Chemical Papers 68 (10) 1298–1304 (2014) DOI: 10.2478/s11696-014-0576-0
ORIGINAL PAPER
Evaluation of antioxidant activity and DNA cleavage protection effect of naphthyl hydroxamic acid derivatives through conventional and fluorescence microscopic methods Priyanka Singh, Deepesh Khare, Rama Pande* School of Studies in Chemistry, Pt. Ravishankar Shukla University, Raipur (C.G.), 492 010, India Received 18 November 2013; Revised 10 February 2014; Accepted 15 February 2014
Antioxidant capacity of N-(1-naphthyl)valerohydroxamic acid (NVHA) and N-(1-naphthyl)phenylacetohydroxamic acid (NPAHA) has been evaluated by a novel approach employing the fluorescence microscopic single molecule observation method. This method allows direct observation of the changes in single DNA molecules. The DNA cleavage protection activity of the compounds was also assessed by the gel electrophoresis method. The applied methods confirmed that both compounds are capable of inhibiting the free radical mediated DNA damage. Free radical scavenging activity was assessed via the 2,2 -diphenyl-1-picrylhydrazyl free radical (DPPH) and lipid peroxidation inhibition methods. The effective concentration causing a 50 % inhibition of the DPPH concentration, EC50 , was found to be 371.54 mM for NVHA and 365.95 mM for NPAHA. Its lipid peroxidation inhibition ability was calculated to be 40.91 % at 371.54 mM for NVHA and 41.14 % at 365.95 mM for NPAHA. These results show the antioxidant potential of the naphthyl hydroxamic acids. c 2014 Institute of Chemistry, Slovak Academy of Sciences Keywords: DNA cleavage protection, fluorescence microscope, free radical scavenging activity, naphthyl hydroxamic acid
Introduction The phenomenon of fluorescence can provide valuable information for the fundamental study of molecular interactions. The development of specific fluorescent dyes has stimulated widespread use of fluorescence microscopy in life sciences to identify cells and submicroscopic cellular components, such as lipids and nucleic acids, with a high degree of specificity. The fluorescence microscope utilizes electronic imaging to acquire information at visually undetectable wavelengths. It has become an indispensable device in various fields of research. It can reveal the presence of a single fluorescing DNA molecule for its further study regarding its interactions with different biomolecules. Rigler and his co-workers were the first to use confocal fluorescence microscopy for the detection of single molecules (Rigler et al., 1993). Since then, a number of methods have been developed to manipulate single DNA molecules (Nie & Zare, 1997).
Free radicals and reactive oxygen species are continuously produced in biological systems during various metabolic processes (Valko et al., 2007). These free radicals oxidize nucleic acids, proteins and lipids and cause degenerative diseases (Ames et al., 1993; Valko et al., 2006). Antioxidant compounds have the capability of scavenging free radicals such as peroxide or hydroxyl radicals, the causative agents of these degenerative diseases (Harman, 1995; Autore et al., 2010), and hence they play a significant role in the prevention of these diseases. Therefore, analysis of the antioxidant activity of new compounds from the medical point of view has received increased attention in recent years (Shirinzadeh et al., 2010). Several methods for the detection of antioxidant capability of compounds are available. Broderick and Cooke (2009) used fluorescence microscopy for the detection of fruit composition, tissues and localization of antioxidants and capsaicinoids in Capsicum peppers. Hyogo et al. (2010) used a fluo-
*Corresponding author, e-mail:
[email protected]
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P. Singh et al./Chemical Papers 68 (10) 1298–1304 (2014)
rescence microscope to study the antioxidant effects of protocatechuic acid, ferulic acid and caffeic acid in human neutrophils. The effect of lipophilic antioxidants on iodoacetic acid (IAA) induced membrane structural aberrations was also observed through a fluorescence microscope by Wu et al. (1996). Behl et al. (1997) used a fluorescence microscope for the detection of intracellular H2 O2 and related peroxides. Sautin et al. (2007) and Nakamura et al. (1998) used a fluorescence microscope to study the adverse effects of the classic antioxidant in adipocytes and cardiac hypertrophy inhibition, respectively. Sato et al. (2005) used the fluorescence microscope single molecule observation technique to study the conformational transition of plasmid DNA, pGEG.GL3 induced by spermine. Oana et al. (2002) studied the lambda DNA cleavage by the restriction enzyme ApaLI in the presence of spermine through a fluorescence microscope. Generally, more than one method is recommended for in vitro interpretation of the antioxidant tendency (Aruoma, 1996; Aruoma & Cuppett, 1997). Therefore, the single molecule observation technique was used along with several conventional methods in the present investigation to study the antioxidant activities of N(1-naphthyl)valerohydroxamic acid (NVHA) and N(1-naphthyl)phenylacetohydroxamic acid (NPAHA). The advantage of this new approach lies in the possibility to observe the changes in the DNA configuration directly by optical imaging through a fluorescence microscope. To the best of our knowledge, this method has not yet been applied for the study of DNA protection against free radical mediated damage in case of hydroxamic acids. NVHA and NPAHA are members of the N-arylhydroxamic acid family showing antitumour/anti-cancer activity (Rajwade et al., 2008, 2009). They are also capable of acting as hydrogenbond donors (Tiwari & Pande, 2006; Patre et al., 2011) and the antioxidant capability and radical scavenging effect mainly depend on the ease with which a particular compound is able to release its H-atom in order to neutralize the free radical. Therefore, the above mentioned facts inspired us to determine the antioxidant ability of the N-arylhydroxamic acid series. Phenylbenzohydroxamic acid (PBHA), the parent compound of the series, has already been reported to show antioxidant activity (Khare et al., 2012). The present investigation is done in continuation of the work going on in our laboratory.
Experimental N-(1-naphthyl)valerohydroxamic acid (NVHA) and N-(1-naphthyl)phenylacetohydroxamic acid (NPAHA) (Fig. 1) were prepared according to the standard procedure (Gupta & Tandon, 1972). Their purity was confirmed by the determination of their melting points, and UV and IR spectral analyses. The data were then compared with the reported literature
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Fig. 1. Structure of N-(1-naphthyl)phenylacetohydroxamic acid (NPAHA) (a) and N-(1-naphthyl)valerohydroxamic acid (NVHA) (b).
values (Gupta & Tandon, 1972). A Varian–EL analyzer apparatus was used for elemental analysis. N-(1-naphthyl)valerohydroxamic acid (NVHA). Formula: C15 H17 NO2 ; Mr = 243.30; M.p. = 102– 103 ◦C; wi (found)/%: C, 74.05; H, 7.04; N, 5.76; O, 13.15; IR, ν˜/cm−1 : 3125 (N—OH), 1634 (C—O). N-(1-naphthyl)phenylacetohydroxamic acid (NPAHA). Formula: C18 H15 NO2 ; Mr = 277.32; M.p. = 115–117 ◦C; wi (found)/%: C, 77.96; H, 5.45; N, 5.05; O, 11.54; IR, ν˜/cm−1 : 3125 (N—OH), 1625 (C—O). Stock solution of the hydroxamic acids was prepared in ethanol and stored in a cool and dark place. 2,2 -Diphenyl-1-picrylhydrazine (DPPH), calf thymus DNA (Ct-DNA) and the fluorescent dye, 4,6-diamidino-2-phenylindole (DAPI), were purchased from Sigma–Aldrich (USA). The λ plasmid DNA and hydrogen peroxide were purchased from Merck (Japan). Ct-DNA was dissolved in a tris-HCl buffer at the final concentration of 1 mM for the stock preparation and stored at 4 ◦C. Tris-HCl buffer solution (0.1 M) was used for further dilution of Ct-DNA. pH was maintained at 7.8 with 0.01 M HCl prepared by a standard procedure. TAE buffer (50 mM) was used for gel electrophoresis. Spectroscopic grade chemicals and doubly distilled deionised water were used throughout the experiments. The absorbance measurements were carried out using an Elico India Biospectrum BL-198 (India) equipped with thermostatic temperature controller Gl-635 using 1.0 cm quartz cells. Gel electrophoresis was performed in a Genei submerged gel electrophoresis system. Fluorescence microscopic images were taken with a Nikon Eclipse (Japan) Ti–S fluorescence microscope equipped with a Nikon DS-Fi2 camera. pH measurements were carried out using an Eutech instruments Oakton (Singapore) digital pH meter, with a combined glass-calomel electrode. DPPH free radical scavenging method Solutions of the compounds and DPPH were prepared in ethanol and they were added to the DPPH solution in different concentrations. Absorbance of the
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reaction mixture was recorded at 517 nm at the beginning of the reaction and after 30 minutes of incubation at room temperature. Ethanol and ethanolic solution of BHA were used as the negative and positive control, respectively. The data thus obtained were presented as a plot of percentage inhibition of DPPH radical vs. concentration of compounds. Percentage inhibition of the DPPH free radical was calculated from Eq. (1) (Končič et al., 2009): A0 − AS Inhibition = 100 % A0
RH = (1)
where A0 and AS are the absorbance of control and test sample, respectively. The radical scavenging activity of the compounds was found to increase with the concentration.
20 mg of linoleic acid and 200 mg of Tween 40 were added to 1 mL of 0.251 g L−1 of β-carotene in chloroform. 50 mL of aerated distilled water was added after chloroform removal. The compound solution was added to it. The reaction mixtures were incubated at 50 ◦C for 2 h. The absorbance was recorded at 470 nm. A blank solution lacking β-carotene was also prepared. The reaction rate was calculated according to the first-order kinetics by the following equation, Eq. (2) (Končič et al., 2009):
A0 At
(2)
where R is the rate of β-carotene bleaching, t is the time in minutes, A0 and At are the initial absorbance and the absorbance after time interval t. Antioxidant activity of the compounds was calculated from Eq. (3): AA = 1 −
kB T 6πηS D
Rt 100 R0
(3)
where AA is the antioxidant activity after time t, Rt and R0 are the rate of inhibition with and without the sample, respectively. Fluorescence microscopic method Hydroxamic acids were added at different concentrations to the solution of λ DNA in the tris-HCl buffer in different concentrations and the reaction mixtures were incubated for 60 min at 37 ◦C. Negative control with λ DNA was also incubated at the above mentioned conditions. The reaction mixtures were treated with H2 O2 solution for 15 minutes. DAPI dye and β-mercaptoethanol (4 vol. %) were added just before the observation to slow down the photobleaching of
(4)
where kB is the Boltzmann constant and η s is the viscosity of the solvent. The diffusion constant D for an individual fluorescent object can be obtained from the slope of the linear relationship using Eq. (5) (Sato et al., 2005): (R(t) − R(t0 ))2 = 4D(t − t0 )
Lipid peroxidation method
1 R = ln t
the dye. Coverslips were placed on top of the samples taken in concavity microslides. The images were captured using an inverted fluorescence microscope 40 × objective. Images were then analyzed by the NisElements Documentation software (Oana et al., 2002; Sato et al., 2005). The hydrodynamic radii, RH , were calculated from these trajectories using Eq. (4), the Stokes–Einstein relation (Sato et al., 2005):
(5)
where R = (Rx , Ry ) is a two-dimensional vector indicating the spatial position of the DNA molecule. Gel electrophoresis method Different concentrations of hydroxamic acids were added to Ct-DNA prepared in the tris-HCl buffer at pH 7.8 and H2 O2 was added. These reaction mixtures, negative control containing Ct-DNA only and the solution of H2 O2 treated Ct-DNA were incubated at 37 ◦C for 1 hour. The solutions were then loaded on 0.8 mass % agarose gel and electrophoresed in a submerged gel electrophoresis system containing a 50 mM TAE buffer at 50 V for about 1 hour. The gels were then stained with 1 mM ethidium bromide dye in a container, visualized by a UV transilluminator and photographed (Ramadas & Leela, 2011).
Results and discussion DPPH free radical scavenging method DPPH method is the easiest and the most common conventional method used for antioxidant assay. It is based on the 2,2 -diphenyl-1-picrylhydrazyl free radical decolorizing ability of a substance. The free radical has deep purple color which changes to yellow on the addition of an antioxidant. The DPPH free radical gives absorption maxima at 517 nm. The decrease in the absorbance of the DPPH radical at 517 nm shows the radical scavenging effect of the compounds. Therefore, the changes in the absorbance maxima of the DPPH free radical were observed in the presence of varying concentration of compounds for the determination of antioxidant properties (Sharma & Kumar, 2011). Percentage inhibition of the DPPH radical is shown in Fig. 2. The effective concentration causing a 50 % inhibition of the DPPH concentration (EC50 )
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arylhydroxamic acids have been reported several times to show considerable hydrogen bond donor capability. The H-atom of the hydroxyl group of the hydroxamic acid functionality can be held responsible for free radicals trapping. NPAHA contains two aromatic rings: naphthyl and phenyl ring, whereas NVHA contains a naphthyl ring and an alkyl group (Fig. 1). The lower phenyl ring exerts a –I effect in NPAHA. The presence of such an effect aids the release of the H-atom. On the contrary, the alkyl chain exerts a +I effect in NVHA and thus hinders the release of the H-atom. Therefore, NPAHA is capable of DPPH free radical inhibition and lipid peroxidation at lower concentrations as compared to NVHA. Fluorescence microscopic method
Fig. 2. DPPH radical scavenging and lipid peroxidation inhibition activity of NPAHA and NVHA as a function of hydroxamic acid concentration. DPPH inhibition by NPAHA (a) and by NVHA (b), lipid peroxidation inhibition by NPAHA (c) and lipid by NVHA (d).
was found to be 371.54 mM for NVHA and 365.95 mM for NPAHA. All experiments were done in triplicates and the data are presented as the mean values. The EC50 value was calculated through an extrapolation of the data obtained. Lipid peroxidation method The antioxidant potential was also analyzed using the lipid peroxidation method. β-Carotene seems to be colored due to the presence of conjugated double bonds. An addition of a linoleic acid free radical causes degradation and discoloration of β-carotene (Ames et al., 1993). Antioxidants interrupt this course of action and prevent lipid peroxidation leading to an inhibition of the new radicals generation. The absorbance was measured at 470 nm in the absence and in the presence of different concentrations of the studied compounds, similarly to the DPPH method, to study the rate of β-carotene degradation. Antioxidant activity was reported as the percentage inhibition of lipid peroxidation after 90 minutes of incubation (AA90 ) (Ames et al., 1993). The negative control had no protective effect against DPPH or β-carotene oxidation. The radical scavenging activity of both these compounds increased with the increasing concentration. The antioxidant activity after 90 minutes, AA90 was calculated at the EC50 concentration (obtained from the DPPH method) and it was found to be 40.91 % at 371.54 mM for NVHA and 41.14 % at 365.95 mM for NPAHA (Fig. 2). As already mentioned, the main characteristic of an antioxidant is its ability to trap free radicals. N-
Fig. 3 shows the images of λ DNA, H2 O2 treated λ DNA and H2 O2 treated compound (400 mM) + λ DNA. The right hand side of the figure shows the effect of NVHA and NPAHA on the hydrodynamic radius, RH , of λ DNA in the presence of H2 O2 . The inset shows the trajectories of H2 O2 damaged λ DNA as well as of λ DNA protected from H2 O2 by 400 mM compound over a period of 6 s. H2 O2 treated λ DNA (Fig. 3b) shows less Brownian motion along with a decrease in the fluorescence intensity. Whereas λ DNA incubated for one hour with the studied compound followed by the H2 O2 treatment (Fig. 3c) showed greater Brownian motion and fluorescence intensity as compared to the negative control. Variation of the hydrodynamic radii, RH , of λ DNA molecules at various concentrations of the compounds is shown in Fig. 3d. The fluorescence microscopic images show λ DNA molecules as small fluorescing granular structures. They were used to study factors such as change in the fluorescence intensity and the mobility of the DNA molecules. The fluorescence intensity of a molecule in the supercoiled configuration seems to be higher. This effect is due to the apparent increase in the local concentration of the dye (Houseal et al., 1989). Fig. 3. shows a decrease in the fluorescence intensity of λ DNA upon the H2 O2 treatment. But the compound pretreated λ DNA molecules seem to be brighter and faster as indicated by the images obtained from the fluorescence microscope, confirming the presence of a different higher-order structure of DNA molecules (Houseal et al., 1989). This was again confirmed by the analysis of the Brownian motion of the respective DNA molecules. The hydrodynamic radius, RH , was calculated to be about 350 nm for native λ DNA molecules. This value is comparable to that of the reported size of the λ DNA molecule (Oana et al., 2002) and it increases to above 410 nm in case of H2 O2 treated λ DNA due to the oxidative damage of the λ DNA molecules caused by OHú radicals. As NPAHA and NVHA have been proved to be effective against DPPH at the NVHA concentration of
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Fig. 3. Fluorescence microscopic images of negative control (a), H2 O2 cleaved λ DNA (b) and λ DNA cleavage protected from H2 O2 by 400 mM NPAHA (c). Effect of compounds NVHA ( ) and NPAHA ( ) on the hydrodynamic radius, RH , of λ DNA in the presence of H2 O2 (d). Inset on the left-hand side shows trajectories of the H2 O2 damaged λ DNA whereas that on the right-hand side shows trajectories of λ DNA protected from H2 O2 by 400 mM NPAHA in the corresponding solutions over a period of 6 s.
•
Fig. 4. H2 O2 induced DNA cleavage protection activity of NPAHA (a) and NVHA (b) as obtained by the agarose gel electrophoresis at different r values. Lane 1 = control DNA, lane 2 = H2 O2 cleaved Ct-DNA and lanes 3–5 = cleaved DNA protected by 100 mM, 200 mM and 300 mM hydroxamic acid, respectively.
371.54 mM and that of NPAHA of 365.95 mM, the microscopic images were taken in the concentration range of 0–400 mM. The RH value decreased from 410 nm to 260 nm and 267 nm in case of H2 O2 damaged λ DNA pretreated with 400 mM NPAHA and NVHA, respectively; i.e., the decrease in the size of H2 O2 damaged λ DNA molecules was around 37.34 %
and 35.66 % for NPAHA and NVHA, respectively. At least 15 DNA molecules were analyzed for each sample solution. This shows that both the studied compounds prevent λ DNA damage from hydroxyl radicals and lead to its supercoiling (Oana et al., 2002; Sato et al., 2005). Distribution of the hydrodynamic radius, RH , of H2 O2 damaged plasmid DNA treated with various
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concentrations of the studied compounds showed their protective activity. Gel electrophoresis method A similar experiment was carried out with Ct-DNA (50 mM) using the gel electrophoresis method to confirm the DNA cleavage protection ability of the studied compounds. Figs. 4a and 4b show the results on the DNA protection effect of the compounds in different concentrations achieved by the gel electrophoresis method. Lane 1 contains control DNA (without H2 O2 and compound), lane 2 contains DNA treated with H2 O2 and lanes 3–5 contain compound pretreated CtDNA attacked by the hydroxyl free radicals generated by the oxidation of H2 O2 (Alam et al., 2013). Lane 1 shows the presence of dark and clean DNA bands as compared to the H2 O2 damaged DNA present in lane 2. Lanes 3–5 show the protective effect of the different concentrations (0–300 mM) of the studied compounds. The hydroxyl (OH) free radical generated from H2 O2 damages the Ct-DNA. The distance covered by DNA as well as the disappearance of bands in the agarose gel signify the damage of DNA by H2 O2 (Figs. 4a and 4b, lane 2). The appearance of DNA bands in lanes 3–5 shows that both the compounds bind to the Ct-DNA and protect it from oxidative damage caused by the hydroxyl free radicals.
Conclusions The presented antioxidant studies show that both NPAHA and NVHA are capable of DPPH free radical scavenging and lipid peroxidation inhibition preventing λ DNA and Ct-DNA against the H2 O2 -mediated oxidative damage. The EC50 values show that NPAHA containing a lower phenyl ring has higher antioxidant potential than NVHA with an alkyl chain instead of the phenyl ring, which is caused by the hydrogen bond donor nature of the hydroxamic acid functionality. The present investigation also presents the usefulness of the fluorescence microscope single molecule observation method in the detection of antioxidant potential. Acknowledgements. Authors are grateful to the University Grants Commission, New Delhi, for providing financial support and the grant for fluorescence microscopy under SAP program [grant number F – 540/9/DRS/2010].
References Alam, M. N., Bristi, N. J., & Rafiquzzaman, M. (2013). Review on in vivo and in vitro methods evaluation of antioxidant activity. Saudi Pharmaceutical Journal, 21, 143–152. DOI: 10.1016/j.jsps.2012.05.002. Ames, B. N., Shigena, M. K., & Hagen, T. M. (1993). Oxidants, antioxidants and the degenerative diseases of aging. The Proceedings of the National Academy of Sciences USA, 90, 7915–7922.
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Aruoma, O. I. (1996). Assessment of potential prooxidant and antioxidant actions. Journal of the American Oil Chemists’ Society, 73, 1617–1625. DOI: 10.1007/bf02517962. Aruoma, O. I., & Cuppett, S. L. (1997). Antioxidant methodology: In vivo and in vitro concepts (pp. 39–51). Champain, IL, USA: AOCS Press. Autore, G., Caruso, A., Marzocco, S., Nicolaus, B., Palladino, C., Pinto, A., Popolo, A., Sinicropi, M. S., Tommonaro, G., & Saturnino, C. (2010). Acetamide derivatives with antioxidant activity and potential anti-inflammatory activity. Molecules, 15, 2028–2038. DOI: 10.3390/molecules15032028. Behl, C., Skutella, T., Lezoualc’h, F., Post, A., Widmann, M., Newton, C. J., & Holsboer, F. (1997). Neuroprotection against oxidative stress by estrogens: Structure–activity relationship. Molecular Pharmacology, 51, 535–541. DOI: 10.1124/mol.51.4.535. Broderick, C. E., & Cooke, P. H. (2009). Fruit composition, tissues and localization of antioxidants and capsaicinoids in Capsicum peppers by fluorescence microscopy. Acta Horticulturae, 841, 85–90. Gupta, V. K., & Tandon, S. G. (1972). Hydroxamic acids. Preparation and properties of N-1-napthylhydroxamic acids. Journal of Chemical & Engineering Data, 17, 248–249. DOI: 10.1021/je60053a008. Harman, D. (1995). Role of antioxidant nutrients in aging: Overview. Age, 18, 51–62. DOI: 10.1007/bf02432519. Houseal, T. W., Bustamante, C., Stump, R. F., & Maestre, M. F. (1989). Real-time imaging of single DNA molecules with fluorescence microscopy. Biophysical Journal, 56, 507–516. DOI: 10.1016/s0006-3495(89)82697-9. Hyogo, A., Kobayashi, T., del Saz, E. G., & Seguchi, H. (2010). Antioxidant effects of protocatechuic acid, ferulic acid and caffeic acid in human neutrophils using a fluorescent substance. International Journal of Morphology, 28, 911–920. DOI: 10.4067/s0717-95022010000300040. Khare, D., Verma, B., & Pande, R. (2012). Antioxidant activity of N-phenylbenzohydroxamic acid. Asian Journal of Pharmaceutical and Clinical Research, 5, 121–124. Konči´c, M., Raji´ c, Z., Petri´ c, N., & Zorc, B. (2009). Antioxidant activity of NSAID hydroxamic acids. Acta Pharmaceutica, 59, 235–242. DOI: 10.2478/v10007-009-0017-8. Nakamura, K., Fushimi, K., Kouchi, H., Mihara, K., Miyazaki, M., Ohe, T., & Namba, M. (1998). Inhibitory effects of antioxidants on neonatal rat cardiac myocyte hypertrophy induced by tumor necrosis factor-α and angiotensin II. Circulation, 98, 794–799. DOI: 10.1161/01.cir.98.8.794. Nie, S., & Zare, R. N. (1997). Optical detection of single molecules. Annuual Review on Biophysics and Biomolecular Structure, 26, 567–596. DOI: 10.1146/annurev.biophys.26.1. 567. Oana, H., Tsumoto, K., Yoshikawa, Y., & Yoshikawa, K. (2002). Folding transition of large DNA completely inhibits the action of a restriction endonuclease as revealed by single-chain observation. FEBS Letters, 530, 143–146. DOI: 10.1016/s0014-5793(02)03448-8. Patre, S., Thakur, P., & Pande, R. (2011). Solubility and hydrophobic descriptors of N-arylhydroxamic acids. International Journal of Bioscience, Biochemistry and Bioinformatics, 1, 267–270. DOI: 10.7763/ijbbb.2011.v1.50. Rajwade, R. P., Pande, R., Mishra, K. P., Kumar, A., & Pandey, B. N. (2008). Quantitative structure – activity relationship (QSAR) of N-arylsubstituted hydroxamic acids as inhibitors of human adenocarcinoma cells A431. Medicinal Chemistry, 4, 237–243. DOI: 10.2174/157340608784325106. Rajwade, R. P., Pande, R., Mishra, K. P., Kumar, A., & Pandey, B. N. (2009). Hydroxamic acids analogous against breast cancer cells: 2D-QSAR and 3D-QSAR stud-
Unauthenticated Download Date | 9/24/15 11:14 PM
1304
P. Singh et al./Chemical Papers 68 (10) 1298–1304 (2014)
ies. QSAR & Combinatorial Sciences, 28, 1500–1508. DOI: 10.1002/qsar.200910023. Ramadas, D., & Leela, S. (2011). Antioxidant effects of 28 kDa antioxidant protein from turmeric (Curcuma longa L). Asian Journal of Pharmaceutical and Clinical Research, 4, 112– 118. ¨ Widengren, J., & Kask, P. (1993). FluoRigler, R., Mets, U., rescence correlation spectroscopy with high count rate and low background: Analysis of translational diffusion. European Biophysics Journal, 22, 169–175. DOI: 10.1007/bf00185777. Sautin, Y. Y., Nakagawa, T., Zharikov, S., & Johnson, R. J. (2007). Adverse effects of the classic antioxidant uric acid in adipocytes: NADPH oxidase-mediated oxidative/nitrosative stress. AJP: Cell Physiology, 293, C584– C596. DOI: 10.1152/ajpcell.00600.2006. Sato, Y. T., Hamada, T., Kubo, K., Yamada, A., Kishida, T., Mazda, O., & Yoshikawa, K. (2005). Folding transition into a loosely collapsed state in plasmid DNA as revealed by singlemolecule observation. FEBS Letters, 579, 3095–3099. DOI: 10.1016/j.febslet.2005.04.072. Sharma, U. S., & Kumar, A. (2011). In vitro antioxidant activity of Rubus ellipticus fruits. Journal of Advanced Pharmaceutical Technology & Research, 2, 47–50. DOI: 10.4103/22314040.79805.
¨ Shirinzadeh, H., Eren, B., Gurer-Orhan, H., Suzen, S., & Ozden, S. (2010). Novel indole-based analogs of melatonin: Synthesis and in vitro antioxidant activity studies. Molecules, 15, 2187– 2202. DOI: 10.3390/molecules15042187. Tiwari, V., & Pande, R. (2006). Molecular descriptors of Narylhydroxamic acids: A tool in drug design. Chemical Biology & Drug Design, 68, 225–228. DOI: 10.1111/j.17470285.2006.00433.x. Valko, M., Rhodes, C. J., Moncol, J., Izakovic, M., & Mazur, M. (2006). Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chemico–Biological Interactions, 160, 1–40. DOI: 10.1016/j.cbi.2005.12.009. Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T. D., Mazur, M., & Telser, J. (2007). Free radicals and antioxidants in normal physiological functions and human disease. The International Journal of Biochemistry & Cell Biology, 39, 44– 84. DOI: 10.1016/j.biocel.2006.07.001. Wu, Y., Sun, F. F., Tong, D. M., & Taylor, B. M. (1996). Changes in membrane properties during energy depletioninduced cell injury studied with fluorescence microscopy. Biophysical Journal, 71, 91–100. DOI: 10.1016/s0006-3495(96) 79243-3.
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