Infrared Spectroscopy as a Tool to Study the Antioxidant Activity of Polyphenolic Compounds in Isolated Rat Enterocytes

Guillermo Barraza-Garza, Hiram Castillo-Michel, Laura A de la Rosa, Alejandro Martinez-Martinez, Jorge A Pérez-León, Marine Cotte, Emilio Alvarez-Parrilla, Guillermo Barraza-Garza, Hiram Castillo-Michel, Laura A de la Rosa, Alejandro Martinez-Martinez, Jorge A Pérez-León, Marine Cotte, Emilio Alvarez-Parrilla

Abstract

The protective effect of different polyphenols, catechin (Cat), quercetin (Qc) (flavonoids), gallic acid (GA), caffeic acid (CfA), chlorogenic acid (ChA) (phenolic acids), and capsaicin (Cap), against H2O2-induced oxidative stress was evaluated in rat enterocytes using Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy and Fourier Transform Infrared Microspectroscopy (FTIRM), and results were compared to standard lipid peroxidation techniques: conjugated dienes (CD) and Thiobarbituric Acid Reactive Substances (TBARS). Analysis of ATR-FTIR and FTIRM spectral data allowed the simultaneous evaluation of the effects of H2O2 and polyphenols on lipid and protein oxidation. All polyphenols showed a protective effect against H2O2-induced oxidative stress in enterocytes, when administered before or after H2O2. Cat and capsaicin showed the highest protective effect, while phenolic acids had weaker effects and Qc presented a mild prooxidative effect (IR spectral profile of biomolecules between control and H2O2-treated cells) according to FTIR analyses. These results demonstrated the viability to use infrared spectroscopy to evaluate the oxidant and antioxidant effect of molecules in cell systems assays.

Figures

Figure 1
Figure 1
Molecular structure of the polyphenolic compounds used in this work. (a) Gallic acid (GA). (b) Caffeic acid (CfA). (c) Chlorogenic acid (ChA). (d) Catechin (Cat). (e) Quercetin (Qc). (f) Capsaicin (Cap).
Figure 2
Figure 2
Cell viability after treatments. Enterocytic cells after isolation from the rat intestine and exposed to their different treatments. (a) Control cell after 1 hour in DMEM plus antibiotics. (b) Cells exposed for 30 min to DMEM medium with 0.5 mM of H2O2 first and then transferred to base DMEM medium, for 30 min each. (c) Cells exposed to DMEM-0.1 mM gallic acid first for 30 min and afterwards transferred to H2O2 for 30 min for each treatment. Measurements of viable cells were made by using 0.1% trypan blue exclusion assay with a Neubauer chamber average of percentage of viability reported in Results. Cell viability and SD noted for each treatment. Scale bar 40 μm.
Figure 3
Figure 3
Infrared spectra obtained using FTIRM and processed with a second derivative. (a) Average spectrums obtained with the control treatment (DMEM) during 1 hr (I) and H2O2-treated cells (DMEM added with 0.5 mM of H2O2) 30 min (II), spectra were obtained with a spectral resolution set to 6 cm−1. (b) Average spectra processed with second derivative using Savitsky-Golay with 21 smoothing points and a second polynomial order. Control treatment cells (DMEM) during 1 hr (I) and H2O2-treated cells (DMEM added with 0.5 mM of H2O2) for 30 min (II). The differences seen in these treatments were analyzed by the PCA analysis to classify them and to know the most important variations in the dataset. There are two major zones of interest: proteins zone (1720–1470 cm−1) and lipids zone (3200–2800 cm−1).
Figure 4
Figure 4
Ratios between chemical bonds related to the lipid peroxidation observed with the FTIR spectra data. Those ratios were as follows: (a) the ratio assigned to 1740 cm−1/2960 cm−1 is related to lipid oxidation, (b) the ratio assigned to 2920 cm−1/2960 cm−1 is related to lipid saturation, (c) the ratio assigned to 3012 cm−1/2960 cm−1 is related to lipid desaturation, and (d) the ratio assigned 1630 cm−1/1650 cm−1 is related to protein aggregation. The dotted line indicates the mean value of the control cells for each ratio; all ratios were compared to the control ratio in each case. PP: polyphenol.
Figure 5
Figure 5
Results obtained with FTIRM infrared spectra data using PCA. (a) Loading plot from the FTIRM samples observed in both protein region (1800–900 cm−1) and lipid region (3200–2800 cm−1) for PC-1 and PC-2. (b) Score plot from the FTIRM samples in the protein wavenumbers (1900–900 cm−1) and the lipid zone of wavenumbers (3200–2800 cm−1) for PC-1 and PC-2. The number of samples used per treatment was 30. Main changes are observed as a shift in amide I (1660 cm−1) and changes in the relations between CH2 (2920 cm−1) and CH3 (2960 cm−1) bonds.
Figure 6
Figure 6
(a) Conjugated dienes (CD) assay. Concentration of CD per million cells observed in the full battery of control and polyphenolic treatments. Values are the mean + SD from six measurements. (b) TBARS assay. Concentration of MDA equivalents per million cells observed in the full battery of control and polyphenolic treatments. Values are the mean + SD from six measurements.

References

    1. Vauzour D., Rodriguez-Mateos A., Corona G., Oruna-Concha M. J., Spencer J. P. E. Polyphenols and human health: prevention of disease and mechanisms of action. Nutrients. 2010;2(11):1106–1131. doi: 10.3390/nu2111106.
    1. Vauzour D. Dietary polyphenols as modulators of brain functions: biological actions and molecular mechanisms underpinning their beneficial effects. Oxidative Medicine and Cellular Longevity. 2012;2012:16. doi: 10.1155/2012/914273.914273
    1. Lü J.-M., Lin P. H., Yao Q., Chen C. Chemical and molecular mechanisms of antioxidants: experimental approaches and model systems. Journal of Cellular and Molecular Medicine. 2010;14(4):840–860. doi: 10.1111/j.1582-4934.2009.00897.x.
    1. Andriantsitohaina R., Auger C., Chataigneau T., et al. Molecular mechanisms of the cardiovascular protective effects of polyphenols. British Journal of Nutrition. 2012;108(9):1532–1549. doi: 10.1017/S0007114512003406.
    1. Tan A. C., Konczak I., Ramzan I., Sze D. M.-Y. Antioxidant and cytoprotective activities of native Australian fruit polyphenols. Food Research International. 2011;44(7):2034–2040. doi: 10.1016/j.foodres.2010.10.023.
    1. Calhau C., Faria A., Keating E., Martel F. Interaction of polyphenols with the intestinal and placental absorption of some nutrients and other compounds. In: Watson R., Preedy V., Zibadi S., editors. Polyphenols in Human Health and Disease. chapter 39. New York, NY, USA: Elsevier-Academic Press; 2014. pp. 523–536.
    1. Birringer M. Hormetics: dietary triggers of an adaptive stress response. Pharmaceutical Research. 2011;28(11):2680–2694. doi: 10.1007/s11095-011-0551-1.
    1. Nakamura Y., Miyoshi N. Electrophiles in foods: the current status of isothiocyanates and their chemical biology. Bioscience, Biotechnology and Biochemistry. 2010;74(2):242–255. doi: 10.1271/bbb.90731.
    1. Vileno B., Jeney S., Sienkiewicz A., Marcoux P. R., Miller L. M., Forró L. Evidence of lipid peroxidation and protein phosphorylation in cells upon oxidative stress photo-generated by fullerols. Biophysical Chemistry. 2010;152(1–3):164–169. doi: 10.1016/j.bpc.2010.09.004.
    1. Gianoncelli A., Vaccari L., Kourousias G., et al. Soft X-ray microscopy radiation damage on fixed cells investigated with synchrotron radiation FTIR microscopy. Scientific Reports. 2015;5 doi: 10.1038/srep10250.10250
    1. Oleszko A., Olsztyńska-Janus S., Walski T., et al. Application of FTIR-ATR spectroscopy to determine the extent of lipid peroxidation in plasma during haemodialysis. BioMed Research International. 2015;2015:8. doi: 10.1155/2015/245607.245607
    1. Vargas-Caraveo A., Castillo-Michel H., Mejia-Carmona G. E., Pérez-Ishiwara D. G., Cotte M., Martínez-Martínez A. Preliminary studies of the effects of psychological stress on circulating lymphocytes analyzed by synchrotron radiation based-Fourier transform infrared microspectroscopy. Spectrochimica Acta—Part A: Molecular and Biomolecular Spectroscopy. 2014;128:141–146. doi: 10.1016/j.saa.2014.02.148.
    1. National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals. Guide for the Care and Use of Laboratory Animals. Washington, Wash, USA: National Academies Press; 2011.
    1. Chougule P., Herlenius G., Hernandez N. M., Patil P. B., Xu B., Sumitran-Holgersson S. Isolation and characterization of human primary enterocytes from small intestine using a novel method. Scandinavian Journal of Gastroenterology. 2012;47(11):1334–1343. doi: 10.3109/00365521.2012.708940.
    1. Nik A. M., Carlsson P. Separation of intact intestinal epithelium from mesenchyme. BioTechniques. 2013;55(1):42–44. doi: 10.2144/000114055.
    1. Bellisola G., Sorio C. Infrared spectroscopy and microscopy in cancer research and diagnosis. American Journal of Cancer Research. 2012;2(1):1–21.
    1. Martin F. L., Kelly J. G., Llabjani V., et al. Distinguishing cell types or populations based on the computational analysis of their infrared spectra. Nature Protocols. 2010;5(11):1748–1760. doi: 10.1038/nprot.2010.133.
    1. Benseny-Cases N., Klementieva O., Cotte M., Ferrer I., Cladera J. Microspectroscopy (μFTIR) reveals co-localization of lipid oxidation and amyloid plaques in human Alzheimer disease brains. Analytical Chemistry. 2014;86(24):12047–12054. doi: 10.1021/ac502667b.
    1. Chwiej J., Dulinska J., Janeczko K., et al. Synchrotron FTIR micro-spectroscopy study of the rat hippocampal formation after pilocarpine-evoked seizures. Journal of Chemical Neuroanatomy. 2010;40(2):140–147. doi: 10.1016/j.jchemneu.2010.03.008.
    1. Petibois C., Drogat B., Bikfalvi A., Déléris G., Moenner M. Histological mapping of biochemical changes in solid tumors by FT-IR spectral imaging. FEBS Letters. 2007;581(28):5469–5474. doi: 10.1016/j.febslet.2007.10.052.
    1. André W., Sandt C., Dumas P., Djian P., Hoffner G. Structure of inclusions of Huntington's disease brain revealed by synchrotron infrared microspectroscopy: polymorphism and relevance to cytotoxicity. Analytical Chemistry. 2013;85(7):3765–3773. doi: 10.1021/ac400038b.
    1. Meade A., Clarke C., Byrne H., Lyng F. Fourier transform infrared microspectroscopy and multivariate methods for radiobiological dosimetry. Radiation Research. 2010;173(2):225–237. doi: 10.1667/RR1836.1.
    1. Chio-Srichan S., Réfrégiers M., Jamme F., Kascakova S., Rouam V., Dumas P. Photosensitizer effects on cancerous cells: a combined study using synchrotron infrared and fluorescence microscopies. Biochimica et Biophysica Acta (BBA)—General Subjects. 2008;1780(5):854–860. doi: 10.1016/j.bbagen.2008.02.004.
    1. Devasagayam T. P. A., Boloor K. K., Ramasarma T. Methods for estimating lipid peroxidation: an analysis of merits and demerits. Indian Journal of Biochemistry and Biophysics. 2003;40(5):300–308.
    1. Alvarez-Parrilla E., De La Rosa L. A., Amarowicz R., Shahidi F. Protective effect of fresh and processed Jalapeño and Serrano peppers against food lipid and human LDL cholesterol oxidation. Food Chemistry. 2012;133(3):827–834. doi: 10.1016/j.foodchem.2012.01.100.
    1. Mac Donal O., Chediack J. G., Caviedes-Vidal E. Isolation of epithelial cells, villi and crypts from small intestine of pigeons (Columba livia) Biocell. 2008;32(3):219–227.
    1. Miliukiene V., Nemeikaite-Čeniene A., Čenas N. Prooxidant cytotoxicity of polyphenolic compounds in primary mice splenocytes: the role of redox potential and lipophilicity. Chemija. 2014;25(4):218–223.
    1. Jain A. K., Thanki K., Jain S. Novel self-nanoemulsifying formulation of quercetin: implications of pro-oxidant activity on the anticancer efficacy. Nanomedicine: Nanotechnology, Biology, and Medicine. 2014;10(5):959–969. doi: 10.1016/j.nano.2013.12.010.
    1. Jamme F., Vindigni J.-D., Méchin V., Cherifi T., Chardot T., Froissard M. Single cell synchrotron FT-IR microspectroscopy reveals a link between neutral lipid and storage carbohydrate fluxes in S. cerevisiae . PloS ONE. 2013;8(9)e74421
    1. Williams R. J., Spencer J. P. E., Rice-Evans C. Flavonoids: antioxidants or signalling molecules? Free Radical Biology and Medicine. 2004;36(7):838–849. doi: 10.1016/j.freeradbiomed.2004.01.001.
    1. Rice-Evans C. A., Miller N. J., Paganga G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biology and Medicine. 1996;20(7):933–956. doi: 10.1016/0891-5849(95)02227-9.
    1. Pignatelli P., Ghiselli A., Buchetti B., et al. Polyphenols synergistically inhibit oxidative stress in subjects given red and white wine. Atherosclerosis. 2006;188(1):77–83. doi: 10.1016/j.atherosclerosis.2005.10.025.
    1. Giftson J. S., Jayanthi S., Nalini N. Chemopreventive efficacy of gallic acid, an antioxidant and anticarcinogenic polyphenol, against 1,2-dimethyl hydrazine induced rat colon carcinogenesis. Investigational New Drugs. 2010;28(3):251–259. doi: 10.1007/s10637-009-9241-9.
    1. Maurya D. K., Devasagayam T. P. A. Antioxidant and prooxidant nature of hydroxycinnamic acid derivatives ferulic and caffeic acids. Food and Chemical Toxicology. 2010;48(12):3369–3373. doi: 10.1016/j.fct.2010.09.006.
    1. Conte A., Pellegrini S., Tagliazucchi D. Synergistic protection of PC12 cells from β-amyloid toxicity by resveratrol and catechin. Brain Research Bulletin. 2003;62(1):29–38. doi: 10.1016/j.brainresbull.2003.08.001.
    1. Jimenez-Lopez J. M., Cederbaum A. I. Green tea polyphenol epigallocatechin-3-gallate protects HepG2 cells against CYP2E1-dependent toxicity. Free Radical Biology and Medicine. 2004;36(3):359–370. doi: 10.1016/j.freeradbiomed.2003.11.016.

Source: PubMed

赞助者和合作者

医疗条件

药物干预

3
订阅