Medicinal Chemistry Research

, Volume 27, Issue 6, pp 1559–1577 | Cite as

Inhibitor discovery from pomegranate rind for targeting human salivary α-amylase

  • Jiachen Sun
  • Shengjie Dong
  • Yueting Wu
  • Hui Zhao
  • Xia Li
  • Wenyuan Gao
Original Research


This study explored the effects of the main active compounds of the pomegranate (Punica granatum L.) rind extract on the activity of the human salivary α-amylase and their molecular inhibitory mechanisms. Four compounds exhibited remarkable inhibitory activities against α-amylase, including (1) rutin, (2) luteolin, (3) quercetin, and (4) kaempferol. The IC50 values were found to be 265.65, 59.67, 99.56, and 139.72 μM for rutin, luteolin, quercetin, and kaempferol, respectively. The kinetic study using the Lineweaver–Burk revealed the four compounds showed a non-competitive inhibition against α-amylase. However, the exact localization of the binding site and the potentiation mechanism at the molecular level are presently unknown. We have performed the “blind docking” of four compounds on the human salivary α-amylase. The molecular modeling demonstrated a high affinity and tight binding capacity of these compounds at the binding site of α-amylase, where the Glu 233 was supposed to play a key role in exerting the inhibition activity of these compounds. The results may provide an important insight for the applications of computational methods in the drug design with treating disorders of carbohydrate metabolism.


Pomegranate rind Human salivary α-amylase Inhibition activity Molecular docking Molecular dynamics simulation 



This work was supported by grants from the National Natural Science Foundation of China (NO. 81173487, 81373904 and 81673535), Science and Technology Program of China (NO. 2014FY111100) and Special Scientific Research of Chinese Medicine Industry (NO. 201307008).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

44_2018_2164_MOESM1_ESM.pdf (1.2 mb)
Supplementary Information


  1. Aghajari N, Feller G, Gerday C, Haser R (2002) Structural basis of α-amylase activation by chloride. Protein Sci 11:1435–1441CrossRefPubMedPubMedCentralGoogle Scholar
  2. Ali H, Houghton PJ, Soumyanath A (2006) α-Amylase inhibitory activity of some Malaysian plants used to treat diabetes; with particular reference to Phyllanthus amarus. J Ethnopharmacol 107:449–455CrossRefPubMedGoogle Scholar
  3. Alonso H, Bliznyuk AA, Gready JE (2006) Combining docking and molecular dynamic simulations in drug design. Med Res Rev 26:531–568CrossRefPubMedGoogle Scholar
  4. Amakura Y, Okada M, Tsuji S, Tonogai Y (2000) High-performance liquid chromatographic determination with photodiode array detection of ellagic acid in fresh and processed fruits. J Chromatogr A 896:87–93CrossRefPubMedGoogle Scholar
  5. Altürk S (2015) Synthesis, spectroscopic characterization, second and third-order nonlinear optical properties, and DFT calculations of a novel Mn (II) complex J Organomet Chem 797:110–119CrossRefGoogle Scholar
  6. Azhari SJ, Mlahi MR, Mostafa MM (2015) Comparative studies between 4-allyl-, 4-phenyl-and 4-ethyl-1-(2-hydroxybenzoyl) thiosemicarbazides and the synthesis, characterization and DFT calculations of binary and ternary complexes derived from 4-ethyl (L 1) and 2, 2’-dipyridyl. Spectrochim Acta Part A 150:949–958CrossRefGoogle Scholar
  7. Baker J, Kessi A, Delley B (1996) The generation and use of delocalized internal coordinates in geometry optimization. J Chem Phys 105:192–212CrossRefGoogle Scholar
  8. Bernfeld P (1955) Amylases, alpha and beta. Methods Enzymol 1:149–158CrossRefGoogle Scholar
  9. Brayer GD, Luo Y, Withers SG (1995) The structure of human pancreatic α-amylase at 1.8 Å resolution and comparisons with related enzymes. Protein Sci 4:1730–1742CrossRefPubMedPubMedCentralGoogle Scholar
  10. Brayer GD, Sidhu G, Maurus R, Rydberg EH, Braun C, Wang Y, Nguyen NT, Overall CM, Withers SG (2000) Subsite mapping of the human pancreatic α-amylase active site through structural, kinetic, and mutagenesis techniques. Biochemistry 39:4778–4791CrossRefPubMedGoogle Scholar
  11. Chakrabarti R, Rajagopalan R (2002) Diabetes and insulin resistance associated disorders: disease and the therapy. Curr Sci 83:1533–1538Google Scholar
  12. Corbeil CR, Williams CI, Labute P (2012) Variability in docking success rates due to dataset preparation. J Comput-Aided Mol Des 26:775–786CrossRefPubMedPubMedCentralGoogle Scholar
  13. Cornish-Bowden A (1974) A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors. Biochem J 137:143–144CrossRefPubMedPubMedCentralGoogle Scholar
  14. Deenmamode JM, Sherwood RA, Sherman DI, Peters TJ (1993) Total pancreatic and salivary serum is α-amylase activities in alcohol misusers in relapse and remission and in alcoholic liver disease. Clin Chim Acta 223:169–172CrossRefPubMedGoogle Scholar
  15. Delley B (1990) An all-electron numerical method for solving the local density functional for polyatomic molecules. J Chem Phys 92:508–517CrossRefGoogle Scholar
  16. Delley B (1991) Analytic energy derivatives in the numerical local-density-functional approach. J Chem Phys 94:7245–7250CrossRefGoogle Scholar
  17. Delley B, Ellis DE (1982) Efficient and accurate expansion methods for molecules in local density models. J Chem Phys 76:1949–1960CrossRefGoogle Scholar
  18. Doig AJ, Williams DH, Oelrichs PB, Baczynskyj L (1990) Isolation and structure elucidation of punicalagin, a toxic hydrolysable tannin, from Terminalia oblongata. J Chem Soc 8:2317–2321Google Scholar
  19. Greenberger NJ, Toskes PP (1991) Harrison’s principles of internal medicine. McGraw-Hill, New York, NYGoogle Scholar
  20. Gyémánt G, Kandra L, Nagy V, Somsák L (2003) Inhibition of human salivary α-amylase by glucopyranosylidene-spiro-thiohydantoin. Biochem Biophys Res Commun 312:334–339CrossRefPubMedGoogle Scholar
  21. Hengesh EJ (1995) Principles of medical chemistry. Williams & Wilkins, BaltimoreGoogle Scholar
  22. Iulek J, Franco OL, Silva M, Slivinski CT, Bloch C, Rigden DJ, de SáInt MFG (2000) Purification, biochemical characterisation and partial primary structure of a new α-amylase inhibitor from Secale cereale (rye). Int J Biochem Cell Biol 32:1195–1204CrossRefPubMedGoogle Scholar
  23. Lansky EP, Newman RA (2007) Punica granatum (pomegranate) and its potential for prevention and treatment of inflammation and cancer. J Ethnopharmacol 109:177–206CrossRefPubMedGoogle Scholar
  24. Lineweaver H, Burk D, Deming WE (1934) The dissociation constant of nitrogen-nitrogenase in Azotobacter. J Am Chem Soc 56:658–666CrossRefGoogle Scholar
  25. Lo Piparo E, Scheib H, Frei N, Williamson G, Grigorov M, Chou CJ (2008) Flavonoids for controlling starch digestion: structural requirements for inhibiting human α-amylase. J Med Chem 51:3555–3561CrossRefPubMedGoogle Scholar
  26. Kandra L, Gyémánt G (2004) Inhibitory effects of tannin on human salivary α-amylase Biochem Biophys Res Commun 319:1265–1271CrossRefPubMedGoogle Scholar
  27. Kordik CP, Reitz AB (1999) Pharmacological treatment of obesity: therapeutic strategies. J Med Chem 42:181–201CrossRefPubMedGoogle Scholar
  28. Kwon YI, Apostolidis E, Shetty K (2008) In vitro studies of eggplant (Solanum melongena) phenolics as inhibitors of key enzymes relevant for type 2 diabetes and hypertension. Bioresour Technol 99:2981–2988CrossRefPubMedGoogle Scholar
  29. Maurus R, Begum A, Kuo HH, Racaza A, Numao S, Andersen C, Tams JW, Vind J, Overall CM, Withers SG, Brayer GD (2005) Structural and mechanistic studies of chloride induced activation of human pancreatic α-amylase. Protein Sci 14:743–755CrossRefPubMedPubMedCentralGoogle Scholar
  30. Maurus R, Begum A, Williams LK, Fredriksen JR, Zhang R, Withers SG, Brayer GD (2008) Alternative catalytic anions differentially modulate human α-amylase activity and specificity. Biochemistry 47:3332–3344CrossRefPubMedGoogle Scholar
  31. Mirsaeedghazi H, Mousavi SM, Emam-Djomeh Z, Rezaei K, Aroujalian A, Navidbakhsh M (2012) Comparison between ultrafiltration and microfiltration in the clarification of pomegranate juice. J Food Process Eng 35:424–436CrossRefGoogle Scholar
  32. Mishra PJ, Ragunath C, Ramasubbu N (2002) The mechanism of salivary amylase hydrolysis: role of residues at subsite S2’. Biochem Biophy Res Commun 292:468–473CrossRefGoogle Scholar
  33. Na M, Kim BY, Osada H, Ahn JS (2009) Inhibition of protein tyrosine phosphatase 1B by lupeol and lupenone isolated from Sorbus commixta. J Enzym Inhib Med Chem 24:1056–1059CrossRefGoogle Scholar
  34. Nauntofte B, Tenevuo JO, Lagerlöf F, Fejerskov O, Kidd E (2003) Dental caries: the disease and its clinical management. Blackwell Munksgard, OxfordGoogle Scholar
  35. Numao S, Maurus R, Sidhu G, Wang Y, Overall CM, Brayer GD, Withers SG (2002) Probing the role of the chloride ion in the mechanism of human pancreatic α-amylase. Biochemistry 41:215–225CrossRefPubMedGoogle Scholar
  36. Parmar HS, Kar A (2007) Antidiabetic potential of Citrus sinensis and Punica granatumpeel extracts in alloxan treated male mice. Biofactors 31:17–24CrossRefPubMedGoogle Scholar
  37. Pinto GP, Bras NF, Perez MA, Fernandes PA, Russo N, Ramos MJ, Toscano M (2015) Establishing the catalytic mechanism of human pancreatic α-amylase with QM/MM methods. J Chem Theory Comput 11:2508–2516CrossRefPubMedGoogle Scholar
  38. Ramasubbu N, Paloth V, Luo Y, Brayer GD, Levine MJ (1996) Structure of human salivary α-amylase at 1.6 Å resolution: implications for its role in the oral cavity. Acta Crystallogr Sect D 52:435–446CrossRefGoogle Scholar
  39. Ramasubbu N, Ragunath C, Mishra PJ (2003) Probing the role of a mobile loop in substrate binding and enzyme activity of human salivary amylase. J Mol Biol 325:1061–1076CrossRefPubMedGoogle Scholar
  40. Ramasubbu N, Ragunath C, Sundar K, Mishra PJ, Gyémánt G, Kandra L (2005) Structure-function relationships in human salivary α-amylase: role of aromatic residues. Biologia 60:47–56Google Scholar
  41. Seeram NP, Adams LS, Henning SM, Niu Y, Zhang Y, Nair MG, Heber D (2005) In vitro antiproliferative, apoptotic and antioxidant activities of punicalagin, ellagic acid and a total pomegranate tannin extract are enhanced in combination with other polyphenols as found in pomegranate juice. J Nutr Biochem 16:360–367CrossRefPubMedGoogle Scholar
  42. Selvan A, Seniya C, Chandrasekaran SN, Siddharth N, Anishetty S, Pennathur G (2010) Molecular dynamics simulations of human and dog gastric lipases: insights into domain movements. FEBS Lett 584:4599–4605CrossRefPubMedGoogle Scholar
  43. Strelow J, Dewe W, Iversen PW, Brooks HB, Radding JA, McGee J, Weidner J (2012) Mechanism of action assays for enzymes. Assay Guidance ManualGoogle Scholar
  44. Tadera K, Minami Y, Takamatsu K, Matsuoka T (2006) Inhibition of alpha-glucosidase and alpha-amylase by flavonoids. J Nutr Sci Vitaminol 52:149–143CrossRefPubMedGoogle Scholar
  45. Takahiro N, Yusuke N, Mitsuru E, Tatsuo Y, Michio O, Takesada M, Kenichi M (1986) Cloning, characterization and nucleotide sequences of two cDNAs encoding human pancreatic trypsinogens. Gene 41:299–304CrossRefGoogle Scholar
  46. Trott O, Olson AJ (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31:455–461PubMedPubMedCentralGoogle Scholar
  47. Van Elswijk DA, Schobel UP, Lansky EP, Irth H, van der Greef J (2004) Rapid dereplication of estrogenic compounds in pomegranate (Punica granatum) using on-line biochemical detection coupled to mass spectrometry. Phytochemistry 65:233–241CrossRefPubMedGoogle Scholar
  48. Vaya J, Mahmood S, Goldblum A, Aviram M, Volkova N, Shaalan A, Musa R, Tamir S (2003) Inhibition of LDL oxidation by flavonoids in relation to their structure and calculated enthalpy. Phytochemistry 62:89–99CrossRefPubMedGoogle Scholar
  49. Woolnough JW, Bird AR, Monro JA, Brennan CS (2010) The effect of a brief salivary alpha-amylase exposure during chewing on subsequent in vitro starch digestion curve profiles. Int J Mol Sci 11:2780–2790CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Tianjin University of Traditional Chinese MedicineTianjinChina
  2. 2.School of Pharmaceutical Science and TechnologyTianjin UniversityTianjinChina
  3. 3.Faculty of General EducationGuangdong Baiyun UniversityGuangzhouChina
  4. 4.Department of PhysicsTianjin Normal UniversityTianjinChina

Personalised recommendations