Purification and characterization of an antioxidant protein (16 kDa) from Terminalia chebula fruit Pratibha Srivastava a,⇑ , Hema N. Raut a , Renuka S. Wagh a , Hemalata M. Puntambekar a , Mahesh J. Kulkarni b a Chemistry Group, Animal Science Division, Agharkar Research Institute, Pune 411004, India b Proteomics Facility, Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008, India article info Article history: Received 23 February 2011 Received in revised form 19 April 2011 Accepted 18 August 2011 Available online 25 August 2011 Keywords: Terminalia chebula fruit Purification Characterization 16 kDa protein molecule of T. chebula Antioxidant and free radical scavenging activity abstract Terminalia chebula fruit is used as folk medicine in India and Southeast Asia. An antioxidant protein was isolated by bioassay guided fractionation of T. chebula fruit by homogenizing in the citrate phosphate buf- fer. The isolated protein (TCP-III) obtained from fruit was purified by gel chromatography and preparative HPLC, showed apparent molecular weight of 16 kDa by SDS–PAGE and MALDI-TOF/MS analyses. Amino acid sequence obtained by LC–MS E analysis showed homology with the predicted protein fragments of Populus trichocarpa, putative uncharacterized protein fragments from Oryza sativa and with fragments of 17 kDa thylakoid lumenal protein from Spinacia oleracea. TCP-III exhibited significant radical scaveng- ing in DPPH, NO, H 2 O 2 and ABTS assays. In addition, TCP-III inhibited oxidation of linoleic acid in b-car- otene bleaching assay, reduced ferric ions and chelated ferrous ions. The present finding demonstrates uniquely, for the first time, characterization of an antioxidant protein from T. chebula fruit. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Oxidative stress in the living organism involves excessive pro- duction of reactive oxygen species (ROS) which plays a significant role in induction of many diseases such as cancer, ageing, athero- sclerosis, asthma and diabetes (Finkel & Holbrook, 2000). Exposure of the living organisms to ROS such as superoxide, hydroxyl radical and hydrogen peroxide is inevitable in cell metabolism as the gen- eration of ATP from molecular oxygen requires electrons (Droge, 2002). The potential targets for ROS in cells are membrane lipids, DNA and proteins. All organisms possess innate defense systems to protect them from free radical mediated oxidative damage. Host defense of living organism constitutes antioxidative enzymes like superoxide dismutase, catalase and peroxidase, which cause re- moval of either superoxide or hydroperoxide. Under the conditions of extreme oxidative stress, however, the host defense does not provide comprehensive protection from ROS, which could lead to oxidative damage related diseases. Antioxidant consumption might be helpful to prevent oxidative stress in the body. The main drawback of synthetic antioxidants such as butylated hydroxyani- sole (BHA) and butylated hydroxyltoluene (BHT) is their toxicity at high doses, (Watts, 1975) which curbs their therapeutic usage. Sev- eral foods derived antioxidants such as b-carotene (Paiva & Russell, 1999) and curcumin (Weber, Hunsaker, Abcouwer, Deck, & Vander Jagt, 2005) have been extensively used to counter the epidemic of oxidative damage induced diseases, but b-carotene imposes high risk of lung cancer in smokers (Paolini et al., 1999). Turmerin from Curcuma longa (Srinivas, Shalini, & Shylaja, 1992), PNP from Phyllanthus niruri (Sarkar, Kinter, Mazumdar, & Sil, 2009) and APC from Murraya koenigii (Ningappa & Srinivas, 2008) are few well- known antioxidative proteins from plants, but literature search indicates that there is still scarcity of dietary antioxidants derived from plant sources. In view of paucity of plant based proteins as an antioxidant, the search for a beneficial antioxidant led to isolation of an important protein from Hirda fruit. Terminalia chebula Retz. (Hirda) is a plant from Combretaceae family, commonly known as the king of medicine and is used in many ayurvedic preparations. The tree is abundant throughout India and Southeast Asia, especially in deciduous forests. Its yellowish-brown fruit is included in Indian 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.08.048 Abbreviations: AA, ascorbic acid; ABTS, 2,2 0 -azino-bis(3-ethylbenzthiazoline-6- sulphonic acid) diammonium salt; APC, antioxidant protein from curry leaves; BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene; DPPH, 2,2-diphenyl-1- picrylhydrazyl; NO, nitric oxide; PNP, Phyllanthus niruri protein; ROS, reactive oxygen species; TBA, thiobarbituric acid; TCA, trichloroacetic acid; TCP, Terminalia chebula protein. ⇑ Corresponding author. Address: Chemistry Group, Animal Science Division, Agharkar Research Institute, G.G. Agarkar Road, Pune 411004, Maharashtra, India. Tel.: +91 20 25653680x351; fax: +91 20 25651542. E-mail address: psri94@yahoo.com (P. Srivastava). Food Chemistry 131 (2012) 141–148 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem