Camel and Horse Milk Casein Hydrolysates Exhibit Angiotensin Converting Enzyme Inhibitory and Antioxidative Effects In Vitro and In Silico

  • Chizoba Paul Ugwu
  • Muawiya Musa Abarshi
  • Sanusi Bello MadaEmail author
  • Babangida Sanusi
  • Humphrey Chukwuemeka Nzelibe


Angiotensin converting enzyme (ACE) and reactive oxygen species are crucial targets for nutritional management of hypertension. The aim of this study is to investigate ACE inhibitory and antioxidative effects of camel and horse milk casein hydrolysates. After casein was isolated from raw camel and horse milk, the purity and molecular weights of the isolated casein were verified using SDS-PAGE prior to digestion with pepsin, trypsin and combined enzymes. The results obtained showed that hydrolysates obtained from pepsin and trypsin combined enzymes exhibited higher ACE-inhibitory activity than individual enzymes. In addition, DPPH radical scavenging activity (%) was significantly (p < 0.05) higher for camel milk casein hydrolysate than horse milk casein hydrolysate. Both hydrolysates displayed significant (p < 0.05) inhibition of lipid peroxidation and hydroxyl radical when compared with standard antioxidant (BHA). Moreover, simulation of casein proteolysis in silico generated fragments with potential ACE inhibitory activity using molecular docking analysis. The present findings suggested that camel and horse milk casein hydrolysates contain bioactive peptides responsible for ACE inhibition and antioxidative effects and thus could be exploited for the treatment and management of hypertension.


Camel milk Horse milk Casein hydrolysates Angiotensin converting enzyme inhibition Antioxidant property 



The authors are grateful to the Department of Biochemistry, Ahmadu Bello University Zaria for providing laboratory facilities to carry out this piece of work.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.


  1. Abdel-Salam AM, Al-Dekheil A, Babkr A, Farahna M, Mousa HM (2013) High fiber probiotic fermented Mare’s milk reduces the toxic effects of mercury in rats. Norteam J Med Sci 2:569–575Google Scholar
  2. Abubakar A, Saito T, Kitazawa H, Kawai Y, Itoh T (1998) Structural analysis of new antihypertensive peptides derived from cheese whey protein by proteinase K digestion. J Dairy Sci 81:3131–3138CrossRefGoogle Scholar
  3. Bamdad F, Shin SH, Suh J, Nimalaratne C, Sunwoo H (2017) Anti-inflammatory and antioxidant properties of casein hydrolysates produced using high hydrostatic pressure combined with proteolytic enzymes. Molecules.
  4. Blois MS (1958) Antioxidant determination by the use of a stable free radical. Nature 181:1199–1200CrossRefGoogle Scholar
  5. Ceriello A (2008) Possible role of oxidative stress in the pathogenesis of hypertension. Diabetes Care 31(2):S181–S184CrossRefGoogle Scholar
  6. Cushman DW, Cheung HS (1971) Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochem Pharmacol 20:1637–1648CrossRefGoogle Scholar
  7. Daliri EB, Deog HO, Byong HL (2017) Bioactive peptides. Foods. Google Scholar
  8. Davies DT, Law AJR (1977) An improved method for the quantitative fractionation of casein mixture using ion-exchange chromatography. J Dairy Res 44:213–221CrossRefGoogle Scholar
  9. Du Y, Zhao Y, Cai W (2014) Hydroxyl radical scavenging activity of peptide from fish intestine protein by hydrolysis with complex enzyme. Adv J Food Sci Technol 6(1):126–129CrossRefGoogle Scholar
  10. Frostegard J, Wu R, Lemne C, Thulin T, Witztum JL, de Faire U (2003) Circulating oxidized low-density lipoprotein is increased in hypertension. Clin Sci 105:615–620CrossRefGoogle Scholar
  11. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A (2005) Protein identification and analysis tools on the ExPASy server. In: Walker JM (ed) The proteomics protocols handbook. Humana, New York, pp 571–607CrossRefGoogle Scholar
  12. Ghasemi S, Khoshgoftarmanesh AH, Hadadzadeh H, Jafari M (2012) Synthesis of Iron-amino acid chelates and evaluation of their efficacy as iron source and growth stimulator for tomato in nutrient solution culture. J Plant Growth Regul 31:498–508CrossRefGoogle Scholar
  13. Ghazi L, Drawz P (2017) Advances in understanding the renin-angiotensin-aldosterone system (RAAS) in blood pressure control and recent pivotal trials of RAAS blockage in heart failure and diabetic nephropathy. F1000 Research, 6, 297Google Scholar
  14. Han Z, Zhang W, Luo W, Li J (2016) Novel antioxidant peptides derived from enzymatic hydrolysates of macadamia protein. J Biosci Med 4:6–14Google Scholar
  15. Hernandez-Ledesma B, Garcia-Nebot MJ, Fernandez-Tome S, Amigo L, Recio I (2014) Dairy protein hydrolysates: peptides for health benefits. Int Dairy J 38:82–100CrossRefGoogle Scholar
  16. Huma N, Rafiq S, Sameen A, Pasha I, Khan MI (2018) Antioxidant potential of buffalo and cow milk cheddar cheeses to tackle human colon adenocarcinoma (Caco2) cells. Asian-Australas J Anim Sci 31(2):287–292CrossRefGoogle Scholar
  17. Jialal I (1998) Evolving lipoprotein risk factors: lipoprotein (a) and oxidized low-density lipoprotein. Clin Chem 44:1827–1832Google Scholar
  18. Kumar R, Chaudhary K, Chauhan JS, Nagpal G, Kumar R, Sharma M, Raghava GPS (2015) An in-silico platform for predicting, screening and designing of antihypertensive peptides. Sci Rep 5:12512CrossRefGoogle Scholar
  19. Kumar D, Chatli MK, Singh R, Mehta N, Kumar P (2016) Enzymatic hydrolysis of camel milk casein and its antioxidant properties. Dairy Sci Technol 96:391–404CrossRefGoogle Scholar
  20. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  21. Lassegue B, Griendling KK (2004) Reactive oxygen species in hypertension: an update. Am J Hypertens 17:852–860CrossRefGoogle Scholar
  22. Li Y, Jiang H, Huang G (2017) Protein hydrolysates as promoters of non-haem iron absorption-review. Nutrients 9:609CrossRefGoogle Scholar
  23. Lin K, Zhang L, Han X, Xin L, Meng Z, Gong P, Cheng D (2018) Yak milk casein as potential precursor of angiotensin-i-converting enzyme inhibitory peptides based on in silico proteolysis. Food Chem 254:340–347CrossRefGoogle Scholar
  24. Mada SB, Reddi S, Kumar N, Kumar R, Kapila S, Kapila R, Trivedi R, Karvande A, Ahmad N (2017a) Antioxidative peptide from milk exhibits anti-osteopenic effects through inhibition of oxidative damage and bone-resorbing cytokines in ovariectomized rats. Nutrition 43–44: 21–31CrossRefGoogle Scholar
  25. Mada SB, Reddi S, Kumar N, Kapila S, Kapila R (2017b) Protective effects of casein-derived peptide VLPVPQK against hydrogen peroxide–induced dysfunction and cellular oxidative damage in rat osteoblastic cells. Hum Exp Toxicol 36(4):096032711667829. Google Scholar
  26. Mada SB, Reddi S, Kumar N, Vij R, Yadav R, Kapila S, Kapila R (2018) Casein-derived antioxidative peptide prevents oxidative stress-induced dysfunction in osteoblast cells. PharmaNutrition 6:169–179CrossRefGoogle Scholar
  27. Meisinger C, Baumert J, Khuseyinova N, Loewel H, Koenig W (2005) Plasma oxidized low-density lipoprotein, a strong predictor for acute coronary heart disease events in apparently healthy, middle-aged men from the general population. Circulation 112:651–657CrossRefGoogle Scholar
  28. Mohanty DP, Mohapatra S, Misra S, Sahu PS (2016) Milk derived bioactive peptides and their impact on human health—a review. Saudi J Biol Sci 23(5):577–583CrossRefGoogle Scholar
  29. Muñoz-Durango N, Fuentes CA, Castillo AE, González-Gómez LM, Vecchiola A, Fardella CE, Kalergis AM (2016) Role of the renin-angiotensin-aldosterone system beyond blood pressure regulation: molecular and cellular mechanisms involved in end-organ damage during arterial hypertension. Int J Mol Sci. Google Scholar
  30. Nawaz KAA, David SM, Murugesh E, Thandeeswaran M, Gopikrishnan K, Mahendran R, Palaniswamy M, Angayarkannia J (2017) Identification and in silico characterization of a novel peptide inhibitor of angiotensin converting enzyme from Pigeon pea (Cajanuscajan). Phytomedicine (17) 30130-7Google Scholar
  31. Nisha N, Sreekumar S, Biju C (2016) Identification of lead compounds with cobra venom detoxification activity in andrographis Paniculata (Burm. F.) Nees Through in Silico Method. Int J Pharm Pharm Sci. (8), 212–217Google Scholar
  32. Palaniswamy M, Angayarkanni J, Nandhini B (2012) Angiotensin converting enzyme inhibitory activity and antioxidant properties of goat milk. Hydrolysates Int J Pharm Pharmaceut Sci 4(4):367–370Google Scholar
  33. Paudel KR, Lee UW, Kim DW (2016) Chungtaejeon, a Korean fermented tea, prevents the risk of atherosclerosis in rats fed with a high-fat atherogenic diet. J Integr Med 14(2):134–142CrossRefGoogle Scholar
  34. Pihlanto A (2006) Antioxidative peptides derived from milk proteins. Int Dairy J 16:1306–1314CrossRefGoogle Scholar
  35. Reddi S, Kumar N, Vij R, Mada SB, Kapila S, Kapila R (2016) Akt drives buffalo casein derived novel peptide mediated osteoblast differentiation. J Nutr Biochem 38:134–144CrossRefGoogle Scholar
  36. Ryan MT, Chopra RK (1976) Determination of protein concentration by biuret method. Biochem Biophys Acta 427:337–349Google Scholar
  37. Sachidanandam K, Fagan SC, Ergul A (2005) Oxidative stress and cardiovascular disease: antioxidants and unresolved issues. Cardiovasc Drug Rev 23:115–132CrossRefGoogle Scholar
  38. Sarma H, Mattaparthi VSK (2018) Unveiling the transient protein-protein interactions that regulate the activity of human lemur tyrosine kinase-3 (LMTK3) domain by cyclin dependent kinase 5 (CDK5) in breast cancer: an in-silico study. Curr Proteomics 15:62–70CrossRefGoogle Scholar
  39. Shanmugam VP, Kapila S, Sonfack TK, Kapila R (2015) Antioxidative peptide derived from enzymatic digestion of buffalo casein. Int Dairy J 42:1–5CrossRefGoogle Scholar
  40. Yahya MA, Alhaj OA, Al-Khalifa AS (2017) Antihypertensive effect of fermented skim camel (Camelus Dromedaries) milk on spontaneously hypertensive rats. Nutr Hospistalaria 34(2):416–421CrossRefGoogle Scholar
  41. Zidane F, Zeder-Lutz G, Altschuh D, Girardet J, Miclo L, Corbier C, Cakir-Kiefer C (2013) Surface plasmon resonance analysis of the binding mechanism of pharmacological and peptidic inhibitors to human somatic angiotensin I-converting enzyme, biochemistry.
  42. Zou T, He T, Li H, Tang H, Xia E (2016) The structure-activity relationship of the antioxidant peptides from natural proteins. Molecules 21:72CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Chizoba Paul Ugwu
    • 1
  • Muawiya Musa Abarshi
    • 1
  • Sanusi Bello Mada
    • 1
    Email author
  • Babangida Sanusi
    • 1
  • Humphrey Chukwuemeka Nzelibe
    • 1
  1. 1.Department of BiochemistryAhmadu Bello UniversityZariaNigeria

Personalised recommendations