduced pressure concentration; and / or, the composite liquid is concentrated to a relative density of 1.1-1.25; and / or, based on the mass percentage of the tangerine peel and kudzu root polypeptide composition, the feeding amount of the maltodextrin is 20-40%; and / or, the method of drying to powder is spray drying; and / or, the inlet temperature of the spray drying is 170-190°C; and / or, the outlet temperature of the spray drying is 70-100°C; and / or, after drying to powder, it is further sieved, and the mesh number of the sieve is 40-70.

5. The method for preparing the Tangerine Peel and Pueraria lobata polypeptide composition according to any one of claims 1 to 4, characterized in that: The sequence of the I9-27 polypeptide is: MPDGPAISALEASPHVENMEMDQEVTTQ.

6. The method for preparing any one of the Citrus Reticulatae and Puerariae Radix polypeptide compositions according to claims 1 to 5, characterized in that: The I9-27 polypeptide is obtained by fermenting the Morchella conicola CCBAS932 strain in one or more steps.

7. A tangerine peel and kudzu root polypeptide composition prepared by the method according to any one of claims 1 to 6.

8. A polypeptide having the sequence: MPDGPAISALEASPHVENMEMDQEVTTQ.

9. A food comprising the composition of claim 7.

10. Use of the tangerine peel and kudzu root polypeptide composition of claim 7 in the preparation of a drug for anti-inflammatory, hypolipidemic, or cell viability enhancement, or the food of claim 9 in the preparation of a food for anti-inflammatory, hypolipidemic, or cell viability enhancement.

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