Peptide Fractions from Chymotrypsin-hydrolyzed Moringa oleifera Seed Proteins Inhibit α-amylase and α-glucosidase in vitro

Oluwafemi Emmanuel Ekun, Augustine Olusegun Olusola, Joseph Adaviruku Sanni, Feyisayo Ishola


This study attempts to investigate the activities of chymotrypsin-digested M. oleifera seed proteins and their peptide fractions on carbohydrate-hydrolyzing enzymes. Proteins from M. oleifera seeds were isolated using isoelectric point precipitation and hydrolyzed using chymotrypsin. The hydrolysates obtained were fractionated into peptide fractions of <1 kD, 1-3 kD and 3-5 kD ranges by means of gel-filtration chromatography. The inhibitory effects of the hydrolysates and their fractions on α-amylase and α-glucosidase were evaluated, and kinetics of inhibition were also determined. Using starch and p-nitrophenyl glucopyranoside as substrates, the hydrolysate and fractions demonstrated concentration-dependent inhibition of α-amylase and α-glucosidase respectively (IC50 of 0.172 ± 0.005 mg mL-1 to 1.312 ± 0.267 mg mL-1, for α-amylase inhibition and IC50 of 0.463 ± 0.008 mg mL-1 to 0.696 ± 0.051 mg mL-1 for α-glucosidase inhibition). Kinetic analysis revealed that selected hydrolysate fractions competitively inhibited α-amylase while displaying a mixed mode of inhibition of α-glucosidase. This study suggests that subjecting M. oleifera seed proteins to proteolysis could yield therapeutic peptide products having immense potentials that could be harnessed to develop novel anti-diabetic agents and additives to food, which could serve as cost effective alternatives to current therapies.
Keywords: Moringa oleifera, hydrolysate, peptide, chymotrypsin, α-amylase, α-glucosidase


Moringa oleifera; hydrolysate; peptide; chymotrypsin; α-amylase; α-glucosidase

Full Text:



Abd-Rani NZ, Husain K. Kumolosasi E (2018) Moringa Genus: A Review of Phytochemistry and Pharmacology. Frontiers in Pharmacology 9(1):1 – 26

Acharya DK, Shah IJ, Gami PN, Shukla RM (2014) Optimization for α- amylase production by Aspergillus oryzae using submerged fermentation technology. Basic Res. J. Microbiol. 1(4): 01-10

Alashi AM, Blanchard CL, Mailer RJ, Agboola SO, Mawson AJ, He R, Malomo SA, Girgih AT, Aluko RE (2014) Blood pressure lowering effects of Australian canola protein hydrolysates in spontaneously hypertensive rats. Food Research International 55: 281-287.

Ali H, Houghton PJ, Soumyanath A (2006) Alpha-amylase inhibitory activity of some Malaysian plants used to treat diabetes, with particular reference to Phyllanthus amarus. J. Ethnopharmacol. 107: 449–455

Anwar F, Latif S, Ashraf M, Gilani AH (2007) Moringa oleifera: A food plant with multiple medicinal uses. Phytother Res 21(1):17–25

Arise RO, Yekeen AA, Ekun OE (2016) In vitro antioxidant and -amylase inhibitory properties of watermelon seed protein hydrolysates. Environmental and Experimental Biology, 14: 163–172.

Arise RO, Idi JJ, Mic-Braimoh IM, Korode E, Ahmed RN, Osemwegie O (2019) In vitro Angiotesin-1-converting enzyme, α-amylase and α-glucosidase inhibitory and antioxidant activities of Luffa cylindrical (L.) M. Roem seed protein hydrolysate, Heliyon, 5(5): e0163

Awosika TO, Aluko RE (2019) Inhibition of the in-vitro activities of α-amylase, α-glucosidase and pancreatic lipase by yellow field pea (Pisum satvum L.) protein hydrolysates. Int. J. Food Sci and Tech. doi:10.1111/ijfs.14087.

Boyer R (2012) Biochemistry Laboratory: Modern Theory and Techniques. 2nd Edition Pearson Education Inc. New Jersey, United States

Divi SM, Bellamkonda R, Dasireddy SK (2012) Evaluation of antidiabetic and antihyperlipedemic potential of aqueous extract of Moringa oleifera in fructose fed insulin resistant and STZ induced diabetic wistar rats: a comparative study, Asian J. Pharm. Clin. Res. 5: 67–72.

Famuwagun AA, Alashi AM., Gbadamosi SO, Taiwo KA, Oyedele, DJ, Adeboye OC, Aluko RE (2020) In Vitro Characterization of Fluted Pumpkin Leaf Protein Hydrolysates and Ultrafiltration of Peptide Fractions: Antioxidant and Enzyme-Inhibitory Properties Pol. J. Food Nutr. Sci., 70(4):429–443

Freire JE, Vasconcelos IM, Moreno FB, Batista AB, Lobo MD, Pereira ML, Lima JP, Almeida RV, Sousa AJ, Monteiro-Moreira AC, Oliveira JT. Grangeiro TB (2015) Mo-CBP3, an Antifungal Chitin-Binding Protein from Moringa oleifera Seeds, Is a Member of the 2S Albumin Family. PLoS ONE, 10, e0119871.

Galvão CA, Silva AF, Custódio MF, Monti R. and Giordano, RC (2001) Controlled Hydrolysis of Cheese Whey Proteins Using Trypsin and -Chymotrypsin. Applied Biochemistry and Biotechnology Vol. 91–93: 761-776

Girgih, AT, Udenigwe CC, Li H, Adebiyi AP, Aluko RE (2011) Kinetics of enzyme inhibition and antihypertensive effects of hemp seed (Cannabis sativa L.) protein hydrolysates. J. Am. Oil Chem. Soc. 88: 1767–1774.

Ibrahim MA, Bester MJ, Neitz AW, Gaspar AR (2018) Structural properties of bioactive peptides with α-glucosidase inhibitory activity. Chemical Biology and Drug Design, 91(2): 370-379.

International Diabetes Federation (2020) Diabetes Atlas. 9th edition Brussels.

Kim YM, Jeong YK, Wang MH, Lee WY, Rhee HI (2005) Inhibitory effect of pine extract on alpha-glucosidase activity and postprandial hyperglycemia. Nutrition. 21(6): 756-61.

Kwaambwa HM, Hellsing MS, Rennie AR, Barker R (2015) Interaction of Moringa oleifera Seed Protein with a Mineral Surface and the Influence of Surfactants. Journal of Colloid and Interface Science.448, 339-346.

Leone A, Spada A, Battezzati A, Schiraldi A, Aristil J, Bertoli S (2016) Moringa oleifera Seeds and Oil: Characteristics and Uses for Human Health. Int. J. Mol. Sci.17: 2141

Lopez-Barrios L, Gutierrez-Uribe JA, Serna-Saldıvar SO (2014) Bioactive Peptides and Hydrolysates from Pulses and Their Potential Use as Functional Ingredients. Journal of Food Science 79(3) 273-283

Mahajan SG, Banerjee A, Chauhan BF, Padh H, Nivsarkar M, Mehta AA (2009) Inhibitory effect of n-butanol fraction of Moringa oleifera Lam. Seeds on ovalbumin- induced airway inflammation in a guinea pig model of asthma. Int. J. Toxicol. 28:519-527.

Majumder K, Wu J (2015) Molecular Targets of Antihypertensive Peptides: Understanding the Mechanisms of Action Based on the Pathophysiology of Hypertension. International Journal of Molecular Sciences, 16(1), 256–283.

Malomo SA, Aluko RE (2016) In vitro acetylcholinesterase inhibitory properties of enzymatic hemp seed protein hydrolysates. Journal of the American Oil Chemists’ Society, 93:411-420

Mune-Mune MA, Nyobe EC, Bassogog CB, Minka SR (2016) A comparison on the nutritional quality of proteins from Moringa oleifera leaves and seeds. Cogent Food & Agriculture 2: 1213618.

Ndong M, Uehara M, Katsumata S, Suzuki K. (2007) Effects of oral administration of Moringa oleifera Lam on glucose tolerance in gotokakizaki and wistar rats. J. Clin. Biochem. Nutr. 40:229-233.

Oboh G, Ademiluyi AO, Faloye YM (2011) Effect of combination on the antioxidant and inhibitory properties of tropical pepper varieties against α-amylase and α-glucosidase activities in vitro. Journal of Medical Foods, 14: 1152–1158.

Olusola AO, Ekun OE (2019a) Alpha-Amylase – Inhibitory Properties and in vitro Antioxidant Potentials of Cowpea Seed Protein Hydrolysates. AASCIT Communications 6(1): 1-12.

Olusola AO, Ekun OE (2019b) Moringa oleifera seed protein hydrolysates inhibit haemoglobin glycation and α-glucosidase activity in-vitro. Global Journal of Medical Research XIX(III) 31-40

Olusola AO, Ekun OE, David TI, Olorunfemi OE, Oyewale MB (2018) Moringa oleifera Seed Protein Hydrolysates: Kinetics of α-amylase Inhibition and Antioxidant Potentials. Global Advanced Research Journal of Medicine and Medical Sciences 7(9) pp. 190-201.

Onuh JO, Girgih AT, Malomo SA, Aluko RE Aliani M (2015) Kinetics of in vitro renin and angiotensin converting enzyme inhibition by chicken skin protein hydrolysates and their blood pressure lowering effects in spontaneously hypertensive rats. Journal of functional food. 14: 133 – 143.

Palmer T, Bonner PL (2007) Enzymes: Biochemistry, Biotechnology and Clinical Chemistry. 2nd edition. Woodhead Publishing. Cambridge, United Kingdom.

Prasad S, Mandal I, Singh S, Mandal B, Venkatramani R, Swaminathan R (2017) Near UV-Visible Electronic Absorption Originating from Charged Amino Acids in a Monomeric Protein. Chem. Sci. 8:5416-5433

Qaisar MN, Chaudhary BA, Sajid MU, Hussain N (2014) α-glucosidase Inhibitory Activity of Dichoromethane and Methanol Extracts of Croton bonpladianum Baill. Tropical Journal of Pharmaceutical Research 13(11): 1833-1836

Rhoades RA, Bell BR (2013) Medical Physiology: Principles for clinical medicine. Lippincott Williams & Wilkins 4th edition. pp 660-663

Shanmugam VP, Kapila S, Sonfack TK, Kapila R. (2015) Antioxidative peptide derived from enzymatic digestion of buffalo casein. International Dairy Journal 42:1-5

Siddhuraju P, Beck K (2003) Antioxidant properties of various solvent extract of total phenolic constituent from three different agrochemical origins of drumstick tree (Moringa Oleifera Lam) J. Agric Food Chem. 15: 2144 – 2155.

Teixeira EM, Carvalho MR, Neves VA, Silva MA, Arantes-Pereira, L (2014) Chemical Characteristics and Fractionation of Proteins from Moringa oleifera Lam. Leaves. Food Chemistry. 147, 51-54

Thundimadathil J (2012) Cancer treatment using peptides: current therapies and future prospects. J. Amino Acids, 2012, 967347.

Tounkara F, Bashari M, Le G, Shi Y (2014) Antioxidant Activities of Roselle (Hibiscus Sabdariffa L.) Seed Protein Hydrolysates and its Derived Peptide Fractions. International Journal of Food Properties 17(9):1998-2011

Ulagesan S, Kuppusamy A. Kim HJ (2018) Antimicrobial and antioxidant activities of protein hydrolysates from terrestrial snail Cryptozona bistrialis. Journal of Applied Pharmaceutical Science 8(12): 12-19

Vlieghe P, Lisowski V, Martinez J, Khrestchatisky M (2010) Synthetic therapeutic peptides: science and market. Drug Discov. Today, 15, 40–56.

Voet D, Voet JG, Pratt, CW (2016). Fundamentals of Biochemistry. 5th edition. United States: Wiley

Wani AA, Sogi DS, Singh P, Wani IA, Shivhare US (2011) Characterisation and Functional Properties of Watermelon (Citrullus lanatus) Seed Proteins. J. Agric. Food Chem. 91: 113- 121.

Yamada A, Sakurai T, Ochi D, Mitsuyama E, Yamauchi K, Abe F (2013) Novel Angiotensin I-Converting Enzyme Inhibitory Peptide Derived from Bovine Casein. Food Chem., 141, 3781–3789.

Yu Z, Liu B, Zhao W, Yin Y, Liu J, Chen F (2012) Primary and secondary structure of novel ACE-inhibitory peptides from egg white protein. Food Chem.133:315–322



  • There are currently no refbacks.

Copyright (c) 2022 Oluwafemi Emmanuel Ekun, Augustine Olusegun Olusola, Joseph Adaviruku Sanni, Feyisayo Ishola

Biology, Medicine, & Natural Product Chemistry
ISSN 2089-6514 (paper) - ISSN 2540-9328 (online)
Published by Sunan Kalijaga State Islamic University & Society for Indonesian Biodiversity.

Creative Commons License
This work is licensed under a CC BY-NC