- Email: [email protected]
Protection of DNA and erythrocytes from free radical induced oxidative damage by black gram (Vigna mungo L.) husk extract
Food and Chemical Toxicology 50 (2012) 1690–1696
Contents lists available at SciVerse ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Protection of DNA and erythrocytes from free radical induced oxidative damage by black gram (Vigna mungo L.) husk extract Talakatta K. Girish, Padmaraju Vasudevaraju, Ummiti J.S. Prasada Rao ⇑ Department of Biochemistry and Nutrition, CSIR Unit, Central Food Technological Research Institute, Mysore 570 020, India
a r t i c l e
i n f o
Article history: Received 3 August 2011 Accepted 28 January 2012 Available online 6 February 2012 Keywords: Black gram husk extract Protection of DNA damage Protection of erythrocyte damage Scatchard plot Melting temperature of DNA Scanning electron microscopy
a b s t r a c t Antioxidants present in various plant tissues exhibit health benefits by scavenging reactive oxygen species generated under various pathophysiological conditions. In the present study, bioactive compounds from black gram husk were extracted with water and the protection of black gram husk (BGH) extract against oxidative damage in DNA and erythrocytes were studied. BGH extract had total polyphenol content of 59 mg of gallic acid equivalents (GAE). The phenolic acids identified in the extract using RP-HPLC were gallic, protocatechuic, gentisic and ferulic acids. The extract showed good antioxidant properties. The IC50 value for DPPH radical scavenging activity was found to be 3.92 lg of GAE. The BGH extract also showed a-glucosidase inhibition and the IC50 value was found to be 2.78 lg of GAE. The oxidative hemolysis caused by hydrogen peroxide in rat erythrocytes was inhibited by BGH extract in a dose dependent manner. The IC50 values for BGH extract and BHA for hemolysis were 11.5 and 14 lg of GAE, respectively. Morphological changes in erythrocyte membrane caused by hydrogen peroxide were protected by BGH extract. As BGH extract exhibited various antioxidant properties in different systems, it could be used as a functional food or nutraceutical product for health benefits. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Reactive oxygen species (ROS) such as hydrogen peroxide, superoxide anion and hydroxyl radical are generated under normal biological systems during aerobic metabolism and these ROS generated are scavenged by endogenous and exogenous cellular mechanisms (Ames et al., 1993). However, excessive generation of ROS takes place under certain pathophysiological conditions, also by exogenous agents like drugs, pollution, UV light and ionizing radiation, and the normal endogenous defense mechanism present in the living organism could not scavenge them completely. Therefore, the excessive ROS produced can damage cellular biomolecules like proteins, lipids and DNA resulting in pathological condition such as cancer, atherosclerosis and neurodegenerative diseases (Rice-Evans and Burdon, 1993; Barnham et al., 2004; Pardo-Andreu et al., 2006). Polyphenols present in various fruits, vegetables and grains are reported to possess good antioxidant properties and may protect cellular damage caused by ROS by scavenging them (Keith and Christy, 2010) and thus, protect against various diseases such as cancer, atherosclerosis, diabetes, inflammation and aging (Peinado et al., 2010). DNA damage is a common event in all living cells and free radical induced damage is a major form of damage. In recent years, there is a deep interest in identifying free radical scavengers
or antioxidants that inhibit DNA from oxidative damage (Peihong and Hongxiang, 2004). Erythrocytes 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 O2 transport associated with redox active hemoglobin molecules, which are potent promoters of reactive oxygen species (Sadrzadeh et al., 1984). Therefore, due to their susceptibility to oxidation, erythrocytes have been used as a cellular model to investigate oxidative damage in biomembranes. Black gram (Vigna mungo L.) belongs to the Leguminosae family, and is used for the preparation of a variety of products. It is mainly used in the form of dhal (cotyledon). During milling of black gram into dhal, husk (seed coat) is a waste by-product that constitutes about 9% of the grain. Legumes are valuable source of nutrients and phytochemicals, and they are distributed in different tissues. In the present study, the antioxidant properties, a-glucosidase inhibition of black gram husk extract (BGH) and its role in the prevention of DNA damage and protection against hydrogen peroxide induced oxidative damage in rat erythrocytes are reported. 2. Materials and methods 2.1. Materials
⇑ Corresponding author. Tel.: +91 821 2514876; fax: +91 821 2517233. E-mail address: [email protected] (U.J.S. Prasada Rao). 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2012.01.043
k-DNA was purchased from Bangalore Genei, India. Agarose, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Folin–Ciocalteu reagent, and trichloroacetic acid (TCA) were purchased from Sisco Research Laboratories.
T.K. Girish et al. / Food and Chemical Toxicology 50 (2012) 1690–1696 FeSO47H2O, H2O2, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, butylated hydroxyanisole (BHA), TRIS base, Na2EDTA, ethidium bromide, a-glucosidase, caffeic acid, p-coumaric acid, cinnamic acid, ferulic acid, gallic acid, gentisic acid, protocatechuic acid, syringic acid, vanillic acid and ethylenediaminetetraacetic acid (EDTA) were purchased from M/s Sigma Chemicals Co. (St. Louis, MO, USA). All other chemicals used were of analytical grade.
2.2. Preparation of aqueous extract of black gram husk and estimation of total polyphenols Black gram husk was separated from the seed according to the method described by Ajila and Prasada Rao (2009). Husk (1 g) was homogenized into paste using mortar and pestle in minimum volume of distilled water. The paste was suspended in 20 ml of water, stirred for 1 h and was centrifuged for 15 min at 8000g. The clear supernatant obtained was freeze-dried and used for further studies. The amount of the extract obtained for 1 g of BGH was found to be 82.6 ± 2.6 mg. Total polyphenol content in the extract was determined according to the method described by Swain and Hillis (1959). The content of total polyphenols in the extract was expressed as gallic acid equivalents (GAE).
1691
2.6. Prevention of k-DNA damage by BGH extract 2.6.1. Agarose gel electrophoresis The prevention of oxidative k-DNA damage by BGH extract was performed as previously described by Ghanta et al. (2007). k-DNA (0.5 lg), with and without BGH extract (5 lg GAE), was incubated with 1 mM FeSO4, 25 mM H2O2 in Tris buffer (10 mM, pH 7.4) in a final reaction volume of 20 ll for 1 h and 2 h at 37 °C. k-DNA was also incubated with 5 lg of BGH extract alone for 1 h. Samples were analyzed on 1% agarose gel prepared in Tris–acetate–EDTA buffer (pH 8.5) at 50 V for 4 h at room temperature. 2.6.2. EtBr binding to DNA by fluorescence analysis BGH extract protection against oxidative k-DNA damage was analyzed by measuring the changes in fluorescence of ethidium bromide (EtBr) binding to DNA. kDNA (1 lg) was incubated with 1 mM FeSO4, 25 mM H2O2 in 10 mM Tris–HCl (pH 7.4) for 1 h for the oxidative k-DNA damage and the protection against oxidative damage of k-DNA was analyzed in the presence of 5 lg of BGH aqueous extract for 1 h. k-DNA was also incubated with 5 lg of BGH extract alone for 1 h to know any DNA damage role by the extract itself. The samples thus prepared were mixed with 5 lg of EtBr and the fluorescence was recorded by exciting at 535 nm and emission at 600 nm.
2.3. Identification of phenolic compounds in aqueous extract by HPLC The BGH aqueous extract was extracted five times with ethyl acetate phase separation followed by drying with anhydrous sodium sulfite. Sodium sulfate was removed by filtration followed by evaporation to dryness and dissolved in methanol. Phenolic acids were separated on a reverse phase Luna C18 column (4.6 250 mm; 5 lm) using HPLC system (Agilent-Model 1200 series) using diode array detector (operating at 280 and 320 nm). A solvent system consisting of water/ methanol/acetic acid (83:15:2) was used as mobile phase (isocratic) at a flow rate of 1 ml/min (Glowniak et al., 1996). Known quantities of phenolic acid standards such as caffeic acid, p-coumaric acid, cinnamic acid, ferulic acid, gallic acid, gentisic acid, protocatechuic acid, syringic acid, vanillic acid were used for identification and quantification of phenolic acids present in the extract.
2.4. Measurement of antioxidant activity 2.4.1. Measurement of reducing power The reducing power of the BGH extract and BHA was determined according to the method described by Yen and Chen (1995). Extract containing 5–20 lg of gallic acid equivalents (GAE) were made up to 500 ll with 0.2 M phosphate buffer (pH 6.6) and mixed with 1 ml of potassium ferricyanide (0.1%) and the mixture was incubated at 50 °C for 20 min. Trichloroacetic acid (500 ll, 10%) was added to the reaction mixture and centrifuged at 3000g for 10 min. The supernatant obtained was mixed with equal volume of distilled water. To this mixture, 300 ll of 1% ferric chloride was added and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicates the increased reducing power. The antioxidant activity of the extract was compared with BHA.
2.4.2. Measurement of free radical scavenging activity The effect of aqueous extract of BGH on DPPH radical was determined according to the method described by Blois (1958) with the modifications described by BrandWilliams et al. (1995). A 100 lM solution of DPPH in methanol was prepared and BGH extract (200 ll) containing 1–5 lg GAE were mixed with 1 ml of DPPH solution. The mixture was shaken vigorously and left in the dark at room temperature for 20 min. The absorbance of the resulting solution was measured at 517 nm. The control contained all the reagents except sample extract/BHA. The capacity to scavenge DPPH radical was calculated using the following equation.
Scavenging activity ð%Þ ¼ 1 ðAs =A0 Þ 100 where A0 is the absorbance at 517 nm of the control and As is the absorbance in the presence of the sample extract/BHA. The results were plotted as the % of scavenging activity against the concentration of the sample. The half-inhibition concentration (IC50) was defined as the amount of GAE required for 50% of free radical scavenging activity. The IC50 value was calculated from the plots as the antioxidant concentration required for providing 50% free radical scavenging activity.
2.5. a-Glucosidase enzyme inhibition The enzyme inhibition assay mixture contained 50 ll of p-nitrophenyl-a-Dglucopyranoside (10 mg in 2 ml phosphate buffer), different concentrations of extract (inhibitor; 10 ll) and the reaction mixture was made up to 2.98 ml with sodium phosphate buffer (pH 6.8; 50 mM), The reaction was initiated by adding 20 ll of a-glucosidase enzyme (2 mg in 1 ml of phosphate buffer; 5.7 U/mg; Sigma Aldrich, USA). The reaction was monitored by increase in absorption at 405 nm and compared with the enzyme reaction without the extract.
2.6.3. Ethidium bromide binding analysis by Scatchard plots k-DNA (0.5 lg) was incubated with 1 mM FeSO4, 25 mM H2O2 in Tris buffer (10 mM, pH 7.4) with and without BGH extract (5 lg GAE) in a reaction volume of 20 ll for 1 h at 37 °C. The fluorescence was measured by titrating with increasing EtBr against constant amount of k-DNA. The maximum amount of EtBr bound per base pair of k-DNA was calculated using Scatchard plots of ‘r’ vs. ‘r/Cf’ in the DNA–EtBr reaction mixture at various titration intervals when increasing amount of EtBr was titrated to constant amount of DNA (Scatchard, 1949; Chatterjee and Rao, 1994). The concentration of bound EtBr in 1 ml dye–DNA mixture (Cb0 ) was calculated by using the following formula. 0
Cb ¼ ½Co0 ðF FoÞ=ðV FoÞ where, Cf0 = concentration of EtBr (pmoles) in the dye complex mixture; F = observed fluorescence of EtBr at any point of dye–DNA mixture; Fo = observed fluorescence of EtBr with no DNA; V = experimental value, ratio of bound EtBr to free EtBr at saturation point. The concentration of free EtBr (Cf0 ) was then calculated by using the formula. 0
Cf ¼ Co0 Cb
0
where, Cf0 , Co0 and Cb0 were expressed in pmoles. The amount of bound EtBr bound per base pair was calculated by r = Cb0 (pmoles)/DNA concentration (pmoles of base pair). A plot was made for r vs. r/Cf and the point where the straight line interacts the axis r was defined as the maximum amount of dye bound per base pair (n), where Cf = Cf0 1012 moles. 2.6.4. Melting temperature studies Thermal denaturation studies were performed to know the k-DNA integrity. kDNA (1 lg) was incubated with 1 mM FeSO4, 25 mM H2O2 in 10 mM Tris–HCl (pH 7.4) for 1 h for monitoring the oxidative k-DNA damage, with and without BGH extract (5 lg GAE), for 1 h. k-DNA also incubated with 5 lg of BGH aqueous extract alone for 1 h. The melting profiles (Tm) of k-DNA were recorded at different temperatures ranging from 25 to 95 °C using the Spectrophotometer (Ultraspec, 4300 probe) equipped with thermo-programmer and data processor (Amersham Pharmacia Biotech, Hong Kong). Tm values were determined graphically from the absorbance vs. temperature plots. 2.7. Preparation of erythrocytes All the animal experiments were carried out with the approval of institutional animal ethical committee. Male wistar rats in the body weight range of 180–220 g were housed in individual polypropylene cages and had free access to food and water. The animals were fed with standard diet. The animals were sacrificed under anesthesia and blood was collected by heart puncture in heparinized tubes. Erythrocytes were isolated and stored according to the method described by Yuan et al. (2005) and Yang et al. (2006). Briefly, blood samples collected were centrifuged (1500g, 5 min) at 4 °C, erythrocytes were separated from the plasma and buffy coat, and were washed three times by using 10 volumes of 20 mM phosphate buffered saline (pH 7.4; PBS). Each time the cell suspension centrifuged at 1500g for 5 min. The supernatant and buffy coats of white cells were carefully removed with each wash. Erythrocytes thus obtained were stored at 4 °C and used within 6 h for further studies. 2.8. In vitro assay of inhibition of rat erythrocyte hemolysis The inhibition of rat erythrocyte hemolysis by the BGH aqueous extract was evaluated according to the procedure described by Tedesco et al. (2000), with slight modifications. The rat erythrocyte hemolysis was performed with hydrogen
1692
T.K. Girish et al. / Food and Chemical Toxicology 50 (2012) 1690–1696
peroxide as free radical initiator. To 200 ll of 10% (v/v) suspension of erythrocytes in PBS, 50 ll of BGH extract with different concentrations (5–25 lg GAE in PBS pH 7.4) was added. To this, 100 ll of 200 lM H2O2 (in PBS pH 7.4) was added. The reaction mixture was incubated at 37 °C for 30 min and was centrifuged at 2000g for 10 min. The absorbance of the resulting supernatant was measured at 410 nm by taking 200 ll of reaction mixture with 800 ll PBS to determine the hemolysis. Likewise, the erythrocytes were treated with hydrogen peroxide and without inhibitors (BGH extract) to obtain a complete hemolysis. The absorbance of the supernatant was measured at the same condition. The inhibitory effect of the extract was compared with standard antioxidant BHA. Percentage of hemolysis was calculated by taking hemolysis caused by 200 lM hydrogen peroxide as 100%. The IC50 values were calculated from the plots as the antioxidant concentration required for the inhibition of 50% hemolysis. 2.9. Evaluation of oxidative damage on erythrocyte ghost membrane proteins by SDS– PAGE The erythrocyte ghost membranes were prepared by following the procedure of Kwant and Seeman (1970) by one step hemolysis. 1 ml of intact erythrocytes was added to 0.25% NaCl in 10 mM sodium phosphate buffer (pH 7.0). The ghost membrane suspension (100 lg protein) was subjected to oxidative damage using 50 ll of 100 lM H2O2 with or without BGH extract (5 lg GAE) incubated for 1 h. The oxidative modification on erythrocytes ghost membrane proteins were determined by SDS–PAGE on 7.5% discontinuous gel according to the method of Laemmli (1970) and the protein bands were visualized with Coomassie brilliant blue. 2.10. Protective effect on erythrocytes structural morphology Erythrocytes (50 ll) were incubated with and without BGH extract 50 ll; (10 lg GAE) and treated with 100 ll of 200 lM H2O2 for 30 min at 37 °C. After incubation, the incubate was centrifuged at 1500g for 10 min and the cell pellets were processed and were fixed in 3% glutaraldehyde on a coverslip (Agrawal and Sultana, 1993; Hoyer and Bucana, 1982). After fixing on the coverslip, the cells were dehydrated in an ascending series of acetone 0 (30–100%). The dried samples were mounted on an aluminum stubb (100–200 A Å) using double sided tape and coated with gold film with a thickness of 10–20 nm using sputter coater (Polaron, E 5000, SEM coating system). The cells were examined under a scanning electron microscope (Model No., LEO 425 VP, Electron microscopy Ltd., Cambridge, UK).
Fig. 1. HPLC chromatogram of BGH extract. (1) gallic acid; (2) protocatechuic acid; (3) gentisic acid; (4) ferulic acid.
Table 1 Free phenolic content in BGH aqueous extract. Phenolic acid
Free phenolic acid contents (lg/g of husk)
Gallic acid Protocatechuic acid Gentisic acid Ferulic acid
88.77 ± 1.22c 38.59 ± 2.19a 69.52 ± 2.22b 416.51 ± 5.39d
Values are mean ± SD (n = 3). Means with different letters within a column for each extract are significantly different at p < 0.05.
2.11. Statistical analysis Three independent experiments were conducted in triplicate and the data were reported as mean ± SD. Duncan’s new multiple range test was used to determine the difference of means, and P < 0.05 was considered to statistically significant (Steel and Torrie, 1980).
3. Results and discussion 3.1. Polyphenol content in BGH aqueous extract and identification of phenolic acids Polyphenols are the major group of compounds that contribute to the antioxidant properties. The total phenolic content in the black gram husk water extract was found to be 58.83 mg GAE/g. The polyphenols in BGH extract were separated on reverse phase C18 column on HPLC. Gallic acid, protocatechuic acid, gentisic acid and ferulic acid were the phenolic acids identified in BGH extract (Fig. 1) Ferulic acid was the major phenolic acid followed by gallic acid and gentisic acid (Table 1). 3.2. Antioxidant activity The antioxidant activity of polyphenols is due to the reactivity of phenol moiety (hydroxyl group on aromatic ring). Polyphenols have the ability to scavenge free radicals via hydrogen donation or electron donation (Shahidi and Wanasundara, 1992). In the present study, two different methods viz., reducing power assay and the DPPH radical scavenging activity were used to measure the antioxidant activity of BGH extract. 3.2.1. Reducing power of BGH extract The reducing power of a compound is related to its electron transfer ability and may, therefore, serve as a significant indicator
Fig. 2. Reducing power of the aqueous BGH extract.
of its antioxidant activity (Meir et al., 1995). The reducing power increased with the concentration of the BGH aqueous extract (Fig. 2). At 20 lg GAE concentration, BGH extract and BHA showed an absorbance of 0.363 and 0.412, respectively. The antioxidant property of the extract is mainly due to the presence of polyphenols. It has been reported that polyphenols are electron donors and could reduce Fe3+/ferricyanide complex to ferrous form (Chung et al., 2002; Yen and Chen, 1995).
T.K. Girish et al. / Food and Chemical Toxicology 50 (2012) 1690–1696
1693
3.2.2. DPPH free radical scavenging effect of BGH aqueous extract Scavenging the stable DPPH radical is another widely used method to evaluate antioxidant activity. DPPH is a stable free radical with characteristic absorption at 517 nm and antioxidants react with DPPH and convert it to 2,2-diphenyl-1-picrylhydrazine. The degree of discoloration indicates the scavenging potential of the antioxidant extract, which is due to the hydrogen donating ability (Van Gadow et al., 1997). The DPPH radical scavenging activity of BGH aqueous extract increased with the increased concentration of phenols in BGH extract, which may be attributed due to their hydrogen donating ability. The IC50 value for BGH extract was found to be 3.92 lg of GAE, while that of BHA was found to be 3.58 lg. Thus, the free radical scavenging activity of BGH extract is comparable to BHA, a synthetic antioxidant. 3.3. a-Glucosidase inhibitory activity of BGH aqueous extract Type-2 diabetes is caused due to impaired secretion of insulin resulting in increased postprandial glucose level. One of the approaches to decrease postprandial glucose level is to inhibit the a-glucosidase enzyme. The inhibition of this enzyme decreases the blood glucose levels and involved in the management of type-2 diabetes. Puls et al. (1977) and Shim et al. (2003) have hypothesized that by inhibiting this enzyme can regulate glucose uptake from intestinal lumen by inhibiting carbohydrate digestion and absorption, leading to normal glucose homeostasis in diabetic subjects. a-Glucosidase inhibitors from natural food sources is an attractive strategy to manage postprandial hyperglycemia. In this study, the extract from black gram husk was studied for their inhibitory activity of the enzyme. The inhibition by the extract was dose dependent (Fig. 3). The results indicated that BGH aqueous extract showed better enzyme inhibitory activity with the IC50 value of 2.78 lg.
Fig. 3. a-Glucosidase inhibition by BGH extract. Values are mean ± SD (n = 3). Means with different letters are significantly different at p < 0.05.
3.4. Inhibition of Fe2+ induced k-DNA damage by BGH aqueous extract 3.4.1. Agarose gel studies As the BGH aqueous extract showed potential antioxidant properties, it was tested for its ability to inhibit DNA damage caused by hydroxyl radicals that are generated by FeSO4 and H2O2 using agarose gel electrophoresis. The hydroxyl radicals attack hydrogen atoms of k-DNA leading to the formation of nicks in DNA which results single strand and double strand breaks in the k-DNA (Balasubramanian et al., 1998). Incubation of k-DNA with FeSO4 and H2O2 for 1 h resulted a decrease in the band intensity by 80% (Fig. 4A, lane 2). Further incubation of k-DNA with FeSO4 and H2O2 for 2 h, caused the total disappearance of k-DNA band indicating complete k-DNA damage in 2 h (Fig. 4B, lane 2). However, in the presence of BGH aqueous extract, the k-DNA damage by FeSO4/H2O2 system was not observed. As can be seen from Fig. 4A and B (lanes 3), DNA treated with FeSO4/H2O2 system for 1 and 2 h in presence of BGH extract, the DNA band intensity is similar to control (Fig. 4A and B, lanes 1) indicating the prevention of DNA damage by the extract. k-DNA in the presence of BGH aqueous extract alone was intact (Fig. 4A and B, lane 4) for 1 and 2 h incubation indicating that extract did not have any DNA damage property. Thus, the results indicate that BGH extract prevents DNA damage against oxidative damage. The preventive capability may be due to free radical scavenging activity of polyphenols present in aqueous BGH extract. 3.4.2. EtBr Binding and titration studies In the previous experiment we have demonstrated that FeSO4/ H2O2 system has caused damage in k-DNA. To further characterize the k-DNA damage by FeSO4/H2O2 system and its protection by
Fig. 4. Gel electrophoresis image of DNA damage inhibition by BGH extract. (A) After 1 h incubation of reaction mixture; (B) after 2 h incubation of reaction mixture. Lanes 1: 0.5 lg DNA alone; Lane 2: 0.5 lg DNA + 1 mM FeSO4 + 25 mM H2O2; Lane 3: 0.5 lg DNA + 0.5 mM FeSO4 + 25 mM H2O2 + 5 lg aqueous BGH; Lane 4: 0.5 lg DNA + 5 lg aqueous BGH extract.
BGH aqueous extract, changes in fluorescence of EtBr upon binding to k-DNA were carried out. EtBr fluorescence in the presence of intact k-DNA was 18.3 (Fig. 5A), whereas it was 12.6 (Fig. 5B) for k-DNA treated with FeSO4 in the presence of H2O2. The significant decrease in the fluorescence intensity in the case of k-DNA treated with FeSO4 and H2O2 was due to the damage of DNA by hydroxyl radicals generated. However, the fluorescence intensity for DNA in the presence of FeSO4/H2O2 and BGH extract was increased to 15.3 (Fig. 5C), and the increase in the fluorescence intensity may be due to the prevention of DNA damage by free radical scavenging activity of polyphenols present in BGH aqueous extract. The fluorescence intensity for DNA in the presence of BGH extract alone was 18.1 (Fig. 5D) and this value is similar to control DNA sample indicating that BGH extract did not damage DNA. In order to further study the k-DNA damage protection by BGH extract, we have measured the differences in binding of EtBr to intact k-DNA and damaged k-DNA. The amount of EtBr molecules bound per base pair (bp) of DNA was determined by EtBr titration studies. Three representative Scatchard plots of ‘r vs. r/Cf’ for k-DNA are shown in Fig. 6. The number of EtBr molecules bound/
1694
T.K. Girish et al. / Food and Chemical Toxicology 50 (2012) 1690–1696 Table 2 Melting temperature (Tm) of DNA. Sample
Tm value
DNA DNA + FeSO4 + H2O2 DNA + BGH extract + FeSO4 + H2O2 DNA + BGH extract
70.46 °C ± 0.50b 51.66 °C ± 1.40a 74.66 °C ± 1.04c 82.73 °C ± 1.10d
Values are mean ± SD (n = 3). Means with different letters within a column for each extract are significantly different at p < 0.05.
Fig. 5. EtBr binding to DNA. (A) k-DNA; (B) k-DNA + FeSO4 + H2O2; (C) kDNA + aqueous BGH extract + FeSO4 + H2O2; (D) k-DNA + aqueous BGH extract. Values are mean ± SD (n = 3). Means with different letters are significantly different at p < 0.05.
DNA alone
4500000000
DNA + FeSO4 +H2O2
4000000000
DNA + BGH + FeSO4 + H2O2
3500000000
r/Cf
3000000000 2500000000 2000000000
Fig. 7. In vitro protective effects of BGH extract against H2O2 induced hemolysis of rat erythrocytes.
1500000000 1000000000 500000000 0
0
0.5
1
1.5
2
r
2.5
3
3.5
4
4.5
Fig. 6. Scatchard plot of ethidium bromide binding to DNA. (d) DNA, (j) DNA + FeSO4 + H2O2, (N) DNA + BGH extract + FeSO4 + H2O2. Fluorescent measurements were done at room temperature setting excitation at 535 nm and emission at 600 nm. The Scatchard plot was drawn using least square method.
bp of DNA (control) was 4.1 (Fig. 6d), whereas it was 2.2 (Fig. 6j) for k-DNA treated with FeSO4/H2O2. The number of EtBr molecules bound per base pair of k-DNA decreased because of oxidative damage of DNA. However, in the presence of aqueous BGH extract, FeSO4/H2O2, the number of EtBr molecules bound per base pair of k-DNA was comparable to the value obtained with intact k-DNA (3.9) (Fig. 6N). Thus, the results confirm that the BGH extract prevents the DNA damage by FeSO4/H2O2. 3.4.3. Melting temperature (Tm) studies The melting temperature study provides an insight on the integrity of DNA. The melting temperature (Tm) of k-DNA was 70.4 °C, where as it was 51 °C, when it was treated with FeSO4/H2O2 (Table 2). Low Tm in the case of k-DNA treated with FeSO4/H2O2 was due to the damage of DNA in presence of hydroxyl radicals. However, the Tm value for k-DNA in presence of BGH extract and FeSO4/H2O2 was 74.5 °C. The higher Tm of k-DNA in the presence of BGH extract indicates that oxidative damage was prevented by the extract. Tm value for DNA in presence of BGH extract alone was found to be 84 °C
indicating k-DNA is stable in presence of aqueous BGH extract. Thus, the studies revealed that BGH extract prevented the oxidative DNA damage caused by free radicals. The mechanism of prevention may be due to the scavenging of free radicals by the polyphenols present in the aqueous extract. 3.5. Inhibition of rat erythrocytes hemolysis Oxidants lyse the erythrocyte and results in the release of hemoglobin pigment into the medium. The measurement of color mediated by hemoglobin at 410 nm will give a measure of oxidant damage in cells. In the present study, H2O2 hemolysis was induced in erythrocytes and studied the effect of BGH extract on its protection against hemolysis. As can be seen from Fig. 7, BGH extract inhibited the rat erythrocytes hemolysis in a dose dependent manner. As compared to with BHA, a synthetic antioxidant, BGH extract showed a better inhibition for erythrocyte inhibition. The IC50 values for the BGH extract and BHA were 11.5 and 14 lg, respectively. BGH extract alone was tested and found that it did not show any harmful effect on erythrocytes. The percentage of hemolysis of BGH alone was found to be 3.03% which is comparable to control (1.64%). 3.6. Protection of oxidative damage on RBC ghost membrane proteins The changes in the protein pattern of membrane protein produced by oxidative stress were observed by SDS–PAGE. Ghost membrane was prepared by hypotonic lysis of normal erythrocytes. Membranes were treated with H2O2 and with or without BGH extract and were analyzed on SDS–PAGE (Fig. 8) (Ajila and
T.K. Girish et al. / Food and Chemical Toxicology 50 (2012) 1690–1696
1695
even after 1 h incubation with H2O2 (Fig. 8, lanes 3, 4). Thus, the results indicate that the BGH extract effectively prevented the membrane proteins from oxidation. 3.7. Protective effect on RBC structural morphology
Fig. 8. SDS–PAGE of RBC membrane proteins showing protective effects of BGH extract against H2O2 induced oxidative damage on erythrocyte ghost membrane. Lane 1: membrane proteins treated with H2O2, Lane 2: membrane proteins (untreated), Lane 3: membrane proteins treated with H2O2 in presence of BGH extract, Lane 4: membrane proteins treated with H2O2 in presence of BHA.
Scanning electron micrographs of erythrocytes treated in vitro with H2O2 and BGH aqueous extract are shown in Fig. 9. Untreated erythrocytes appeared as typical discocytes while exposure to H2O2 resulted in a significant change in the cell shape and distinct echinocyte formation. The morphological changes induced by oxidative system were prevented when the cells were treated with BGH extract and BHA. Oxidative damage to cell membrane leads to alterations in cell rigidity and shape and results in the formation of echinocytes. The cell damage thus occurred ultimately affects the functioning of erythrocytes (Linderkamp et al., 1997). According to bilayer couple hypothesis, the changes in erythrocyte shape induced by foreign molecules are due to differential expansion of two monolayers of the red cell membrane (Sheetz and Singer, 1974; Lim et al., 2002) Thus, stomatocytes are formed when the compounds inserts into the inner monolayer, whereas speculated-shaped echinocytes are produced when it locates into the outer moiety. The results in the present study indicate that BGH extract prevents the morphological changes induced by oxidants in erythrocytes. 4. Conclusions The present study shows that the black gram husk aqueous extract is rich in polyphenols. The extract exhibited antioxidant properties, inhibited a-glucosidase activity. It also protected DNA and erythrocyte from oxidative damage. As the black gram husk extract showed various antioxidant properties in different systems, it could be used as a functional food ingredient or nutraceutical product for health benefits. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements Authors are thankful to Dr. P.V. Salimath for encouragement in this study. The authors also thank Mr. K. Anbalagan for his assistance in scanning electron microscopy. References
Fig. 9. Scanning electron micrograph of normal erythrocytes and protective effect BGH and BHA against H2O2 induced oxidative damage on RBC 10,000.
Prasada Rao, 2008). Upon treatment with oxidant, most of the bands present above actin bands were diminished (Fig. 8, lane 1). However, in presence of BGH extracts and BHA, protein bands 1, 2, 3, 4 and actin bands were clearly present in the membranes,
Agrawal, D., Sultana, P., 1993. Biochemical and structural alterations in rat erythrocytes due to hexachlorocyclohexane exposure. Food Chem. Toxicol. 31, 443–448. 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. Ajila, C.M., Prasada Rao, U.J.S., 2009. Purification and characterization of black gram (Vigna mungo) husk peroxidase. J. Mol. Catal. B Enzym. 60, 36–44. Ames, B.N., Shigenaga, M.K., Hagen, T.M., 1993. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. USA 90, 7915–7922. Balasubramanian, B., Pogozelski, W.K., Tullius, T.D., 1998. DNA strand breaking by the hydroxyl radical is governed by the accessible surface areas of the hydrogen atoms of the DNA backbone. Proc. Natl. Acad. Sci. USA 95, 9738–9743. Barnham, K.J., Masters, C.L., Bush, A.I., 2004. Neurodegenerative diseases and oxidative stress. Nature Rev. Drug Disc. 3, 205–214. Blois, M.S., 1958. Antioxidant determinations by the use of a stable free radical. Nature 181, 1199–1200. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity. Lebensm. Wiss. Technol. 28, 25–30. Chatterjee, B., Rao, G.R.K., 1994. Superhelical density of goat mitochondrial DNA: fluorescent studies. Indian J Biochem. Biophys. 31, 77–79.
1696
T.K. Girish et al. / Food and Chemical Toxicology 50 (2012) 1690–1696
Chung, Y.C., Chang, C.T., Chao, W.W., Lin, C.F., Chou, S.T., 2002. Antioxidant activity and safety of the 50% ethanolic extract from red bean fermented by Bacillus subtillus IMR-NK1. J. Agric. Food Chem. 50, 2454–2458. Ghanta, S., Banerjee, A., Poddar, A., Chattopadhyay, S., 2007. Oxidative DNA damage preventive activity and antioxidant potential of Stevia rebaudiana (Bertoni) Bertoni, a natural sweetener. J. Agric. Food Chem. 55, 10962–10967. Glowniak, K., Zgorka, G., Kozyra, M., 1996. Solid-phase extraction and reversed phase high-performance liquid chromatography of free phenolic acids in some Echinacea species. J. Chromatogr. 730, 25–29. Hoyer, L.C., Bucana, C., 1982. Principles of immuno-electron microscopy. In: Marchalories, J.J., Warr, G.W. (Eds.), Antibodies as a tool. John Wiley, London, pp. 257–258. Keith, R.M., Christy, L.A., 2010. Polyphenols as dietary supplements: a double-edged sword. Nutr. Dietary Suppl. 2, 1–12. Kwant, W.O., Seeman, P., 1970. The erythrocyte ghost is a perfect osmometer. J. Gen. Physiol. 55, 208–219. Laemmli, U.K., 1970. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature 227, 680–685. Lim, G., Wortis, M., Mukhopadhyay, R., 2002. Stomatocyte-discocyte echinocyte sequence of the human red blood cell: evidence for the bilayer-couple hypothesis from membrane mechanics. Proc. Natl. Acad. Sci. USA 99, 16766– 16769. Linderkamp, O., Kiau, U., Ruef, P., 1997. Cellular and membrane deformability of red blood cells in protein infants with and without growth retardation. Clin. Hemorheol. Microcirc. 17, 279–283. Meir, S., Kanner, J., Akiri, B., Hadas, S.P., 1995. Determination and involvement of aqueous reducing compounds in oxidative defensive system of various senescing leaves. J. Agric. Food Chem. 43, 1813–1815. Pardo-Andreu, G.L., Philip, S.J., Riaño, A., Sánchez, C., Viada, C., Núñez-Sellés, A.J., Delgado, R., 2006. Mangifera indica L. (Vimang) protection against serum oxidative stress in elderly humans. Arch. Med. Res. 37, 158–164. Peihong, F., Hongxiang, L., 2004. Effects of polyphenols from grape seeds on oxidative damage to cellular DNA. Mol. Cellular Biochem. 267, 67–74. Peinado, J., Lo´pez de Lerma, N., Peinado, R.A., 2010. Synergistic antioxidant interaction between sugars and phenolics from a sweet wine. Eur. Food Res. Technol. 231, 363–370.
Puls, W., Keup, U., Krause, H.P., Thomas, G., Hoffmeister, F., 1977. Glucosidase inhibition. A new approach to the treatment of diabetes, obesity, and hyperlipoproteinaemia. Naturwissenschaften 64, 536–537. Rice-Evans, C., Burdon, R., 1993. Free radical-lipid interactions and their pathological consequences. Progress in Lipid Res. 32, 71–110. Sadrzadeh, S.M., Graf, E., Panter, S.S., Hallaway, P.E., Eaton, J.W., 1984. Hemoglobin. A biologic fenton reagent. J. Biol. Chem. 259, 14354–14356. Scatchard, G., 1949. The attraction of proteins for small molecules and ions. Ann. N Y Acad. Sci. 51, 660–672. Shahidi, F., Wanasundara, P.K.J.P.D., 1992. Phenolic antioxidants. Crit. Rev. Food Sci. Nutr. 32, 67–103. Sheetz, M.P., Singer, S.J., 1974. Biological membranes as bilayer couples. A molecular mechanism of drug-erythrocyte induced interactions. Proc. Natl. Acad. Sci. USA 71, 4457–4461. Shim, Y.J., Doo, H.K., Ahn, S.Y., Kim, Y.S., Seong, J.K., Park, I.S., Min, B.H., 2003. Inhibitory effect of aqueous extract from the gall of Rhus chinensis on alpha-glucosidase activity and postprandial blood glucose. J. Ethnopharmacol. 85, 283–287. Steel, R.G.D., Torrie, J.H., 1980. Principles and procedures of statistics. McGraw, New York. Swain, T., Hillis, W.E., 1959. The phenolic constituents of Prunus domestica. 1. The quantitative analysis of phenolic constituents. J. Sci. Food Agric. 10, 63–68. 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 blosod cells. J. Nutr. Biochem 11, 114–119. Van Gadow, A., Joubert, E., Hannsman, C.T., 1997. Comparison of the antioxidant activity of aspalathin with that of other plant phenols of roolbos tea (Aspalathus linearis), a-tocoferol, BHT, and BHA. J. Agric. Food Chem. 45, 632–638. 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. Yen, G.C., Chen, H.Y., 1995. Antioxidant activity of various tea extracts in relation to their antimutagenicity. J. Agric. Food Chem. 43, 27–32. Yuan, X., Wang, J., Yao, H., Chen, F., 2005. Free radical-scavenging capacity and inhibitory activity on rat erythrocytes hemolysis of feruloyl oligosaccharides from wheat bran insoluble dietary fiber. Lebensm. Wiss. Technol. 38, 877–883.
Contents lists available at SciVerse ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Protection of DNA and erythrocytes from free radical induced oxidative damage by black gram (Vigna mungo L.) husk extract Talakatta K. Girish, Padmaraju Vasudevaraju, Ummiti J.S. Prasada Rao ⇑ Department of Biochemistry and Nutrition, CSIR Unit, Central Food Technological Research Institute, Mysore 570 020, India
a r t i c l e
i n f o
Article history: Received 3 August 2011 Accepted 28 January 2012 Available online 6 February 2012 Keywords: Black gram husk extract Protection of DNA damage Protection of erythrocyte damage Scatchard plot Melting temperature of DNA Scanning electron microscopy
a b s t r a c t Antioxidants present in various plant tissues exhibit health benefits by scavenging reactive oxygen species generated under various pathophysiological conditions. In the present study, bioactive compounds from black gram husk were extracted with water and the protection of black gram husk (BGH) extract against oxidative damage in DNA and erythrocytes were studied. BGH extract had total polyphenol content of 59 mg of gallic acid equivalents (GAE). The phenolic acids identified in the extract using RP-HPLC were gallic, protocatechuic, gentisic and ferulic acids. The extract showed good antioxidant properties. The IC50 value for DPPH radical scavenging activity was found to be 3.92 lg of GAE. The BGH extract also showed a-glucosidase inhibition and the IC50 value was found to be 2.78 lg of GAE. The oxidative hemolysis caused by hydrogen peroxide in rat erythrocytes was inhibited by BGH extract in a dose dependent manner. The IC50 values for BGH extract and BHA for hemolysis were 11.5 and 14 lg of GAE, respectively. Morphological changes in erythrocyte membrane caused by hydrogen peroxide were protected by BGH extract. As BGH extract exhibited various antioxidant properties in different systems, it could be used as a functional food or nutraceutical product for health benefits. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Reactive oxygen species (ROS) such as hydrogen peroxide, superoxide anion and hydroxyl radical are generated under normal biological systems during aerobic metabolism and these ROS generated are scavenged by endogenous and exogenous cellular mechanisms (Ames et al., 1993). However, excessive generation of ROS takes place under certain pathophysiological conditions, also by exogenous agents like drugs, pollution, UV light and ionizing radiation, and the normal endogenous defense mechanism present in the living organism could not scavenge them completely. Therefore, the excessive ROS produced can damage cellular biomolecules like proteins, lipids and DNA resulting in pathological condition such as cancer, atherosclerosis and neurodegenerative diseases (Rice-Evans and Burdon, 1993; Barnham et al., 2004; Pardo-Andreu et al., 2006). Polyphenols present in various fruits, vegetables and grains are reported to possess good antioxidant properties and may protect cellular damage caused by ROS by scavenging them (Keith and Christy, 2010) and thus, protect against various diseases such as cancer, atherosclerosis, diabetes, inflammation and aging (Peinado et al., 2010). DNA damage is a common event in all living cells and free radical induced damage is a major form of damage. In recent years, there is a deep interest in identifying free radical scavengers
or antioxidants that inhibit DNA from oxidative damage (Peihong and Hongxiang, 2004). Erythrocytes 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 O2 transport associated with redox active hemoglobin molecules, which are potent promoters of reactive oxygen species (Sadrzadeh et al., 1984). Therefore, due to their susceptibility to oxidation, erythrocytes have been used as a cellular model to investigate oxidative damage in biomembranes. Black gram (Vigna mungo L.) belongs to the Leguminosae family, and is used for the preparation of a variety of products. It is mainly used in the form of dhal (cotyledon). During milling of black gram into dhal, husk (seed coat) is a waste by-product that constitutes about 9% of the grain. Legumes are valuable source of nutrients and phytochemicals, and they are distributed in different tissues. In the present study, the antioxidant properties, a-glucosidase inhibition of black gram husk extract (BGH) and its role in the prevention of DNA damage and protection against hydrogen peroxide induced oxidative damage in rat erythrocytes are reported. 2. Materials and methods 2.1. Materials
⇑ Corresponding author. Tel.: +91 821 2514876; fax: +91 821 2517233. E-mail address: [email protected] (U.J.S. Prasada Rao). 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2012.01.043
k-DNA was purchased from Bangalore Genei, India. Agarose, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Folin–Ciocalteu reagent, and trichloroacetic acid (TCA) were purchased from Sisco Research Laboratories.
T.K. Girish et al. / Food and Chemical Toxicology 50 (2012) 1690–1696 FeSO47H2O, H2O2, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, butylated hydroxyanisole (BHA), TRIS base, Na2EDTA, ethidium bromide, a-glucosidase, caffeic acid, p-coumaric acid, cinnamic acid, ferulic acid, gallic acid, gentisic acid, protocatechuic acid, syringic acid, vanillic acid and ethylenediaminetetraacetic acid (EDTA) were purchased from M/s Sigma Chemicals Co. (St. Louis, MO, USA). All other chemicals used were of analytical grade.
2.2. Preparation of aqueous extract of black gram husk and estimation of total polyphenols Black gram husk was separated from the seed according to the method described by Ajila and Prasada Rao (2009). Husk (1 g) was homogenized into paste using mortar and pestle in minimum volume of distilled water. The paste was suspended in 20 ml of water, stirred for 1 h and was centrifuged for 15 min at 8000g. The clear supernatant obtained was freeze-dried and used for further studies. The amount of the extract obtained for 1 g of BGH was found to be 82.6 ± 2.6 mg. Total polyphenol content in the extract was determined according to the method described by Swain and Hillis (1959). The content of total polyphenols in the extract was expressed as gallic acid equivalents (GAE).
1691
2.6. Prevention of k-DNA damage by BGH extract 2.6.1. Agarose gel electrophoresis The prevention of oxidative k-DNA damage by BGH extract was performed as previously described by Ghanta et al. (2007). k-DNA (0.5 lg), with and without BGH extract (5 lg GAE), was incubated with 1 mM FeSO4, 25 mM H2O2 in Tris buffer (10 mM, pH 7.4) in a final reaction volume of 20 ll for 1 h and 2 h at 37 °C. k-DNA was also incubated with 5 lg of BGH extract alone for 1 h. Samples were analyzed on 1% agarose gel prepared in Tris–acetate–EDTA buffer (pH 8.5) at 50 V for 4 h at room temperature. 2.6.2. EtBr binding to DNA by fluorescence analysis BGH extract protection against oxidative k-DNA damage was analyzed by measuring the changes in fluorescence of ethidium bromide (EtBr) binding to DNA. kDNA (1 lg) was incubated with 1 mM FeSO4, 25 mM H2O2 in 10 mM Tris–HCl (pH 7.4) for 1 h for the oxidative k-DNA damage and the protection against oxidative damage of k-DNA was analyzed in the presence of 5 lg of BGH aqueous extract for 1 h. k-DNA was also incubated with 5 lg of BGH extract alone for 1 h to know any DNA damage role by the extract itself. The samples thus prepared were mixed with 5 lg of EtBr and the fluorescence was recorded by exciting at 535 nm and emission at 600 nm.
2.3. Identification of phenolic compounds in aqueous extract by HPLC The BGH aqueous extract was extracted five times with ethyl acetate phase separation followed by drying with anhydrous sodium sulfite. Sodium sulfate was removed by filtration followed by evaporation to dryness and dissolved in methanol. Phenolic acids were separated on a reverse phase Luna C18 column (4.6 250 mm; 5 lm) using HPLC system (Agilent-Model 1200 series) using diode array detector (operating at 280 and 320 nm). A solvent system consisting of water/ methanol/acetic acid (83:15:2) was used as mobile phase (isocratic) at a flow rate of 1 ml/min (Glowniak et al., 1996). Known quantities of phenolic acid standards such as caffeic acid, p-coumaric acid, cinnamic acid, ferulic acid, gallic acid, gentisic acid, protocatechuic acid, syringic acid, vanillic acid were used for identification and quantification of phenolic acids present in the extract.
2.4. Measurement of antioxidant activity 2.4.1. Measurement of reducing power The reducing power of the BGH extract and BHA was determined according to the method described by Yen and Chen (1995). Extract containing 5–20 lg of gallic acid equivalents (GAE) were made up to 500 ll with 0.2 M phosphate buffer (pH 6.6) and mixed with 1 ml of potassium ferricyanide (0.1%) and the mixture was incubated at 50 °C for 20 min. Trichloroacetic acid (500 ll, 10%) was added to the reaction mixture and centrifuged at 3000g for 10 min. The supernatant obtained was mixed with equal volume of distilled water. To this mixture, 300 ll of 1% ferric chloride was added and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicates the increased reducing power. The antioxidant activity of the extract was compared with BHA.
2.4.2. Measurement of free radical scavenging activity The effect of aqueous extract of BGH on DPPH radical was determined according to the method described by Blois (1958) with the modifications described by BrandWilliams et al. (1995). A 100 lM solution of DPPH in methanol was prepared and BGH extract (200 ll) containing 1–5 lg GAE were mixed with 1 ml of DPPH solution. The mixture was shaken vigorously and left in the dark at room temperature for 20 min. The absorbance of the resulting solution was measured at 517 nm. The control contained all the reagents except sample extract/BHA. The capacity to scavenge DPPH radical was calculated using the following equation.
Scavenging activity ð%Þ ¼ 1 ðAs =A0 Þ 100 where A0 is the absorbance at 517 nm of the control and As is the absorbance in the presence of the sample extract/BHA. The results were plotted as the % of scavenging activity against the concentration of the sample. The half-inhibition concentration (IC50) was defined as the amount of GAE required for 50% of free radical scavenging activity. The IC50 value was calculated from the plots as the antioxidant concentration required for providing 50% free radical scavenging activity.
2.5. a-Glucosidase enzyme inhibition The enzyme inhibition assay mixture contained 50 ll of p-nitrophenyl-a-Dglucopyranoside (10 mg in 2 ml phosphate buffer), different concentrations of extract (inhibitor; 10 ll) and the reaction mixture was made up to 2.98 ml with sodium phosphate buffer (pH 6.8; 50 mM), The reaction was initiated by adding 20 ll of a-glucosidase enzyme (2 mg in 1 ml of phosphate buffer; 5.7 U/mg; Sigma Aldrich, USA). The reaction was monitored by increase in absorption at 405 nm and compared with the enzyme reaction without the extract.
2.6.3. Ethidium bromide binding analysis by Scatchard plots k-DNA (0.5 lg) was incubated with 1 mM FeSO4, 25 mM H2O2 in Tris buffer (10 mM, pH 7.4) with and without BGH extract (5 lg GAE) in a reaction volume of 20 ll for 1 h at 37 °C. The fluorescence was measured by titrating with increasing EtBr against constant amount of k-DNA. The maximum amount of EtBr bound per base pair of k-DNA was calculated using Scatchard plots of ‘r’ vs. ‘r/Cf’ in the DNA–EtBr reaction mixture at various titration intervals when increasing amount of EtBr was titrated to constant amount of DNA (Scatchard, 1949; Chatterjee and Rao, 1994). The concentration of bound EtBr in 1 ml dye–DNA mixture (Cb0 ) was calculated by using the following formula. 0
Cb ¼ ½Co0 ðF FoÞ=ðV FoÞ where, Cf0 = concentration of EtBr (pmoles) in the dye complex mixture; F = observed fluorescence of EtBr at any point of dye–DNA mixture; Fo = observed fluorescence of EtBr with no DNA; V = experimental value, ratio of bound EtBr to free EtBr at saturation point. The concentration of free EtBr (Cf0 ) was then calculated by using the formula. 0
Cf ¼ Co0 Cb
0
where, Cf0 , Co0 and Cb0 were expressed in pmoles. The amount of bound EtBr bound per base pair was calculated by r = Cb0 (pmoles)/DNA concentration (pmoles of base pair). A plot was made for r vs. r/Cf and the point where the straight line interacts the axis r was defined as the maximum amount of dye bound per base pair (n), where Cf = Cf0 1012 moles. 2.6.4. Melting temperature studies Thermal denaturation studies were performed to know the k-DNA integrity. kDNA (1 lg) was incubated with 1 mM FeSO4, 25 mM H2O2 in 10 mM Tris–HCl (pH 7.4) for 1 h for monitoring the oxidative k-DNA damage, with and without BGH extract (5 lg GAE), for 1 h. k-DNA also incubated with 5 lg of BGH aqueous extract alone for 1 h. The melting profiles (Tm) of k-DNA were recorded at different temperatures ranging from 25 to 95 °C using the Spectrophotometer (Ultraspec, 4300 probe) equipped with thermo-programmer and data processor (Amersham Pharmacia Biotech, Hong Kong). Tm values were determined graphically from the absorbance vs. temperature plots. 2.7. Preparation of erythrocytes All the animal experiments were carried out with the approval of institutional animal ethical committee. Male wistar rats in the body weight range of 180–220 g were housed in individual polypropylene cages and had free access to food and water. The animals were fed with standard diet. The animals were sacrificed under anesthesia and blood was collected by heart puncture in heparinized tubes. Erythrocytes were isolated and stored according to the method described by Yuan et al. (2005) and Yang et al. (2006). Briefly, blood samples collected were centrifuged (1500g, 5 min) at 4 °C, erythrocytes were separated from the plasma and buffy coat, and were washed three times by using 10 volumes of 20 mM phosphate buffered saline (pH 7.4; PBS). Each time the cell suspension centrifuged at 1500g for 5 min. The supernatant and buffy coats of white cells were carefully removed with each wash. Erythrocytes thus obtained were stored at 4 °C and used within 6 h for further studies. 2.8. In vitro assay of inhibition of rat erythrocyte hemolysis The inhibition of rat erythrocyte hemolysis by the BGH aqueous extract was evaluated according to the procedure described by Tedesco et al. (2000), with slight modifications. The rat erythrocyte hemolysis was performed with hydrogen
1692
T.K. Girish et al. / Food and Chemical Toxicology 50 (2012) 1690–1696
peroxide as free radical initiator. To 200 ll of 10% (v/v) suspension of erythrocytes in PBS, 50 ll of BGH extract with different concentrations (5–25 lg GAE in PBS pH 7.4) was added. To this, 100 ll of 200 lM H2O2 (in PBS pH 7.4) was added. The reaction mixture was incubated at 37 °C for 30 min and was centrifuged at 2000g for 10 min. The absorbance of the resulting supernatant was measured at 410 nm by taking 200 ll of reaction mixture with 800 ll PBS to determine the hemolysis. Likewise, the erythrocytes were treated with hydrogen peroxide and without inhibitors (BGH extract) to obtain a complete hemolysis. The absorbance of the supernatant was measured at the same condition. The inhibitory effect of the extract was compared with standard antioxidant BHA. Percentage of hemolysis was calculated by taking hemolysis caused by 200 lM hydrogen peroxide as 100%. The IC50 values were calculated from the plots as the antioxidant concentration required for the inhibition of 50% hemolysis. 2.9. Evaluation of oxidative damage on erythrocyte ghost membrane proteins by SDS– PAGE The erythrocyte ghost membranes were prepared by following the procedure of Kwant and Seeman (1970) by one step hemolysis. 1 ml of intact erythrocytes was added to 0.25% NaCl in 10 mM sodium phosphate buffer (pH 7.0). The ghost membrane suspension (100 lg protein) was subjected to oxidative damage using 50 ll of 100 lM H2O2 with or without BGH extract (5 lg GAE) incubated for 1 h. The oxidative modification on erythrocytes ghost membrane proteins were determined by SDS–PAGE on 7.5% discontinuous gel according to the method of Laemmli (1970) and the protein bands were visualized with Coomassie brilliant blue. 2.10. Protective effect on erythrocytes structural morphology Erythrocytes (50 ll) were incubated with and without BGH extract 50 ll; (10 lg GAE) and treated with 100 ll of 200 lM H2O2 for 30 min at 37 °C. After incubation, the incubate was centrifuged at 1500g for 10 min and the cell pellets were processed and were fixed in 3% glutaraldehyde on a coverslip (Agrawal and Sultana, 1993; Hoyer and Bucana, 1982). After fixing on the coverslip, the cells were dehydrated in an ascending series of acetone 0 (30–100%). The dried samples were mounted on an aluminum stubb (100–200 A Å) using double sided tape and coated with gold film with a thickness of 10–20 nm using sputter coater (Polaron, E 5000, SEM coating system). The cells were examined under a scanning electron microscope (Model No., LEO 425 VP, Electron microscopy Ltd., Cambridge, UK).
Fig. 1. HPLC chromatogram of BGH extract. (1) gallic acid; (2) protocatechuic acid; (3) gentisic acid; (4) ferulic acid.
Table 1 Free phenolic content in BGH aqueous extract. Phenolic acid
Free phenolic acid contents (lg/g of husk)
Gallic acid Protocatechuic acid Gentisic acid Ferulic acid
88.77 ± 1.22c 38.59 ± 2.19a 69.52 ± 2.22b 416.51 ± 5.39d
Values are mean ± SD (n = 3). Means with different letters within a column for each extract are significantly different at p < 0.05.
2.11. Statistical analysis Three independent experiments were conducted in triplicate and the data were reported as mean ± SD. Duncan’s new multiple range test was used to determine the difference of means, and P < 0.05 was considered to statistically significant (Steel and Torrie, 1980).
3. Results and discussion 3.1. Polyphenol content in BGH aqueous extract and identification of phenolic acids Polyphenols are the major group of compounds that contribute to the antioxidant properties. The total phenolic content in the black gram husk water extract was found to be 58.83 mg GAE/g. The polyphenols in BGH extract were separated on reverse phase C18 column on HPLC. Gallic acid, protocatechuic acid, gentisic acid and ferulic acid were the phenolic acids identified in BGH extract (Fig. 1) Ferulic acid was the major phenolic acid followed by gallic acid and gentisic acid (Table 1). 3.2. Antioxidant activity The antioxidant activity of polyphenols is due to the reactivity of phenol moiety (hydroxyl group on aromatic ring). Polyphenols have the ability to scavenge free radicals via hydrogen donation or electron donation (Shahidi and Wanasundara, 1992). In the present study, two different methods viz., reducing power assay and the DPPH radical scavenging activity were used to measure the antioxidant activity of BGH extract. 3.2.1. Reducing power of BGH extract The reducing power of a compound is related to its electron transfer ability and may, therefore, serve as a significant indicator
Fig. 2. Reducing power of the aqueous BGH extract.
of its antioxidant activity (Meir et al., 1995). The reducing power increased with the concentration of the BGH aqueous extract (Fig. 2). At 20 lg GAE concentration, BGH extract and BHA showed an absorbance of 0.363 and 0.412, respectively. The antioxidant property of the extract is mainly due to the presence of polyphenols. It has been reported that polyphenols are electron donors and could reduce Fe3+/ferricyanide complex to ferrous form (Chung et al., 2002; Yen and Chen, 1995).
T.K. Girish et al. / Food and Chemical Toxicology 50 (2012) 1690–1696
1693
3.2.2. DPPH free radical scavenging effect of BGH aqueous extract Scavenging the stable DPPH radical is another widely used method to evaluate antioxidant activity. DPPH is a stable free radical with characteristic absorption at 517 nm and antioxidants react with DPPH and convert it to 2,2-diphenyl-1-picrylhydrazine. The degree of discoloration indicates the scavenging potential of the antioxidant extract, which is due to the hydrogen donating ability (Van Gadow et al., 1997). The DPPH radical scavenging activity of BGH aqueous extract increased with the increased concentration of phenols in BGH extract, which may be attributed due to their hydrogen donating ability. The IC50 value for BGH extract was found to be 3.92 lg of GAE, while that of BHA was found to be 3.58 lg. Thus, the free radical scavenging activity of BGH extract is comparable to BHA, a synthetic antioxidant. 3.3. a-Glucosidase inhibitory activity of BGH aqueous extract Type-2 diabetes is caused due to impaired secretion of insulin resulting in increased postprandial glucose level. One of the approaches to decrease postprandial glucose level is to inhibit the a-glucosidase enzyme. The inhibition of this enzyme decreases the blood glucose levels and involved in the management of type-2 diabetes. Puls et al. (1977) and Shim et al. (2003) have hypothesized that by inhibiting this enzyme can regulate glucose uptake from intestinal lumen by inhibiting carbohydrate digestion and absorption, leading to normal glucose homeostasis in diabetic subjects. a-Glucosidase inhibitors from natural food sources is an attractive strategy to manage postprandial hyperglycemia. In this study, the extract from black gram husk was studied for their inhibitory activity of the enzyme. The inhibition by the extract was dose dependent (Fig. 3). The results indicated that BGH aqueous extract showed better enzyme inhibitory activity with the IC50 value of 2.78 lg.
Fig. 3. a-Glucosidase inhibition by BGH extract. Values are mean ± SD (n = 3). Means with different letters are significantly different at p < 0.05.
3.4. Inhibition of Fe2+ induced k-DNA damage by BGH aqueous extract 3.4.1. Agarose gel studies As the BGH aqueous extract showed potential antioxidant properties, it was tested for its ability to inhibit DNA damage caused by hydroxyl radicals that are generated by FeSO4 and H2O2 using agarose gel electrophoresis. The hydroxyl radicals attack hydrogen atoms of k-DNA leading to the formation of nicks in DNA which results single strand and double strand breaks in the k-DNA (Balasubramanian et al., 1998). Incubation of k-DNA with FeSO4 and H2O2 for 1 h resulted a decrease in the band intensity by 80% (Fig. 4A, lane 2). Further incubation of k-DNA with FeSO4 and H2O2 for 2 h, caused the total disappearance of k-DNA band indicating complete k-DNA damage in 2 h (Fig. 4B, lane 2). However, in the presence of BGH aqueous extract, the k-DNA damage by FeSO4/H2O2 system was not observed. As can be seen from Fig. 4A and B (lanes 3), DNA treated with FeSO4/H2O2 system for 1 and 2 h in presence of BGH extract, the DNA band intensity is similar to control (Fig. 4A and B, lanes 1) indicating the prevention of DNA damage by the extract. k-DNA in the presence of BGH aqueous extract alone was intact (Fig. 4A and B, lane 4) for 1 and 2 h incubation indicating that extract did not have any DNA damage property. Thus, the results indicate that BGH extract prevents DNA damage against oxidative damage. The preventive capability may be due to free radical scavenging activity of polyphenols present in aqueous BGH extract. 3.4.2. EtBr Binding and titration studies In the previous experiment we have demonstrated that FeSO4/ H2O2 system has caused damage in k-DNA. To further characterize the k-DNA damage by FeSO4/H2O2 system and its protection by
Fig. 4. Gel electrophoresis image of DNA damage inhibition by BGH extract. (A) After 1 h incubation of reaction mixture; (B) after 2 h incubation of reaction mixture. Lanes 1: 0.5 lg DNA alone; Lane 2: 0.5 lg DNA + 1 mM FeSO4 + 25 mM H2O2; Lane 3: 0.5 lg DNA + 0.5 mM FeSO4 + 25 mM H2O2 + 5 lg aqueous BGH; Lane 4: 0.5 lg DNA + 5 lg aqueous BGH extract.
BGH aqueous extract, changes in fluorescence of EtBr upon binding to k-DNA were carried out. EtBr fluorescence in the presence of intact k-DNA was 18.3 (Fig. 5A), whereas it was 12.6 (Fig. 5B) for k-DNA treated with FeSO4 in the presence of H2O2. The significant decrease in the fluorescence intensity in the case of k-DNA treated with FeSO4 and H2O2 was due to the damage of DNA by hydroxyl radicals generated. However, the fluorescence intensity for DNA in the presence of FeSO4/H2O2 and BGH extract was increased to 15.3 (Fig. 5C), and the increase in the fluorescence intensity may be due to the prevention of DNA damage by free radical scavenging activity of polyphenols present in BGH aqueous extract. The fluorescence intensity for DNA in the presence of BGH extract alone was 18.1 (Fig. 5D) and this value is similar to control DNA sample indicating that BGH extract did not damage DNA. In order to further study the k-DNA damage protection by BGH extract, we have measured the differences in binding of EtBr to intact k-DNA and damaged k-DNA. The amount of EtBr molecules bound per base pair (bp) of DNA was determined by EtBr titration studies. Three representative Scatchard plots of ‘r vs. r/Cf’ for k-DNA are shown in Fig. 6. The number of EtBr molecules bound/
1694
T.K. Girish et al. / Food and Chemical Toxicology 50 (2012) 1690–1696 Table 2 Melting temperature (Tm) of DNA. Sample
Tm value
DNA DNA + FeSO4 + H2O2 DNA + BGH extract + FeSO4 + H2O2 DNA + BGH extract
70.46 °C ± 0.50b 51.66 °C ± 1.40a 74.66 °C ± 1.04c 82.73 °C ± 1.10d
Values are mean ± SD (n = 3). Means with different letters within a column for each extract are significantly different at p < 0.05.
Fig. 5. EtBr binding to DNA. (A) k-DNA; (B) k-DNA + FeSO4 + H2O2; (C) kDNA + aqueous BGH extract + FeSO4 + H2O2; (D) k-DNA + aqueous BGH extract. Values are mean ± SD (n = 3). Means with different letters are significantly different at p < 0.05.
DNA alone
4500000000
DNA + FeSO4 +H2O2
4000000000
DNA + BGH + FeSO4 + H2O2
3500000000
r/Cf
3000000000 2500000000 2000000000
Fig. 7. In vitro protective effects of BGH extract against H2O2 induced hemolysis of rat erythrocytes.
1500000000 1000000000 500000000 0
0
0.5
1
1.5
2
r
2.5
3
3.5
4
4.5
Fig. 6. Scatchard plot of ethidium bromide binding to DNA. (d) DNA, (j) DNA + FeSO4 + H2O2, (N) DNA + BGH extract + FeSO4 + H2O2. Fluorescent measurements were done at room temperature setting excitation at 535 nm and emission at 600 nm. The Scatchard plot was drawn using least square method.
bp of DNA (control) was 4.1 (Fig. 6d), whereas it was 2.2 (Fig. 6j) for k-DNA treated with FeSO4/H2O2. The number of EtBr molecules bound per base pair of k-DNA decreased because of oxidative damage of DNA. However, in the presence of aqueous BGH extract, FeSO4/H2O2, the number of EtBr molecules bound per base pair of k-DNA was comparable to the value obtained with intact k-DNA (3.9) (Fig. 6N). Thus, the results confirm that the BGH extract prevents the DNA damage by FeSO4/H2O2. 3.4.3. Melting temperature (Tm) studies The melting temperature study provides an insight on the integrity of DNA. The melting temperature (Tm) of k-DNA was 70.4 °C, where as it was 51 °C, when it was treated with FeSO4/H2O2 (Table 2). Low Tm in the case of k-DNA treated with FeSO4/H2O2 was due to the damage of DNA in presence of hydroxyl radicals. However, the Tm value for k-DNA in presence of BGH extract and FeSO4/H2O2 was 74.5 °C. The higher Tm of k-DNA in the presence of BGH extract indicates that oxidative damage was prevented by the extract. Tm value for DNA in presence of BGH extract alone was found to be 84 °C
indicating k-DNA is stable in presence of aqueous BGH extract. Thus, the studies revealed that BGH extract prevented the oxidative DNA damage caused by free radicals. The mechanism of prevention may be due to the scavenging of free radicals by the polyphenols present in the aqueous extract. 3.5. Inhibition of rat erythrocytes hemolysis Oxidants lyse the erythrocyte and results in the release of hemoglobin pigment into the medium. The measurement of color mediated by hemoglobin at 410 nm will give a measure of oxidant damage in cells. In the present study, H2O2 hemolysis was induced in erythrocytes and studied the effect of BGH extract on its protection against hemolysis. As can be seen from Fig. 7, BGH extract inhibited the rat erythrocytes hemolysis in a dose dependent manner. As compared to with BHA, a synthetic antioxidant, BGH extract showed a better inhibition for erythrocyte inhibition. The IC50 values for the BGH extract and BHA were 11.5 and 14 lg, respectively. BGH extract alone was tested and found that it did not show any harmful effect on erythrocytes. The percentage of hemolysis of BGH alone was found to be 3.03% which is comparable to control (1.64%). 3.6. Protection of oxidative damage on RBC ghost membrane proteins The changes in the protein pattern of membrane protein produced by oxidative stress were observed by SDS–PAGE. Ghost membrane was prepared by hypotonic lysis of normal erythrocytes. Membranes were treated with H2O2 and with or without BGH extract and were analyzed on SDS–PAGE (Fig. 8) (Ajila and
T.K. Girish et al. / Food and Chemical Toxicology 50 (2012) 1690–1696
1695
even after 1 h incubation with H2O2 (Fig. 8, lanes 3, 4). Thus, the results indicate that the BGH extract effectively prevented the membrane proteins from oxidation. 3.7. Protective effect on RBC structural morphology
Fig. 8. SDS–PAGE of RBC membrane proteins showing protective effects of BGH extract against H2O2 induced oxidative damage on erythrocyte ghost membrane. Lane 1: membrane proteins treated with H2O2, Lane 2: membrane proteins (untreated), Lane 3: membrane proteins treated with H2O2 in presence of BGH extract, Lane 4: membrane proteins treated with H2O2 in presence of BHA.
Scanning electron micrographs of erythrocytes treated in vitro with H2O2 and BGH aqueous extract are shown in Fig. 9. Untreated erythrocytes appeared as typical discocytes while exposure to H2O2 resulted in a significant change in the cell shape and distinct echinocyte formation. The morphological changes induced by oxidative system were prevented when the cells were treated with BGH extract and BHA. Oxidative damage to cell membrane leads to alterations in cell rigidity and shape and results in the formation of echinocytes. The cell damage thus occurred ultimately affects the functioning of erythrocytes (Linderkamp et al., 1997). According to bilayer couple hypothesis, the changes in erythrocyte shape induced by foreign molecules are due to differential expansion of two monolayers of the red cell membrane (Sheetz and Singer, 1974; Lim et al., 2002) Thus, stomatocytes are formed when the compounds inserts into the inner monolayer, whereas speculated-shaped echinocytes are produced when it locates into the outer moiety. The results in the present study indicate that BGH extract prevents the morphological changes induced by oxidants in erythrocytes. 4. Conclusions The present study shows that the black gram husk aqueous extract is rich in polyphenols. The extract exhibited antioxidant properties, inhibited a-glucosidase activity. It also protected DNA and erythrocyte from oxidative damage. As the black gram husk extract showed various antioxidant properties in different systems, it could be used as a functional food ingredient or nutraceutical product for health benefits. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements Authors are thankful to Dr. P.V. Salimath for encouragement in this study. The authors also thank Mr. K. Anbalagan for his assistance in scanning electron microscopy. References
Fig. 9. Scanning electron micrograph of normal erythrocytes and protective effect BGH and BHA against H2O2 induced oxidative damage on RBC 10,000.
Prasada Rao, 2008). Upon treatment with oxidant, most of the bands present above actin bands were diminished (Fig. 8, lane 1). However, in presence of BGH extracts and BHA, protein bands 1, 2, 3, 4 and actin bands were clearly present in the membranes,
Agrawal, D., Sultana, P., 1993. Biochemical and structural alterations in rat erythrocytes due to hexachlorocyclohexane exposure. Food Chem. Toxicol. 31, 443–448. 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. Ajila, C.M., Prasada Rao, U.J.S., 2009. Purification and characterization of black gram (Vigna mungo) husk peroxidase. J. Mol. Catal. B Enzym. 60, 36–44. Ames, B.N., Shigenaga, M.K., Hagen, T.M., 1993. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. USA 90, 7915–7922. Balasubramanian, B., Pogozelski, W.K., Tullius, T.D., 1998. DNA strand breaking by the hydroxyl radical is governed by the accessible surface areas of the hydrogen atoms of the DNA backbone. Proc. Natl. Acad. Sci. USA 95, 9738–9743. Barnham, K.J., Masters, C.L., Bush, A.I., 2004. Neurodegenerative diseases and oxidative stress. Nature Rev. Drug Disc. 3, 205–214. Blois, M.S., 1958. Antioxidant determinations by the use of a stable free radical. Nature 181, 1199–1200. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity. Lebensm. Wiss. Technol. 28, 25–30. Chatterjee, B., Rao, G.R.K., 1994. Superhelical density of goat mitochondrial DNA: fluorescent studies. Indian J Biochem. Biophys. 31, 77–79.
1696
T.K. Girish et al. / Food and Chemical Toxicology 50 (2012) 1690–1696
Chung, Y.C., Chang, C.T., Chao, W.W., Lin, C.F., Chou, S.T., 2002. Antioxidant activity and safety of the 50% ethanolic extract from red bean fermented by Bacillus subtillus IMR-NK1. J. Agric. Food Chem. 50, 2454–2458. Ghanta, S., Banerjee, A., Poddar, A., Chattopadhyay, S., 2007. Oxidative DNA damage preventive activity and antioxidant potential of Stevia rebaudiana (Bertoni) Bertoni, a natural sweetener. J. Agric. Food Chem. 55, 10962–10967. Glowniak, K., Zgorka, G., Kozyra, M., 1996. Solid-phase extraction and reversed phase high-performance liquid chromatography of free phenolic acids in some Echinacea species. J. Chromatogr. 730, 25–29. Hoyer, L.C., Bucana, C., 1982. Principles of immuno-electron microscopy. In: Marchalories, J.J., Warr, G.W. (Eds.), Antibodies as a tool. John Wiley, London, pp. 257–258. Keith, R.M., Christy, L.A., 2010. Polyphenols as dietary supplements: a double-edged sword. Nutr. Dietary Suppl. 2, 1–12. Kwant, W.O., Seeman, P., 1970. The erythrocyte ghost is a perfect osmometer. J. Gen. Physiol. 55, 208–219. Laemmli, U.K., 1970. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature 227, 680–685. Lim, G., Wortis, M., Mukhopadhyay, R., 2002. Stomatocyte-discocyte echinocyte sequence of the human red blood cell: evidence for the bilayer-couple hypothesis from membrane mechanics. Proc. Natl. Acad. Sci. USA 99, 16766– 16769. Linderkamp, O., Kiau, U., Ruef, P., 1997. Cellular and membrane deformability of red blood cells in protein infants with and without growth retardation. Clin. Hemorheol. Microcirc. 17, 279–283. Meir, S., Kanner, J., Akiri, B., Hadas, S.P., 1995. Determination and involvement of aqueous reducing compounds in oxidative defensive system of various senescing leaves. J. Agric. Food Chem. 43, 1813–1815. Pardo-Andreu, G.L., Philip, S.J., Riaño, A., Sánchez, C., Viada, C., Núñez-Sellés, A.J., Delgado, R., 2006. Mangifera indica L. (Vimang) protection against serum oxidative stress in elderly humans. Arch. Med. Res. 37, 158–164. Peihong, F., Hongxiang, L., 2004. Effects of polyphenols from grape seeds on oxidative damage to cellular DNA. Mol. Cellular Biochem. 267, 67–74. Peinado, J., Lo´pez de Lerma, N., Peinado, R.A., 2010. Synergistic antioxidant interaction between sugars and phenolics from a sweet wine. Eur. Food Res. Technol. 231, 363–370.
Puls, W., Keup, U., Krause, H.P., Thomas, G., Hoffmeister, F., 1977. Glucosidase inhibition. A new approach to the treatment of diabetes, obesity, and hyperlipoproteinaemia. Naturwissenschaften 64, 536–537. Rice-Evans, C., Burdon, R., 1993. Free radical-lipid interactions and their pathological consequences. Progress in Lipid Res. 32, 71–110. Sadrzadeh, S.M., Graf, E., Panter, S.S., Hallaway, P.E., Eaton, J.W., 1984. Hemoglobin. A biologic fenton reagent. J. Biol. Chem. 259, 14354–14356. Scatchard, G., 1949. The attraction of proteins for small molecules and ions. Ann. N Y Acad. Sci. 51, 660–672. Shahidi, F., Wanasundara, P.K.J.P.D., 1992. Phenolic antioxidants. Crit. Rev. Food Sci. Nutr. 32, 67–103. Sheetz, M.P., Singer, S.J., 1974. Biological membranes as bilayer couples. A molecular mechanism of drug-erythrocyte induced interactions. Proc. Natl. Acad. Sci. USA 71, 4457–4461. Shim, Y.J., Doo, H.K., Ahn, S.Y., Kim, Y.S., Seong, J.K., Park, I.S., Min, B.H., 2003. Inhibitory effect of aqueous extract from the gall of Rhus chinensis on alpha-glucosidase activity and postprandial blood glucose. J. Ethnopharmacol. 85, 283–287. Steel, R.G.D., Torrie, J.H., 1980. Principles and procedures of statistics. McGraw, New York. Swain, T., Hillis, W.E., 1959. The phenolic constituents of Prunus domestica. 1. The quantitative analysis of phenolic constituents. J. Sci. Food Agric. 10, 63–68. 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 blosod cells. J. Nutr. Biochem 11, 114–119. Van Gadow, A., Joubert, E., Hannsman, C.T., 1997. Comparison of the antioxidant activity of aspalathin with that of other plant phenols of roolbos tea (Aspalathus linearis), a-tocoferol, BHT, and BHA. J. Agric. Food Chem. 45, 632–638. 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. Yen, G.C., Chen, H.Y., 1995. Antioxidant activity of various tea extracts in relation to their antimutagenicity. J. Agric. Food Chem. 43, 27–32. Yuan, X., Wang, J., Yao, H., Chen, F., 2005. Free radical-scavenging capacity and inhibitory activity on rat erythrocytes hemolysis of feruloyl oligosaccharides from wheat bran insoluble dietary fiber. Lebensm. Wiss. Technol. 38, 877–883.