Purification and characterization of ∼35 kDa antioxidant protein from curry leaves (Murraya koenigii L.)

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Toxicology in Vitro 22 (2008) 699–709 www.elsevier.com/locate/toxinvit

Purification and characterization of 35 kDa antioxidant protein from curry leaves (Murraya koenigii L.) Mylarappa Bantaganahalli Ningappa a,b,*, Leela Srinivas a b

a Adichunchanagiri Biotechnology and Cancer Research Institute, B.G.Nagara-571448, Karnataka, India Jawaharlal Nehru Centre for Advanced Scientific Research, Molecular Biology and Genetics Unit, Jakkur, Bangalore-64, India

Received 7 May 2007; accepted 9 November 2007 Available online 17 November 2007

Abstract Curry leaves (Murraya koenigii L. Spreng, Rutaceae) is an herbal species used in traditional medicine in eastern Asia. In this study, the antioxidant protein was purified by homogenization of curry leaves powder in Tris buffer, 65% ammonium sulphate precipitation and gel filtration on Sephadex G-75 column which resulted in three peaks (PI, PII and PIII). PII protein inhibited lipid peroxidation in human RBC ghost at 100 lg by 80%, whereas PI and PIII at 600 lg showed moderate antioxidant activity (40–50%). Homogeneity of PII was confirmed by rechromatographing on Sephadex G-75 and reverse phase HPLC. The antioxidant protein (PII) from curry leaves (APC) showed apparent molecular weight of 35 kDa by SDS–PAGE and MALDI/MS analysis. The protein nature of APC was established by the presence of amino acids and loss of antioxidant activity on heat and protease treatment. The APC at 0.8 lM inhibited lipoxygenase activity by 71%, effectively prevented diene, triene and tetraene lipids formation at 3 lM, and scavenged about 85% hydroxyl and DPPH radicals at 150-fold lesser concentration compared to BHA and a-tocopherol (400 lM). In addition, APC reduced cytochrome c and ferric ion, chelated ferrous ion, and inhibited ferrous sulfate: ascorbate-induced fragmentation and sugar oxidation to 80–90%. Thus, the present study demonstrates the in vitro characterization of antioxidant protein from the curry leaves (M. koenigii L.) Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Curry leaves; Antioxidant protein; Purification and characterization; Antioxidant and free radical scavenging activities

1. Introduction Reactive oxygen species have been implicated in several diseases like cancer, asthma, arthritis, etc. Production of reactive oxidants such as superoxide, hydroxyl radical and hydrogen peroxide in living cells is an inevitable process of normal oxygen metabolism (Droge, 2002). Peroxidative agents like lipoxygenases, free metal cations like

Abbreviations: APC, antioxidant protein from curry leaves; TBARs, thiobarbituric acid reactive substances; BHA, butylated hydroxyl anisole; LOX, lipoxygenase; NDGA, nordihydroguaiaretic; DPPH, 11-diphenyl-2picrylhydrazyl; AZT, azidothymidine. * Corresponding author. Address: Jawaharlal Nehru Centre for Advanced Scientific Research, Molecular Biology and Genetics Unit, Jakkur, Bangalore-64, India. Tel.: +91 9242357689; fax: +91 80 22082766. E-mail address: [email protected] (M.B. Ningappa). 0887-2333/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2007.11.009

iron and copper, and ultraviolet and ionizing radiations generate free radicals deleterious to the body. These radicals bring about their effect by oxidizing the various machineries vital for the survival of a life form. The most prominent ill effects are the oxidation of phospholipids in the lipid bilayer of cell membrane and side chain modification of proteins rendering the protein dysfunctional and oxidative damage of DNA leading to dreaded conditions of carcinogenesis as a direct effect of induced mutations (Lee et al., 2004). All organisms have antioxidant systems that are able to control and counter the onslaught of free radical mediated oxidative damage. The first line of defense constitutes antioxidative enzymes like superoxide dismutase, catalase and peroxidase, which bring about their effects by removal of either superoxide or hydroperoxide. There are other proteins such as ferritin, transferrin and lactoferrin, involved in the

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sequestration of iron, ceruloplasmin and albumin involved in copper storage (Haenen and Bast, 2002). Under the conditions of severe oxidative stress, however, cellular defenses do not provide complete protection from attack of reactive oxidants, which could lead to oxidative damage related-diseases. This situation calls for an external supply of antioxidants to counter this trend. The main disadvantage of synthetic antioxidants (butylated hydroxyl anisole and butylated hydroxyl toluene) is their toxicity at fairly high doses (Watts, 1975), which limits their therapeutic usage. Therefore, dietary sources have been recognized as safer and effective antioxidants in the context of their efficiency and non-toxicity. The intake of fruits and vegetables containing high amounts of antioxidative nutraceuticals has been associated with the balance of the free radicals/antioxidants status, which helps to minimize the oxidative stress in the body and to reduce the risks of cancers and cardiovascular diseases (Kaur and Kapoor, 2001). Several food derived antioxidants such as b-carotene, curcumin, ascorbate, etc. have been extensively used to counter the menace of oxidative damage induced diseases. It has been reported that antioxidant like b-carotene imposes high risk of lung cancer in smokers (Paolini et al., 1999). The usage of safer dietary antioxidant as extraneous source of antioxidants is gaining in popularity recently. Turmerin, an antioxidant protein from turmeric (Curcuma longa) is known to be an effective antioxidant (Srinivas and Shalini, 1991; Srinivas et al., 1992). Evidence also exists which shows that efficacious anti HIV therapy at less toxic doses of AZT could be achieved in the presence of turmerin compared to curcumin as demonstrated by in vitro studies (Cohly et al., 2003). It has also been shown that the protein hydrolysate from soybean inhibits linoleic acid peroxidation (Yamaguchi et al., 1975). Turmerin and soybean protein hydrolysate, being antioxidant proteins from plant sources, calls for more such antioxidants, which highlight the role of proteins as dietary antioxidants. With the above scenario, search for such a beneficial agents led to a new antioxidant protein from curry leaves. Plants of the genus Murraya have been widely used in traditional medicine in eastern Asia, Australia, and South Africa, as tonics for dysentery, fever, influenza stomachic, stimulants, carminative and the bites of poisonous animals and as source of flavors. In this study, systematic search was conducted to isolate and characterize the active antioxidant protein (35 kDa) from curry leaves (APC). The antioxidant properties of APC were studied in comparison with standard antioxidants such as BHA, a-tocopherol and ascorbate. 2. Materials and methods 2.1. Plant material Curry leaves (Murraya koenigii L.) were obtained from the garden maintained by Adichunchanagiri Biotechnology

and Cancer Research Institute (ABCRI), B.G.Nagara571448, Karnataka state, India in the months of July to September 2003. The identity of the plant was confirmed by G.R. Shivamurthy, Taxonomist, University of Mysore, India. The herbarium of the plant was deposited at ABCRI against voucher No. ABCRI 7/2003. The curry leaves were dried at ambient temperature for 15–20 days. After complete drying, the leaves were grounded to fine powder using domestic electric grinder [MASTER 17 (CM/L-736889), Soni Appliances, Mumbai, India]. 2.2. Chemicals/materials Ammonium sulphate, butylated hydroxyl anisole, atocopherol, acrylamide, bisacrylamide, N,N,N0 ,N0 -tetramethylethylenediamine (TEMED), thiobarbituric acid (TBA), calf thymus DNA, ferrous sulphate, ascorbic acid, polyvinyl pyrrolidone, polyethyleneimine, 2-deoxy ribose, ferric chloride, linoleic acid, were purchased from Sigma (St. Louis, USA). Sephadex G-75 and Sephadex G-25 were procured from Pharmacia, Sweden. All other chemicals unless otherwise mentioned were procured from Merck (Dermastadt, Germany). Shimadzu UV-1601 spectrophotometer (Tokyo, Japan) was used for calorimetric analysis and Kubota 6800 (Kubota Co., Osaka, Japan) was used for centrifugation. 2.3. Purification of the protein Five gram of curry leaves powder was homogenized in 20 ml of 10 mM Tris buffer pH 7.0 and the volume was made up to 100 ml with the same buffer, and 200 mg of polyvinyl pyrrolidone was added to remove polyphenols. The suspension was incubated overnight at 4 °C with constant stirring. The homogenate was filtered through Whatman No. I filter paper and centrifuged at 13,000 rpm at 4 °C for 20 min. The supernatant was treated with 0.1% polyethyleneimine to precipitate nucleotides. The resultant pellet was discarded and the supernatant was brought to 65% saturation of ammonium sulphate. The pellet was dissolved in 20 mM Tris pH 7.4 and desalted on Sephadex G-25 equilibrated with the same buffer. The protein was concentrated to required volume using centrikon of 10 kDa cutoff and loaded on to Sephadex G-75 column (Vo = 34 ml, Vt = 104 ml, flow rate of 1.5 ml/5 min) equilibrated with Tris–HCl buffer (20 mM, pH 7.4). Elution was carried out with the same buffer and monitored at 280 nm and protein content of each fraction was estimated by Bradford’s (1976) method. Each peak fractions (Fig. 1a) were pooled separately and concentrated to required volume using centrikon (10 kDa). The antioxidant activity of these protein samples at various concentrations ranging from 20 to 1000 lg was tested by TBARs assay (Shimazaki et al., 1984). The active peak II protein (fractions from 29 to 33) was further fractionated on Sephadex G-75 column (Fig. 1b) by eluting with Tris–HCl buffer (20 mM, pH 7.4) to arrive at homogenous preparation. The protein obtained

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Fig. 1. (a) Gel filtration profile of ammonium sulphate precipitated protein of crude buffer extract of curry leaves on Sephadex G-75 column. Three peaks (PI, PII and PIII) eluted from the column and antioxidant activity of each peak was determined by TBARs assay; (b) The active peak II fractions rechromatographed on Sephadex G-75 column. The chromatogram shows single peak. The results are shown as means ± SD (n = 6).

by this process was designated as ‘‘antioxidant protein from curry leaves” (APC).

mated according to the method described by Sadasivam and Manickam (1997a,b).

2.4. Characterization of the protein

2.4.4. Effect of temperature on antioxidant activity of the protein APC (500 lg) was incubated at 45 °C, 60 °C and 95 °C for 20 min in 1 ml of Tris–HCl buffer (20 mM, pH 7.4). Aliquots of samples were then tested for their antioxidant activity by TBARs assay.

2.4.1. Tests for homogeneity The homogeneity and the molecular weight of the protein were assessed according to the method of Laemmli (1970). SDS–PAGE was performed with 10% resolving and 4% stacking gels. The APC (10 lg) obtained from gel filtration (Fig. 1b) was subjected to reverse phase HPLC on Vydac C18 column (Shimadzu LC10AVP HPLC with a dual wavelength detector) and eluted by gradient elution using 70% acetonitrile in water with 0.1% trifluoroacetic acid, at a flow rate of 1 ml/min for 65 min. Fractions were monitored at 280 nm. 2.4.2. MALDI/MS analysis The APC was mixed with matrix solution (synapinic acid in 50% acetonitrile and 0.1% TFA) and the mixture was spotted on the MALDI target plate. The spectrum was acquired in positive ion mode at 20 kV in linear mode on a MALDI TOF TOF Bruker Ultraflex II. 2.4.3. Estimations The protein content at various stages of purification was determined by Bradford’s (1976) method; the ninhydrin test was done according to the method described by Sadasivam and Manickam (1997c). Sulphydryl group was estimated by the Ellman’s (1959) method. The presence of fatty acid derivatives was estimated by Folch lipid extraction method and analyzed by thin layer chromatography (Folch et al., 1957). The total sugar was estimated by the phenol–sulphuric acid method (Dubois et al., 1956), total phenolic content was determined by the Folin–Ciocalteau reagent (Kujala et al., 2000); chlorophyll content was esti-

2.4.5. Effect of protease on antioxidant activity of the protein The effect of non-specific protease Staphylococcus aureus (S) was assessed on APC. APC (500 lg) was incubated at 37 °C for 1 h with 20 lg of S. aureus (S) in 20 mM Tris– HCl buffer pH 6.8. The incubation mixture contained APC in the presence or absence of proteolytic enzyme in a ratio of 25:1 w/w. The reaction was arrested by keeping the tubes in ice. Aliquots of samples were then tested for their antioxidant activity by TBARs assay. 2.5. Antioxidant activity (TBARs assay) A simple spectrophotometric assay for evaluating antioxidant activity was based on the inhibition of peroxidation in RBC ghost (Shimazaki et al., 1984). An assessment of oxidation was achieved by measurement of thiobarbituric acid reactive substances (Dahle et al., 1962). The human erythrocyte ghost was isolated according to the method of Dodge et al. (1963). 100 l1 of ghost suspension (300 lg membrane protein equivalent) was subjected to peroxidation by 10:100 lmol of ferrous sulphate and ascorbic acid (Fenton, 1984; Srinivas et al., 1992) in final volume of 1 ml of Tris buffered saline (20 mM, pH 7.4,150 mM NaCl). The reaction mixture was treated with or without crude extract (200–2000 lg/ml), 65% ammonium sulphate precipitated protein, peak I, peak II and peak III at various

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concentrations ranging from 50 to 1000 lg/ml. Butylated hydroxy anisole (BHA) was used as positive control. The contents were incubated for 1 h at 37 °C. The reaction was terminated by the addition of 10 ll of 5% phenol and 1 ml of 1% trichloroacetic acid (TCA). To each system 1 ml of 1% thiobarbituric acid (TBA) was added, the contents were kept in a boiling water bath for 15 min, cooled and centrifuged at 6000 rpm for 10 min. The absorbance of supernatants was measured colorimetrically at 535 nm. Appropriate blanks were included for each measurement. The negative control without any test sample was considered as 100% peroxidation. The % inhibition of lipid peroxidation was determined accordingly by comparing the absorbance of the test samples with negative control. 2.6. Inhibition of lipoxygenase activity The assay was carried out by monitoring the appearance of linoleic acid hydroperoxide at 234 nm (Axelrod et al., 1981; Narayan et al., 1999). One unit of the enzyme activity is defined as the amount of enzyme required to form 1 lmol of the product per minute at 30 °C under the assay conditions. 25 nM soybean lipoxygenase enzyme was pretreated with APC (0.2–1 lM) in 2.7 ml Tris buffer (20 mM, pH 7.4) for 5 min at ambient temperature. The reaction was initiated by the addition of 50 lM linoleic acid and final volume was made to 3 ml with Tris–HCl buffer (20 mM, pH 7.4). The absorbance was recorded for 3 min at 234 nm. Similarly, experiments were done with standard inhibitors such as NDGA and caffeic acid (Inhibitors dissolved in a minimum quantity of DMSO at less than 0.1% concentration). The relative activity was expressed as percentage ratio of enzyme activity in the presence of inhibitors or APC to the enzyme activity in the absence of enzyme inhibitor (negative control) at the end of 3 min of enzyme reaction time. Inhibition was expressed as percentage relative to negative control. 2.7. Diene–triene–tetraene conjugation The extent of lipid peroxidation was measured by the release of dienes, trienes and tetraenes. Conjugation indicates the initial stages of oxidative lipid peroxidation of unsaturated fatty acids such as linoleic acid (conjugated diene: 233 nm), linolenic acid (conjugated triene: 268 nm) and arachidonic acid (conjugated tetraene: 314 nm) (Stoffel and Ahrens, 1958; Srinivas et al., 1992). Ghost suspension (300 lg of protein equivalent) was subjected to peroxidation with ferrous sulphate: ascorbic acid (10:100 lmol) with or without APC (1.5, 3 lM) and BHA (400 lM) in final volume of 1 ml Tris buffer (20 mM, pH 7.4). The peroxidized ghost lipid was extracted with four volumes of chloroform: methanol [2:1 v/v]. The lower organic phase was evaporated under nitrogen and solubilized in 3 ml of hexane. The solutions were scanned between 210 and 340 nm

against a hexane blank. The control was without any antioxidant or test sample. 2.8. Hydroxyl radical scavenging activity Deoxyribose assay was used to determine the hydroxyl radical scavenging activity in an aqueous medium (Halliwell et al., 1987). The reaction mixture containing FeCl3 (100 lM), EDTA (104 lM), H2O2 (1 mM) and 2-deoxyD-ribose (2.8 mM) were mixed with or without APC at various concentrations (0.5–2.5 lM) in 1 ml final reaction volume made with potassium phosphate buffer (20 mM pH 7.4) and incubated for 1 h at 37 °C. The mixture was heated at 95 °C in water bath for 15 min followed by the addition of 1 ml each of TCA (2.8%) and TBA (0.5% TBA in 0.025 M NaOH containing 0.02% BHA). Finally the reaction mixture was cooled on ice and centrifuged at 5000 rpm for 15 min. Absorbance of supernatant was measured at 532 nm. All readings were corrected for any interference from brown color of the extract or antioxidant by including appropriate controls. The negative control without any antioxidant or APC was considered 100% deoxyribose oxidation. The % hydroxyl radical scavenging activity of test sample was determined accordingly in comparison with negative control. 2.9. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity DPPH radical scavenging activity was assessed according to the method of Shimada et al. (1992). The APC at various concentrations ranging from 0.5 to 2.5 lM was mixed in 1 ml of freshly prepared 0.5 mM DPPH ethanolic solution and 2 ml of 0.1 M acetate buffer pH 5.5. The resulting solutions were then incubated at 37 °C for 30 min and measured colorimetrically at 517 nm. BHA (400 lM) was used as positive control under the same assay conditions. Negative control was without any inhibitor or APC. Lower absorbance at 517 nm represents higher DPPH scavenging activity. The % DPPH radical scavenging activity of APC was calculated from the decrease in absorbance at 517 nm in comparison with negative control. 2.10. Ferric ion reducing capacity The reducing power was determined according to the method of Wang et al. (2003) with minor modifications. 100 ll of potassium ferricyanide solution (4 mM) was mixed with 200 ll of phosphate buffer (20 mM, pH 6.5) with or without APC at the concentrations ranging from 0.5 to 2.5 lM. The contents were incubated at 50 °C for 20 min. 200 ll of 10% TCA was added to the reaction mixture and centrifuged at 5000 rpm. The resulting supernatant was taken and mixed with 100 ll of ferric chloride solution (2 mM) and final volume was made up to 1 ml with water and then incubated at ambient temperature

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for 10 min. BHA (400 lM) and ascorbic (1 mM) were served as positive controls. The absorbance was recorded at 700 nm. Absorbance increases with increase in reducing capacity. 2.11. Cytochrome c reduction The cytochrome c reducing capacity of APC was determined according to the method of Suter and Richter (2000). Unaltered cytochrome c has characteristic spectra with kmax of 550 nm due to its active heme group, which contains ferrous ion. When subjected to oxidation by oxygen saturated phosphate buffer (0.1 mM, pH 7), the peak at 550 nm will diminish quantitatively. In the assay, cytochrome c (15 lM) was subjected to oxidation by 2.9 ml of oxygen saturated phosphate buffer (0.1 mM, pH 7), then APC (1.5, 3 lM)/ascorbic acid (1 mM)/BHA (400 lM) was added. Final volume was made to 3 ml with the same buffer and incubated at ambient temperature for 30 min. Absorbance at 550 nm was measured. Absorbance increases with increase in reduction of oxidized cytochrome c. Appropriate controls were maintained. 2.12. Ferrous ion chelating activity Ferrous ion chelating activity was measured according to the method of Suter and Richter (2000) with minor modifications. The reaction solution containing ferrous chloride (200 lM)and potassium ferricyanide (400 lM) with or without APC (0.5–2.5 lM)/ EDTA (60 lM) was made to 1 ml with double distilled water and mixed. The reaction mixture was incubated at 20 °C for 10 min. Formation of the potassium hexacyanoferrate complex was measured at 700 nm in Shimadzu spectrophotometer. The assay was carried out at 20 °C to prevent Fe2+ oxidation. Lower absorbance indicated higher iron chelating capacity. The negative control was without any chelating compound or test sample. The % ferrous ion chelating activity was calculated from the decrease in absorbance at 700 nm in comparison with negative control. 2.13. Protective effect of APC on fenton reactant induced DNA sugar damage Oxidative DNA sugar damage was determined according to the method of Sultan et al. (1995) with minor modifications. The reaction mixture contained 1 mg of calf thymus DNA was treated with ferrous sulphate and ascorbate (5:50 lmol) (Fenton, 1984; Srinivas et al., 1992) in total volume of 1 ml of potassium phosphate buffer (20 mM, pH 7.4) with or without APC (1–5 lM)/BHA (400 lM). Reaction mixture was incubated at 37°C for 1 h in water bath shaker. The colour was developed by adding 1 ml of 2.8% TCA and 1 ml of 1% TBA and boiled for 20 min, cooled on ice and centrifuged at 5000 rpm for

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10 min. The absorbance was read at 535 nm. The negative control without any test sample or antioxidant was considered as 100% oxidation. The % inhibition of DNA oxidation was calculated by comparing the absorbance of the test sample with negative control. 2.14. 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 means ± SD. P < 0.05 was considered as statistically significant as comparing to relevant controls. 3. Results 3.1. Purification of the antioxidant protein The maximum antioxidant activity was recovered in the fraction precipitating at 65% ammonium sulphate saturation from the Tris buffer extract of curry leaves. The pellet was dissolved in 20 mM Tris pH 7.4 and further passed through Sephadex G-25 column to remove ammonium salts and other low molecular components such as free sugars or polyphenols, which could contribute to the antioxidant activity. Fractionation of protein from curry leaves (20 mg) on Sephadex G-75 column by eluting with tris buffer (20 mM, pH 7.4) resulted in three peaks (PI, PII and PIII) as monitored at 280 nm (Fig. 1a). The protein content of fractions was also simultaneously estimated by Bradford method, which corresponded with the peaks at A280 curves. Inhibitory effect of each fraction from the peaks against ferrous sulphate: ascorbate (10:100 lmol) induced human RBC ghost lipid peroxidation was determined by TBARs assay and the activity was expressed by protein concentration. The fractions falling under peak II (from 29 to 33) displayed the maximal antioxidant activity and it was designated as active peak II. The fractions of each peak were pooled separately, concentrated by centrikon of 10 kDa cutoff to required volume and concentration. Table 1 shows the antioxidant activity of ammonium sulphate precipitated protein, peak I, peak II and peak III in comparison with BHA, studied using RBC ghost as model system. The 65% ammonium sulphate precipitated protein showed 64% antioxidant activity when used at 1000 lg concentration in the assay. The peak II protein exhibited highest antioxidant activity (80%) at 100 lg whereas Peak I and peak III protein showed moderate inhibition of 40% and 50% at 600 lg, respectively. The antioxidant activity of peak II (100 lg) was found to be more efficient than BHA (400 lM) that showed only 64% antioxidant activity. The active peak II protein was rechromatographed on Sephadex G-75 column resulted in single peak (Fig. 1b) indicating that homogeneous preparation of the antioxidant protein from curry leaves (APC) has been accomplished.

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Table 1 Summary of purification antioxidant protein from curry leaves (Murraya koenigii L.) Purification steps

Total proteina (mg)

1. Buffer extract 39 ± 4 2. 65% (NH4)2SO4 21 ± 2 precipitation 3. Gel filtration on Sephadex G-75 Peak I 10 ± 2

Yield (%)

Protein (lg/ml)

% Inhibition

100 53 ± 4

2000 1000

51 ± 4 64 ± 2

25 ± 3

400 600 50 100 400 600 BHA (72 lg)

34 ± 3 52 ± 2 56 ± 2 81 ± 2 27 ± 2 43 ± 2 64 ± 3

Peak II

8±2

22 ± 2

Peak III

2 ± 0.21

17 ± 2

Standard antioxidant

Inhibition of lipid peroxidation

Antioxidant activity of curry leaves proteins at the concentrations ranging from 0 to 500 lg/1 ml was determined using RBC ghost as model system by TBARs assay as described in the methods. The results are shown as means ± SD (n = 6). BHA, butylated hydroxy anisole. a Data refer to the protein obtained during the various stages of purification from the 5 g of curry leaves dried material.

3.2. Characterization of antioxidant protein from curry leaves (APC) The purified antioxidant protein from curry leaves showed approximate molecular weight of 35 kDa by SDS–PAGE (Fig. 2a) and an exact molecular mass of 35.28 kDa by MALDI/MS (Fig. 2b). The protein purity was further confirmed by reverse phase HPLC that showed single peak (Fig. 2c). Fig. 3 shows the susceptibility of APC to proteolytic enzyme and heat. Untreated APC (3 lM) showed 80%

Fig. 3. Effect of heat and protease treatment on antioxidant activity of APC. The aliquots of APC (500 lg) was preheated at 45 °C, 65 °C and 95 °C and digested with protease S as described in materials and methods. Antioxidant activity of APC (3 lM) was determined by TBARs assay. The results are shown as means ± SD (n = 6). APC, antioxidant protein from curry leaves.

antioxidant activity, while preheat treatment of APC (3 lM) at 45 °C, 65 °C and 95 °C decreased antioxidant activity to 64%, 39% and 8%, respectively, suggesting that antioxidant protein is destabilized above 45 °C. Treatment of APC (3 lM) with non-specific protease (S. aureus S.) resulted in a significant loss of antioxidant activity (only 6% antioxidant activity is remained after the treatment). The biochemical tests proved that APC is devoid of total sugars, fatty acid derivatives, polyphenols and chlorophyll. Antioxidant protein tested proved positive for ninhydrin. These results suggest that antioxidant principle was a protein. The composition of SH groups were tested by Ellman’s reagent (Ellman, 1959) and 9 lmol of sulfhydryl groups/g of APC was detected. The data are shown in Table 2.

Fig. 2. (a) SDS–PAGE of purified antioxidant protein from curry leaves. Lane 1, Molecular weight marker; Lane 2, the purified antioxidant protein (peak II). The purified protein antioxidant of curry leaves migrated as a single sharp protein band in the region of 35 kDa; (b) the chromatogram of purified protein by MALDI/MS analysis, shows single peak at 35.322 kDa; (c) the reverse phase HPLC chromatogram of through C18 hydrophobic column, showing a single peak.

M.B. Ningappa, L. Srinivas / Toxicology in Vitro 22 (2008) 699–709 Table 2 Biochemical characterization of antioxidant protein from curry leaves Analysis

Result

Amino acids Sulphydryl groups Lipid residue Polyphenols Total sugars Chlorophyll

Positive 9 ± 1.2 lmol/g of APC Negative Negative Negative Negative

Test for amino acids, lipid residue, polyphenols, chlorophyll, total sugars, chlorophyll and estimation of sulphydryl groups were carried out as described in methods. The results are shown as means ± SD (n = 6). APC, antioxidant protein from curry leaves.

3.3. Antioxidant properties 3.3.1. Inhibition of lipoxygenase activity Fig. 4a shows that APC inhibits lipoxygenase activity in a dose dependent manner saturating at 79% inhibition. The effective concentration of APC for saturation was 0.8 lM. The known LOX inhibitors NDGA (at 1000 lM) (Tapel et al., 1953) and caffeic acid (at 500 lM) (Koshihara et al., 1984) showed inhibition of lipoxygenase enzyme activity by 69% and 61%, respectively. 3.3.2. Diene–triene–tetraene conjugation Fig. 4b shows the release of diene, triene and tetraene conjugated lipids in ferrous sulphate: ascorbate (fenton reactant) treated human erythrocyte membrane. As seen from the figure, APC at 3 lM decreased the release of conjugated lipids to the levels comparable to BHA (400 lM). 3.3.3. Hydroxyl radical scavenging activity Fig. 5a shows that APC scavenges hydroxyl radicals in a dose dependent manner saturating at 87%. The effective concentration to achieve saturation was 3 lM for APC.

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Both BHA and a-tocopherol at 400 lM scavenged hydroxyl radicals by 80–90%. 3.3.4. DPPH radical scavenging activity Fig. 5b shows that APC exhibits DPPH radical scavenging activity of 42% at 1.8 lM and 80% at 3 lM. BHA and a-tocopherol at 400 lM each showed DPPH radical scavenging activity of 75–80%. 3.3.5. Ferric ion reducing power As shown in Fig. 6a, reducing power of APC at 2.5 lM was found to be 1.42 absorbance at 700 nm, which was higher than BHA (400 lM) and comparable to ascorbic acid (1000 lM). 3.3.6. Cytochrome c reduction Fig. 6b shows that APC (2.5 lM) reduces oxidized cytochrome c by 100% at 20 min. Ascorbic acid at 1000 lM showed 100% cytochrome c reduction. 3.3.7. Ferrous ion chelation The chelation of Fe2+ ions by the purified protein was tested in a competition assay with potassium ferricyanide and the activity was monitored by loss of absorption at 700 nm (iron–hexacyanoferrate complex). As shown in Fig. 7a, APC exhibited maximum chelating effect of 90% at 2 lM and its iron chelating effect is comparable to EDTA (60 lM). 3.3.8. Protection of DNA sugar damage To confirm the chelating ability, the inhibitory effect of APC against iron dependent oxidation of calf thymus DNA sugar was tested by TBARs assay. As shown in Fig. 7b, APC offered effective protection against ferrous sulphate: ascorbate-induced DNA sugar oxidation by 93% at 5 lM as compared to BHA that showed 90% protection at 400 lM.

Fig. 4. (a) Inhibition of lipoxygenase activity by APC; (b) inhibition of diene–triene–tetraene formation by APC. The results are shown as means ± SD (n = 6). APC, antioxidant protein from curry leaves; NDGA, nordihydroguaiaretic.

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Fig. 5. (a) Hydroxyl radical scavenging activity of APC; (b) DPPH radical scavenging activity of APC. The results are shown as means ± SD (n = 6). APC, antioxidant protein from curry leaves; BHA, butylated hydroxyl anisole.

Fig. 6. (a) Ferric ion reducing power of APC; (b) cytochrome c reduction by APC. The results are shown as means ± SD (n = 6). APC antioxidant protein from curry leaves; BHA, butylated hydroxyl anisole.

Fig. 7. (a) Ferrous ion chelation by APC; (b) protective effect of APC on fenton reactant induced DNA sugar damage. The results are shown as means ± SD (n = 6). APC, antioxidant protein from curry leaves; BHA, butylated hydroxyl anisole; EDTA, N,N,N0 ,N0 -tetramethylethylenediamine.

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4. Discussion Curry leaves (M. koenigii L.), one of the major spices, has been consumed in India for its characteristic flavor and aroma. Polyphenol-enriched ethyl alcohol:water (1:1) extract isolated from curry leaves have been shown to be more effective than BHA and a-tocopherol in their antioxidant properties (Ningappa et al., 2008). Literature evidence also reveals that curry leaves possess antioxidant effects in rats fed with high fat diet (Khan et al., 1997), anti-carcinogenic effects in dimethylhydrazine-treated rats (Khanum et al., 2000) and hypoglycemic effects in alloxan induced diabetic rats (Yadav et al., 2002). The present study was carried out to isolate and characterize active antioxidant protein from the buffer extract of curry leaves (M. koenigii L.). The active protein was isolated and purified to homogeneity after homogenization, ammonium sulphate precipitation and gel filtration chromatography. SDS–PAGE and MALDI/MS analysis of protein demonstrated approximate molecular weight of 35 kDa. Heat treatment and protease digestion destroyed the antioxidant activity of the protein indicating its characteristic nature. The APC contains amino acids as evidenced by ninhydrin test and it was found to be devoid of fatty acid derivatives, polyphenols, total sugars and chlorophyll. Furthermore, 9 lmol of sulfhydryl groups/g of APC was detected. Hence, the presence of SH groups in the APC could be additive along with the other unknown functional groups of APC for effective antioxidant property. It has been reported that SH group act as a free radical scavenger in plants and animal tissue (Patterson and Rhoades, 1988) and SH group of cysteine facilitates the antioxidant activity of glutathione (Selvam and Devaraj, 1996). The effect of APC on downstream inflammatory enzyme viz lipoxygenase was studied. Lipoxygenase is one of the key enzymes in the metabolism of arachidonic acid to pro-inflammatory leukotrienes and it causes lipid peroxidation with the release of hydroxy ecosatetraenoic acid and hydroperoxy ecosatetraenoic acids that are responsible for membrane mediated DNA damage (Narayan et al., 1999). The results indicate that APC is an efficient inhibitor of lipoxygenase at 1000- and 500-fold lesser concentrations compared to NDGA and caffeic acid, respectively. The mechanism of inhibitory effect of APC on lipoxygenase activity may be due to binding of Fe2+ ion [as evidenced by Fe2+ ion chelating activity (Fig. 7a)], present in the enzyme as the prosthetic group (Axelrod et al., 1981) or non-specific interaction. Hence, the APC could play a modulating role in the cellular oxidative stress induced by enzymatic lipid peroxidation. Similar studies have been reported that anthocyanin isolated from carrot cell culture inhibited in vitro lipoxygenase catalyzed lipid peroxidation (Narayan et al., 1999). Keeping the above antioxidant activity in view, the analogy was further extended. The antioxidant activity was confirmed by diene–triene–tetraene conjugation method as a sensitive index of lipid peroxidation. Conjugated diene

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reacts with oxygen to form a peroxyl radical which in turn abstracts a hydrogen bond from another lipid molecule forming lipid hydroperoxide and other lipid oxidation products like aldehydes and alkenals (malonaldehyde and 4-hydroxy-2-nonenal) (Buege and Aust, 1978; Rice-Evans and Burdon, 1993). This initiates a chain reaction of lipid peroxidation of variety of molecules at the cellular level. The experimental results indicate that APC significantly decreased the release of diene, triene and tetraene conjugation at 133-fold lesser concentration than BHA indicating that the APC is effective inhibitor of lipid peroxidation. The indirect evidence of the scavenging activity of APC on Fe3+-dependent hydroxyl radical generation was confirmed using a direct approach with DPPH radicals (Chang et al., 2001). Hydroxyl radicals are known to be the most reactive of all the reduced forms of dioxygen and are thought to initiate cell damage in vivo (Rollet-Labelle et al., 1998). The experimental results indicate that the APC effectively scavenges hydroxyl and DPPH radicals compared to BHA and a-tocopherol. Thus APC could be an effective scavenger of free radicals. The extracts of spices and herbs may well act as electron donors and can react with free radicals to convert them to more stable products and terminate radical chain reactions and it has been shown that the antioxidation effect exponentially increased as a function of the development of the reducing power (Tanaka et al., 1988). The APC exhibited maximum reducing power at 150- and 400-fold lesser concentration compared to BHA and ascorbic acid, respectively. The reducing power might be due to their hydrogen donating ability (Shimada et al., 1992). Cytochrome c, a major electron transport protein of the respiratory chain, was used as a model protein to investigate direct reductive capacity of APC as mechanism of antioxidant property (Suter and Richter, 2000). The results indicate that APC reduces the cytochrome c far more effectively at a concentration which is three orders of magnitude lesser than ascorbic acid. It is self evident that the strong reductive power of antioxidants may also affect ions, especially Fe2+ and Cu2+. Iron is an essential mineral for normal physiology, but excess of it may result in cellular injury. If they undergo the fenton reaction, these reduced metals may form highly reactive hydroxyl radicals and thereby contribute to oxidative stress (Hippeli and Elstner, 1999). Since ferrous ions are the most effective pro-oxidants in food system, the good chelating effect would be beneficial and removal of free iron from circulation could be promising approach to prevent oxidative stress induced diseases. These processes can be delayed by iron chelation and deactivation. When iron is chelated, it may lose pro-oxidant properties. The APC was found to be capable of binding Fe2+ ions and it effectively inhibited ferrous sulphate: ascorbate (fenton reactant) induced DNA sugar oxidation. These results indicate that APC could exert a protective effect under conditions of the fenton reaction. Similar studies have reported that iron chelating protein act as a cytoprotective antioxidant (Balla et al., 1992).

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