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Cytoprotective Properties of Antioxidant Protein from Curry leaves (Murraya koenigii L.) against Oxidative Stress Induced Damage in Human Erythrocytes

Mylarappa B Ningappa1,2*, Dinesha Ramadas1, Dinesha Nanjegowda1, Kiruthika Balasubramanian1, Khusdeep Chahal3, Sachin Patil4 and Leela Srinivas1

1Adichunchanagiri Biotechnology and Cancer Research Institute, Central Research Laboratory, Adichunchanagiri Institute of Medical Sciences, Balagangadharanatha Nagara, Mandya District, Karnataka, India

2Department of Surgery, University of Pittsburgh, Pittsburgh, PA, USA

3UAB School of Medicine, Huntsville Regional Campus, Huntsville, AL 35801, USA

4Presence Medical Center, University of Illinois and Hospitalist (Christie Clinic), Urbana, IL, USA

*Corresponding Author:
Mylarappa B. Ningappa
Research Associate, Department of Surgery
University of Pittsburgh, Rangos Research Center
530 45th street, Pittsburgh, PA, United States
Tel: 4126573269
E-mail: [email protected]

Received date: December 27, 2015; Accepted date: February 05, 2016; Published date: February 08, 2016

Citation: Ningappa MB, Ramadas D, Nanjegowda D, Balasubramanian K, Chahal K, et al. (2016) Cytoprotective Properties of Antioxidant Protein from Curry leaves (Murraya koenigii L.) against Oxidative Stress Induced Damage in Human Erythrocytes. Med chem 6:075-080. doi:10.4172/2161-0444.1000328

Copyright: © 2016 Ningappa MB, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Oxidative stress induced by reactive oxygen species (ROS) cause lipid peroxidation at the human erythrocyte membrane with subsequent alterations in the ATPases function. In this study, potential cytoprotective activities of previously reported ”Antioxidant Protein from Curry leaves (APC)’’ in erythrocytes against reactive oxygen (ROS) species generated by pro-oxidants in vitro. The APC prevented red blood cell lysis induced by pro-oxidants; Fe: As (2:20 μmole), Hydrogen peroxide (0.2 mM), and tertiary butyl hydroperoxide (1 mM) upto 97.5, 82.5 and 63%, respectively. Further, APC prevented Fe:As induced K+ leakage in red blood cells up to 95%. The inhibition offered by APC on K+ leakage was comparable to inhibition offered by quinine sulphate, a known K+ channel blocker. Interestingly, the APC at dose dependently restored Na+K+ ATPase and Ca2+Mg2+ ATPase activities of erythrocyte membrane when altered by ROS. The restoration of ATPase activity by APC was two times more than standard antioxidants BHA and α-tocopherol. In conclusion, Curry leaves protein antioxidant is an effective antioxidant in preventing membrane damage and associated functions mediated by reactive oxygen species. It can be further developed as an effective bioprotective antioxidant agent to cellular components.

Keywords

Antioxidant protein of curry leaves; Human erythrocytes; Lipid peroxidation; Fenton reactants; Oxidative hemolysis; K+ leakage; Na+K+ ATPase; Ca2+Mg2+ ATPase

Abbreviations

APC: Antioxidant Protein of Curry leaves; RBC: Red Blood Cells; BHA: Butylated hydroxyanisole; Fe: As, Ferrous sulphate and Ascorbic acid mixture; t-BOOH: Tetra butyl hydroperoxide; TBA: Thiobarbituric acid; TBARs: Thiobarbituric acid reactive substances; MDA: Malondialdehyde; ROS: Reactive oxygen species

Introduction

Oxidative stress is detrimental to cells, as reactive oxygen species (ROS) can cause oxidative damage to lipids, proteins, DNA and other macromolecules [1-4]. Elevated levels of ROS have been implicated in etiology of many diseases like cancer, neurodegenerative disorders, cardiovascular diseases, atherosclerosis, cataract and inflammation [5-10].

Lipid peroxidation by ROS at the membrane levels generates variety of reactive substances such as aldehydes, including malondialdehyde (MDA) [11,12]. These may potentially affect membrane permeability and functioning of ion pumps and structure function relationship of membrane bound proteins resulting in the inability of the cell to maintain its ionic environment. Several structural and functional damages are caused to RBC (Red Blood Cells) by the exposure to MDA [13,14].

Under oxidative stress, hemolysis of RBCs takes place due to the action of ROS [15,16]. Further, intra cellular K+ concentration is maintained by the cell to accomplish essential physiological task. The ROS cause disruption of cellular membrane proteins and leakage of intracellular contents [17,18]. The Na+K+ ATPAse and Ca2+Mg2+ ATPases are membrane bound enzymes which are involved in maintenance of ion concentration within cells. The altered activities of ATPases indicate the extent of damage to cell membranes. It has been reported that there is a permanent inhibition of ATPase activities by free radicals via lipid peroxidation [19]. A limiting factor which may control /prevent the extent of damage to cells may be the level of exogenously derived antioxidants. Dietary antioxidants have been shown to protect the cells from damage caused by oxidative stress and to fortify the defense system against degenerative diseases [8].

As reported earlier, an Antioxidant Protein of molecular weight ~ 35 KDa from Curry leaves (APC) [20]. In this study, we tested ability of APC to prevent Fenton reactants (Ferrous sulphate: Ascorbate system) induced oxidative membrane damage to RBCs. The use of human RBCs as a model for oxidative damage is because the liability of erythrocyte membrane to lipid peroxidation induced by peroxidation in vitro reflects the liability of other cell membranes to oxidative damage in vivo relating to oxidative stress [21,22]. This study for the first time shows the cytoprotective effect of APC against fenton reactant mediated alterations in RBC membrane, ATPases and K+ channels.

Materials and Methods

Materials

Ferrous sulphate, Ascorbate, Acrylamide, thiobarbituric acid, Ascorbate, α-tocopherol, O-phenanthroline, Adenosine 5`triphosphate (5`ATP) and all other chemicals were purchased from Sigma Chemical Co., USA. All other chemicals unless otherwise mentioned were of analytical grade and procured from Merck (Darmastadt, Germany). Solvents were distilled prior to use. Blood samples were obtained from the healthy male volunteers of Adichunchanagiri Biotechnology and cancer research institute (ABCRI), Mandya district, Karnataka, India.

Plant material

Curry leaves (Murraya koenigii L.) were obtained from a garden maintained by Adichunchanagiri Biotechnology and Cancer Research Institute (ABCRI), BG Nagara, Mandya district, India. Prof. GR Shivamurthy, Taxonomist, University of Mysore, India authenticated the plant. The plant was deposited at ABCRI against voucher No. ABCRI, 7/2007.

Purification of antioxidant protein of curry leaves (APC)

The APC was purified according to the method of Ningappa and Srinivas [20]. Briefly, curry leaves were washed, shade dried and powdered. Five grams of curry leaves powder was homogenized in 20 ml of 10 mM Tris buffer, pH 7.0, with addition of polyvinylpyrrolidone to remove polyphenols. The suspension was incubated overnight at 4°C with constant stirring, then filtered and centrifuged at 13,000 rpm at 4°C for 20 min. The supernatant was treated with 0.1% polyethyleneimine to precipitate nucleotides. The resulting pellet was discarded and supernatant brought to 65% saturation with ammonium sulphate. The pellet was dissolved in 20 mM Tris buffer, pH 7.4 (NH4SO4 extract). The NH4SO4 extract (21 mg) was loaded onto a Sephadex G-75 column (14 × 73 cm), pre-equilibrated and eluted with 20 mM Tris buffer, pH 7.4, at a flow rate of 1.5 ml/5 min. Protein elution was monitored at 280 nm using a spectrophotometer. The antioxidant activity of these protein samples at various concentrations ranging from 20-1000 μg was tested by TBARs assay. Active peak II (8 mg), pooled and lyophilised was further fractionated on a Sephadex G-75 column by eluting with 20 mM Tris buffer, pH 7.4, to give a homogenous preparation, APC, which showed maximum antioxidant activity.

Preparation of human erythrocytes

Human red blood cells (RBC) were separated from heparinized blood that was collected from healthy volunteers. The blood was centrifuged at 2500 rpm for 10 min to separate the RBCs from plasma. RBCs were washed three times with phosphate-buffered saline (PBS) at pH 7.4. During the last washing the cells were centrifuged at 2500 rpm for 10 min to obtain a constantly packed RBC-cell suspension.

Erythrocyte susceptibility to oxidation and protection by APC

100 μl of freshly prepared RBC suspension) was preincubated with antioxidants such as APC (0-125 μg), Trolox (0-125 μg), and α-tocopherol (0-125 μg) for 20 min at 37°C, then ferrous sulphate: ascorbate (2:20 μmole) or hydrogen peroxide (0.2 mM) or t-BOOH (1 mM) was added and final volume of 1 ml with saline. Reaction mixture was incubated at 37°C for 3 hr, centrifuged at 2500 rpm for 10 min and the extent of hemolysis was measured using spectrophotometer at 540 nm. Appropriate controls were taken and percentage inhibition of oxidative hemolysis by antioxidants using the formula.

image

Preparation of human erythrocyte ghost (membrane)

Human blood was collected from healthy male volunteers. Erythrocyte ghost, free of hemoglobin and superoxide dismutase, was prepared by the method of Dodge et al. [23]. Blood was centrifuged at 2500 rpm for 15 min, the supernatant obtained was discarded and the RBC pellet was taken and kept as RBC suspension for further assays. The RBC pellet was washed three to five times with isotonic phosphate buffer (310 milli osmolar, pH 7.4) centrifuged at 2500 rpm at 4°C for 20 min. The RBC pellet obtained was suspended in hypotonic phosphate buffer and incubated overnight at room temperature for hypotonic hemolysis to take place. The contents were centrifuged at 12000 rpm at 4°C for 20 min, to remove unlysed RBC cells. Supernatant was collected and centrifuged at 12000 rpm for 20 min and washed in 0.9% saline. Erythrocyte ghost was suspended in 0.9% NaCl in aliquots and stored at -20°C for further use. The protein content of ghost was estimated by Bradford`s method [24].

Inhibition of lipid peroxidation induced by Fenton reactants (Fe:As) by APC and standard antioxidants

Erythrocyte membrane (200 μg membrane protein) were preincubated with or without various concentrations of APC (0-25 μg), BHA (400 μM), α-tocopherol (400 μM) in 0.5 ml of TBS, pH 7.4 at 37°C for 20 min. Then Ferrous sulphate: ascorbate (2: 20 μmole) were added, final volume made upto 1 ml with TBS, pH 7.4 and incubated at 37°C for 60 min. To each supernatant 1 ml of 10% TCA and 1 ml of 10% TBA was added, heated for 95°C for 15 min, cooled, centrifuged at 500 rpm for 10 min and supernatant was read at 535 nm. MDA equivalents were calculated using molecular extinction coefficient of MDA (1.56 × 105 M-1cm-1).

Intracellular K+ leakage in erythrocytes and protection by APC in comparison with standard antioxidants

To 100 μl of RBC suspension, ferrous sulphate: ascorbate (2:20 μmole) was added and final volume made upto 1 ml with saline. At various time intervals, the amount of K+ leakage from cells into the medium was measured by a K+ specific micro electrode (Micro electrodes Inc. London-derry-New Hampshire, 03053, USA) connected to a pH meter. Calibration was done with 10-5, 10-4, 10-3, 10-2 and 10-1 M KCl solution. The amount of K+ released was expressed as μM K+.

For studying inhibitory effect of antioxidants on Fe:As induced K+ ion leakage in erythrocytes. 100 μl ml RBC suspension was preincubated with or without different concentrations of APC (0-120 μg), BHA (0-120 μg), α-tocopherol (0-120 μg), Quinine sulphate (1 μM) and O-phenathroline (10 mM) for 20 min at 37°C. To this Fe:As (2:20 μmole) was added and final volume was made upto 1 ml with saline. The amount of K+ ion leakage was monitored at 20th min as described above. Suitable controls of solvents or antioxidants or inhibitors alone were maintained. μM K+ ion leakage induced by pro-oxidant without inhibitor or any antioxidants was expressed as 100% and % inhibition of K+ ion leakage is calculated accordingly.

Restoration of Na+K+ ATPase activity by APC in comparison with standard antioxidants

Na+K+ ATPase activity of RBC membranes was determined following Ames [25-27]. Erythrocyte membranes (200 μg of membrane protein) were preincubated with or without various concentrations of APC (0-100 μg), BHA (0-100 μg) α-tocopherol (0-100 μg) and O-phenathroline (10 mM) in 0.5 ml of TBS, pH 7.4 at 37°C for 20 min. Then Fe: As (2: 20 μmole) were added, final volume made upto 1 ml with TBS, pH 7.4 and incubated at 37°C for 60 min. The supernatant discarded and pellet was dissolved and incubated in 0.5 ml of reaction mix (Tris 50 mM, NaCl 350 mM, KCl 35 mM, MgCl2 7.5 mM, EDTA 0.5 mM, pH 7.0) for 10 min at 37°C. At the end of the incubation period, ATP (15 mM) was added and further incubated at 37°C for 60 min, Reaction was stopped by adding 0.1 ml of 10% TCA, kept in ice water for 10 min. Na+K+ ATPase activity of RBC membranes were estimated by the inorganic phosphorous liberated (Pi). 700 μl of Ammonium molybdate reagent was added followed by addition of 40 μl of ANSA reagent, incubated at 37°C for 60 min. The blue colour developed was read at 690 nm. Appropriate controls were done. The phosphorous content was calculated from calibration curve (absorbance versus phosphorous content) and enzyme activity was expressed as μmole Pi/ mg membrane protein/hr.

Restoration of Ca2+Mg2+ ATPase activity by antioxidants

Ca2+Mg2+ ATPase activity of RBC membranes was determined following Ames [25-27]. Erythrocyte membranes (200 μg of membrane protein) were preincubated with or without various concentrations of APC (0-100 μg), BHA (0-100 μg), α-tocopherol (0-100 μg) and o-phenanthroline (10 mM) in 0.5 ml of TBS, pH 7.4 at 37C for 20 min. Then Fe: As (2: 20 μmole) were added, final volume made upto 1 ml with TBS, pH 7.4 and incubated at 37°C for 60 min. The supernatant discarded and pellet was dissolved and incubated in 0.5 ml of reaction mix (Tris 50 mM, NaCl 350 mM, KCl 35 mM, MgCl2 7.5 mM, EDTA 0.5 mM, pH 7.0) for 10 min at 37°C. At the end of the incubation period, ATP (15 mM) was added and further incubated at 37°C for 60 min, Reaction was stopped by adding 0.1 ml of 10% TCA, kept in ice water for 10 min. Ca2+Mg2+ ATPase activity of RBC membranes were estimated by the inorganic phosphorous liberated (Pi). 700 μl of Ammonium molybdate reagent was added followed by addition of 40 μl of ANSA reagent, incubated at 37C for 60 min. The blue colour developed was read at 690 nm. Appropriate controls were done. The phosphorous content was calculated from calibration curve (absorbance versus phosphorous content) and enzyme activity was expressed as μmole Pi/ mg membrane protein/hr.

Statistical analysis

Statistical analysis was done in SPSS (Windows version 10.0.1 Software Inc., New York) using a one-sided student`s t-test. All results refer to mean ± SD. P<0.05 was considered as statistically significant as comparing to relevant controls.

Results and Discussion

Lipid peroxidation by ROS at membrane levels, affect the structure and function of membranes. High levels of ROS have been implicated in several oxidative damage related diseases [6-8]. The limiting factor that may prevent the extent of damage could be the quantum of exogenously derived antioxidants [8]. The present study reports the cytoprotective effects of APC [20] against oxidative membrane damage in RBC cells. In this study, RBC`s are used to test the extent of lipid peroxidation, as liability of erythrocyte membrane to lipid peroxidation in vitro reflect the liability of other cell membranes to oxidative damage in vivo, relating to oxidative stress [21,22]. Further, in vivo erythrocytes are highly exposed to oxygen and are site for radical formation under pathological conditions [28]. Anemia of chronic inflammatory diseases appears to be caused, in part by oxidative damage to erythrocytes.

A number of factors have been considered relevant to the oxidative lysis of RBC`s and in addition, it has been reported that hemolysis finally depends on the integrity of membrane proteins [15,16]. Hence it was important to study the protective role of APC to prevent oxidative hemolysis. When APC was tested for its efficiency to prevent the oxidative RBC lysis induced by pro-oxidants, there was a dose dependent prevention by APC of membrane lysis upon fenton reactants; Fe;As (2:20 μmole), hydrogen peroxide (0.2 mM) and t-BOOH (1 mM) induction (Figure 1). At 125 μg APC exhibits maximum protection to the extent of 97.5% against Fe:As induced oxidative hemolysis, on the other hand α-tocopherol and BHA at 400 μM exhibited protection by 92% and 85%, respectively (Figure 2). Upon hydrogen peroxide and t-BOOH induced oxidative hemolysis, APC at 125 μg showed about 63% protection against hydrogen peroxide induced hemolysis and about 82.5% against t-BOOH induced hemolysis. On the other hand, BHA and α-tocopherol showed 83% and 94% against hydrogen peroxide induced hemolysis and about 78% and 90% against t-BOOH induced hemolysis (Figure 2). The results indicate that APC is a potent protectant against oxidative lysis of erythrocytes when compared to standard antioxidants BHA and α-tocopherol. Similar studies have reported that oolong tea extracts and melatonin shown to inhibit hemolysis under oxidative stress [28-30]. The results suggest that oxidative membrane damage could be efficiently protected by antioxidants.

medicinal-chemistry-Dose-dependent-protective

Figure 1: Dose dependent protective effect of APC on pro-oxidant induced oxidative hemolysis. 100 μl of RBC suspension was preincubated with APC (0- 125 μg) at 37°C for 20 min. Then Fe:As (2:20 μmole) or hydrogen peroxide (0.2 mM) or t-BOOH (1 mM) added in 1 ml saline, incubated at 37°C for 180 min. Oxidative hemolysis induced by pro-oxidants in the absence of antioxidants was expressed as 100% lysis. Data represent the mean ± SD (n=6).

medicinal-chemistry-Protective-effect-oxidant

Figure 2: Protective effect of APC on pro-oxidant induced oxidative hemolysis: comparison with standard antioxidants. 100 μl of RBC suspension preincubated with APC (125 μg) or BHA (400 μM) or α -tocopherol (400 1M) at 37°C for 20 min. Then Fe:As (2:20 μmole) or hydrogen peroxide (0.2 mM) or t-BOOH (1 mM) added in 1 ml saline, incubated at 37°C for 180 min. Oxidative hemolysis induced by pro-oxidants in the absence of antioxidants was expressed as 100% lysis. Data represent the mean ± SD (n=6).

The K+ ion leakage is one of the results of membrane damage. In order to accomplish essential physiological task, virtually all cells accumulate K+ and excludes Na+ from the cytoplasm. The oxidation of membrane lipids, with the formation of aldehydes results in the disruption of cellular membrane proteins and cause leakage of intracellular contents [17,18]. In this study, when erythrocyte suspension was treated with Fe:As (2:20 μmole) the maximum damage was at 20th min, indicated by leakage of K+ ions in the medium. When antioxidants were tested in terms of their potency to prevent K+ ion leakage in erythrocytes, it was found that APC, BHA and α-tocopherol dose dependently prevented Fe:As induced K+ leakage to (Figure 3). APC at 120 μg inhibited K+ leakage in erythrocyte membrane upto 95%, whereas BHA and α-tocopherol at 120 μg inhibited up to 81% and 89%, respectively (Table 1). APC was more potent than standard antioxidants in inhibiting K+ leakage and inhibition is comparable to inhibition offered by quinine sulphate (upto 90%), a known K+ channel blocker. In addition, O-phenanthroline (lipophilic iron chelator) offered 92% inhibition against Fe:As induced K+ leakage (Table 1). Inhibition of lipid peroxidation by iron chelators was shown to completely prevent cell death indicating that iron induced peroxidative damage was responsible for causing irreversible injury. The comparison studies with OH radical quenchers (BHA and α-tocopherol) and lipophilic iron chelator (O-phenanthroline) indicate that APC is effective inhibitors of OH radical formation and iron induced peroxidative damage.

medicinal-chemistry-inhibition-induced-leakage

Figure 3: Dose dependent inhibition of Fe:As induced K+ leakage by APC and standard antioxidants. 100 μl of RBC suspension was pre-incubated with or without antioxidants at indicated concentrations for 20 min at 37°C. Fe:As (2: 20 μmole) were added and final volume made upto 1 ml with saline. The amount of K+ ion leakage was measured at 20th min with K+ specific microelectrode connected to pH meter. Amount of K+ released is expressed as μM K+/106 cells. Data represent the mean ± SD (n=6).

Antioxidants/Inhibitors Function Concentration % inhibition of K+ leakage  in erythrocytes (1 × 106 cells)
No Antioxidants/ Inhibitors     0
Quinine sulphate K+ channel Blocker 1 μM 90 ± 1.9
O-phenanthroline iron chelator 10 mM 92 ± 1.5
APC Antioxidant 120 μg 95 ± 1.1
BHA Antioxidant 120 μg 81 ± 1.7
α-tocopherol 1O2 quencher 120 μg 89 ± 1.3

Table 1: Inhibitory effect of APC on pro-oxidant induced K+ leakage in erythrocyte.

Na+K+ ATPase and Ca2+Mg2+ ATPase are membrane bound enzymes, which are involved in maintenance of respective monovalent and divalent cation concentrations within cells. The altered activities of ATPases possibly show the extent of damage in cell membrane. It has been reported that there is a permanent inhibition of ATPase activity by oxygen free radicals via lipid peroxidation [19]. When the effect of antioxidants including APC was tested on Na+K+ ATPase and Ca2+Mg2+ upon induction by Fe:As (2:20 μmole), it was observed that all the antioxidants tested APC, BHA, and α-tocopherol dose dependently restored Na+K+ ATPase (Figure 4) and Ca2+Mg2+ ATPase activities (Figure 5) of erythrocyte membrane. The activities of isolated RBC membranes (Untreated membranes) were 8.5 ± 1.0 and 11.2 ± 1.2 μmole/mg membrane protein/hr for Na+K+ ATPase and Ca2+Mg2+ ATPase activities, respectively. Treatment of RBC membranes with Ferrous sulphate: ascorbate (2:20 μmole) resulted in significant inhibition of ATPase activities, the activities were found to be 2.5 ± 0.08 and 3.4 ± 0.07 for Na+K+ ATPase and Ca2+Mg2+ ATPase activities, respectively. ATPase activities in presence of APC (100 μg), BHA (100 μg) and α-tocopherol (100 μg) were found to be 7.9 ± 0.3, 6.6 ± 0.55 and 7.4 ± 0.35 for Na+K+ ATPase (Table 2) and 9.9 ± 0.9, 7.3 ± 0.6 and 8.4 ± 0.55 for Ca2+Mg2+ ATPase activities (Table 2). In comparison, the decrease in ion pump ATPase activity was significantly prevented by the pretreatment of O-phenanthroline (10 mM), which showed 8.4 ± 1.2 and 10.3 ± 0.9 for Na+K+ ATPase and Ca2+Mg2+ ATPase activities, respectively (Table 2). This suggests that if iron is chelated, inhibition of ion pump ATPases activities could be diminished. Overall ATPase activities in presence of antioxidants were comparable to activities of untreated RBC membrane Based on the results obtained it can be concluded that APC is an effective antioxidant in restoration of ATPases activities upon oxidative damage to membrane.

medicinal-chemistry-Restoration-antioxidants-Erythrocyte

Figure 4: Restoration of Na+K+ ATPase activity by APC and standard antioxidants. Erythrocyte membrane (200 μg) preincubated with APC (0-100 μg) or BHA (0-100 μg) or α-tocopherol (0-100 μg) in 0.5 ml TBS, pH 7.4 and incubated at 37°C for 30 min. Fe:As (2:20 umole) were added, final volume made upto 1 ml with TBS, Incubated at 37°C for 60 min. following incubation Na+K+ pump ATPase of membrane were determined and enzyme activity is expressed as μmole Pi/mg protein/hr. Data represent the mean ± SD (n=6).

medicinal-chemistry-Restoration-Erythrocyte-membrane

Figure 5: Restoration of Ca2+Mg2+ ATPase activity by APC and standard antioxidants. Erythrocyte membrane (200 μg) preincubated with APC (0-100 μg) or BHA (0-100 μg) or α -tocopherol (0-100 μg) in 0.5 ml TBS, pH 7.4 and incubated at 37°C for 30 min. Fe:As (2:20 umole) were added, final volume made upto 1 ml with TBS, Incubated at 37°C for 60 min. following incubation Ca2+Mg2+ pump ATPase of membrane were determined and enzyme activity is expressed as μmole Pi/mg protein/hr. Data represent the mean ± SD (n=6).

Antioxidants Final Concentration Na+K+ATPase activity* Ca2+Mg2+ ATPase activity*
Untreated RBC Membrane   8.5 ± 1.0 11.2 ± 1.2
RBC membrane + Fe:As (2:10 μmole) + APC   2.5 ± 0.1a 3.4 ± 0. a
RBC membrane + Fe:As (2:10 μmole) + APC 100 μg 7.9 ± 0.3b 9.9 ± 0.9b
RBC membrane + Fe:As (2:10 μmole) + APC 100 μg 6.6 ± 0.5c 7.3 ± 0.6c
RBC membrane + Fe:As (2:10 μmole) + APC 100 μg 7.4 ± 0.3b 8.4 ± 0.5b
RBC membrane + Fe:As (2:10 μmole) + APC 10 mM 8.4 ± 1.2b 10.3 ± 0.9b

Table 2: Restoration of ATPase activities by APC; Comparison with standard antioxidants.

Studies have shown that end products of lipid peroxidation, MDA is capable of cross-linking membrane components containing amino groups [17,31]. It has been reported that one potential mechanism for iron mediated damage of Ca2+ pump ATPase is through cross-linking by MDA [32]. The incubation of RBC membrane (200 μg) with Fe:As (2:20 μmole) as for ATPase analysis was found to induce membrane lipid peroxidation as evidenced by the formation of TBARs of about 1.56 nmole of MDA equivalents/mg membrane protein/hr. The treatment of APC (0-25 μg), BHA (0-25 μg) and α-tocopherol (0-25 μg) found to limit the formation of TBARs in a dose dependent manner (Figure 6) for protection of ion pump ATPases (Figures 4 and 5). The above results indicate that ferrous sulphate: ascorbate induced formation of TBARs in RBC ghost which closely parallels with inhibition of both ion pump ATPases (Na+K+ ATPase and Ca2+Mg2+ ATPase) and pretreatment of APC, BHA and α-tocopherol significantly ameliorated the oxidative damage of RBC membrane by reducing MDA levels and restoring ATPases activities towards normalcy. The mechanism by which APC does it probably is by directly scavenging radicals or chelating the ferrous ions (Fenton reaction) [33].

medicinal-chemistry-Inhibition-lipid-peroxidation

Figure 6: Inhibition of lipid peroxidation by APC and standard antioxidants. RBC ghost (200 μg) pre-incubated with antioxidants, treated with Fe:As (10:100 μmole) in 1 ml of TBS, pH 7.4. TBARs determined and calculated as equivalents of MDA using molecular extinction coefficient of 1.56 × 105 M-1cm-1 and expressed as nmole of MDA equivalents/mg membrane protein/hr. Data represent the mean ± SD (n=6).

In conclusion, the Curry leaves protein antioxidant is an effective antioxidant in preventing membrane damage and associated functions mediated by reactive oxygen species. It could be further developed as an effective bioprotective antioxidant agent to cellular components.

Acknowledgement

This work was supported by Research Fellowship awarded to Mylarappa B Ningappa from University of Mysore, India. The authors acknowledge the Adichunchanagiri Mahasamstana Mutt and Shikshana Trust for providing facilities at Adichunchanagiri Biotechnology and Cancer Research Institute for carrying out this work.

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  1. Fizzah
    Posted on Sep 29 2016 at 7:27 pm
    The research study for the first time shows the cytoprotective effect of APC against fenton reactant mediated alterations in RBC membrane, ATPases and K+ channels. In general, this article is informative and provides useful and comprehensive information.
 

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