A multifunctional polypeptide and its application

Abstract

The present invention discloses a multifunctional polypeptide and its application. The polypeptide is polypeptide I or polypeptide II. The amino acid sequence of polypeptide I is shown as VLPF; the amino acid sequence of polypeptide II is shown as HYF. The present invention screened two highly active polypeptides from Zhongdian yak casein, which exhibit anti-inflammatory and antioxidant activities and promote cell proliferation.

Description

Technical Field

[0001] The present invention relates to the field of polypeptides, and in particular to a multifunctional polypeptide and applications thereof. Background Art

[0002] Chronic inflammation and oxidative stress are core drivers of numerous pathological processes, including neurodegenerative diseases (such as Alzheimer's disease), metabolic syndrome, cardiovascular disease, and skin aging. While traditional anti-inflammatory drugs (such as nonsteroidal anti-inflammatory drugs and glucocorticoids) can effectively suppress inflammatory responses, long-term use can easily lead to side effects such as gastrointestinal damage and immunosuppression. Commonly used antioxidants in clinical practice (such as vitamin C and N-acetylcysteine) suffer from low bioavailability and a single target site, making precise regulation difficult.

[0003] In recent years, bioactive peptides have become a research hotspot due to their high selectivity, low toxicity and easy modification. Studies have shown that some naturally derived peptides (such as lactoferrin peptides and marine collagen peptides) can function by inhibiting the NF-κB / NLRP3 inflammasome pathway or activating the Nrf2 / ARE antioxidant pathway, but their strength and stability are limited by the inherent defects of the natural sequence. Although existing synthetic peptide strategies (such as amino acid substitution and cyclization modification) can partially improve activity, they mostly focus on a single function (anti-inflammatory or antioxidant), ignoring the synergistic amplification effect of inflammation and oxidative stress, resulting in limited practical application effects. Therefore, the development of peptide molecules that have both efficient anti-inflammatory and antioxidant synergistic effects, stable structures and can be prepared on a large scale is still a technical problem that needs to be overcome in this field. Summary of the Invention

[0004] In response to the deficiencies of the prior art, the present invention provides a multifunctional polypeptide and its application. The present invention screened and obtained two highly active polypeptides, which have anti-inflammatory and antioxidant activities and can also promote cell proliferation.

[0005] To solve the above technical problems, the technical solution of the present invention is as follows: a multifunctional polypeptide, the polypeptide is polypeptide I or polypeptide II, the amino acid sequence of polypeptide I is shown as VLPF; the amino acid sequence of polypeptide II is shown as HYF.

[0006] As a further description of the above scheme: the polypeptide screening process is as follows:

[0007] The Zhongdian yak casein was subjected to simulated gastrointestinal digestion to obtain a crude extract rich in active peptides. The crude extract was then ultrafiltered using an ultrafiltration membrane to separate peptides <3 kDa. The filtrate was then freeze-dried into a powder for later use. The peptides were identified by LC-MS / MS and scored using the Peptide Ranker database. Peptides with a score >0.8 were considered to have potential activity.

[0008] Target peptides were screened from multiple peptides with scores greater than 0.8, and the anti-inflammatory and antioxidant activities of antimicrobial peptides were predicted using the BIOPEP-UWM database. Multifunctional active peptides were identified through in vitro antioxidant and anti-inflammatory activity assays.

[0009] The present invention also provides the use of the multifunctional polypeptide in preparing antioxidant and / or anti-inflammatory products.

[0010] The present invention also provides the use of the multifunctional polypeptide in promoting cell proliferation.

[0011] The present invention also provides a cell culture medium, wherein the polypeptide is added to the culture medium to promote the proliferation of microorganisms or cells.

[0012] Compared with the prior art, the present invention has the following beneficial effects: the present invention screens out two highly active polypeptides from Zhongdian yak casein, which have anti-inflammatory and antioxidant activities and can also promote cell proliferation. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Identification of peptides with molecular weight <3 kDa in Zhongdian yak casein; A is the ion chromatogram of LC-MS / MS identification; B is the molecular weight distribution of peptides; C is the number of peptides with a score >0.8;

[0014] Figure 2 The effects of 7 peptides on the survival rate of RAW264.7 cells;

[0015] Figure 3 The effects of 7 peptides on the survival rate of Caco2 cells;

[0016] Figure 4 The effects of 7 peptides on the survival rate of HepG2 cells;

[0017] Figure 5 is the DPPH scavenging activity of 7 peptides;

[0018] Figure 6 The ABTS scavenging activity of 7 peptides;

[0019] Figure 7 The effects of seven peptides on NO secretion in RAW264.7 cells;

[0020] Figure 8 The effect of two synthetic peptides on the mRNA expression of inflammatory response-related genes (iNOS);

[0021] Figure 9 The following are the effects of two synthetic peptides on cell proliferation. DETAILED DESCRIPTION

[0022] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solutions.

[0023] 1. Preparation of milk-derived bifunctional active peptides

[0024] The milk of Zhongdian yak cattle was collected from Shangri-La City, Diqing Prefecture, Yunnan Province. The pH was adjusted to 4.6-4.8 by adding acid to obtain casein sedimentation. The Zhongdian yak casein was subjected to simulated gastrointestinal digestion. After simulated gastrointestinal digestion, a crude extract rich in active peptides was obtained. The crude extract of Zhongdian yak casein was then ultrafiltered using a 3kDa ultrafiltration membrane. The filtrate (<3kDa) was collected and vacuum-freezed into powder for later use. The peptide fragments were then identified by LC-MS / MS ( Figure 1 A).

[0025] like Figure 1 As shown in Figure B, a total of 386 peptides were identified, among which peptides with a molecular weight of less than 0.5 kDa accounted for the highest proportion, accounting for 35.23% of the total peptides, followed by peptides with a molecular weight ranging from 1.0 kDa to 1.5 kDa, accounting for 21.50% of the total peptides, indicating that after simulated gastrointestinal digestion, yak casein was enzymatically hydrolyzed into a large number of small molecular peptides; Figure 1 C. The peptides were scored using the Peptide Ranker database. It is generally believed that peptides with a score > 0.8 have potential activity. A total of 204 peptides had a score > 0.8, of which 136 were < 0.5 kDa and 60 were between 0.5 kDa and 1 kDa, indicating that small molecule peptides may have stronger activity. At the same time, there are a large number of new active short peptides in yak casein.

[0026] 2. Screening of milk-derived bifunctional active peptides based on database

[0027] Target anti-inflammatory peptides were screened from 204 peptides with scores greater than 0.8. The anti-inflammatory activity of antimicrobial peptides was predicted using the BIOPEP-UWM database. A total of 7 peptides were predicted to have anti-inflammatory and antioxidant activities (as shown in Table 1). The toxicity of the peptides was then predicted using the online database "ToxinPred". It was found that all 7 potential active peptides were non-toxic. Subsequently, the 7 peptides were chemically synthesized, and the bifunctional active peptides were identified through in vitro antioxidant and anti-inflammatory activity assays.

[0028] Table 1 Basic information of 7 potential active peptides

[0029] Serial number sequence Molecular weight (Da) Score toxicity 1 FLGY 498.24783 0.91 Non-Toxin 2 LGLF 448.26857 0.92 Non-Toxin 3 LGYF 498.24783 0.93 Non-Toxin 4 VLPF 474.28422 0.84 Non-Toxin 5 LPFP 472.26857 0.96 Non-Toxin 6 YGF 385.16377 0.97 Non-Toxin 7 HYF 465.20122 0.88 Non-Toxin

[0030] 3. Effects of peptides on the proliferation activity of different cells

[0031] 3.1 Proliferative activity of peptides on RAW264.7 cells

[0032] The MTT assay was used to detect the effect of different concentrations of synthetic peptides (400-25 μg / mL) on the survival rate of RAW264.7 cells after 24 hours of treatment. Figure 2 As shown, the synthetic peptide FLGY can significantly inhibit cell growth within the concentration range of 400 μg / mL, and the cell proliferation rate is lower than 85%. The cell proliferation rates of the other 6 peptides are all greater than 85%, indicating that they are not cytotoxic.

[0033] 3.2 Proliferative activity of peptides on Caco2 cells

[0034] The MTT assay was used to detect the effect of different concentrations of synthetic peptides (400-25 μg / mL) on the survival rate of Caco2 cells after 24 hours. Figure 3 As shown, the cell proliferation rates of the seven polypeptides were all greater than 85%, indicating that they were not cytotoxic.

[0035] 3.3 Proliferative activity of peptides on HepG2 cells

[0036] The MTT assay was used to detect the effect of different concentrations of synthetic peptides (400-25 μg / mL) on the survival rate of Caco2 cells after 24 hours. Figure 4 As shown, the cell proliferation rates of the seven polypeptides were all greater than 85%, indicating that they were non-cytotoxic and could promote the proliferation of Caco2 cells.

[0037] 4. Screening of milk-derived bifunctional active peptides based on in vitro experiments

[0038] 4.1 Antioxidant activity assay

[0039] like Figure 5 As shown, the scavenging rates of DPPH free radicals by the seven peptides were all above 20% when the concentration was 0.125-2 mg / mL, indicating that all seven peptides could scavenge DPPH free radicals. Among them, the scavenging rate of DPPH free radicals by peptide HYF reached 80% when the concentration was 2 mg / mL.

[0040] like Figure 6 As shown, the ABTS free radical scavenging rates of all seven peptides at concentrations of 0.125-2 mg / mL were all above 20%, indicating that all seven peptides can scavenge ABTS free radicals. Among them, peptides FLGY, LGYF, and HYF achieved ABTS free radical scavenging rates exceeding 60% at a concentration of 2 mg / mL, and the ABTS free radical scavenging rate of HYF peptide reached over 80%. These experimental results indicate that all seven peptides have antioxidant activity, and that peptides FLGY, LGYF, and HYF exhibit superior antioxidant activity compared to the other four peptides.

[0041] 4.2 Anti-inflammatory activity assay

[0042] In order to clarify the anti-inflammatory activity of the seven peptides, the Griess method was used to detect the effects of the synthetic peptides at concentrations of 400 to 25 μg / mL on NO production in LPS-stimulated RAW264.7 cells. Figure 7 As shown, compared with the control (3.87 μM), the NO level in the cell culture fluid of the LPS model group was significantly increased (22.54 μM) (P < 0.001). Compared with the LPS model group, the two synthetic peptides (VLPF and HYF) at different concentrations were able to reduce the NO level in the cells (P < 0.01). Cells secrete a large amount of NO when they are inflammatory. The synthetic peptides VLPF and HYF can significantly reduce the NO produced in the LPS-induced inflammation model. At a concentration of 400 μg / mL, the NO inhibition rate exceeded 50%, indicating that they have good anti-inflammatory activity.

[0043] 4.3 Determination of anti-inflammatory mechanisms

[0044] In NO synthesis, iNOS is the most important enzyme that leads to excessive NO production, which in turn leads to inflammatory responses. In addition, iNOS expressed in cells can regulate the secretion of inflammatory cytokines, including IL-6, IL-1β, and TNF-α. The expression level of iNOS was determined by qPCR. Figure 8 It can be seen that compared with the LPS group, the mRNA expression level of iNOS gene was significantly decreased after treatment with synthetic peptides VLPF and HYF (P < 0.05), suggesting that synthetic peptides VLPF and HYF regulate the secretion of NO at the transcriptional level.

[0045] 5. Peptides VLPF and HYF can act as nutrients to promote cell proliferation

[0046] Adding fetal bovine serum (FBS) to the cell basal culture medium (DMEM) can provide hormones that maintain the exponential growth of cells, nutrients that are absent or very small in the basal culture medium, and major low-molecular nutrients. Figure 9 It can be seen that the DMEM+FBS basal medium was supplemented with serum, and the other groups were supplemented with different concentrations of polypeptides in the basal medium. There was no significant difference in growth between the cells in the polypeptide group and the cells in the fetal bovine serum group (P>0.05). There was a significant difference in cell proliferation between the cells in the polypeptide group and the basal medium (DMEM) (P<0.05), indicating that the polypeptides VLPF and HYF can provide nutrients for cell growth, and the nutrients provided are similar to those of fetal bovine serum, and can be added to cell culture media as nutrients to promote cell or microbial growth.

[0047] It should be understood that the above-described specific embodiments of the present invention are merely illustrative or illustrative of the principles of the present invention and do not constitute limitations of the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included within the scope of protection of the present invention. In addition, the appended claims are intended to cover all variations and modifications that fall within the scope and metes and bounds of the appended claims, or equivalents thereof.

Claims

1. Use of a multifunctional polypeptide in the preparation of an anti-inflammatory product, characterized in that: The multifunctional polypeptide is polypeptide I or polypeptide II, the amino acid sequence of polypeptide I is shown in SEQ ID No. 1; the amino acid sequence of polypeptide II is shown in SEQ ID No.

2.

2. Use of a multifunctional polypeptide in promoting cell proliferation, characterized in that: The multifunctional polypeptide is polypeptide I or polypeptide II, the amino acid sequence of polypeptide I is shown in SEQ ID No. 1; the amino acid sequence of polypeptide II is shown in SEQ ID No. 2.

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