Food Chemistry 131 (2012) 141148

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Food Chemistry
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Purication 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 b

Chemistry Group, Animal Science Division, Agharkar Research Institute, Pune 411004, India Proteomics Facility, Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008, India

a r t i c l e

i n f o

a b s t r a c t
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 buffer. The isolated protein (TCP-III) obtained from fruit was puried by gel chromatography and preparative HPLC, showed apparent molecular weight of 16 kDa by SDSPAGE and MALDI-TOF/MS analyses. Amino acid sequence obtained by LCMSE 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 signicant radical scavenging in DPPH, NO, H2O2 and ABTS assays. In addition, TCP-III inhibited oxidation of linoleic acid in b-carotene bleaching assay, reduced ferric ions and chelated ferrous ions. The present nding demonstrates uniquely, for the rst time, characterization of an antioxidant protein from T. chebula fruit. 2011 Elsevier Ltd. All rights reserved.

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 Purication Characterization 16 kDa protein molecule of T. chebula Antioxidant and free radical scavenging activity

1. Introduction Oxidative stress in the living organism involves excessive production of reactive oxygen species (ROS) which plays a signicant role in induction of many diseases such as cancer, ageing, atherosclerosis, 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 generation 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 removal of either superoxide or hydroperoxide. Under the conditions

Abbreviations: AA, ascorbic acid; ABTS, 2,20 -azino-bis(3-ethylbenzthiazoline-6sulphonic acid) diammonium salt; APC, antioxidant protein from curry leaves; BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene; DPPH, 2,2-diphenyl-1picrylhydrazyl; 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). 0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.08.048

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 hydroxyanisole (BHA) and butylated hydroxyltoluene (BHT) is their toxicity at high doses, (Watts, 1975) which curbs their therapeutic usage. Several 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 wellknown 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 benecial 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

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Pharmacopoeia under the category astringent. The medicinal properties of T. chebula have been amply documented in the ancient Indian literature (Chattopadhyay & Bhattacharyya, 2007). It is used to treat digestive tract diseases, urinary diseases, heart diseases, parasitic infections, fever, atulence and constipation. The fruit is an important constituent of triphala formulation, which is a composite mixture of T. chebula, T. bellerica and Emblica ofcinalis. Antioxidant properties of aqueous (Lee et al., 2005), ethanolic (Subramaniyan, Chennam, & Devi, 2005) and methanolic (Walia, Kumar, & Arora, 2007) extracts of T. chebula are reported in literature but there is no report of an antioxidant protein from T. chebula fruit till the date. Therefore, a logical search was conducted to isolate and characterize the antioxidant protein (TCPIII) from T. chebula fruit. The antioxidant properties of TCP-III were compared with standard antioxidants such as BHA, BHT and ascorbic acid. 2. Materials and methods 2.1. Plant material Fruit of T. chebula were obtained from the market and authenticated by Botany Group of Agharkar Research Institute, Pune, India. Shade dried fruit were powdered using an electric grinder. 2.2. Chemicals/materials 2,2-Azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt (ABTS), bisacrylamide, 2,2-diphenyl-1-picrylhydrazyl (DPPH), DEAE Sepharose, Ellmans reagent, ethylenediaminetetraacetic acid (EDTA), ferrozine, Griess reagent, linoleic acid, polyvinylpyrrolidone, Sephadex G-100, N,N,N0 ,N0 -tetramethylene diamine (TEMED) and Tween-20 were procured from Fluka-Sigma Aldrich (USA). Ascorbic acid, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), FolinCoicalteau reagent, sodium nitroprusside, thiobarbituric acid (TBA) and trichloroacetic acid (TCA) were procured from Molychem, India. All other chemicals unless otherwise mentioned were procured from Merck (Germany). Schimadzu UV-2501 PC spectrophotometer (Tokyo, Japan) was used for colorimetric analyses. Centrifuge (Kubota 7780, Japan), Freeze Drier, (Modulyod-230), Analytical, Preparative HPLC (Waters, USA) and Voyager- De-STR (Applied Biosystems, USA) MALDI-TOF/MS, LCMSE (Waters, USA) were used for centrifugation, lyophilization, purication, MS and amino acid sequence analysis respectively. 2.3. Purication of the protein Hirda fruit powder (40 g) was suspended in 200 ml of 20 mM citrate phosphate buffer of pH 6.8 and 800 mg of polyvinylpyrrolidone was added to remove polyphenols. The mixture was kept at 4 C for 24 h. The homogenate was ltered through Whatman lter paper No.1 and centrifuged at 10,000 rpm at 4 C for 15 min. The protein quantication of the supernatant (350 lg/ml, BSA equivalent) was carried out using Bradfords method (Bradford, 1976) and analyzed by analytical HPLC. The pellet obtained after treatment with 40% ammonium sulfate was dissolved in 40 ml 0.1 M NaCl. It was loaded on Sephadex G-100 column (3 50 cm) preequilibrated with 0.1 M NaCl and eluted in same solution. The fractions were collected at 280 nm using BIO-RAD Econo UV detector. The pooled fractions were mixed and lyophilized up to 40 ml. It was puried by gel chromatography using DEAE Sepharose (3 30 cm) column pre-equilibrated with 0.01 M TrisHCl buffer of pH 6.0. Elution was carried out using stepwise gradient of NaCl (0.1, 0.2, 0.3, 0.4, 0.5 and 1 M). The pooled fractions were analyzed

by analytical HPLC with UV detector at 280 nm and further puried by preparative HPLC using X Bridge C18, 5 lm, 4.6 100 mm, reverse phase column. The column was eluted with water: acetonitrile (95:5) containing 0.01% TFA and the ow rate was maintained at 1.5 ml/min. The peaks were resolved at 280 nm. The isolated proteins designated as T. chebula proteins, TCP-I, TCP-II and TCP-III. Crude buffer extract, ammonium sulfate precipitate, fraction obtained from gel chromatography and pure proteins (1 ml each) were tested for antioxidant activity by DPPH method. 2.4. Characterization of the protein 2.4.1. Test for homogeneity The homogeneity and molecular weight of proteins were assessed according to the method of Laemmli (1970). SDSPAGE was performed with 15% resolving and 4% stacking gels. A set of molecular weight marker proteins (6205 kDa) was run in the gel to determine molecular weight of the proteins. 2.4.2. MALDI/MS analysis Mass spectral analysis was performed on a Voyager-De-STR (Applied Biosystems, USA) MALDI-TOF/MS. A nitrogen laser (337 nm) was used for desorption and ionization. A spectrum was acquired in the range of 10100 kDa, in linear mode with delayed ion extraction and with an accelerating voltage of 25 kV. The low mass ion gate was set at 4500 Da. All the analyses were performed in four replications. The instrument was calibrated with myoglobulin and bovine serum albumin. 2.4.3. Nano LCMSE analysis Proteins puried by preparative HPLC were identied by nano-LCMSE approach using nanoAcquity-Synapt-HDMS system (Waters Corporation, Milford, MA, USA). Protein eluted was reduced and alkylated by using DDT and iodoacetamide respectively, and digested with trypsin. Peptides (4 ll) were loaded on the nano-LC. Peptides were trapped on a 5 lm symmetry C18 column (180 lm 20 mm) and washed for 3 min at 5 ll/ min with mobile phase A (0.1% formic acid). Peptides were then separated and eluted for MS analysis using a 60 min reverse phase gradient at 400 nl/min (550% ACN over 35 min) on a BEH 130 C18 1.7 lM 100 lM 100 mm nanoAcquity UPLC column. The column temperature was set at 35 C. The reference ([Glu1]-brinopeptide B, 500 fmol/ml) was constantly infused by the nanoAcquity auxiliary pump at a constant ow rate of 500 nl/min at interval of 20 s. The eluted peptides spectra were acquired by Synapt-HDMS (Q-TOF) with following parameters. Sample was analyzed in positive V mode in a mass range of 502000 m/z with a scan time of 1.5 s. The on-line eluted peptides were analyzed at both low collision energy (4 eV) and high collision energy (1535 eV). LCMSE data were processed with ProteinLynx GlobalServer v2.4 (Waters Corporation, Milford, MA, USA) and searched against with database of owering plants (UniProtKB) for protein identication. 2.4.4. Estimations Protein content at various stages of purication was determined by Bradfords (1976) method. Ninhydrin test was done with the method described by Sadashivam and Manickam (1997a) and sulphydryl group was estimated by Ellmans (1959) method. The presence of fatty acid derivatives was estimated by Folch lipid extraction method and analyzed by thin layer chromatography (Folch, Less, & Sloane-Stanley, 1957). Total sugar was estimated by phenolsulfuric acid method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956) and total phenolic content was determined by FolinCoicalteau reagent (Kujala, Loponen, Klika, & Pihlaja, 2000).

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The chlorophyll content was estimated according to the method described by Sadashivam and Manickam (1997b). 2.4.5. Effect of temperature on antioxidant activity of the protein TCP-III (100 lg) was incubated at 45 C, 60 C and 95 C for 20 min in 1 ml of TrisHCl buffer (20 mM, pH 7.4). Aliquots of samples were tested for antioxidant activity by DPPH method. 2.4.6. Effect of protease on antioxidant activity of the protein TCP-III (500 lg) was incubated at 37 C for 1 h with 20 lg of proteinase K from Staphylococcus aureus (S) in 20 mM TrisHCl buffer of pH 6.8. The reaction was quenched by placing the tubes in ice. Aliquots of samples were then tested for their antioxidant activity using DPPH assay.

2.5. Antioxidant activity (DPPH assay) DPPH radical scavenging activity was measured according to the method of Shimada, Fujikawa, Yahara, and Nakamura (1992). Protein fractions (TCP-III, 100 ll) of various concentrations ranging from 6 to 30 lM in deionised water were mixed with 2 ml of 0.05 M acetate buffer, 1.9 ml methanol and 1 ml of 0.5 mM solution of DPPH in methanol. Blank contained 100 ll of the protein fractions of different concentrations without DPPH and control was without test sample. The mixture was shaken immediately after addition of DPPH and allowed to stand at room temperature in dark and decrease in absorbance at 517 nm was measured at a 0 min and after every 30 min until the reaction reached a plateau. BHA (3 mM) was used as a standard. Samples were analyzed in

Fig. 1. Purication and characterization of T. chebula fruit protein (a) HPLC chromatogram of crude aqueous extract of T. chebula fruit. (b) HPLC chromatogram of puried TCPIII. (c) SDSPAGE of puried antioxidant protein from Hirda fruit. Lane 1, puried TCP-I; Lane 2, puried antioxidant protein (TCP-III, 16 kDa). Lane 3, molecular weight marker, Lane 4, crude aqueous extract of Hirda fruit; (d) chromatogram of puried protein (TCP-III) by MALDI/MS analysis (e) effect of heat and protease treatment on antioxidant activity of TCP-III.

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triplicates. The inhibitory percentage of DPPH was calculated as per the formula: % Scavenging effect = [(A0 (A Ab))/A0] 100; where, A0 = absorbance of control; A = absorbance of sample and Ab = absorbance of blank. 2.6. ABTS radical cation decolourisation assay ABTS assay (Re et al., 1999) was used to evaluate the scavenging ability of test sample. ABTS radical cations were generated by reacting ABTS solution (7 mM, 3 ml) with ammonium persulphate (2.45 mM, 15 ml) in deionised water. The mixture was allowed to stand in the dark at room temperature for 16 h before use. The test solutions (630 lM) in deionised water, standard solutions of BHA (3 mM) and BHT (3 mM) in methanol were prepared. Test sample (500 ll) was added to 300 ll ABTS solution and nal volume was adjusted to 1 ml with methanol. Control was prepared by adding methanol (700 ll) to ABTS solution (300 ll) while blank was without ABTS solution. All the samples were analyzed in triplicates. Absorbance was read at 745 nm and the percentage inhibition was calculated using the formula: % Inhibition = [(A0 (A Ab))/ A0] 100; where, A0 = absorbance of control; A = absorbance of test solution and Ab = absorbance of blank. 2.7. Nitric oxide scavenging assay Nitric oxide radical scavenging activity was performed as per the procedure reported by Jain and Agrawal (2008). Test solutions (50 ll) of various concentrations (630 lM) and 50 ll of 10 mM sodium nitroprusside in 0.5 ml phosphate buffer saline (20 mM, pH 7.4) were mixed and incubated at 25 C for 150 min. Following the incubation, 125 ll of Griess reagent was added and incubated further for 10 min at room temperature. The same reaction mixture without test sample served as control and blank was without Griess reagent. Ascorbic acid (2 mM) was used as standard and samples were run in triplicates. The colour developed was measured at 546 nm. The scavenging effect was measured using the formula: % Scavenging activity = [(Ac As)/Ac] 100; where, Ac = absorbance of control and As=absorbance of sample/standard. 2.8. Deoxyribose degradation assay The scavenging of hydroxyl radical was measured by employing the method of Halliwell, Gutteridge, and Aruoma (1987) with minor modications. Stock solutions of EDTA (1 mM), FeCl3 (10 mM), ascorbic acid (1 mM), H2O2 (10 mM) and deoxyribose (10 mM) were prepared in deionised water. Reaction mixture was prepared by adding 0.05 ml EDTA, 0.005 ml FeCl3, 0.05 ml H2O2, 0.18 ml deoxyribose, 500 ll TCP-III protein of concentrations varying from 6 to 30 lM, 165 ll phosphate buffer (50 mM, pH 7.4) and 50 ll ascorbic acid. The mixture was incubated at 37 C for 1 h. The incubated mixture (250 ll) was mixed with 250 ll 10% TCA, 250 ll 2% TBA in 0.025 M NaOH and heated at 80 C for 1 h to develop a pink chromogen, which was measured at 532 nm. BHT (3 mM) was used as a standard. The control was without the test sample and the blank contained deoxyribose and buffer. The inhibition effect on hydroxyl radicals was calculated as follows: % Hydroxyl radical scavenging capacity = (1 As/Ac) 100, where Ac = absorbance of control and As = absorbance of sample/ standard. 2.9. b-Carotene bleaching assay Antioxidant activity was measured using the method of Jayaprakasha, Singh, and Sakariah (2001) with minor modications. b-Carotene (3.34 mg) in chloroform (1 ml) was mixed with linoleic acid (40 mg) and Tween-20 (400 mg). The chloroform layer was

removed at 40 C under vacuum. The resulting mixture was immediately diluted up to 100 ml with 0.01 M H2O2. Aliquots (2 ml) of this emulsion were transferred into different test tubes containing 0.1 ml test sample of different concentrations (630 lM) in deionised water. BHA (3 mM) was used as a standard. Control contained 0.2 ml methanol and 4 ml of the above emulsion and the blank was prepared as mentioned previously, but without b-carotene linoleic acid emulsion. The tubes were placed in a water bath at 50 C. Absorbance of all the samples at 470 nm was measured at zero time and after every 20 min until the disappearance of the colour of b-carotene in the control. Antioxidant activity was measured by following formula: Antioxidant activity 1 A0 At =A A 0 t 100, where A0 and A0 are the absorbance values measured at initial incubation time for sample/standard and control respectively. At and A are the absorbance values at different time intervals for the t sample/standard and control, respectively.

2.10. Ferric ion reducing capacity The reducing power was determined according to the method of Wang, Yen, Ling, and Wu (2003) with minor modications. Potassium ferricyanide solution (100 ll, 4 mM) in deionised water was mixed with 200 ll phosphate buffer (20 mM, pH 6.5) and test sample (100 ll) of different concentrations (630 lM). The contents were incubated at 50 C for 20 min. TCA (200 ll, 10%) was added to the reaction mixture and centrifuged at 5000 rpm. The resulting supernatant was mixed with 100 ll ferric chloride solution (2 mM) and the nal volume was made up to 1 ml with water and then incubated at ambient temperature for 10 min. Blank was without test sample. BHA (3 mM) and ascorbic acid (2 mM) served as positive controls. The absorbance was recorded at 700 nm.

2.11. Ferrous ion chelating activity Ferrous ion chelating activity of the protein was measured according to the procedure described by Decker and Welch (1990). TCP-III protein (100 ll) of different concentrations (6 30 lM) was mixed with 370 ll deionised water and was reacted with 10 ll ferrous chloride (2 mM) and 20 ll ferrozine (5 mM). Blank was without ferrous chloride and ferrozine. The reaction mixture was kept at room temperature for 20 min and absorbance at 562 nm was recorded along with EDTA as a positive control. Chelating activity of TCP-III on ferrous ions was calculated as per the formula: % Chelation = [(Ac As)/Ac] 100; where Ac = absorbance of control without test sample; As = absorbance of standard/sample.

Table 1 Summary of purication of antioxidant protein (TCP-III) from Hirda fruit. Purication steps Total protein (mg) 70 5 55 2 35 3 20 4
a

Yield (%) 100 78 3 50 4 28 3

Protein content (lg/ ml) 350 200 200 400

DPPH assay % Inhibition 55 3 68 4 71 5 90 5

Buffer extract 40% Ammonium sulfate precipitation Gel ltration on Sephadex G-100 Gel ltration using DEAE Sepharose (peak III)

Antioxidant activity of protein from Hirda fruit at the concentrations ranging from 200500 lg/ml was determined using DPPH assay, as described in methods. The results are shown as means SD (n = 3). a Data refer to the protein obtained during various stages of purication from 40 g of dried Hirda fruit.

P. Srivastava et al. / Food Chemistry 131 (2012) 141148 Table 2 LCMS sequencing of TCP-III protein from T. chebula fruit and its alignment with plant database search (UniProtKB). S. no. 1 Protein ID P82537 Protein Thylakoid lumenal 17 kDa protein fragment OS Spinacia oleracea PE 1 SV 1 Predicted protein fragment OS Populus trichocarpa GN POPTRDRAFT 726662 PE 4 SV 1 PLGS score 168.3923 Peptides 3 Theoretical peptides 4 Coverage (%) 30 T. chebula protein (TCP-III) sequence with m/z value (K)GFLPVIDKK(D) (1016.623) (L)PVIDKK(D) (699.437) (K)GFLPVIDK(K) (870.58) (K)FITLVGR(G) (805) (R)LWLSLLALLCLLSFALFGLK(F) (2291.38) (K)FITL(V) (475.2912) (S)FALFGLK(F) (795.4882) (K)LPNRELILEAK(A) (1295.76) (R)ALALALAHLATRR(T) (1376.85) (L)ILEAK(A) (573.35) (N)RELILEAK(A) (971.57)

145

B9HVA7

165.8836

11

25

A2XW92

Putative uncharacterized protein OS Oryza sativa subsp. indica GN OsI 16918 PE 4 SV 1

162.6417

19

11

a
% Effectiveness

100 80 60 40 20 0

b
% Effectiveness

102 100 98 96 94 92 90 6 12 18 24 30 BHA BHT

12

18

24

30

BHA

Concentration (M)

Concentration ( M)

d
% Inhibition

70 60 50 40 30 20 10 0

c 100
% Inhibition
80 60 40 20 0

12

18

24

30

AA

12

18

24

30

BHT

Concentration (M)

Concentration (M)

Fig. 2. Free radical scavenging activity. (a) DPPH radical scavenging activity of TCP-III (630 lM) and BHA (3 mM). The results are shown as means SD (n = 3) and q < 0.05. (b) ABTS radical cation scavenging activity of TCP-III (630 lM), BHA (3 mM) and BHT (3 mM). The results are shown as means SD (n = 3) and q < 0.01. (c) Nitric oxide scavenging activity of TCP-III (630 lM) and AA (2 mM). The results are shown as means SD (n = 3) and q < 0.05. (d) Deoxyribose degradation assay of TCP-III (630 lM) and BHT (3 mM). The results are shown as means SD (n = 3) and q < 0.02.

2.12. Statistical analysis Statistical analysis was done in MS Excel (Microsoft Windows XP) using one sided Students t-test. All results refer to means SD and q < 0.05 was considered as statistically signicant as compared to relevant controls. 3. Results 3.1. Purication of antioxidant protein

protein by preparative HPLC resulted in three major peaks. The purity of peaks was conrmed by analytical HPLC (Fig. 1b) and the peak which eluted at 2.154 min was designated as TCP-III. The protein content of the peaks was estimated by Bradfords method. Table 1 shows antioxidant activity of crude extract, fractions from Sephadex column and peak III using DPPH radical scavenging assay. Crude extract showed 55% antioxidant activity. Peak III exhibited highest antioxidant activity (90%), peak II showed 26% activity whereas peak I did not show any activity. 3.2. Characterization of antioxidant protein (TCP-III) from Hirda fruit

The homogenate of Hirda fruit obtained in the citrate phosphate buffer after centrifugation, showed 55% antioxidant activity by DPPH radical scavenging assay. HPLC analysis of the homogenate showed three major peaks along with some minor peaks (Fig. 1a). The fraction precipitating at 40% ammonium sulfate saturation showed 68% antioxidant activity. Purication of the crude

The protein purity, conrmed by reverse phase HPLC showed a single peak (Fig. 1b). Puried antioxidant protein (TCP-III) from Hirda fruit showed approximate molecular weight of 16 kDa by SDSPAGE (Fig. 1c) and an exact molecular mass of 16.267 kDa by MALDI-TOF/MS analysis (Fig. 1d).

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0.05

Absorbance at 470 nm

0.04

TCP III
0.03 0.02 0.01 0 0 20 40 60 80 100

scavenging activity of TCP-III at 30 lM concentration was 90% at 0 min which was more than the activity shown by standard BHA (3 mM) at zero and 30 min also. Analysis by Students t-test showed q < 0.05, which indicated that results are signicant. 3.3.2. ABTS radical cation decolourisation assay A comparison of % inhibition of ABTS radical cation versus concentration of TCP-III is shown in Fig. 2b. It is clearly demonstrated from the gure that TCP-III showed signicant radical cation scavenging activity at all concentrations (630 lM), and it was always higher than both the standards, BHA (3 mM) and BHT (3 mM). The results of Students t-test showed q < 0.01, which indicated that readings are signicant. 3.3.3. Nitric oxide scavenging assay Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates NO radicals, which interact with oxygen to produce nitrite ions that can be determined with Griess reagent. TCP-III protein showed concentration dependent moderate nitric oxide scavenging activity and maximum scavenging effect (52%) were achieved at 30 lM concentration as shown in Fig. 2c. The results of Students t-test showed q < 0.05 indicated that readings are signicant. 3.3.4. Deoxyribose degradation assay TCP-III protein scavenged hydroxyl radicals in dose dependent manner (Fig. 2d) and maximum activity (65%) was found at 30 lM concentration while BHT (3 mM) scavenged 54% hydroxyl radicals. The activity shown by TCP-III is superior to standard BHT. The analysis by Students t-test showed q < 0.02 indicated that results are signicant. 3.3.5. b-Carotene linoleic acid assay Fig. 3 represents the graph of absorbance of TCP-III, BHA and control versus time at 470 nm. In the case of the control, there was a fast decrease in absorbance while for BHA the absorbance was almost constant up to 100 min. TCP-III showed a similar pattern but there was a slight decrease in absorbance in comparison to BHA. Oxidation of linoleic acid was signicantly inhibited by TCP-III, but it was less effective when compared to BHA. The results of Students t-test showed q < 0.01 indicated that readings are signicant. 3.3.6. Ferric ion reducing capacity In this assay, an increase in absorbance indicates high reducing power of the test sample. Reducing capacity of TCP-III increased with increase in concentration (630 lM), which was measured by absorbance at 700 nm. As shown in Fig. 4a, reducing power of TCP-III at all concentrations was found to be much better than

BHA Control

Time (min)
Fig. 3. Graph of absorbance at 470 nm versus time showing inhibition of linoleic acid oxidation by TCP-III (630 lM) and BHA (3 mM) in b-carotene bleaching assay. The results are shown as means SD (n = 3).

Protein (TCP-III) eluted from preparative HPLC was further characterized by LCMSE analysis. The data obtained from LC MSE analysis was searched against the database of proteins from all owering plants (UniProtKB), as there is no sequenced genome available for T. chebula. LCMSE analysis led to identication of 11 peptide sequences. Amongst them four sequences showed homology to predicted protein fragment from Populus trichocarpa, four sequences showed homology with putative uncharacterized protein from Oryza sativa indica and three sequences showed homology with 17 kDa Thylakoid lumenal protein from Spinacia oleracea. Data is shown in Table 2. Puried protein (TCP-III) showed susceptibility to heat and proteolytic enzyme (Fig. 1e). Untreated TCP-III (18 lM) showed 80% antioxidant activity, while preheat treatment of TCP-III (18 lM) at 45 C, 65 C and 95 C decreased the antioxidant activity to 60%, 35% and 8% respectively, indicating that antioxidant protein is destabilized above 45 C. The biochemical tests proved that TCP-III is devoid of total sugars, fatty acid derivatives, polyphenols and chlorophyll. TCP-III showed positive test for sulphydryl group and for ninhydrin indicating that antioxidant principle is a protein (TCP-III). 3.3. Antioxidant properties of TCP-III 3.3.1. DPPH radical scavenging activity DPPH scavenging activity of TCP-III increased with the increase in concentration from 6 to 30 lM at 0 min and 50% decrease in absorbance of DPPH was seen at 18 lM concentration at 0 min itself. As shown in Fig. 2a, TCP-III exhibited 42% activity at 6 lM and 90% activity at 24 lM concentration. The scavenging activity remained constant above 24 lM concentration. DPPH radical

a
Absorbance at 700 nm

0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0

b 120
100

% Chelation

80 60 40 20 0
EDTA TCP III

12

18

24

30

BHA

AA

12

18

24

30

Concentration (M)

Concentration ()

Fig. 4. (a) Ferric ion reducing power of TCP-III (630 lM), BHA (3 mM) and AA (2 mM). The results are shown as means SD (n = 3) and q < 0.01. (b) Ferrous ion chelating assay of TCP-III (630 lM); the results are shown as means SD (n = 3) and q < 0.05.

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BHA (3 mM) and ascorbic acid (2 mM). The results of Students ttest showed q < 0.01, which indicated that readings are signicant. 3.3.7. Ferrous ion chelating activity The chelation of Fe2+ ions by TCP-III protein was tested with ferrozine and the activity was monitored by reduction in absorption at 700 nm. TCP-III protein showed moderate ability of ferrous ion chelation and reached 15% at 30 lM concentration (Fig. 4b). The analysis of Students t-test showed q < 0.05 indicated that results are signicant. 4. Discussion The present study was carried out to isolate and characterize active antioxidant protein from the buffer extract of T. chebula fruit. The active protein was isolated and puried to homogeneity after gel chromatography and preparative HPLC. SDSPAGE and MALDI-TOF/MS analyses of protein demonstrated its approximate molecular weight of 16 kDa. Protease digestion of the protein (TCP-III) destroyed the antioxidant activity, which conrmed that the active principle is proteinaceous in nature. TCP-III contains amino acids as evidenced by ninhydrin test, and it was found to be devoid of sugars, polyphenols, lipids, fatty acid derivatives and chlorophyll which conrmed that the obtained active principle is protein. Ellmans test detected presence of sulphydryl groups in TCP-III. LCMS/MS analysis of TCP-III led to identication of 11 sequences (Table 2). Amongst the 11 sequences, four sequences showed homology to predicted protein fragments from P. trichocarpa, four sequences showed homology with putative uncharacterized protein from O. sativa indica and three sequences showed homology with 17 kDa thylakoid lumenal protein from S. oleracea. Peptide fragments obtained by LCMS analysis showed that TCP-III contained leucine, lysine, alanine and proline amino acids. Literature search revealed that antioxidant activity of some proteins is attributed to the presence of Leu- His-Pro amino acid sequence (Pea-Ramos, Xiong, & Arteaga, 2004). It has been reported that SH group acts as a free radical scavenger in plant and animal tissue and SH group of cysteine facilitates antioxidant activity of glutathione (Selvam & Devaraj, 1996). Presence of Ser, Thr and Cys amino acids having free hydroxyl and thiol groups in the peptide fragments of TCP-III, may also contribute to antioxidant potential of the protein (TCP-III) along with other unknown functional groups. Oxidative stress inducers could cause a signicant increase in the rate of formation of ROS, such as the superoxide anion radical, hydroxyl radical, NO radical and Hydrogen peroxide. These could also cause enhanced lipid peroxidation and oxidation of proteins as well as DNA. TCP-III effectively scavenges free radicals such as DPPH, ABTS, NO and H2O2 radicals at the lower concentrations than the standard BHA and BHT. These results offer scientic evidence for the use of Hirda fruit in the indigenous system. Along with free radical scavenging activity, TCP-III signicantly inhibited the oxidation of lenoleic acid in b-carotene bleaching assay. It also showed the tendency to reduce ferric ions and moderate ferrous ions binding capacity as demonstrated in the ferric ion reducing and ferrous ion chelating assays. The outcome of present study reveals that antioxidant protein from Hirda fruit exhibits multiple antioxidative properties. The mode of action of antioxidant protein TCP-III could probably be counteracting or quenching of reactive oxygen species, thereby reducing the potential of prooxidants to attack the cellular components. Therefore, effective dose of antioxidant protein can be useful for efcient prevention of oxidative stress. In conclusion, the 16 kDa protein molecule isolated from T. chebula fruit possesses potential antioxidant and free radical scavenging properties,

although the actual mechanism of its protective action is not clearly known. Further studies are necessary to fully characterize this active principle, both structurally and functionally, to arrive at a clear picture of its role as an antioxidant molecule and the work is in progress.

Acknowledgement The authors are thankful to Botany department, Agharkar Research Institute, Pune for authentication of plant material. Hema N. Raut and Renuka S. Wagh are thankful to Director, Agharkar Research Institute, Pune for providing infrastructure.

References
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 7, 248254. Chattopadhyay, R. R., & Bhattacharyya, S. K. (2007). Terminalia chebula: An update. Pharmacognosy Reviews, 1, 151156. Decker, E. A., & Welch, B. (1990). Role of ferritin as lipid oxidation catalyst in muscle food. Journal of Agricultural and Food Chemistry, 38, 674677. Droge, W. (2002). Free radicals in the physiological control of cell function. Physiological Reviews, 282, 4795. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugar and related substances. Analytical Chemistry, 28, 350356. Ellman, G. L. (1959). Tissue sulfhydryl groups. Archives of Biochemistry and Biophysics, 82, 7277. Finkel, T., & Holbrook, N. J. (2000). Oxidants, oxidative stress and biology of ageing. Nature, 408, 239247. Folch, J., Less, M., & Sloane-Stanley, G. H. (1957). A simple method for the isolation and purications of total lipids from animal tissues. Journal of Biological Chemistry, 226, 497509. Halliwell, B., Gutteridge, J. M. C., & Aruoma, O. I. (1987). The deoxyribose method: A simple assay for the determination of rate constants for reactions of hydroxyl radicals. Analytical Biochemistry, 165, 215219. Jain, P. K., & Agrawal, R. K. (2008). Antioxidant and free radical scavenging properties of developed mono- and polyherbal formulations. Asian Journal of Experimental Science, 22, 213220. Jayaprakasha, G. K., Singh, R. P., & Sakariah, K. K. (2001). Antioxidant activity of grape seed (Vitis vinifera) extracts on peroxidation models in vitro. Food Chemistry, 73, 285290. Kujala, T. S., Loponen, J. M., Klika, K. D., & Pihlaja, K. (2000). Phenolics and betacyanins in red beetroot (Beta vulgaris) root: Distribution and effect of cold storage on the content of total phenolics and three individual compounds. Journal of Agricultural and Food Chemistry, 48, 53385342. Laemmli, V. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680685. Lee, H. S., Won, N. H., Kim, K. H., Lee, H., Jun, W., & Lee, K. W. (2005). Antioxidant effects of aqueous extract of Terminalia chebula in vivo and in vitro. Biological & Pharmaceutical Bulletin, 28, 16391644. Ningappa, M. B., & Srinivas, L. (2008). Purication and characterization of 35 kDa antioxidant protein from curry leaves (Murraya koenigii L.). Toxicology in vitro, 22, 699709. Paiva, S. A., & Russell, R. M. (1999). Beta carotene and other carotenoids as antioxidants. Journal of the American College of Nutrition, 18, 426433. Paolini, M., Cantelli-Forti, G., Perocco, P., Pedulli, G. F., Abdel-Rahman, S. Z., & Legator, M. S. (1999). Co-carcinogenic effects of beta-carotene. Nature, 398, 760761. Pea-Ramos, E. A., Xiong, Y. L., & Arteaga, G. E. (2004). Fractionation and characterisation for antioxidant activity of hydrolysed whey protein. Journal of the Science of Food and Agriculture, 84, 19081918. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology & Medicine, 26, 12311237. Sadashivam, S., & Manickam, A. (1997a). Identication of proteins. Biochemical methods (2nd ed.). New Age International Publishers, p. 33. Sadashivam, S., & Manickam, A. (1997b). Estimation of chlorophyll. Biochemical methods (2nd ed.). New Age International Publishers, p. 190. Sarkar, M. K., Kinter, M., Mazumdar, B., & Sil, P. C. (2009). Purication and characterisation of a novel antioxidant protein molecule from Phyllanthus niruri. Food Chemistry, 114, 14051412. Selvam, R., & Devaraj, S. (1996). Oxalate binding to rat kidney mitochondria: Induction by oxidized glutathione. Indian Journal of Biochemistry and Biophysics, 33, 6265. Shimada, K., Fujikawa, K., Yahara, K., & Nakamura, T. (1992). Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. Journal of Agricultural and Food Chemistry, 40, 945948.

148

P. Srivastava et al. / Food Chemistry 131 (2012) 141148 Wang, L., Yen, J. H., Ling, H. L., & Wu, M. J. (2003). Antioxidant effect of methanol extracts from lotus plumule and blossom (Nelumbo nucifera Gertn.). Journal of Food and Drug Analysis, 11, 6066. Watts, P. (1975). BHA isomers and hyperplasia. Food and Chemical Toxicology, 23, 639640. Weber, W. M., Hunsaker, L. A., Abcouwer, S. F., Deck, L. M., & Vander Jagt, D. L. (2005). Anti-oxidant activities of curcumin and related enones. Bioorganic & Medicinal Chemistry, 13, 38113820.

Srinivas, L., Shalini, V. K., & Shylaja, M. (1992). Turmerin: A water-soluble antioxidant peptide from turmeric (Curcuma longa). Archives of Biochemistry and Biophysics, 292, 617623. Subramaniyan, S., Chennam, S., & Devi, S. (2005). Antioxidant activity of ethanolic extract of Terminalia chebula fruit against isoproterenol-induced oxidative stress in rats. Indian Journal Biochemistry and Biophysics, 42, 246249. Walia, H., Kumar, S., & Arora, S. (2007). Analysis of antioxidant activity of methanol extract/fractions of Terminalia chebula Retz. Journal of Chinese Clinical Medicine, 2, 361370.