Octapeptide with antioxidant activity
By developing an octapeptide with an amino acid sequence of SPDIFNPQ, the shortcomings of food-borne peptides in antioxidant applications were solved, and the oxidative stress damage protection against HepG2 cells was achieved, demonstrating its application potential in functional food additives and drugs.
Patent Information
- Application Number
- CN202510521710.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-17
- Publication Date
- 2025-07-29
AI Technical Summary
It is difficult for the prior art to effectively utilize food-borne polypeptides as antioxidants to remove excess reactive oxygen species and prevent and treat oxidative stress-related diseases.
An octapeptide with an amino acid sequence of SPDIFNPQ was developed to predict its antioxidant activity through bioinformatics tools, and to verify its protective effect on oxidative stress damage in human liver cancer cell models.
The octapeptide significantly alleviates oxidative stress damage in HepG2 cells at nontoxic doses, shows good antioxidant activity, and has the potential to develop as a functional food additive or drug.
Smart Images
Figure CN120383654A_ABST
Abstract
Description
Technical Field:
[0001] The present invention relates to the field of peptide biotechnology, and particularly to an octapeptide with antioxidant activity. Background Art:
[0002] The excessive accumulation of reactive oxygen species (ROS) can trigger oxidative stress responses. Under normal physiological conditions, the body's antioxidant defense system can maintain the dynamic balance of ROS levels. However, with aging and the stimulation of external environmental factors (such as pollution, radiation, improper diet, mental stress, etc.), the balance between ROS generation and clearance is disrupted, leading to oxidative stress damage to cells and thus inducing various chronic diseases such as Alzheimer's disease, hypertension, Parkinson's syndrome, and cancer. Research shows that antioxidant peptides, as supplementary antioxidants, can effectively scavenge excessive ROS and reduce oxidative stress damage, demonstrating good application potential in the prevention and treatment of oxidative stress-related diseases. In recent years, food-derived antioxidant peptides have received increasing research attention due to their wide sources, high safety, low toxicity, and good biological activities. Developing food-derived polypeptides as dietary antioxidants has important application value and market prospects. Summary of the Invention:
[0003] The present invention provides an octapeptide with antioxidant activity, whose amino acid sequence is: amino terminus - SPDIFNPQ - carboxyl terminus, and it can be attempted to be developed as an antioxidant. Description of the Drawings:
[0004] In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the drawings, where:
[0005] Figure 1 is the mass spectrometry diagram of the antioxidant octapeptide SPDIFNPQ.
[0006] Figure 2 is the toxicity assessment of SPDIFNPQ in HepG2 cells.
[0007] Figure 3 is the protective effect of SPDIFNPQ on oxidative stress damage in HepG2 cells. Detailed Embodiments:
[0008] A series of polypeptide sequences were isolated and identified from the enzymatic hydrolysate of soy protein isolate. Using bioinformatics tools such as AnOxPP and PeptideRanker, SPDIFNPQ was predicted to have antioxidant activity. Human hepatoma cell line HepG2 was selected as the cell model in the antioxidant experiment. Whether SPDIFNPQ was toxic to HepG2 was mainly evaluated. The cells were pre-incubated for 24 h at a non-toxic dose, and then treated with 175 μM H2O2 for 2 h to simulate oxidative stress injury. The protective effect of the invented peptide on cell oxidative stress injury was evaluated by cell viability.
[0009] The specific operation was as follows: Alcalase was used to hydrolyze soy protein isolate. The hydrolysis conditions were: 51.2 °C, pH 12, substrate concentration 5.273%, enzyme-to-substrate ratio 12.343%, and hydrolysis for 3 h. After hydrolysis, the enzyme was inactivated at 90 °C for 10 min, and then centrifuged at 12000 rpm for 10 min. The supernatant was freeze-dried for later use.
[0010] Liquid chromatography tandem mass spectrometry was used to identify the polypeptide sequences in the hydrolysate. Chromatographic separation: The mobile phase A for liquid chromatography was 0.1% formic acid aqueous solution, and the mobile phase B was 0.1% formic acid acetonitrile aqueous solution (acetonitrile was 84%). The liquid chromatography column (0.15 mm * 150 mm, RP-C18, Column Technology Inc.) was equilibrated with 95% of mobile phase A. The sample was loaded onto Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, DE) by an autosampler and then separated by the liquid chromatography column. The relevant liquid phase gradient settings were as follows:
[0011] 0 min - 50 min, the linear gradient of mobile phase B was from 4% to 50%;
[0012] 50 min - 54 min, the linear gradient of mobile phase B was from 50% to 100%;
[0013] 54 min - 60 min, mobile phase B was maintained at 100%.
[0014] Mass spectrometry identification: After separation by capillary high performance liquid chromatography, the enzymatic hydrolysate was analyzed by a Q Exactive mass spectrometer (ThermoFisher). The analysis duration was 60 min. The detection mode was positive ion. The mass-to-charge ratios of polypeptides and polypeptide fragments were collected according to the following method: 10 fragment spectra (MS2 scan) were collected after each full scan.
[0015] Data analysis: The original file of the mass spectrometry test (Raw File) was retrieved from the corresponding database using the software MaxQuant 1.5.5.1, and finally the sequence identification results were obtained.
[0016] AnOxPP was used to predict potential antioxidant activity. Peptides with a prediction score greater than 0.999 were selected, and PeptideRanker was used for bioactivity prediction with a threshold set at 0.7. Innovagen and ToxinPred were used to predict the water solubility and toxicity of the polypeptides, respectively. The prediction results for SPDIFNPQ were: AnOxPP value of 0.9999, PeptideRanker value of 0.70199, good water solubility, and no toxicity.
[0017] HepG2 cells were seeded in 96-well plates (1×10 4 cells / well), incubated at 37 °C for 24 h, then the medium was replaced with fresh medium containing different concentrations of the synthetic peptide, and incubated for another 12 h. Then the medium was removed, and 100 μL of fresh medium containing 10 μL of CCK-8 reagent was added to each well and incubated at 37 °C for 2 h. The absorbance was measured at 450 nm. For the oxidative stress protection assay, HepG2 cells were pre-incubated with the polypeptide for 12 h and then exposed to 175 μM H2O2 for 2 h. The cell viability was expressed as the ratio of the absorbance value of the synthetic peptide-treated group to that of the control group.
[0018] The results showed that the peptide of the present invention had a good effect of reducing oxidative stress damage in HepG2 cells, and its sequence was: amino terminus - SPDIFNPQ - carboxyl terminus.
[0019] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Obviously, those skilled in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.
Claims
1. An octapeptide with antioxidant activity, characterized in that Its amino acid sequence is: amino terminus - SPDIFNPQ - carboxyl terminus.