Fractionation of Dioscorea bulbifera bulbil protein, its antioxidative potential and inhibitory effects on Carbohydrate-hydrolyzing enzymes

Document Type : Original Article

Authors

Biochemistry and Molecular Biology Department, Faculty of Sciences, Obafemi Awolowo University, IleIfe, Nigeria.

Abstract

The study fractionated Dioscorea bulbifera bulbil proteins, determined the fractions inhibitory potential against α-amylase and α-glucosidase activity, and evaluated their antioxidative activity in-vitro. Dioscorea bulbifera bulbil flesh was homogenized in Phosphate Buffer Saline (pH7.2), centrifuged at 10,000 xg for 30 min and supernatant was subjected to ammonium sulphate fractionation. Three fractions (25%, 60% and 90%) were produced and their hemagglutinating activity, blood group and sugar specificity were determined. Ability of the fractions to inhibit α-amylase and α-glucosidase activity was investigated. Antioxidative properties were evaluated using DPPH, Nitric oxide and hydroxyl radicals scavenging assay. Their reducing power, total antioxidant capacity and ferrous chelating activity were also evaluated. Data obtained revealed the presence of lectin in the crude protein extract, 25% and 60% protein fractions. The lectin agglutinated rabbit blood erythrocyte better than human blood groups erythrocyte. Protein fraction (60%) has no hemagglutinating activity towards human blood erythrocyte. Mannose completely inhibited the hemagglutinating activity.  Alpha-amylase and α-glucosidase activities were inhibited by 25% and 60% protein fractions with no significant difference. But their inhibitory action was better than that of acarbose. All the free radicals tested were scavenged by the fractions to various degrees. Sixty percent protein fraction could not scavenge nitric oxide but it was a better scavenger of hydroxyl radical. No activity was recorded for 90% protein fraction. The results showed the anti-diabetic potential of the protein fractions and can be harnessed for possible development of therapeutics agents for the treatment of diabetes and oxidative stress associated ailments.

Highlights

Olayemi Oludele ODEKANYIN (Google Scholar)

Keywords


1.    Kumar S, Narwal S, Kumar V, Prakash O. α-glucosidase inhibitors from plants: A natural approach to treat diabetes. Pharmacogn Rev. 2011 Jan;5(9):19-29. doi: 10.4103/0973-7847.79096. PMID: 22096315; PMCID: PMC3210010.
2.    Galicia-Garcia U, Benito-Vicente A, Jebari S, Larrea-Sebal A, Siddiqi H, Uribe KB, et al. Pathophysiology of Type 2 Diabetes Mellitus. Int J Mol Sci. 2020 Aug 30;21(17):6275. doi: 10.3390/ijms21176275. PMID: 32872570; PMCID: PMC7503727. 
3.    Alwan AD, Galea G, Stuckler D. Development at risk: addressing noncommunicable diseases at the United Nations high-level meeting. Bull World Health Organ. 2011 Aug 1;89(8):546-546A. doi: 10.2471/BLT.11.091074. PMID: 21836748; PMCID: PMC3150770.
4.    Kim KY, Nam KA, Kurihara H, Kim SM. Potent alpha-glucosidase inhibitors purified from the red alga Grateloupia elliptica. Phytochemistry. 2008 Nov;69(16):2820-5. doi: 10.1016/j.phytochem.2008.09.007. Epub 2008 Oct 23. PMID: 18951591.
5.    Holman RR, Cull CA, Turner RC. A randomized double-blind trial of acarbose in type 2 diabetes shows improved glycemic control over 3 years (U.K. Prospective Diabetes Study 44). Diabetes Care. 1999 Jun;22(6):960-4. doi: 10.2337/diacare.22.6.960. Erratum in: Diabetes Care 1999 Nov;22(11):1922. PMID: 10372249.
6.    Asmat U, Abad K, Ismail K. Diabetes mellitus and oxidative stress-A concise review. Saudi Pharm J. 2016 Sep;24(5):547-553. doi: 10.1016/j.jsps.2015.03.013. Epub 2015 Mar 21. PMID: 27752226; PMCID: PMC5059829. 
7.    World Health Organization. Global reports on diabetes. 2016. Available from:   https://www.who.int/publications/i/item/9789241565257.
8.    Wahab NA, Abdullah N, Aminudin N. Characterisation of potential antidiabetic-related proteins from Pleurotus pulmonarius (Fr.) Quél. (grey oyster mushroom) by MALDI-TOF/TOF mass spectrometry. Biomed Res Int. 2014;2014:131607. doi: 10.1155/2014/131607. Epub 2014 Aug 28. PMID: 25243114; PMCID: PMC4163432.  
9.    Oliveira AEA, Machado OLT, Gomes VM, Neto JX, Pereira AC, Vieira JGH, Fernandes KV,  Xavier‐Filho J. Jack bean seed coat contains a protein with complete sequence homology to bovine insulin. Protein and Peptide Letters. 1999 Feb 1;6(1): 15-22
10.    Li Q, Ye XL, Zeng H, Chen X, Li XG. [Study on the extraction technology and hypoglycemic activity of lectin from Trichosanthes kirilowi]. Zhong Yao Cai. 2012 Mar;35(3):475-9. Chinese. PMID: 22876690.
11.    da Rocha AA, Araújo TF, da Fonseca CS, da Mota DL, de Medeiros PL, Paiva PM, Coelho LC, Correia MT, Lima VL. Lectin from Crataeva tapia Bark Improves Tissue Damages and Plasma Hyperglycemia in Alloxan-Induced Diabetic Mice. Evid Based Complement Alternat Med. 2013;2013:869305. doi: 10.1155/2013/869305. Epub 2013 Nov 13. PMID: 24324521; PMCID: PMC3845403.
12.    Paula PC, Sousa DO, Oliveira JT, Carvalho AF, Alves BG, Pereira ML, Farias DF, Viana MP, Santos FA, Morais TC, Vasconcelos IM. A Protein Isolate from Moringa oleifera Leaves Has Hypoglycemic and Antioxidant Effects in Alloxan-Induced Diabetic Mice. Molecules. 2017 Feb 11;22(2):271. doi: 10.3390/molecules22020271. PMID: 28208654; PMCID: PMC6155657.
13.    Subasini U, Thenmozhi S, Sathyamurthy D, Vetriselvan S, Victor RG, Dubey GP. Pharmacognostic and phytochemical investigations of Dioscorea bulbifera L. Int J Pharm Life Sci. 2013; 4(5): 2693 – 2700.
14.    Nguelefack TB, Dutra RC, Paszcuk AF, Andrade EL, Tapondjou LA, Calixto JB. Antinociceptive activities of the methanol extract of the bulbs of Dioscorea bulbifera L. var sativa in mice is dependent of NO-cGMP-ATP-sensitive-K(+) channel activation. J Ethnopharmacol. 2010 Apr 21;128(3):567-74. doi: 10.1016/j.jep.2010.01.061. Epub 2010 Feb 10. PMID: 20152893.
15.    Ghosh S, Ahire M, Patil S, Jabgunde A, Bhat Dusane M, Joshi BN, Pardesi K, Jachak S, Dhavale DD, Chopade BA. Antidiabetic Activity of Gnidia glauca and Dioscorea bulbifera: Potent Amylase and Glucosidase Inhibitors. Evid Based Complement Alternat Med. 2012;2012:929051. doi: 10.1155/2012/929051. Epub 2011 Jul 9. PMID: 21785651; PMCID: PMC3140190.
16.    Ghosh S, More P, Derle A, Patil AB, Markad P, Asok A, Kumbhar N, Shaikh ML, Ramanamurthy B, Shinde VS, Dhavale DD, Chopade BA. Diosgenin from Dioscorea bulbifera: novel hit for treatment of type II diabetes mellitus with inhibitory activity against α-amylase and α-glucosidase. PLoS One. 2014 Sep 12;9(9):e106039. doi: 10.1371/journal.pone.0106039. PMID: 25216353; PMCID: PMC4162539.
17.    Arya P, Munshi M, Kumar P. Diosgenin: Chemistry, extraction, quantification and health benefits. Food Chem Adv. 2023; 2:100170.
18.    Lowry OH, Rosebrough N, Farr A, Randall R. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265-75. PMID: 14907713.
19.    Wang H, Gao J, Ng TB. A new lectin with highly potent antihepatoma and antisarcoma activities from the oyster mushroom Pleurotus ostreatus. Biochem Biophys Res Commun. 2000 Sep 7;275(3):810-6. doi: 10.1006/bbrc.2000.3373. PMID: 10973803.
20.    Ademiluyi AO, Oboh G. Soybean phenolic-rich extracts inhibit key-enzymes linked to type 2 diabetes (α-amylase and α-glucosidase) and hypertension (angiotensin I converting enzyme) in vitro. Exp Toxicol Pathol. 2013 Mar;65(3):305-9. doi: 10.1016/j.etp.2011.09.005. Epub 2011 Oct 17. PMID: 22005499. 
21.    Shai LJ, Magano SR, Lebelo SL, Mogale AM. Inhibitory effects of five medicinal plants on rat alpha-glucosidase: Comparison with their effects on yeast alpha-glucosidase. J Med Plants Res. 2011; 5(13):2863-7.
22.    Brand-Williams W, Cuvelier ME, Berset CL. Use of a free radical method to evaluate antioxidant activity. LWT-Food Sci Technol. 1995; 28(1):25-30
23.    Halliwell B, Wasil M, Grootveld M. Biologically significant scavenging of the myeloperoxidase-derived oxidant hypochlorous acid by ascorbic acid. Implications for antioxidant protection in the inflamed rheumatoid joint. FEBS Lett. 1987 Mar 9;213(1):15-7. doi: 10.1016/0014-5793(87)81456-4. PMID: 3030805.
24.    Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem. 1982 Oct;126(1):131-8. doi: 10.1016/0003-2697(82)90118-x. PMID: 7181105.
25.    Prieto P, Pineda M, Aguilar M. Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. Anal Biochem. 1999 May 1;269(2):337-41. doi: 10.1006/abio.1999.4019. PMID: 10222007.
26.    Benzie IF, Strain JJ. Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods Enzymol. 1999;299:15-27. doi: 10.1016/s0076-6879(99)99005-5. PMID: 9916193.
27.    Singh N, Rajini PS. Free radical scavenging activity of an aqueous extract of potato peel. Food Chem. 2004; 85(4):611-6.
28.    Saha RK, Acharya S, Jamiruddin M, Roy P, Islam MM, Shovon SS. Antimicrobial effects of a crude plant lectin isolated from the stem of Tinospora tomentosa. J Phytopharmacol. 2014; 3:44-51
29.    Celestine AA, David OI. Comparative nutritional and phytochemical evaluation of the aerial and underground tubers of air potato (Dioscorea bulbifera). Available in Abakaliki, Ebonyi State, Nigeria. Br J Appl Sci Technol. 2015; 11(4): 1-7.
30.    Libra MA, Gonnety JT, Ahi AP, Dabonne S, Ahipo ED, Kouame LP. Physicochemical changes in bulbils of two cultivars of Dioscorea bulbifera during the ripening period. Adv J Food Sci Technol. 2011; 3(5):327-31
31.    Manukumar HM, Madhu CS. Comparative evaluation of fractional efficiency on antioxidant activity of red gram (Cajanus cajan) seed coat crude protein extracts. Int J Recent Sci Res. 2013; 4(9): 1395-1399.
32.    Odekanyin OO, Akeredolu EO. Comparative Investigation of the Biological Activity of the Pterocarpus osun and Bosqueia angolensis Seeds Protein Extracts. Int J Curr Res Biosci Plant Biol. 2016; 3(9):16-26. 
33.    Ohizumi Y, Gaidamashvili M, Ohwada S, Matsuda K, Kominami J, Nakamura-Tsuruta S, Hirabayashi J, Naganuma T, Ogawa T, Muramoto K. Mannose-binding lectin from yam (Dioscorea batatas) tubers with insecticidal properties against Helicoverpa armigera (Lepidoptera: Noctuidae). J Agric Food Chem. 2009 Apr 8;57(7):2896-902. doi: 10.1021/jf8040269. PMID: 19271711.
34.    Sharma M, Hotpet V, B R S, A S K, Swamy BM, Inamdar SR. Purification, characterization and biological significance of mannose binding lectin from Dioscorea bulbifera bulbils. Int J Biol Macromol. 2017 Sep;102:1146-1155. doi: 10.1016/j.ijbiomac.2017.04.085. Epub 2017 May 1. PMID: 28472687.
35.    Odekanyin O. O. and Akanni SI. Hemagglutinin from the Root Tuber of Dioscorea preussii Pax Exhibit Antioxidative Prowess and Haemolysis Inhibition. Turk J Pharma. Sci. 2023 DOI:10.4274/tjps.galenos.2023.62293(Article in Press)
36.    Akinyoola KA, Odekanyin OO, Kuku A, Sosan MB. Anti-insect potential of a lectin from the tuber, Dioscorea mangenotiana towards Eldana saccharina (Lepidoptera: Pyralidae). J Agric Biotechnol Sustain Dev. 2016; 8(3):16-26. DOI:10.5897/JABSD2015.0249
37.    Chan YS, Ng TB. A lectin with highly potent inhibitory activity toward breast cancer cells from edible tubers of Dioscorea opposita cv. nagaimo. PLoS One. 2013;8(1):e54212. doi: 10.1371/journal.pone.0054212. Epub 2013 Jan 21. PMID: 23349827; PMCID: PMC3549954.
38.    Harijono H, Estiasih T, Apriliyanti MW, Afriliana A, Kusnadi J. Physicochemical and Bioactives Characteristics of Purple and Yellow Water Yam (Dioscorea alata) Tubers. Inter J PharmTech Res. 2013; 5(4):1691-701.
39.    Araki T, Funatsu J, Kuramoto M, Konno H, Torikata T. The complete amino acid sequence of yam (Dioscorea japonica) chitinase. A newly identified acidic class I chitinase. J Biol Chem. 1992 Oct 5;267(28):19944-7. PMID: 1400311.
40.    Govindappa M, Sadananda T, Ramachandra YL, Chandrappa CP, Rai PS, Prasad SK. In vitro and in vivo antidiabetic activity of lectin (N-acetyl-galactosamine, 64 kDa) isolated from endophytic fungi, Alternaria species from Viscum album on alloxan induced diabetic rats. Integr Diabetes Obesity. 2015; 1(1):11-9.
41.    Olodude SO, Odekanyin OO, Fatokun AB. Evaluation of antioxidant and carbohydrate-hydrolysing enzymes inhibitory activities of Trilepisium madagascariense Ficalho seeds hemagglutinin. IOSR J Pharm Biol Sci. 2017; 12(6):29-41.
42.    Bhagyawant SS, Narvekar DT, Gupta N, Bhadkaria A, Gautam AK, Srivastava N. Chickpea (Cicer arietinum L.) Lectin Exhibit Inhibition of ACE-I, α-amylase and α-glucosidase Activity. Protein Pept Lett. 2019;26(7):494-501. doi: 10.2174/0929866526666190327130037. PMID: 30919768.
43.    Tran N, Pham B, Le L. Bioactive Compounds in Anti-Diabetic Plants: From Herbal Medicine to Modern Drug Discovery. Biology (Basel). 2020 Aug 28;9(9):252. doi: 10.3390/biology9090252. PMID: 32872226; PMCID: PMC7563488.
44.    Chan CH, Ngoh GC, Yusoff R. A brief review on anti diabetic plants: Global distribution, active ingredients, extraction techniques and acting mechanisms. Pharmacogn Rev. 2012 Jan;6(11):22-8. doi: 10.4103/0973-7847.95854. PMID: 22654401; PMCID: PMC3358964. 
45.    Svensson B, Fukuda K, Nielsen PK, Bønsager BC. Proteinaceous alpha-amylase inhibitors. Biochim Biophys Acta. 2004 Feb 12;1696(2):145-56. doi: 10.1016/j.bbapap.2003.07.004. PMID: 14871655.
46.    Poovitha S, Sai MS, Parani M. Protein extract from the fruit pulp of Momordica dioica shows anti-diabetic, anti-lipidemic and antioxidant activity in diabetic rats. J Funct Foods. 2017; 33:181-7.
47.    Mbiantcha M, Kamanyi A, Teponno RB, Tapondjou AL, Watcho P, Nguelefack TB. Analgesic and Anti-Inflammatory Properties of Extracts from the Bulbils of Dioscorea bulbifera L. var sativa (Dioscoreaceae) in Mice and Rats. Evid Based Complement Alternat Med. 2011;2011:912935. doi: 10.1155/2011/912935. Epub 2010 Sep 8. PMID: 20953397; PMCID: PMC2952333.
48.    Wang JM, Ji LL, Branford-White CJ, Wang ZY, Shen KK, Liu H, Wang ZT. Antitumor activity of Dioscorea bulbifera L. rhizome in vivo. Fitoterapia. 2012 Mar;83(2):388-94. doi: 10.1016/j.fitote.2011.12.001. Epub 2011 Dec 9. PMID: 22178682.
49.    Kuete V, Betrandteponno R, Mbaveng AT, Tapondjou LA, Meyer JJ, Barboni L, Lall N. Antibacterial activities of the extracts, fractions and compounds from Dioscorea bulbifera. BMC Complement Altern Med. 2012 Nov 23;12:228. doi: 10.1186/1472-6882-12-228. PMID: 23176193; PMCID: PMC3528471.
50.    Maithili V, Dhanabal SP, Mahendran S, Vadivelan R. Antidiabetic activity of ethanolic extract of tubers of Dioscorea alata in alloxan induced diabetic rats. Indian J Pharmacol. 2011 Jul;43(4):455-9. doi: 10.4103/0253-7613.83121. PMID: 21845005; PMCID: PMC3153713.
51.    Go HK, Rahman MM, Kim GB, Na CS, Song CH, Kim JS, Kim SJ, Kang HS. Antidiabetic Effects of Yam (Dioscorea batatas) and Its Active Constituent, Allantoin, in a Rat Model of Streptozotocin-Induced Diabetes. Nutrients. 2015 Oct 15;7(10):8532-44. doi: 10.3390/nu7105411. PMID: 26501316; PMCID: PMC4632431.
52.    Zong A, Guan Q, Ma W, Liu L, Jia M, Du F, Xu T. Antidiabetic Effect of Chinese yam (Dioscorea opposita Thunb.) in High‐Fat Diet/Streptozotocin‐Induced Diabetic Rats. Starch‐Stärke. 2023; 75(7-8):2200281. https://doi.org/10.1002/star.202200281
53.    Adomėnienė A, Venskutonis PR. Dioscorea spp.: Comprehensive Review of Antioxidant Properties and Their Relation to Phytochemicals and Health Benefits. Molecules. 2022 Apr 17;27(8):2530. doi: 10.3390/molecules27082530. PMID: 35458730; PMCID: PMC9026138.
54.    Ghosh S, Derle A, Ahire M, More P, Jagtap S, Phadatare SD, et al. Phytochemical analysis and free radical scavenging activity of medicinal plants Gnidia glauca and Dioscorea bulbifera. PLoS One. 2013 Dec 18;8(12):e82529. doi: 10.1371/journal.pone.0082529. PMID: 24367520; PMCID: PMC3867356. 
55.    Liu Y, Li H, Fan Y, Man S, Liu Z, Gao W, Wang T. Antioxidant and Antitumor Activities of the Extracts from Chinese Yam (Dioscorea opposite Thunb.) Flesh and Peel and the Effective Compounds. J Food Sci. 2016 Jun;81(6):H1553-64. doi: 10.1111/1750-3841.13322. Epub 2016 Apr 27. PMID: 27122252.
56.    Adebayo SA, Ondua M, Shai LJ, Lebelo SL. Inhibition of nitric oxide production and free radical scavenging activities of four South African medicinal plants. J Inflamm Res. 2019 Aug 8;12:195-203. doi: 10.2147/JIR.S199377. PMID: 31496781; PMCID: PMC6691489.
57.    Xiao S, Yuan Z, Huang Y. The Potential Role of Nitric Oxide as a Therapeutic Agent against SARS-CoV-2 Infection. Int J Mol Sci. 2023 Dec 5;24(24):17162. doi: 10.3390/ijms242417162. PMID: 38138990; PMCID: PMC10742813.
58.    Flannagan RS, Heit B, Heinrichs DE. Antimicrobial Mechanisms of Macrophages and the Immune Evasion Strategies of Staphylococcus aureus. Pathogens. 2015 Nov 27;4(4):826-68. doi: 10.3390/pathogens4040826. PMID: 26633519; PMCID: PMC4693167. 
59.    Liu TZ, Chin N, Kiser MD, Bigler WN. Specific spectrophotometry of ascorbic acid in serum or plasma by use of ascorbate oxidase. Clin Chem. 1982 Nov;28(11):2225-8. PMID: 7127767.
60.    Moon JK, Shibamoto T. Antioxidant assays for plant and food components. J Agric Food Chem. 2009 Mar 11;57(5):1655-66. doi: 10.1021/jf803537k. PMID: 19182948. 
61.    Zhao C, Zhou B, Miao J, Li X, Jing S, Zhang D, Yijia Wang J, Li X, Huang L, Gao W. Multicomponent analysis and activities for evaluation of Dioscorea oppositifolia and Dioscorea hamiltonii. Food Agric. Immunol. 2019 ;30(1):1148-61.
62.    Adedayo BC, Ademiluyi AO, Oboh G, Akindahunsi AA. Interaction of aqueous extracts of two varieties of Yam tubers (Dioscorea spp.) on some key enzymes linked to type 2 Diabetes in vitro. Int J Food Sci Technol. 2012; 47: 703–709. 
63.    Gulcin İ, Alwasel SH. Metal ions, metal chelators and metal chelating assay as antioxidant method. Processes. 2022; 10(1):132.
64.    Nikolova M. Screening of radical scavenging activity and polyphenol content of Bulgarian plant species. Pharmacognosy Res. 2011 Oct;3(4):256-9. doi: 10.4103/0974-8490.89746. PMID: 22224049; PMCID: PMC3249785.