Acta Physiologiae Plantarum

, Volume 35, Issue 3, pp 901–909 | Cite as

Novel isoforms of proteinaceous α-amylase inhibitor (α-AI) from seed extract of Albizia lebbeck

  • Faiyaz K. Shaikh
  • Prafull P. Gadge
  • Ashok A. Shinde
  • Bhimrao V. Jaiwal
  • Keshav D. Shinde
  • Monohar V. Padul
  • Manvendra S. Kachole
Original Paper


Proteinaceous inhibitors of digestive α-amylase occur naturally in leguminous seeds and find applications in agriculture and clinical studies. We have detected and isolated eight novel α-amylase inhibitor isoforms in the seed extract of Albizia lebbeck. They are designated as AL-αAI-1 to AL-αAI-8. These isoforms specifically inhibit human salivary α-amylase and porcine pancreatic α-amylase. The occurrence and profile of α-amylase inhibitor isoforms were revealed by 7 % native-PAGE containing 0.1 % starch. The apparent molecular weights of native bands of AL-αAIs were 97.4, 68.6, 61.0, 57.2, 56.0, 54.7, 51.1, and 47.7 kDa, respectively. Partial purification of potent α-amylase inhibitor was achieved using ammonium sulfate fractionation and gel filtration chromatography on G-100 Sephadex column followed by preparative gel electrophoresis. SDS-PAGE analysis of partially purified AL-αAI showed two polypeptide bands of ~35.8 and ~32.6 kDa. All these isoforms showed effective resistance to in vitro proteolysis by pepsin, trypsin, and chymotrypsin. These inhibitors are stable over a wide range of pH and temperature and have optimum activity at pH 7 and at 37 °C. The finding and information obtained in the present investigation about novel isoforms of α-amylase inhibitors from A. lebbeck could be important and may find applications in clinical studies to modulate starch digestion and glycemic index.


Proteinaceous α-amylase inhibitor Albizia lebbeck Human salivary α-amylase Porcine pancreatic α-amylase 



α-Amylase inhibitor


Albizia lebbeck α-amylase inhibitor


Bromophenol blue


Bovine serum albumin


3,5-Dinitrosalicylic acid


Human salivary α-amylase


Polyacrylamide gel electrophoresis


Porcine pancreatic α-amylase





First author of the manuscript is grateful to University Grant Commission (UGC), Government of India, New Delhi for providing financial assistance in the form of Maulana Azad national fellowship (MANF).


  1. Anthonamma K, Prasad SHKR, Rajasekhar D, Swapna NL, prasad M (2010) In vitro antimicrobial efficacy of solvent extracts of seeds of Albizzia lebbeck (L.) Benth. Int J Adv Pharm Sci 1:281–283Google Scholar
  2. Baruah CC, Gupta PP, Patnaik GK, Misra-Bhattacharya S, Goel RK, Kulshreshtha DK, Dubey MP, Dhawan BN (2000) Immunomodulatory effect of Albizzia Lebbeck. Pharm Biol 38(3):161–166Google Scholar
  3. Bernfeld P (1955) α-amylases, alpha and beta. In: Colowick SP, Kaplan NO (eds) Methods in enzymology, vol 1. Academic Press, New York, pp 149–158CrossRefGoogle Scholar
  4. Carlson GL, Li BU, Bass P, Olsen WA (1983) A bean alpha-amylase inhibitor formulation (starch blocker) is ineffective in man. Science 219(4583):393–395PubMedCrossRefGoogle Scholar
  5. Celleno L, Tolaini MV, Damore A, Perricone NV, Preuss HG (2007) A dietary supplement containing standardized Phaseolus vulgaris extract influences body composition of overweight men and women. Int J Med Sci 41:45–52CrossRefGoogle Scholar
  6. Chintawar SD, Somani RS, Kasture VS, Kasture SB (2002) Nootropic activity of Albizzia lebbeck in mice. MAPA 24(6):3451Google Scholar
  7. Chokshi D (2006) Toxicity studies of blockal, a dietary supplement containing phase 2 starch neutralizer (Phase 2), a standardized extract of the common white kidney bean (Phaseolus vulgaris). Int J Toxicol 25:361–371PubMedCrossRefGoogle Scholar
  8. Davis BJ (1964) Disc electrophoresis-II. Method and application to human serum proteins. Ann NY Acad Sci 121:404–427PubMedCrossRefGoogle Scholar
  9. Dayler CSA, Mendes PAM, Prates MV, Bloch C Jr, Franco OL, Grossi-de-Sa MF (2005) Identification of a novel bean α-amylase inhibitor with chitinolytic activity. FEBS Lett 579:5616–5620PubMedCrossRefGoogle Scholar
  10. Fontanini D, Capocchi A, Saviozzi F, Galleschi L (2007) Simplified electrophoretic assay for human salivary α-amylase inhibitor detection in cereal seed flours. J Agric Food Chem 55:4334–4339PubMedCrossRefGoogle Scholar
  11. Fowler MJ (2007) Diabetes treatment part 2: oral agents for glycemic management. Clin Diabetes 25:134–144Google Scholar
  12. Franco OL, Rigden DJ, Melo FR, Bloch C Jr, Silva CP, Grossi-de-Sa MF (2000) Activity of wheat alpha amylase inhibitors towards bruchid alpha amylases and structural explanation of observed specificities. Eur J Biochem 267:1466–1473CrossRefGoogle Scholar
  13. Franco DLR, Melo FR, Grossi-de Sá MF (2002) α-Amylase inhibitors and their interaction with insect α-amylase. Structure, function and potential crop protection. Eur J Biochem 269:397–412PubMedCrossRefGoogle Scholar
  14. Gibbs B, Ali I (1998) Characterization of a purified alpha-amylase inhibitor from white kidney beans (Phaseolus vulgaris). Food Res Int 31:217–225CrossRefGoogle Scholar
  15. Giri AP, Kachole MS (1998) α-Amylase inhibitors of pigeonpea (Cajanus Cajan) seeds. Phytochemistry 47:197–202PubMedCrossRefGoogle Scholar
  16. Gupta RS, Chaudhary R, Yadav RK, Verma SK, Dobhal MP (2005) Effect of Saponins of Albizia lebbeck Benth. Bark on the reproductive system of male albino rats. J Ethnopharmacol 96(1–2):31–36PubMedCrossRefGoogle Scholar
  17. Guzman-Partida AM, Jatomea-Fino O, Robles-Burgueno MR, Ortega-Nieblas M, Vazquez-Moreno L (2007) Characterization of a-amylase inhibitor from Palo Fierro seeds. Plant Physiol Biochem 45:711–715PubMedCrossRefGoogle Scholar
  18. Huang WY, Teita NW (1982) Determinations of α-amylase isoenzymesin serum by use of a selective inhibitor. Clin Chem 28:1525–1527PubMedGoogle Scholar
  19. Ishimoto M, Kitamura K (1989) Growth inhibitory effects of an α-amylase inhibitor from kidney bean, Phaseolus vulgaris (L.) on three species of bruchids (Coleoptera: bruchidae). Appl Entomol Zool 24:281–286Google Scholar
  20. Kim YM, Jeong YK, Wang MH, Lee WY, Rhee HI (2005) Inhibitory effect of pine extract on alpha-glucosidase activity and postprandial hyperglycemia. Nutrition 21:756–761PubMedCrossRefGoogle Scholar
  21. Kluh I, Horn M, Hyblova J, Hubert J, Doleckova- Maresova L, Voburka Z, Kudlikova I, Kocourek F, Mares M (2005) Inhibitory specificity and insecticidal selectivity of α-amylase inhibitor from Phaseolus vulgaris. Phytochem 66:31–39CrossRefGoogle Scholar
  22. Krishna BB, Patrícia BP, Raúl AL, Maria FG, Carlos B Jr, Jorge ATM, Betania FQ, Eliane FN, Octávio LF (2007) Molecular identification of four different α-amylase inhibitors from Baru (Dipteryx alata) seeds with activity toward insect enzymes. J Biochem Mol Biol 40(4):494–500CrossRefGoogle Scholar
  23. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of Bacteriophage T4. Nature 227:680–685Google Scholar
  24. Layer P, Carlson GL, DiMagno EP (1985) Partially purified white bean amylase inhibitor reduces starch digestion in vitro and inactivates intraduodenal amylase in humans. Gastroenterology 88:1895–1902PubMedGoogle Scholar
  25. LeBerre-Anton V, Bompard-Gilles C, Payan F, Rouge P (1997) Characterization and functional properties of the alpha-amylase inhibitor (alpha A-1) from kidney bean (Phaseolus vulgaris) seeds. Biochim Biophys Acta 1343:31–40CrossRefGoogle Scholar
  26. Leiner IE, Donatrucci DA, Tarcza JC (1984) Starch blockers: a potential source of trypsin inhibitors and lectins. Am J Clin Nutr 39:196–200Google Scholar
  27. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  28. Marshall JJ, Lauda CM (1975) Purification and properties of phaseolamine, an inhibitor of α-amylase, from the kidney bean, Phaseolus vulgaris. J Biol Chem 250:8030–8037PubMedGoogle Scholar
  29. Matsui T, Tanaka T, Tamura S, Toshima A, Miyata Y, Tanaka K et al (2007) Alpha-glucosidase inhibitory profile of catechins and theaflavins. J Agric Food Chem 55:99–105PubMedCrossRefGoogle Scholar
  30. Nagaraj RH, Pattabiraman TN (1985) Purification and properties of α-amylase inhibitor specific for human pancreatic amylase from proso (Panicium miliaceum) seeds. J Biosci 7(3 & 4):257–268CrossRefGoogle Scholar
  31. Ndemanisho E, Kimoro B, Mtengeti E, Muhikambele V (2006) The potential of Albizia lebbeck as a supplementary feed for goats in Tanzania. Agrofor Syst 67:85–91CrossRefGoogle Scholar
  32. O’Donnell MD, FitzGerald O, McGeeney KF (1977) Differential serum α-amylase determination by use of an inhibitor, and design of a routine procedure. Clin Chem 23:560–566PubMedGoogle Scholar
  33. Obiro WC, Zhang T, Jiang B (2009) Starch blocking stability of the Phaseolus vulgaris alpha-amylase inhibitor (α-AI1). Am J Food Tech 1:9–19Google Scholar
  34. Octavio L, Rigden D (2002) Plant α-amylase inhibitors and their interaction with -α-amylases. Eur J Biochem 269:397–412CrossRefGoogle Scholar
  35. Olorunsanya AO, Egbewande OO, Ibrahim H, Adeyemo MM (2010) Growth Performance and Carcass Analysis of Broiler Chickens Fed Graded Levels of Toasted Albizia lebbeck Seed Meal. Pak J Nutr 9(9):873–876CrossRefGoogle Scholar
  36. Payan F (2004) Structural basis for the inhibition of mammalian and insect alpha-amylases by plant protein inhibitors. Biochim Biophys Acta 1696:171–180PubMedCrossRefGoogle Scholar
  37. Pratibha N, Saxena VS, Amit A, D’Souza P, Bagchi M, Bagchi D (2004) Anti inflammatory activities of Aller-7, a novel polyherbal formulation for allergic rhinitis. Int J Tissue React 26(1–2):43–51PubMedGoogle Scholar
  38. Qian M, Nahoum V, Bonicel J, Bischoff H, Henrissat B, Payan F (2001) Enzyme-catalyzed condensation reaction in a mammalian a-amylase highresolution structure analysis of an enzyme-inhibitor complex. Biochemistry 40:7700–7709PubMedCrossRefGoogle Scholar
  39. Resmi CR, Venukumar MR, Latha MS (2006) Antioxidant activity of Albizzia lebbeck (Linn.) Benth in alloxan rats. Indian J Physiol Pharma 50(3):297–302Google Scholar
  40. Richardson M (1991) Methods in Plant Biochemistry. In: Rogers LJ (ed), vol 5. Academic Press, New York, pp 259–305Google Scholar
  41. Saha A, Ahmed M (2009) The analgesic and anti-inflammatory activities of the extract of Albizia lebbeck in animal model. Pak J Pharm Sci 22(1):74–77PubMedGoogle Scholar
  42. Schroeder HE, Gollasch S, Moore AE, Tabe LM, Craig S, Hardie DC, Chrispeels MJ, Spencer D, Higgins TJV (1995) Bean α-amylase inhibitor confers resistance to the pea weevil (Bruchus pisorum) in transgenic peas (Pisum satiVum L.). Plant Physiol 107:1233–1239PubMedGoogle Scholar
  43. Sitthipong P (2005) Amylase, maltase and sucrase inhibitors from red kidney bean (Phaseolus vulgaris). Prince of Songkla University, Faculty of ScienceGoogle Scholar
  44. Sopade PA, Gidley MJ (2009) A rapid in vitro digestibility assay based on glucometry for investigating kinetics of starch digestion. Starch/Stärke 61:245–255CrossRefGoogle Scholar
  45. Svensson B, Fukuda K, Nielsen PK, Bønsager BC (2004) Proteinaceous α-amylase inhibitors. Biochim Biophys Acta 1696:145–156PubMedCrossRefGoogle Scholar
  46. Tormo MA, Gil-Exojo I, Romero de Tejada A, Campillo JE (2004) Hypoglycaemic and anorexigenic activities of α-amylase inhibitor from white kidney beans (Phaseolus vulgaris) in Wistar rats. Br J Nutr 92:785–790PubMedCrossRefGoogle Scholar
  47. Valencia-Jimenez JA, Bustillo AE, Ossa GA, Chrispeels MJR (2000) Amylases of the coffee berry borer (Hypothenemus hampei) and their inhibition by two plant amylase inhibitors. Insect Biochem Mol Biol 30:207–213CrossRefGoogle Scholar
  48. Xiaoyan H, Jiangui L, Qiughua S, Jusong Z, Xiaoling H, Hao M (2009) Characterization of a novel legumin alpha amylase inhibitor from chickpea (Cicer arietinum L.) seeds. Biosci Biotechnol Biochem 73(5):1200–1202CrossRefGoogle Scholar
  49. Yamada T, Hattori K, Ishimoto M (2001) Purification and characterization of two α-amylase inhibitors from seeds of tepary bean (Phaseolus acutifolius A. Gray). Phytochemistry 58:59–66PubMedCrossRefGoogle Scholar
  50. Yoshikawa H, Kotaru M, Tanaka C, Ikeuchi T, Kawabata M (1999) Characterization of kintoki bean (Phaseolus vulgaris) alpha-amylase inhibitor: inhibitory activities against human salivary and porcine pancreatic alpha-amylases and activity changes by proteolytic digestion. J Nutr Sci Vitaminol 45:797–802PubMedCrossRefGoogle Scholar

Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2012

Authors and Affiliations

  • Faiyaz K. Shaikh
    • 1
  • Prafull P. Gadge
    • 1
  • Ashok A. Shinde
    • 1
  • Bhimrao V. Jaiwal
    • 1
  • Keshav D. Shinde
    • 1
  • Monohar V. Padul
    • 1
  • Manvendra S. Kachole
    • 1
  1. 1.Department of BiochemistryDr. Babasaheb Ambedkar Marathwada UniversityAurangabadIndia

Personalised recommendations