Potential in vitro antioxidant and protective effects of Mesua ferrea Linn. bark extracts on induced oxidative damage

Industrial Crops and Products 47 (2013) 186–198

Contents lists available at SciVerse ScienceDirect

Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop

Potential in vitro antioxidant and protective effects of Mesua ferrea Linn. bark extracts on induced oxidative damage K.P. Rajesh a , H. Manjunatha a,∗ , V. Krishna a , B.E. Kumara Swamy b a Department of PG Studies and Research in Biotechnology and Bioinformatics, Jnanasahyadri, Kuvempu University, Shankaraghatta 577451, Shivamogga, Karnataka, India b Department of PG Studies and Research in Industrial Chemistry, Jnanasahyadri, Kuvempu University, Shankaraghatta 577451, Shivamogga, Karnataka, India

a r t i c l e

i n f o

Article history: Received 19 October 2012 Received in revised form 6 March 2013 Accepted 9 March 2013 Keywords: Mesua ferrea L. Ghost membrane Free iron Cyclic voltammetry In-gel heme staining Antioxidant

a b s t r a c t Free radicals and activated oxygen radical species are generated as a result of aerobic metabolism. Imbalance, if any in the antioxidant defense system against free radicals can degrade biomolecules inside the living system. In this present study, antioxidant arbitrated protective activity of Mesua ferrea L. is reported on induced oxidative damage in erythrocytes, Hb and DNA. Both, MCE (M. ferrea chloroform extract) and MEE (M. ferrea ethanol extract) exhibited significant antioxidant activity while MEE showed >90% protection to erythrocytes, Hb and DNA by virtue of high total phenolics (1.005 ± 0.005 mg EGA mg−1 ) and total flavonoids (514.8 ␮g mg−1 ) whereas MCE showed <90% but >70% antioxidant protective activity probably due to 0.596 ± 0.002 mg EGA mg−1 total phenolics and 275.9 ␮g EQ mg−1 total flavonoid content. It indicates that M. ferrea Linn. possesses significant protective activity against induced oxidative stress by acting as a strong antioxidant and potential electro-catalyst during the electrochemical oxidation of H2 O2 . Furthermore, HPLC of MCE and MEE revealed well known various good antioxidant molecules such as gallic acid, ellagic acid, coumaric acid, vanillic acid, rutin, quercetin, myricetin and kaempferol. Thus, M. ferrea Linn. bark was found to be having a good antioxidant property. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Aerobic metabolic pathways in the human body normally generate reactive oxygen species (ROS) and free radicals. Aerobic organisms have developed their own efficient enzymatic and nonenzymatic self-defensive network during the course of evolution against oxidative stress to maintain cellular homeostasis (Halliwell and Gutteridge, 2007). If the array of these defensive networks becomes unable to maintain cellular homeostasis under some circumstances, the exogenous supply of antioxidants is obligatory to restore the homeostasis. This reactive species, if produced in excess, extensively cause the oxidative damage to the cellular biomolecules (Droge, 2002) and eventually contribute to the pathogenesis of numerous oxidative stress related diseases including cancer, aging, heart failure, diabetes, lung disease, neurodegenerative disorders and rheumatoid arthritis, etc. (Halliwell and Gutteridge, 2007). The antioxidants play a crucial role in scavenging the active free radicals before they attack biologically vital

∗ Corresponding author. Tel.: +91 8282 256198. E-mail addresses: [email protected] (K.P. Rajesh), [email protected], [email protected] (H. Manjunatha). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.03.008

molecules by donating hydrogen atom to maintain the cellular homeostasis. Generally, erythrocytes have been used as a cellular model to investigate oxidative damage, because they are considered as prime targets for free radical attack owing to the presence of both high membrane concentration of polyunsaturated fatty acids (PUFA) and the redox active protein hemoglobin (Hb), which is the potent promoter of ROS (Sadrazadeh et al., 1984; Sukalski et al., 1997). These erythrocytes undergo hemolysis and change in their shape when it get exposed to harsh conditions of H2 O2 , which indicates the oxidative damage on the erythrocytes. Bio-molecules are vulnerable to exorbitant oxidative stress condition in vivo. Therefore Hb experiences irreversible structural changes involving iron/heme oxidation, heme-adduct products formation and amino acid oxidation when induced by H2 O2 (Vallelian et al., 2008) which further results in the formation of potentially toxic oxidized iron species, as well as heme and protein radical(s). Numerous synthetic and natural compounds have been reported to possess antioxidant property, but only limited number of compounds were accepted and mentioned in the list of GRAS (generally regarded as safe), as the synthetic molecules have the potential to cause serious adverse side effects. In this context, natural antioxidants have become one of the major areas of scientific research. The plant kingdom offers a wide range of medicinal plants.

K.P. Rajesh et al. / Industrial Crops and Products 47 (2013) 186–198

Many herbal and plant infusions of medicinal plants were frequently used as domestic medicines, which were proved to possess antioxidant and pharmacological properties contributed by multiple compounds in them. These beneficial properties are attributed to various polyphenols present in the plant (Ajila and Prasada Rao, 2008). The biological, pharmacological and medicinal properties of these plants and their constituents have been consistently and extensively investigated by many scientists till date (Krishnaiah et al., 2011). India is blessed with a wide variety of medicinal plants and their practice in Ayurveda with broad spectrum of application to treat various diseases is well known. Mesua ferrea L. belongs to Clusiaceae, commonly called as Naga kesara (Sanskrit), Naga sampige (Kannada), and cobra saffron (English) is a medium sized to hefty evergreen tree commonly found in Western Ghats up to an altitude of 1500 m, is rich with polyphenols and flavonoids (Ghani, 2003). The aim of the present study is to evaluate an in vitro antioxidant property and protective effect of M. ferrea L. bark extracts against induced oxidative damage in normal erythrocytes, Hb and DNA (pUC19).

2. Materials and methods 2.1. Chemicals Petroleum ether, chloroform, ethanol, butylated hydroxyl anisole (BHA), acrylamide, N,N -methylenebisacrylamide, sulfanilamide, sodium nitroprusside (SNP), trichloroacetic acid (TCA), Folin–Ciocalteu reagent were purchased from Merck (Germany). Catechin, quercetin, thiobarbituric acid (TBA), 2,2-diphenyl-1picrylhydrazyl (DPPH), nicotinamide adenine dinucleotide (NADH), ferrozine, nitro blue tetrazolium disodium salt (NBT), phenazine methosulfate (PMS), O-dianisidine, ammonium persulfate (APS), tetramethylethylenediamine (TEMED), (␣-napthyl)-ethylenediamine, 2,2 -azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) and Coomassie brilliant blue G-250 were obtained from Sigma–Aldrich (St. Louis, MO, USA). Gallic acid, ascorbic acid, ferrous chloride, hemoglobin and potassium persulfate were procured from Himedia (India). pUC19 and agarose were procured from GeNeiTM (Bangalore, Karnataka, India). All the chemicals used were of analytical grade. Water was purified on a Milli-Q system from Millipore (Millipore, Bedford, MA, USA). 2.2. Plant material and preparation of plant extract Stem bark of M. ferrea L. was collected from the Agumbe reserve forest area of Western Ghats, Shivamogga (Dist.), Karnataka, India with the permission of Forest Department (Megaravalli, Karnataka). Stem bark was cleansed thoroughly, shade dried and pulverized mechanically (sieve no. 10/44) and defatted in petroleum ether using Soxhlet apparatus. Further, hot extraction was carried out with defatted material (600 g) successively with chloroform (1.5 L, 45 ◦ C, ≈15 cycles) and ethanol (2 L, 50 ◦ C, ≈15–17 cycles). The chloroform extract and ethanol extract was concentrated in vacuo at 40 and 50 ◦ C, respectively. And named as M. ferrea chloroform extract (yield: 1.33%) (MCE) and M. ferrea ethanolic extract (yield: 14.46%) (MEE), respectively and stored in desiccator to avoid oxidation until further studies. 2.3. Qualitative phytochemical screening The extracts were qualitatively examined for various secondary metabolites using standard tests as described by Khanna and Kannabiran (2006).

187

2.4. Quantitative analysis of crude extracts 2.4.1. Determination of total phenol content Total phenol content in MCE and MEE was measured by the Folin–Ciocalteu method (Chang et al., 2002). Briefly, 1 ml of MCE and MEE (50 ␮g) was mixed with Folin–Ciocalteu reagent (2 ml) (diluted 1:10, v/v) followed by the addition of 2 ml of sodium carbonate (7.5%, w/v) and mixed, allowed to stand for 90 min at room temperature and absorbance was measured against the blank at 750 nm using spectrophotometer (Systronics, PC based double beam spectrophotometer 2202). Total phenol content of the extract was expressed in terms of equivalent to gallic acid (EGA, mg mg−1 of dry mass). 2.4.2. Determination of total flavonoid Total flavonoid content of MCE and MEE was determined according to the modified method of Zhishen et al. (1999). Briefly, 5 ml of extract (200 ␮g) was mixed with 300 ␮l of 5% sodium nitrite and 300 ␮l of 10% aluminum chloride followed by the addition of 2 ml of 1 M sodium hydroxide after the incubation of reaction mixture at room temperature for 6 min. The volume in each test tube was made up to 10 ml by adding 2.4 ml of millipore water. Absorbance was measured at 510 nm against the blank. Total flavonoid content of the extract was expressed in terms of equivalent to catechin (mg mg−1 of dry mass). 2.5. Determination of in vitro antioxidant activity 2.5.1. Total antioxidant capacity Total antioxidant capacity of MCE and MEE was performed by phosphomolybdenum method (Prieto et al., 1999). 300 ␮l of MCE and MEE at different concentrations (50, 100, 200 ␮g) was combined with 3 ml of reagent mixture (4 mM ammonium molybdate, 0.6 M sulfuric acid and 28 mM of sodium phosphate). Test tubes were kept for incubation at 95 ◦ C for 90 min and allowed to cool. Absorbance of the content was measured at 695 nm against blank. Antioxidant capacity of each extract is expressed as equivalents of ascorbic acid. 2.5.2. Total reductive capability Total reductive capacity of MCE and MEE was determined according to the method of Oyaizu (1986). 1 ml of MCE and MEE at different concentrations (50, 100, 200 ␮g) were mixed with phosphate buffer (2.5 ml, 0.2 M pH 6.5) and potassium ferricyanide (2.5 ml, 1%). The mixture was incubated at 50 ◦ C for 20 min. At the end of the incubation period, trichloroacetic acid (2.5 ml, 10%) was added and centrifuged at 3000 rpm for 10 min. To the 2.5 ml of supernatant, 2.5 ml of millipore water and ferric chloride (0.5 ml, 0.1%) was added. The absorbance of the reaction mixture was measured at 700 nm against blank. Increased absorbance of the reaction mixture indicated increased reducing power. Total reducing capacity of each extract is expressed as equivalents of quercetin. 2.5.3. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity MCE and MEE were screened for free radical scavenging activity by DPPH method (Braca et al., 2001). Free radical scavenging activity of the extracts was carried out on the basis scavenging ability of stable DPPH radical. Each extract (MCE and MEE) at different concentrations were added to 3 ml of 0.004% DPPH in 95% ethanol and the mixtures were incubated at room temperature in dark condition for 30 min. The scavenging activity of extracts against DPPH radical was determined by measuring the absorbance at 517 nm. DPPH radical scavenging activity of BHT was assayed for comparison. Radical scavenging activity was calculated using the formula: % inhibition = [(Acontrol − Atest )/Acontrol ] × 100, where Acontrol is the

188

K.P. Rajesh et al. / Industrial Crops and Products 47 (2013) 186–198

absorbance of the control reaction and Atest is the absorbance of the extract reaction. IC50 value was calculated using the formula: IC50 = [(C/I) × 50], where C is the sum of extracts concentrations used to test and I is the sum of percentage of inhibition at different concentrations. 2.5.4. Superoxide radical scavenging assay Superoxide anion radical scavenging activity of MCE and MEE was measured according to the method of Nishimiki et al. (1972) with slight modifications. All the reagents were prepared in phosphate buffer (pH 7.4). 1 ml of NBT (156 ␮M), 1 ml of NADH (468 ␮M) and 2 ml of extracts at different concentrations were added to each test tube. The reaction was initiated by adding 100 ␮l of PMS (60 ␮M) and incubated at 25 ◦ C for 5 min followed by the measurement of absorbance at 560 nm against blank. Decreased absorbance of the reaction mixture indicated increased super oxide anion radical scavenging activity. Ascorbic acid was taken as reference standard. The percentage inhibition was calculated using the following formula, % inhibition = [(Acontrol − Atest )/Acontrol ] × 100, where Acontrol is the absorbance of the control reaction and Atest is the absorbance of the extract reaction. IC50 value was calculated using the formula: IC50 = [(C/I) × 50], where C is the sum of extracts concentrations used to test and I is the sum of percentage of inhibition at different concentrations. 2.5.5. Lipid peroxidation inhibition assay TBA reacts with malondialdehyde (MDA) to form a diadduct, a pink chromogen, which can be detected according to the method of Halliwell and Guttridge (1989). 10% of rat liver (CPCSEA Reg. No. 144/1999) homogenate in 0.15 M potassium chloride was prepared. 0.5 ml of liver homogenate and 1 ml of MCE and MEE at different concentrations were taken in test tubes. Lipid peroxidation was induced by adding ferrous sulfate (50 ␮l, 0.07 M) and incubated at room temperature for 30 min. The reaction was stopped by adding chilled acetic acid (1.5 ml, 20%, pH 3.5) containing 20% TCA followed by the addition of TBA (50 ␮l, 0.8% TBA in 1.1% SDS). The content was mixed thoroughly and incubated in boiling water bath for 60 min. After cooling, 5 ml of butanol was added and centrifuged at 3000 rpm for 10 min. Absorbance of the organic supernatant was measured at 532 nm. Percentage of inhibition was calculated using the following formula, % inhibition = [(Acontrol − Atest )/Acontrol ] × 100, where Acontrol is the absorbance of the control reaction and Atest is the absorbance of the extract reaction. IC50 value was calculated using the formula: IC50 = [(C/I) × 50], where C is the sum of extracts concentrations used to test and I is the sum of percentage of inhibition at different concentrations. 2.5.6. Nitric oxide radical scavenging activity Nitric oxide radical scavenging activity of MCE and MEE was determined according to Marcocci et al. (1994). The reaction mixture containing sodium nitroprusside (2 ml, 10 mM in 0.5 M phosphate buffer, pH 7.4) and 250 ␮l of extract at different concentration was incubated at 25 ◦ C for 150 min. Aliquot (0.5 ml) of reaction mixture was added to test tube having sulfanilamide (1 ml, 1% in 5% phosphoric acid) and incubated at 25 ◦ C for 5 min. 1 ml of 0.1% (␣-napthyl)-ethylenediamine was added to the reaction mixture and incubated for 30 min at 25 ◦ C. Absorbance of the reaction mixture was measured at 546 nm. The percentage of nitric oxide radical scavenging was calculated using the formula, % inhibition = [(Acontrol − Atest )/Acontrol ] × 100, where Acontrol is the absorbance of the control reaction and Atest is the absorbance of the extract reaction. IC50 value was calculated using the formula: IC50 = [(C/I) × 50], where C is the sum of extracts concentrations used to test and I is the sum of percentage of inhibition at different concentrations.

2.5.7. Hydroxyl radical scavenging activity The hydroxyl radical scavenging activity was determined according to the method of Klein et al. (1991). MCE and MEE at different concentrations were taken in different test tube and evaporated to dryness followed by the addition of ironEDTA solution (1 ml, 0.13% ferrous ammonium sulfate and 0.26% EDTA), EDTA (0.5 ml, 0.018%), and DMSO (1 ml, 0.85%, v/v in 0.1 M phosphate buffer, pH 7.4) and the reaction was initiated by adding ascorbic acid (0.5 ml, 0.22%). Test tubes were capped tightly and heated on a water bath at 80–90 ◦ C and the reaction was terminated by the addition of chilled TCA (1 ml, 17.5%, w/v) after 15 min of incubation, 3 ml of Nash reagent (75 g of ammonium acetate, 3 ml of glacial acetic acid, and 2 ml of acetyl acetone were mixed and made up to 1 L by water) was added and left at room temperature for 15 min for color development. The intensity of the yellow color was measured at 412 nm against blank. The percentage hydroxyl radical scavenging was calculated by the formula, % inhibition = 1 − [Asample /Ablank ] × 100, where Asample is the absorbance of the reaction mixture contains extract, Ablank is the absorbance of the blank. IC50 value was calculated using the formula: IC50 = [(C/I) × 50], where C is the sum of extracts concentrations used to test and I is the sum of percentage of inhibition at different concentrations.

2.5.8. Metal chelating activity Metal chelating activity of MCE and MEE was measured by according to the method of Dinis et al. (1994). About 3 ml of extracts and EDTA at different concentrations were taken in different test tubes followed by the addition of ferrous chloride (50 ␮l, 2 mM) and ferrozine (20 ␮l, 5 mM). Tubes were allowed to stand for 10 min at room temperature. Absorbance of reaction mixture was measured at 562 nm against blank. EDTA was used as standard for comparison. Percentage of metal chelating activity was calculated by using the following formula, % inhibition = [(Acontrol − Atest )/Acontrol ] × 100, where Acontrol is the absorbance of the control reaction and Atest is the absorbance of the extract reaction. IC50 value was calculated using the formula: IC50 = [(C/I) × 50], where C is the sum of extracts concentration used to test and I is the sum of percentage of inhibition at different concentrations.

2.5.9. ABTS radical scavenging activity MCE and MEE were screened for ABTS radical scavenging activity according to modified method of Re et al., 1999. ABTS radical scavenging activity of the extracts was compared to BHA standard. The ABTS radical was generated by mixing equal volume (v/v) of 7 mM ABTS and 2.6 mM potassium persulfate and incubated in dark for overnight at room temperature. Prior to assay, 1 ml of ABTS was mixed with methanol (1:60, v/v) to get working reaction mixture of ABTS. 150 ␮l of each extract (MCE and MEE) at different concentrations were added to test tubes and volume in each test tube was made up to 3000 ␮l by adding reaction mixture of ABTS and the mixtures were incubated at room temperature in dark condition for 2 h. The scavenging activity of extracts against ABTS radical was determined by measuring the absorbance at 734 nm. Radical scavenging activity was calculated using the formula: % inhibition = [(Acontrol − Atest )/Acontrol ] × 100, where Acontrol is the absorbance of the control reaction and Atest is the absorbance of the extract reaction. IC50 value was calculated using the formula: IC50 = [(C/I) × 50], where C is the sum of extracts concentrations used to test and I is the sum of percentage of inhibition at different concentrations.

K.P. Rajesh et al. / Industrial Crops and Products 47 (2013) 186–198

2.6. Protective effect of MCE and MEE on induced oxidative stress in erythrocytes Protective effect of MCE and MEE on hydrogen peroxide induced oxidative damage in erythrocytes was evaluated by inhibition of erythrocyte hemolysis, oxidation to erythrocyte ghost membrane proteins and induced changes in erythrocytes structural morphology. IC50 value was calculated using the formula: IC50 = [(C/I) × 50], where C is the sum of extracts concentration used to test and I is the sum of percentage of inhibition at different concentration. 2.6.1. In vitro inhibition of erythrocyte hemolysis assay 2.6.1.1. Preparation of erythrocytes. For erythrocyte isolation, 2 ml of human blood from healthy individual was collected in heparinized tube using sterile syringe. Erythrocytes were isolated and stored according to the method described by Yang et al. (2006). Briefly, blood samples were centrifuged at 3000 rpm for 10 min at 4 ◦ C, erythrocytes were separated from plasma and thicker buffy coat, washed 3–5 times by centrifugation at 3000 rpm (4 ◦ C) for 5 min in 10 volumes of 10 mM phosphate buffered saline (pH 7.4). The supernatant and buffy coats of white cells were carefully removed with each wash. Washed erythrocytes were stored at 4 ◦ C and used within 6 h for further studies. 2.6.1.2. Inhibition of erythrocyte hemolysis. The inhibition of induced erythrocyte hemolysis by MCE and MEE was evaluated according to the method described by Tedesco et al. (2000) with slight modifications. 50 ␮l of MCE and MEE at different concentration was added to a series of test tubes containing 100 ␮l of 5% (v/v) suspension of erythrocytes in PBS. To each tube 100 ␮l of 100 ␮M H2 O2 (in 0.1 M PBS pH 7.4) was added. Simultaneously, one negative control was maintained by adding erythrocytes suspension and 100 ␮l of l00 ␮M H2 O2. The reaction mixture was gently shaken while being incubated at 37 ◦ C for 3 h and diluted with 8 ml of PBS and centrifuged at 3000 rpm for 10 min. Absorbance of the supernatant was measured at 540 nm to determine the percentage of hemolysis. The inhibitory effect of the extracts was compared with standard antioxidant BHA. To assess the possible hemolysis, if any by MCE and MEE, erythrocytes was incubated with 2000 ␮g of extracts for 1 h and determined. Percentage of hemolysis was calculated by taking hemolysis caused by 100 ␮M H2 O2 as 100%. IC50 value was calculated using the formula: IC50 = [(C/I) × 50], where C is the sum of extracts concentrations used to test and I is the sum of percentage of inhibition at different concentration. 2.6.2. Protective effect on erythrocytes structural morphology To evaluate protective effect of MCE and MEE, 50 ␮l of 5% (v/v) suspension of erythrocytes in PBS was added to all test tubes followed by the addition of extracts (1000 ␮g ml−1 ) and 100 ␮l of H2 O2 (100 ␮M, PBS pH 7.4). Simultaneously, negative control (H2 O2 alone) and positive control (erythrocyte suspension alone) were maintained for comparative evaluation. The mixture was incubated at 37 ◦ C for 1 h. After incubation, the mixture was centrifuged at 3500 rpm for 10 min and the cell pellets were observed under microscope at 100× for morphological changes and photographs were taken by Sony Cyber-shot 10.2 camera. 2.6.3. Evaluation of inhibition of oxidative damage on erythrocyte ghost membrane proteins by SDS-PAGE 2.6.3.1. Preparation of erythrocyte ghost membranes. Erythrocyte ghost membrane was prepared by hypotonic lysis of erythrocytes according to the method of Fairbanks et al. (1971) with slight modification. Briefly, ten volume of chilled lysis buffer (5 mM phosphate buffer pH 8.0, with 1 mM EDTA) was added to freshly isolated erythrocytes and incubated for 10 min on ice bath. Later, the mixture

189

was centrifuged for 30 min at 20,000 rpm (4 ◦ C) and the centrifugation was repeated with fresh buffer until the pale pink colored erythrocyte ghost membrane appears at the bottom. Isolated ghost membranes were used immediately for the following study. 2.6.3.2. Inhibition of oxidative damage on erythrocyte ghost membrane proteins. The interference of hydrogen peroxide free radical induced oxidative damage on erythrocyte ghost membrane proteins by MCE and MEE was determined by SDS-PAGE (Carini et al., 2000). Oxidation of membrane proteins was initiated by adding 100 ␮l H2 O2 (200 ␮M) to the reaction mixture containing 50 ␮g of ghost membrane protein (as estimated by Lowry et al., 1951) and with or without extracts at two different concentrations (500 and 1000 ␮g) and incubated for 1 h. Besides, appropriate control was maintained along with the extracts. SDS-PAGE was performed using 10% discontinuous gel at room temperature under constant voltage of 50 V. The protein bands were visualized by staining with Ezee blue. 2.7. Protective effect of MCE and MEE on oxidative damage in hemoglobin Protective effect of MCE and MEE on hydrogen peroxide free radical induced oxidative damage in hemoglobin was assessed by free iron release analysis, absorbance measurement, in-gel heme staining and reducing SDS-PAGE using 0.1 M phosphate buffer at physiological pH. 2.7.1. Free iron release analysis Free iron release upon oxidation was measured using ferrozine as described previously by Carter (1971). Inhibitory effect of extracts on free iron release upon Hb oxidation was estimated. 100 ␮l of hemoglobin solution (1%), 400 ␮l of phosphate buffer, 50 ␮l of extract at different concentrations (0–250 ␮g), 100 ␮l of H2 O2 (10 mM) and 100 ␮l of ascorbic acid (100 mM) was taken in test tube, mixed well and incubated at room temperature for 5 min. 50 ␮l of ammonium acetate (16%) and 50 ␮l of ferrozine (16 mM) were added to the reaction mixture. After 5 min of incubation at room temperature, the reaction mixture was centrifuged at 4 ◦ C, 5000 rpm for 10 min and absorbance was measured at 562 nm. The amount of free iron release was calculated by employing standard curve prepared by Mohr’s salt. To assess possible Hb cleavage induced by extracts, 100 ␮l of Hb alone was incubated with 250 ␮g of each extract and maintained as mentioned above. 2.7.2. Absorbance measurement Absorbance measurement was done by using Ocean Optics DH 2000 UV-Vis spectrophotometer (USA). Reaction mixture was prepared in 1 ml of phosphate buffer supplemented with fixed amount of Hb (0.63 ␮M), H2 O2 (10 ␮M in phosphate buffer), O-dianisidine (0.02 M) and different concentration of extracts (10–60 ␮g). Reaction mixture was incubated for 10 min at 25 ◦ C. Absorbance changes were measured by scanning the reaction mixture from 200 to 800 nm. 2.7.3. Non-reducing in-gel heme staining and reducing SDS-PAGE Hydrogen peroxide mediated Hb degradation was carried out with or without extract as mentioned above (Section 2.7.1). Small aliquot of reaction mixture (up to 10 ␮g of Hb) was incubated with gel loading buffer containing 63 mM Tris–HCl (pH 6.8), SDS (2%, w/v), glycerol (10%, w/v), and bromophenol blue (0.0025%, w/v) with lack of ␤-mercaptoethanol because of its interference with heme – staining dye. Prior to electrophoresis technique; gel, buffer and electrophoresis unit was equilibrated at 4 ◦ C according to Laemmli (1970) and non-reducing gel electrophoresis was performed at the same temperature for about 5 h at a constant

190

K.P. Rajesh et al. / Industrial Crops and Products 47 (2013) 186–198

voltage (50 V) on discontinuous SDS gel (12%). Gel was developed by staining for heme by following slight modification of Thomas et al. (1976) procedure. Briefly, gels were washed with 3:7 (v/v) of methanol and 0.25 M sodium acetate (pH 5.0) for about 10–15 min and consequently, incubated with freshly prepared heme staining solution (7:3, v/v mixture of 0.25 M sodium acetate, pH 5.0 and 12 mM O-dianisidine in methanol) for 20 min in dark at room temperature. Bands were developed by adding 1.25 M H2 O2 (5 min) and washed for 30 min with 8:1:1 (v/v/v) mixture of water, acetic acid and methanol. Reducing gel electrophoresis was performed as mentioned above under reducing condition using reducing agent ␤-mercaptoethanol (10%) and the samples were boiled at 100 ◦ C for 5 min. Electrophoresis was performed at 4 ◦ C for 5 h at 50 V and reduced gels were developed by staining with Coomassie brilliant blue G-250. 2.8. DNA protection activity of MCE and MEE DNA protection activity of MCE and MEE was determined by agarose gel electrophoresis using pUC19. 1 ␮g of DNA with 5 ␮l of TAE, 5 ␮l of extract at different concentrations (10 and 20 ␮g) was incubated for 30 min at room temperature with 5 ␮l of Fenton reagent (30 mM H2 O2 , 50 mM ascorbic acid, 20 mM ferric chloride) as oxidizing agent. pUC19 was separated on 1% agarose gel with ethidium bromide at room temperature under constant voltage (50 V). Gel was documented and DNA protection activity was analyzed based on the mobility of DNA bands corresponding to control. 2.9. Cyclic voltammetry studies 2.9.1. Apparatus and preparation of bare carbon electrode Cyclic voltammetry study was performed with Model CHI-660c electrochemical work station, equipped with personal computer. A conventional three electrode cell was employed throughout the experiments. The electrochemical cell contained carbon paste electrode (CPE) as a working electrode, platinum counter electrode (PCE) and saturated calomel electrode (SCE) as reference. Bare carbon paste electrode was prepared by mixing of 70% graphite powder with 30% silicon oil in pestle and mortar to produce a homogenous mixture. The paste was packed into the homemade cavity (3 mm in diameter) and then smoothed on a butter paper. The electrical contact was provided by a copper wire connected to the paste at the end of the tube and cyclic voltamogram was produced using 10 mm H2 O2 with and without extracts. 2.10. HPLC–UV analysis Phenolic acids and flavonoids from MCE and MEE were analyzed by HPLC (Model LC-10ATVP. Shimadzu Corp, Kyoto, Japan) on a reversed phase Shimpak C18 column (5 ␮m, 250 mm × 4.6 mm). Phenolic content in both the extracts were detected using octadecylsilyl silica gel as stationary phase. Solvent system consisting of [A] phosphoric acid:water (0.5:99.5, v/v), [B] acetonitrile was used as mobile phase at a flow rate of 1 ml min−1 . Phenolic acid standards such as gallic acid, p-coumaric acid, ellagic acid, ferulic acid, mandelic acid and vanillic acids were employed for identification of phenolic acids present in MCE and MEE by comparing the retention time under similar experimental conditions. The detector used for analysis was UV detector at 220 nm. Flavonoid content in both the extracts was detected using octadecylsilyl silica gel as stationary phase. Solvent system consisting of methanol, water and phosphoric acid (50:49.6:0.4, v/v) was used as mobile phase at a flow rate of 0.5 ml min−1 . Rutin, quercetin, myricetin, kaempferol, luteolin were used as reference

standard to identify the flavonoids in MCE and MEE. The detector used for analysis was UV detector at 350 nm. 2.11. Statistical analysis All the experiments were performed in triplicate and results are expressed as mean ± SEM. Statistical analysis was done by GraphPad Prism 5. 3. Results and discussion Many synthetic antioxidant molecules have shown toxic and/or mutagenic effects (Chen et al., 1992) so there is a potential need for naturally occurring antioxidants with no or minimal side effects. Numerous plants have been proven to possess free radical scavenging or antioxidant activity (Krishnaiah et al., 2011) and several researchers are currently working extensively on ethnic based medicinal plants to prove the scientific basis to impart wide dissemination of the medicinal usage of plants all over the world. Several studies have shown potential benefits of antioxidants like flavonoids, polyphenolics, and vitamins from plant sources which play a vital role in evading cells and biomolecules from oxidative stress (Tedesco et al., 2000). The present study showed the antioxidant activity of M. ferrea L. and its protective efficacy against induced oxidative damage in erythrocytes, Hb and DNA (pUC19). 3.1. Qualitative and quantitative analysis of crude extracts The preliminary phytochemical analysis of MCE and MEE revealed the presence of various antioxidant molecules like flavonoids, quinones, saponins, phenolics and tannins. Total polyphenolic content in extracts was expressed as equivalent to gallic acid (EGA) and is found to be 0.596 ± 0.002 and 1.005 ± 0.005 mg mg−1 of dry extract, respectively. Analysis of flavonoid has revealed high concentration in MEE (514.82 ␮g mg−1 ) followed by MCE, which is found to be 275.93 ␮g mg−1 of dry extract. 3.2. Determination of in vitro antioxidant activity Total antioxidant capacity of both extracts was performed by phosphomolybdenum method as described by Prieto et al. (1999) Antioxidant capacity of individual extracts are expressed as equivalents of ascorbic acid. Among the extracts, MEE showed significant activity over MCE (Fig. 1). The extracts have shown remarkable antioxidant and reductive capability, hence it can serve as free radical inhibitors or scavengers. Both the extracts were screened for free radical scavenging ability by DPPH and ABTS radical scavenging method. It is based on the measurement of hydrogen donating ability of antioxidant molecules present in the extracts to reduce purple colored DPPH and blue colored ABTS radical to colorless. Among MCE and MEE, MEE showed better radical scavenging activity (IC50 ; DPPH: 12.70 ± 0.033 ␮g ml−1 , ABTS: 4.36 ± 0.02 ␮g ml−1 ) whereas MCE showed moderate activity (IC50 ; DPPH; 57.82 ± 0.307 ␮g ml−1 ABTS: 10.49 ± 0.03 ␮g ml−1 ) compared to standard (IC50 ; DPPH: 87.97 ± 0.359 ␮g ml−1 , ABTS: 7.07 ± 0.08 ␮g ml−1 ). The significant DPPH scavenging activity of MEE could be attributed to the presence of more phenolic content compared to MCE. MCE and MEE were screened for superoxide radical scavenging, lipid peroxidation inhibition, nitric oxide radical scavenging, hydroxyl radical scavenging, H2 O2 scavenging and metal chelating activity. The results of above activity of MCE and MEE were illustrated in Tables 1 and 2. The screenings of above activities have revealed the contribution of different types of antioxidant

K.P. Rajesh et al. / Industrial Crops and Products 47 (2013) 186–198

191

Fig. 1. Plots of (A) total antioxidant activity and (B) total reductive capability of MCE and MEE. The results shown are averages of three independent experiments.

molecules toward dose dependent property of MCE and MEE. Thus, the relationship between the concentration of extracts and antioxidant activity is linear.

3.3. Protective effect of MCE and MEE on oxidative damage in erythrocytes The antioxidants capacity and efficacy of molecules can be assessed more accurately by the effect of these molecules on the level of oxidation in biological fluids and tissues, such as plasma, erythrocytes, urine from humans and experimental animals. The easy accessibility, finite life span and relative simplicity of erythrocytes make them an attractive model to study the oxidative stress. A preliminary study on the toxic effect of extracts did not show harmful effect on erythrocytes. Inhibitory effect of MCE and MEE at different concentrations on H2 O2 induced hemolysis of erythrocytes for 3 h are shown in Fig. 2. When human erythrocytes were incubated with reactive species H2 O2 , 100% hemolysis was observed due to induced stress but they were stable in presence of

plant individual extracts. Both the extracts showed dose dependent erythrocyte protective activity. However, as shown in Fig. 2A moderate hemolysis was observed due to H2 O2 oxidative stress in presence of MCE (IC50 1001 ± 3.097 ␮g ml−1 ) while MEE showed protective effect (IC50 534.3 ± 3.680 ␮g ml−1 ) significantly equivalent to BHA (IC50 522 ± 3.255 ␮g ml−1 ). Fig. 2 shows microphotographs of human erythrocytes under H2 O2 induced oxidative stress condition, since it clearly illustrates cellular morphology. Normal erythrocytes (Fig. 2B) appeared as typical disc like structure had undergone significant change in cell shape during H2 O2 induced oxidative stress (Fig. 2C). The morphological changes induced by H2 O2 were protected when the cells were treated with MCE and MEE (Fig. 2D and E). This is due to significant scavenging of H2 O2 by the action of multiple antioxidant molecules present in the extracts. Erythrocytes are more fragile in a hostile condition of H2 O2 exposure; it generates a covalent complex of spectrin and hemoglobin as well as myriad of cellular changes including alteration in membrane deformability, hemolysis, phospholipid organization, cell surface characteristics and surface pressure alteration in erythrocytes. It is due to change in lipid interaction as a

192

K.P. Rajesh et al. / Industrial Crops and Products 47 (2013) 186–198

Table 1 In vitro antioxidant activity of MCE and MEE. Sl. no.

Activity

Concentration of extract in ␮g.

% of inhibition

MCE

MEE

MCE

MEE

1.

DPPH radical scavenging activity

25 50 75 100 125

05 10 15 20 25

28.2 48.9 70.4 85.1 91.7

± ± ± ± ±

0.56 0.96 0.25 1.01 0.35

21.5 43.4 58.9 80.8 90.7

± ± ± ± ±

0.81 0.17 0.08 0.66 1.05

2.

Superoxide radical scavenging activity

100 200 300 400 500

30 60 90 120 150

25.1 46.7 55.5 62.3 84.4

± ± ± ± ±

1.66 0.87 0.52 0.26 0.43

13.4 34.6 49.9 68.1 92.1

± ± ± ± ±

0.08 0.52 1.40 1.48 0.96

3.

Lipid peroxidation inhibition

1000 1500 2000 2500 3000

250 500 750 1000 1250

40.6 48.4 59.2 65.9 72.3

± ± ± ± ±

0.65 0.74 0.14 0.65 1.43

18.1 23.6 31.8 45.6 58.2

± ± ± ± ±

0.01 0.12 1.62 0.66 1.39

4.

Nitric oxide radical scavenging activity

050 100 150 200 250

050 100 150 200 250

14.1 30.5 44.2 69.5 72.8

± ± ± ± ±

0.47 0.36 0.31 0.47 0.73

58.6 64.2 72.4 74.8 76.4

± ± ± ± ±

0.95 0.58 0.03 0.06 0.16

5.

Hydroxyl radical scavenging assay

100 200 300 400 500

050 100 150 200 250

2.4 13.6 21.1 35.9 52.8

± ± ± ± ±

0.42 1.62 2.65 3.67 3.19

5.2 12.3 17.1 28.8 48.7

± ± ± ± ±

3.07 3.21 1.10 3.96 1.28

6.

Metal chelating

1000 2000 3000 4000 5000

1000 2000 3000 4000 5000

61.1 68.1 70.7 75.9 79.3

± ± ± ± ±

3.32 0.18 0.55 0.22 0.70

13.3 33.3 48.5 66.1 83.7

± ± ± ± ±

0.85 1.9 0.7 0.8 0.4

7

ABTS radical scavenging activity

05 10 15 20 25

02 04 06 08 10

32.8 55.5 83.5 87.3 98.8

± ± ± ± ±

0.29 0.48 0.48 0.19 0.19

36.4 54.3 64.5 91.9 96.6

± ± ± ± ±

4.6 5.3 1.9 1.0 0.4

The results shown are averages of three independent experiments, values are mean ± SEM.

consequence of H2 O2 induced alteration in lipid structure from unsaturated to saturated fatty acids (Snyder et al., 1985) and it also induces an echinocytic type of shape alteration and peripheral membrane damage in erythrocytes (Ajila and Prasada Rao, 2008). Several studies have shown phenolic content of plant extracts showing a significant role in preventing the ROS effects on erythrocytes. Tedesco et al. (2000) and Ajila and Prasada Rao (2008) reported phenolic content in red wine and mango peel extract showing protective property against erythrocyte hemolysis and H2 O2 induced oxidation of ghost membrane protein by restoring the erythrocytes cell shape and integrity. Interestingly, the high amount of polyphenolic content present in MCE and MEE may have

contributed to the protective effect by counteracting the influence of H2 O2 on erythrocytes. Erythrocyte ghost membrane protein pattern after oxidative damage is illustrated in Fig. 2F. Ghost membrane was prepared by hypotonic lysis of purified erythrocytes and treated with H2 O2 and MCE and MEE individually. Analysis of reaction mixture clearly revealed that, in lane 2, 3 4 and 5, the membrane protein integrity was maintained due to scavenging of H2 O2 by MCE and MEE when compared to lane 6 which comprises H2 O2 treated ghost membrane alone as a result protein integrity was entirely disordered. The extract coating around the membrane bilayer and the resulting restriction on its fluidity may hinder the diffusion of stress

Table 2 IC50 values of MCE and MEE for in vitro antioxidant activity. Sl. no.

Activity

MCE (␮g)

1 2 3 4 5 6 7

DPPH radical scavenging activity Superoxide radical scavenging assay Lipid peroxidation inhibition assay Nitric oxide radical scavenging activity Hydroxyl radical scavenging activity Metal chelating activity ABTS radical scavenging activity

57.82 272.9 1746 162.1 6183 2111 10.49

The results shown are averages of three independent experiments, values are mean ± SEM.

± ± ± ± ± ± ±

0.3 1.8 3.8 2.9 23.2 12.6 0.03

MEE (␮g) 12.70 87.15 1064 108.4 3338 3059 4.36

± ± ± ± ± ± ±

0.03 0.1 2.0 0.4 17.6 9.8 0.02

Standard (␮g) 87.97 ± 95.82 ± – – – 28.92 ± 7.07 ±

0.3 1.2

0.1 0.08

K.P. Rajesh et al. / Industrial Crops and Products 47 (2013) 186–198

193

Fig. 2. (A) In vitro protective effect of MCE and MEE against H2 O2 induced oxidative stress of human erythrocytes compare to BHA as standard. (B–E) Microscopic observation of (B) normal erythrocytes, (C) erythrocytes + H2 O2 , (D) erythrocytes + H2 O2 + MCE and (E) erythrocytes + H2 O2 + MEE (100×). (F) SDS-PAGE electrophoresis study of ghost membrane (Gm) showing protective effect of MCE and MEE on oxidative stress induced damage. (1) Normal ghost membrane, (2) Gm + 500 ␮g of MCE + H2 O2 , (3) Gm + 1000 ␮g of MCE + H2 O2 , (4) Gm + 500 ␮g of MEE + H2 O2 , (5) Gm + 1000 ␮g of MEE + H2 O2 , (6) Gm treated with H2 O2 .

inducers and its consequent damaging effects. This conclusion can also imply that this restriction could apply to the diffusion of free radicals into cell membranes and the subsequent decrease in the kinetics of free radical reactions (Suwalskya et al., 2007). 3.4. Protective effect of MCE and MEE on oxidative damage in hemoglobin Hemoglobin is the potent promoter of ROS, also sensitive to H2 O2 as degrades heme with the release of free iron metal ions, which has been confirmed by free iron release analysis using ferrozine method. These free iron ions act as pro-oxidants, initiate free radicals generation and lipid peroxidation (Puppo and Halliwell, 1988; Sadrazadeh et al., 1984) and highly toxic as it can react rapidly with H2 O2 and damages blood vessels, produces vasodilation leading to hypo-tensions and metabolic acidosis (Crichton et al., 2002; Ong and Halliwell, 2004). Hb protective activity of MCE and MEE was carried out in presence of H2 O2 . This analysis was conducted with constant amount of Hb and H2 O2 by varying the concentration

of extracts (50–250 ␮g). Both the extracts showed significant dose dependent protective activity to heme degradation as illustrated in Fig. 3A, the linear decrease in free iron release upon reaction with ascorbic acid. To demonstrate the Hb protective activity, UV absorption spectral and SDS-PAGE analysis was carried out, which showed maximum absorption spectrum at 410 nm. As depicted in Fig. 3B and C, the decreased absorption peak was proportionate with increased concentration of extract, which indicates the inhibition of free iron release by heme degradation of Hb. In spectrum analysis, the yellow color spectral line represents the complete heme degradation of Hb due to induced oxidative stress by H2 O2 . The inhibition of heme destruction in Hb by H2 O2 in presence of extracts was consistently monitored by decrease in the absorbance peaks. Furthermore, to reinforce free iron release and UV absorption spectral study, inhibition of heme destruction by MCE and MEE was confirmed by in gel heme staining. Fig. 3D and E illustrates the gradual change in the heme content as the concentration of extracts increases by comparing the band intensity with the H2 O2 treated

194

K.P. Rajesh et al. / Industrial Crops and Products 47 (2013) 186–198

Fig. 3. (A) Plot represents the linear decrease in free iron released. (B and C) UV absorbance spectra of the Hb in presence of (B) MCE and (C) MEE. (D and E) In-gel heme staining and reducing SDS-PAGE.

Hb (lane 2). We performed reducing SDS-PAGE analysis followed by Coomassie brilliant blue 250 staining to confirm protein aggregation. It is observed that, as the concentration of extracts increases, protein aggregation was preserved whereas in the lane 2, it was entirely disordered due to H2 O2 influence.

extracts. MCE and MEE showed <90% but >70% and >90% of protection, respectively. This result shows that, polyphenols present in both the extracts have the capacity to quench the free radicals generated in the reaction, thereby protecting the DNA from the induced oxidative damage by Fenton reagent.

3.5. DNA protection activity of MCE and MEE

3.6. Cyclic voltammetry studies

DNA protection activity was observed by both MCE and MEE against Fenton’s induced DNA damage in pUC 19 (Fig. 4). Significant protection to pUC 19 was observed in presence of both the

MCE and MEE showed protective ability to erythrocytes, Hb and DNA under H2 O2 induced oxidative stress condition. To substantiate the beneficial activity of extracts, a new advanced

K.P. Rajesh et al. / Industrial Crops and Products 47 (2013) 186–198

195

Table 3 Quantitative HPLC analysis of MCE and MEE. Sl. no.

1 2 3 4 5

Phenolic acids

Flavonoids

Standard

MCE (mg g−1 )

MEE (mg g−1 )

Standard

MCE (mg g−1 )

MEE (mg g−1 )

Gallic acid Coumaric acid Ellagic acid Hydroxy benzoic acid Vanillic acid

215.01 2.50 2.50 – 0.20

412.90 – 9.35 7.90 –

Rutin Quercetin Myricetin Kaempferol Luteolin

45.6 348.2 – 0.90 –

63.3 753.9 60.1 – –

electrochemical method cyclic voltammetry was conducted to add a note on mechanism of action of MCE and MEE. Fig. 5 shows cyclic voltamogram for reaction mixture (pH 7.4) at bare carbon paste electrode of scan rate 100 mV/s. All the potentials were measured with respect to standard calomel electrode. Fig. 5B shows cyclic voltamogram for H2 O2 and MCE at different concentration in PBS (pH 7.4) of scan rate 100 mV/s. Addition of MCE into the electrochemical cell leads to enhancement in the redox peak current, which acts as electro catalyst for the electrochemical oxidation of H2 O2 with increase in the scan rate and MCE concentration, the oxidation peak current also increases linearly, suggesting overall electrode process is diffusion controlled. Parallel electrochemical measurement was also performed for MEE and obtained the similar results with electro catalyst property (Fig. 5C).

antioxidant property (Megala and Geetha, 2010). HPLC was employed to characterize the important antioxidant molecules present in MCE and MEE as shown in Table 3 with various antioxidant molecules.

3.7. HPLC–UV analysis The analyses of phenolics and flavonoids have been considered as important for the medicinal plant to evaluate its

Fig. 4. DNA protection ability of MCE and MEE. (1) 1 ␮g of normal pUC 19, (2) DNA treated with 5 ␮l of Fenton’s reagent, (3) DNA + 5 ␮l of Fenton’s reagent + 10 ␮g of MCE, (4) DNA + 5 ␮l of Fenton’s reagent + 20 ␮g of MCE, (5) DNA + 5 ␮l of Fenton’s reagent + 10 ␮g of MEE, (6) DNA + 5 ␮l of Fenton’s reagent + 20 ␮g of MEE, (7) DNA + 5 ␮l of Fenton’s reagent + 10 ␮g of gallic acid, (8) DNA + 5 ␮l of Fenton’s reagent + 20 ␮g of gallic acid.

Fig. 5. (A) shows cyclic voltamogram for H2 O2 and MCE at different concentration (50–250 ␮g) in PBS (pH 7.4) and (B) shows cyclic voltamogram for H2 O2 and MEE at different concentration (50–250 ␮g) in PBS (pH 7.4).

196

K.P. Rajesh et al. / Industrial Crops and Products 47 (2013) 186–198

Fig. 6. (A) RP-HPLC chromatogram of phenolic acids of MCE, (B) RP-HPLC chromatogram of phenolic acids of MEE, (C) RP-HPLC chromatogram of flavonoids of MCE and (D) RP-HPLC chromatogram of flavonoids of MEE.

Gallic acid, coumaric acid, ellagic, vanillic acid and three unknown peaks were detected in MCE at 220 nm (Fig. 6A) with retention time of 1.99, 3.42, 3.9 and 12.01 min, respectively, among them, gallic acid was the most abundant (215.01 mg g−1 ) phenolic constituent present in MCE. MEE showed peaks at 220 nm with retention time of 1.93, 3.76, 5.54 and 9.42 min (Fig. 6B). UV spectra of these peaks together with the analysis of retention time of standard molecules under identical experimental conditions indicate the presence of important antioxidant phenolic acids;

gallic acid, coumaric acid, ellagic acid and hydroxy benzoic acid, respectively. UV spectral peaks of MCE and MEE at 350 nm with the analysis of retention time of standard flavonoids showed the presence of rutin, quercetin and kaempferol in MCE with retention time 1.86, 2.6, and 7.4 min, respectively. MEE showed the presence of rutin, quercetin and myricetin (Fig. 6C and D). The presence of various antioxidant molecules was responsible for the antioxidant and protective activity of M. ferrea.

K.P. Rajesh et al. / Industrial Crops and Products 47 (2013) 186–198

197

Fig. 7. Protective ability of MCE and MEE against induced oxidative damage. (A) Pathogenesis of various stress related disorders via oxidative damage to erythrocytes, Hb and DNA. (B) Protective ability of MCE and MEE against induced damage by acting as antioxidant and electro catalyst.

4. Conclusion Overall result of the investigation accentuates the antioxidant and protective activity against induced oxidative damage in erythrocytes, DNA and Hb of M. ferrea (Fig. 7). The study has gathered substantial evidence of M. ferrea L. antioxidant property and also confirms the popular use of plant in ethnic medicine. Thus, M. ferrea L. can be a valuable source of antioxidant agent in pharmaceuticals. Conflicts of interest Authors declare that there are no conflicts of interest. Acknowledgments This work was supported by a grant from Department of Biotechnology (BT/PR13255/GBD/27/239/2009), Ministry of Science and Technology, Govt. of India, New Delhi. First author (K.P.R) thanks DBT for Junior Research Fellowship. Second author (H.M) express sincere thanks to DBT for RGYI award. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.indcrop. 2013.03.008. References Ajila, C.M., Prasada Rao, U.J.S., 2008. Protection against hydrogen peroxide induced oxidative damage in rat erythrocytes by Mangifera indica L. peel extract. Food Chem. Toxicol. 46, 303–309. Braca, A., Tommasi, N.D., Bari, L.D., Pizza, C., Politi, M., Morelli, I., 2001. Antioxidant principles from Bauhinia terapotensis. J. Nat. Prod. 64, 892–895.

Carini, M., Aldini, G., Bombardeelli, E., Morazzoni, P., Maffei Facino, R., 2000. UVB–induced hemolysis of rat erythrocytes: protective effect of procyanidins from grape seeds. Life Sci. 67, 1799–1814. Carter, P., 1971. Spectrophotometric determination of serum iron at the submicrogram level with a new reagent (ferrozine). Anal. Biochem. 40, 450–458. Chang, C., Yang, M., Wen, H., Chern, J., 2002. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. J. Food Drug Anal. 10, 178–182. Chen, C., Pearson, M.A., Gray, I.J., 1992. Effects of synthetic antioxidants (BHA, BHT and PG) on the mutagenicity of IQ-like compounds. Food Chem. 43, 177–183. Crichton, R.R., Wilmet, S., Legssyer, R., Ward, R.J., 2002. Molecular and cellular mechanisms of iron homeostasis and toxicity in mammalian cells. J. Inorg. Biochem. 91, 9–18. Action of phenolic derivatives Dinis, T., Madeira, V., Almeida, L., 1994. (acetaminophen, salicylate, and 5-amino salicylate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Arch. Biochem. Biophys. 315 (1), 161–169. Droge, W., 2002. Free radicals in the physiological control of cell function. Physiol. Rev. 282, 47–95. Fairbanks, G., Steck, T.L., Wallach, D.F.H., 1971. Electrophoretic analysis of the major polypeptide of the human erythrocyte membrane. Biochemistry 10, 2606–2617. Ghani, A., 2003. Medicinal Plants of Bangladesh with Chemical Constituents and Uses, second ed. Asiatic Society of Bangladesh, Bangladesh. Halliwell, B., Gutteridge, J.M.C., 2007. Free Radicals in Biology and Medicine, fourth ed. Oxford University Press, Oxford. Halliwell, B., Guttridge, J.M.C., 1989. Free Radicals in Biology and Medicine, second ed. Japan Scientific Societies Press, Tokyo, Japan. Khanna, V.G., Kannabiran, K., 2006. Larvicidal effect of Hemidesmus indicus, Gymnema sylvestre and Eclipta prostrata against Culex qinquifaciatus mosquito larvae. Afr. J. Biotechnol. 6, 307–311. Klein, S.M., Cohen, G., Cederbaum, A.I., 1991. Production of formaldehyde during metabolism of dimethyl sulphoxide by hydroxyl radical generating system. Biochemistry 20, 6006–6012. Krishnaiah, D., Sarbatl, R., Nithyanandam, R., 2011. A review of the antioxidant potential of medicinal plant species. Food Bioprod. Process. 89, 217–233. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lowry, O.H., Rosenberg, N.I., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin–phenol reagent. J. Biol. Chem. 193, 265–275. Marcocci, L., Packer, L., Sckaki, A., Albert, G.M., 1994. Antioxidant action of Ginko biloba extract EGB 761. Methods Enzymol. 234, 462–475. Megala, J., Geetha, A., 2010. Free radical-scavenging and H+ , K+ -ATPase inhibition activities of Pithecellobium dulce. Food Chem. 121, 1120–1128.

198

K.P. Rajesh et al. / Industrial Crops and Products 47 (2013) 186–198

Nishimiki, M., Rao, N.A., Yagi, K., 1972. The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem. Biophys. Res. Commun. 46, 849–853. Ong, W.Y., Halliwell, B., 2004. Iron, atherosclerosis, and neurodegeneration: a key role for cholesterol in promoting iron-dependent oxidative damage? Ann. N.Y. Acad. Sci. 1012, 51–64. Oyaizu, M., 1986. Studies on product of browning reaction prepared from glucose amine. Jpn. J. Nutr. 44, 307–315. Prieto, P., Pineda, M., Aguilar, M., 1999. Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. Anal. Biochem. 269, 337–341. Puppo, A., Halliwell, B., 1988. Formation of hydroxyl radicals from hydrogen peroxide in the presence of iron. Is hemoglobin a biological Fenton catalyst? Biochem. J. 249, 185–190. 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 Radic. Biol. Med. 26, 1231–1237. Sadrazadeh, S.M.H., Graf, E., Panter, S.S., Hallaway, P.E., Eaten, J.W., 1984. Hemoglobin, a biological Fenton reagent. J. Biol. Chem. 259, 14354–14356. Snyder, L.M., Fortier, N.L., Trainor, J., Jacobs, J., Lob, L., Lubin, B., Chiu, D., Shohet, S., Mohandas, N., 1985. Effect of hydrogen peroxide exposure on normal human erythrocyte deformability, morphology, surface characteristics, and spectrin–hemoglobin cross-linking. J. Clin. Invest. 76, 1971–1977.

Sukalski, K.A., LaBerge, T.P., Johnson, W.T., 1997. In vivo oxidative modification of erythrocyte membrane proteins in copper deficiency. Free Radic. Biol. Med. 22, 835–842. Suwalskya, M., Orellana, P., Avellob, M., Villenac, F., 2007. Protective effect of Ugni molinae Turcz against oxidative damage of human erythrocytes. Food Chem. Toxicol. 45, 130–135. Tedesco, I., Russo, M., Russo, P., Lacomino, G.G., Russo, G.L., Carrasturo, A., Faruolo, C., Mojo, L., Palumbo, R., 2000. Antioxidant effect of red wine polyphenols on red blood cells. J. Nutr. Biochem. 11, 114–119. Thomas, P.E., Ryan, D., Levin, W., 1976. An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels. Anal. Biochem. 75, 168–176. Vallelian, F., Pimenova, T., Pereira, C.P., Abraham, B., Mikolajczyk, M.G., Schoedon, G., Zenobi, R., Alayash, A.I., Buehler, P.W., Schaer, D.J., 2008. The reaction of hydrogen peroxide with hemoglobin induces extensive ␣-globin crosslinking and impairs the interaction of hemoglobin with endogenous scavenger pathways. Free Radic. Biol. Med. 45, 1150–1158. Yang, H.L., Chen, S.C., Chang, N.W., Chang, J.M., Lee, M.L., Tasi, P.E., Fu, H.H., Kao, W.W., Chiang, H.C., Wang, H.H., Hseu, Y.C., 2006. Protection from oxidative damage using Bidens pilosa extracts in normal human erythrocytes. Food Chem. Toxicol. 44, 1513–1521. Zhishen, J., Mengcheng, T., Jianming, W., 1999. The determination of flavonoid content in mulberry and their scavenging effects on superoxide radicals. Food Chem. 64, 555–559.