Inhibitory effect of low-molecular-weight peptides (0–3 kDa) from Spirulina platensis on H₂O₂-induced oxidative damage in L02 human liver cells

Abstract

Spirulina platensis protein hydrolysates were prepared by digesting protein extracts with papain, and the hydrolysates were separated into 30, 10, and 3 kDa weights using membrane ultrafiltration. The 0–3 kDa low-molecular-weight Spirulina peptides (LMWSPs) proved the highest chemical antioxidant activity by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability, hydroxyl radical (·OH) scavenging activities and total antioxidant capacity. Cellular antioxidant ability of LMWPs fractions against 2000 μg/mL H₂O₂ induced oxidative damage of L02 cells were investigated. The MTT assay results displayed that LMWSPs at different concentrations (0–1000 μg/mL) had proliferation effect on the L02 cells and that treatment of the L02 cells with the 1000 μg/mL LMWSPs (0–3 kDa) significantly prevented H₂O₂-induced oxidative damage compared with control cells. Moreover, the 2′,7′-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe assay showed that the levels of ROS and NO were significantly lower in the experimental group that was treated with the peptides for 24 h than in the control group. Furthermore, using the corresponding kits, the treatment inhibited the reduction of SOD activity and the increase of MDA contents in the L02 cells. Therefore, LMWSPs (0–3 kDa) may have potential applications in antioxidant and liver health products.

Keywords

Spirulina platensis peptides / Antioxidant activity / H₂O₂-induced damage / L02 cell

References

[1] Akpinar M, Yumusak N. Production of bioactive proteins and peptides from the diatom Nitzschia laevis and comparison of their in vitro antioxidant activities with those from Spirulina platensis and Chlorella vulgaris. Int J Food Sci Technol, 2018, 53: 676-682.

[2] Alahyaribeika S, Sharifia SD, Tabandehb F, Honarbakhsha S, Ghazanfaria S. Stability and cytotoxicity of DPPH inhibitory peptides derived from biodegradation of chicken feather. Protein Expr Purif, 2021, 177: 105748.

[3] Bayram B, Ozgur A, Tutar L, Tutar Y. Tumor targeting of polymeric nanoparticles conjugated with peptides, saccharides, and small molecules for anticancer drugs. Curr Pharm Des, 2017, 23: 5349-5357.

[4] Checa J, Aran JM. Reactive oxygen species: drivers of physiological and pathological processes. J Inflamm Res, 2020, 13: 1057-1073.

[5] Cheng FY, Lai IC, Lin LC, Sakata R. The in vitro antioxidant properties of alcalase hydrolysate prepared from silkie fowl (Gallus gallus) blood protein. Anim Sci J, 2016, 87: 921-928.

[6] Dong ZY, Tian G, Xu ZG, Li MY, Xu M, Zhao YJ, Ren H. Antioxidant activities of peptide fractions derived from freshwater mussel protein using ultrasound-assisted enzymatic hydrolysis. Czech J Food Sci, 2017, 35: 328-338.

[7] Farvin KHS, Andersen LL, Otte J. Antioxidant activity of cod (Gadus morhua) protein hydrolysates: fractionation and characterisation of peptide fractions. Food Chem, 2016, 204: 409-419.

[8] Gan BH, Cai XG, Javor S, Köhler T, Reymond JL. Synergistic effect of propidium iodide and small molecule antibiotics with the antimicrobial peptide dendrimer G3KL against gram-negative bacteria. Molecules, 2020, 25: 5643.

[9] Georgieva E, Ivanov D, Zhelev Z, Bakalov R, Gulubov M, Aoki I. Mitochondrial dysfunction and redox imbalance as a diagnostic marker of “free radical diseases”. Anticancer Res, 2017, 37: 5373-5381.

[10] Hu RJ, Dunmire KM, Truelock CN, Paulk CB, Aldrich G, Li YH. Antioxidant performances of corn gluten meal and DDGS protein hydrolysates in food, pet food, and feed systems. J Agric Food Res, 2020, 2: 100030.

[11] Hu XM, Wang YM, Zhao YQ. Antioxidant peptides from the protein hydrolysate of Monkfish (Lophius litulon) muscle: purification, identification, and cytoprotective function on HepG2 cells damage by H₂O₂. Mar Drugs, 2020, 18: 3.

[12] Ito N, Fukushima S, Tsuda H. Carcinogenicity and modification of the carcinogenic response by BHA, BHT, and other antioxidants. Crit Rev Toxicol, 1985, 15: 109-150.

[13] Jha S, Mandal P, Bhattacharyya P, Ghosh A. Free-radical scavenging properties of low molecular weight peptide(s) isolated from S1 cultivar of mulberry leaves and their impact on Bombyx mori (L.) (Bombycidae). J Anim Sci Biotechnol, 2014, 5: 1-9.

[14] Jian WJ, Tu LY, Wu LL, Xiong HJ, Pang J, Sun YM. Physicochemical properties and cellular protection against oxidation of degraded Konjac glucomannan prepared by γ-irradiation. Food Chem, 2017, 231: 42-50.

[15] Kleekayai T, Gouic AVL, Deracinois B, Cudennec B, FitzGerald RJ. In vitro characterisation of the antioxidative properties of whey protein hydrolysates generated under pH- and non pH-controlled conditions. Foods, 2020, 9: 582.

[16] Kong BH, Peng XY, Xiong YLL, Zhao XH. Protection of lung fibroblast MRC-5 cells against hydrogen peroxide-induced oxidative damage by 0.1–2.8 kDa antioxidative peptides isolated from whey protein hydrolysate. Food Chem, 2012, 135: 540-547.

[17] Lee SJ, Cheong SH, Kim YS, Hwang JW, Kwon HJ, Kang SH, Moon SH, Jeon BT, Park PJ. Antioxidant activity of a novel synthetic hexa-peptide derived from an enzymatic hydrolysate of duck skin by-products. Food Chem Toxicol, 2013, 62: 276-280.

[18] Lu J, Ren DF, Xue YL, Sawano Y, Miyakawa T, Tanokura M. Isolation of an antihypertensive peptide from alcalase digest of Spirulina platensis. J Agric Food Chem, 2010, 58: 7166-7171.

[19] Moneim AEA. Oxidant/antioxidant imbalance and the risk of Alzheimer’s disease. Curr Alzheimer Res, 2015, 12: 335-349.

[20] Nimse SB, Pal D. Free radical, natural antioxidants, and their reaction mechanisms. RSC Adv, 2015, 5: 27986-28006.

[21] Ray PD, Huang BW, Tsuji Y. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cell Signal, 2012, 24: 981-990.

[22] Singh AJ, Kukreti R, Saso L, Kukreti S. Oxidative stress: a key mdulator in neurodegenerative diseases. Molecules, 2019, 24: 1583.

[23] Stadtman ER. Role of oxidant species in aging. Curr Med Chem, 2004, 11: 1105-1112.

[24] Stemoukhov AA. Histamine-dependent changes in free radical processes during coronary heart disease. Bull Exp Biol Med, 2001, 132: 1157-1159.

[25] Sun YJ, Chang R, Li PY, Li BS. Isolation and characterization of an antibacterial peptide from protein hydrolysates of Spirulina platensis. Eur Food Res Technol, 2018, 244: 1509.

[26] Supriyadi A, Arum LS, Nugraha AS, Ratnadewi AAI, Siswoyo TA. Revealing antioxidant and antidiabetic potency of Melinjo (Gnetum Gnemon) seed protein hydrolysate at different stages of seed maturation. Curr Res Nutr Food Sci, 2019, 7: 479-487.

[27] Tao J, Zhao YQ, Wang B, Chi CF. Bioactive peptides from cartilage protein hydrolysate of spotless smoothhound and their antioxidant activity in vitro. Mar Drugs, 2018, 16: 100.

[28] Tian R, Feng J, Huang G, Tian B, Zhang Y, Jiang LZ, Sui XN. Ultrasound driven conformational and physicochemical changes of soy protein hydrolysates. Ultrason Sonochem, 2020, 68: 105202.

[29] Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem Biol Interact, 2006, 160: 1-40.

[30] Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol, 2007, 39: 44-84.

[31] Verdasco-Martín C, Echevarrieta L, Otero C. Advantageous preparation of digested proteic extracts from Spirulina platensis biomass. Catalysts, 2019, 9: 145.

[32] Wang SY, Jiao HJ. Scavenging capacity of berry crops on superoxide radicals, hydrogen peroxide, hydroxyl radicals, and singlet oxygen. J Argic Food Chem, 2000, 48: 5677-5684.

[33] Wang Z, Zhang X. Inhibitory effects of small molecular peptides from Spirulina (Arthrospira) platensis on cancer cell growth. Food Funct, 2015, 7: 781-788.

[34] Wang ZJ, Zhang XW. Characterization and antitumor activity of protein hydrolysates from Arthrospira platensis (Spirulina platensis) using two-step hydrolysis. J Appl Phycol, 2016, 28: 3379-3385.

[35] Wang ZJ, Zhang XW. Inhibitory effects of small molecular peptides from Spirulina (Arthrospira) platensis on cancer cell growth. Food Funct, 2016, 7: 781-788.

[36] Xia T, Yao J, Zhang J, Zheng Y, Song J, Wang M. Protective effects of Shanxi aged vinegar against hydrogen peroxide-induced oxidative damage in LO2 cells through Nrf2-mediated antioxidant responses. RSC Adv, 2017, 7: 17377-17386.

[37] Xu Y, Galanopoulos M. Effect of enzymatic hydrolysis using endo-and exo-proteases on secondary structure, functional, and antioxidant properties of chickpea protein hydrolysates. J Food Meas Charact, 2019, 14: 343-352.

[38] Zhuang YL, Sun LP, Zhao X, Hou H, Li BF. Investigation of gelatin polypeptides of jellyfish (Rhopilema esculentum) for their antioxidantactivity in vitro. Food Technol Biotech, 2010, 48: 222-228.

[39] Zhang M, Huang TS, Mu TH. Production and characterisation of antioxidant peptides from sweet potato protein by enzymatic hydrolysis with radio frequency pretreatment. Int J Food Sci Technol, 2020, 55: 2352-2358.

Funding

National Key R&D Program of China(2019YFD1002400)

Beijing Nova Program(Z201100006820048)

National promotion project of scientific and tenchnological achievements in forestry and grassland(2020133135)