Standardization of a protocol to extract and analyze chlorophyll a and carotenoids in Gracilaria tenuistipitata Var. Liui. Zhang and Xia (Rhodophyta)

Torres, Priscila Bezerra; Chow, Fungyi; Furlan, Cláudia Maria; Mandelli, Fernanda; Mercadante, Adriana; Santos, Déborah Yara Alves Cursino dos

doi:10.1590/s1679-87592014068106201

Abstracts

Chlorophyll a and carotenoids are important pigments in photosynthesis. Several studies have been published describing extraction and analysis protocols of these pigments, mainly in vascular plant species. This study standardizes an extraction and analysis protocol of these substances in Gracilaria tenuistipitata var. liui, a red seaweed. Apical portions grown in vitro were triturated in liquid nitrogen. Extracts were prepared in 1.5 mL solvent and centrifuged. Quantitative and qualitative analyses of pigments were performed by UV/visible light spectrophotometry and high performance liquid chromatography (HPLC) and HPLC coupled to mass spectrometry (HPLC-MS). The parameters assessed were: minimum biomass, best extraction solvent, and number of extraction steps. Methanol was the most efficient solvent, and 50 mg fresh biomass was the amount of sample indicated, submitted to one single extraction step. No significant differences were observed in levels of these pigments by UV-visible light spectrophotometry and HPLC. However, HPLC or HPLC-MS are required to identify the different carotenoids present in this seaweed species.

Gracilaria tenuistipitata; Chlorophyll a; Carotenoids; UV-visible light spectrophotometry; HPLC

Clorofila a e carotenoides são importantes pigmentos da fotossíntese. Na literatura são encontrados vários protocolos de extração e análise desses pigmentos utilizando, principalmente, plantas vasculares. O objetivo deste estudo foi padronizar uma metodologia de extração e análise dessas substâncias em uma macroalga vermelha, Gracilaria tenuistipitata var. liui. Amostras de talos gametofíticos cultivados in vitro foram trituradas em nitrogênio líquido, extraídas em 1,5 mL de solvente, centrifugadas e os pigmentos analisados quantitativamente e qualitativamente através de espectrofotometria de UV/visível, cromatografia liquida de alta eficiência (CLAE) e CLAE-acoplada a espectrometria de massas (CLAE-EM). Foram testados os parâmetros massa mínima, solvente para extração e número de extrações. Dentre os solventes testados, o metanol foi o mais eficiente, sendo 50mg de material fresco a massa mínima indicada para ser submetida a somente uma extração. Não foram encontradas diferenças significativas na quantificação desses pigmentos comparando-se os dados obtidos em espectrofotometria de UV/visível com os de CLAE. No entanto, para a identificação dos diferentes carotenoides e suas quantificações são necessárias CLAE ou CLAE-EM.

Gracilaria tenuistipitata; Clorofila a; Carotenoides; Espectrometria UV-visível; CLAE

References

ARVAYO-ENRÍQUEZ, H.; MONDACA-FERNÁNDEZ, I.; GORTÁREZ-MOROYOQUI, P.; LÓPEZ-CERVANTES, J.; RODRÍGUEZ-RAMÍREZ, R. Carotenoids extraction and quantification: a review. Anal. Methods, v. 5, p. 2916-2924, 2013.
BORSARELLI, C.; MERCADANTE, A. Thermal and photochemical degradation of carotenoids. In: LANDRUM, J. T. (Ed.). Carotenoids: physical, chemical, and biological functions and properties. Boca Raton: Taylor & Francis Group, 2010. p. 229 - 253.
CARNICAS, E.; JIMÉNEZ, C.; NIELL, F. X. Effects of changes of irradiance on the pigment composition of Gracilaria tenuistipitata var. liui Zhang et Xia. J. Photochem. Photobiol., B, v. 50, n. 2/3, p. 149 - 158, 1999.
EDWARDS, P. Illustrated guide to the seaweeds and sea grasses in the vicinity of Porto Aransas, Texas. Contrib. Mar. Sci., v. 15, p. 1-228, 1970.
FARIA, A. F.; HASEGAWA, P. N.; CHAGAS, E. A.; PIO, R.; PURGATTO, E.; MERCADANTE, A. Z. Cultivar influence on carotenoid composition of loquats from Brazil. J. Food Compos. Anal., v. 22, n. 3, p. 196 - 203, 2009.
GAUTHIER-JAQUES, A.; BORTLIK, K.; HAU, J.; FAY, L. B. Improved method to track chlorophyll degradation. J. Agric. Food Chem., v. 49, n. 3, p. 1117-1122, 2001.
GUEDES, A. C.; AMARO, H. M.; MALCATA, F. X. Microalgae as sources of carotenoids. Mar. Drugs, v. 9, n. 4, p. 625 - 644, 2011.
ISHIDA, B. K.; CHAPMAN, M. H. Carotenoid extraction from plants using a novel, environmentally friendly solvent. J. Agric. Food Chem., v. 57, n. 3, p. 1051 - 1059, 2009.
JEFFREY, S. W.; HUMPHREY, G. New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton. Biochem. Physiol. Pflanz., v. 649, n. 1, p. 105 - 149, 1975.
KELMAN, D.; POSNER, E. K.; MCDERMID, K. J.; TABANDERA, N. K.; WRIGHT, P. R.; WRIGHT, A. D. Antioxidant activity of Hawaiian marine algae. Mar. Drugs, v. 10, n. 2, p. 403 - 16, 2012.
KHACHIK, F. Analysis of carotenoids in nutritional studies. In BRITTON, G.; LIAAEN-JENSEN, S.; PFANDER, D. H. (Eds.). Nutrition and health Basel: Boston: Berlin: Birkhäuser, 2009. p. 7 - 44. (Carotenoids; v. 5).
KOPEC, R. E.; COOPERSTONE, J. L.; CICHON, M. J.; SCHWARTZ, S. J. Analysis methods of carotenoids. In: XU, Z.; HOWARD, L. R. (Eds.). Analysis of antioxidant-rich phytochemicals Hoboken: Chichester: Wiley-Blackwell, 2012. p.105 - 149.
KOPECKY, J.; SCHOEFS, B.; LOEST, K.; STYS, D.; PULZ, O. Microalgae as a source for secondary carotenoid production: a screening study. Arch. Hydrobiol., Suppl., v. 133, p. 153-168, 2000.
LICHTENTHALER, H. K.; BUSCHMANN, C. Chlorophylls and carotenoids: measurement and characterization by UV-VIS. In: WROLSTAD, R. E.; ACREE, T. E.; DECKER, E. A.; PENNER, M. H.; REID, D. S.; SCHWARTZ, S. J.; SHOEMAKER, C. F.; SPORNS, P. Current protocols in food analytical chemistry New York: John Wiley, 2001. p. F4.3.1 - F4.3.8.
MARINHO-SORIANO, E. Effect of depth on growth and pigment contents of the macroalgae Gracilaria bursa-pastoris. Rev. Bras. Farmacogn., v. 22, n. 4, p. 730 - 735, 2012.
PINTO, E.; CARVALHO, A. P.; CARDOZO, K. H. M.; MALCATA, F. X.; ANJOS, F. M.; COLEPICOLO, P. Effects of heavy metals and light levels on the biosynthesis of carotenoids and fatty acids in the macroalgae Gracilaria tenuistipitata (var. liui Zhang & Xia). Rev. Bras. Farmacogn., v. 21, n. 2, p. 349 - 354, 2011.
RAVEN, P. H.; EVERT, R. F.; EICHHORN, S. E. Biologia vegetal 7. ed. Rio de Janeiro: Guanabara Koogan, 2007. 830 p.
ROSSO, V. V.; MERCADANTE, A. Z. Identification and quantification of carotenoids, by HPLC-PDA-MS/MS, from Amazonian fruits. J. Agric. Food Chem., v. 55, n. 13, p. 5062 - 5072, 2007.
SCHMIDT, É. C.; MARASCHIN, M.; BOUZON, Z. L. Effects of UVB radiation on the carragenophyte Kappaphycus alvarezii (Rhodophyta, Gigartinales): changes in ultrastructure, growth, and photosynthetic pigments. Hydrobiologia, v. 649, n. 1, p. 171 - 182, 2010.
VAN BREEMEN, R. B.; DONG, L.; PAJKOVIC, N. D. Atmospheric pressure chemical ionization tandem mass spectrometry of carotenoids. Int. J. Mass Spectrom., v. 312, p. 163 - 172, 2011.
WELLBURN, A. R. The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J. Plant Physiol., v. 144, n. 3, p. 307-313, 1994.
WRIGHT, S. W.; JEFFREY, S. W.; MANTOURA, R. F. C. Evaluation of methods and solvents for pigment extraction. In: JEFFREY, S. W.; MANTOURA, R. F. C.; WRIGHT, S. W. (Eds.). Phytoplankton pigments in oceanography: guidelines to modern methods. Paris: UNESCO, 1997. p. 261 - 282. (Monographs on oceanographic methodology; 10).
URSI, S.; PLASTINO, E. M. Crescimento in vitro de linhagens de coloração vermelha e verde clara de Gracilaria birdiae (Gracilariales, Rhodophyta) em dois meios de cultura: análise de diferentes estádios reprodutivos. Rev. Bras. Bot., v. 24, n, 4, supl., p. 587 - 594, 2001.

Publication Dates

Publication in this collection
03 June 2014
Date of issue
Mar 2014

History

Received
07 Aug 2013
Accepted
18 Mar 2014
Reviewed
09 Mar 2014

This work is licensed under a Creative Commons Attribution 4.0 International License.

[1] ARVAYO-ENRÍQUEZ, H.; MONDACA-FERNÁNDEZ, I.; GORTÁREZ-MOROYOQUI, P.; LÓPEZ-CERVANTES, J.; RODRÍGUEZ-RAMÍREZ, R. Carotenoids extraction and quantification: a review. Anal. Methods, v. 5, p. 2916-2924, 2013.

[2] BORSARELLI, C.; MERCADANTE, A. Thermal and photochemical degradation of carotenoids. In: LANDRUM, J. T. (Ed.). Carotenoids: physical, chemical, and biological functions and properties. Boca Raton: Taylor & Francis Group, 2010. p. 229 - 253.

[3] CARNICAS, E.; JIMÉNEZ, C.; NIELL, F. X. Effects of changes of irradiance on the pigment composition of Gracilaria tenuistipitata var. liui Zhang et Xia. J. Photochem. Photobiol., B, v. 50, n. 2/3, p. 149 - 158, 1999.

[4] EDWARDS, P. Illustrated guide to the seaweeds and sea grasses in the vicinity of Porto Aransas, Texas. Contrib. Mar. Sci., v. 15, p. 1-228, 1970.

[5] FARIA, A. F.; HASEGAWA, P. N.; CHAGAS, E. A.; PIO, R.; PURGATTO, E.; MERCADANTE, A. Z. Cultivar influence on carotenoid composition of loquats from Brazil. J. Food Compos. Anal., v. 22, n. 3, p. 196 - 203, 2009.

[6] GAUTHIER-JAQUES, A.; BORTLIK, K.; HAU, J.; FAY, L. B. Improved method to track chlorophyll degradation. J. Agric. Food Chem., v. 49, n. 3, p. 1117-1122, 2001.

[7] GUEDES, A. C.; AMARO, H. M.; MALCATA, F. X. Microalgae as sources of carotenoids. Mar. Drugs, v. 9, n. 4, p. 625 - 644, 2011.

[8] ISHIDA, B. K.; CHAPMAN, M. H. Carotenoid extraction from plants using a novel, environmentally friendly solvent. J. Agric. Food Chem., v. 57, n. 3, p. 1051 - 1059, 2009.

[9] JEFFREY, S. W.; HUMPHREY, G. New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton. Biochem. Physiol. Pflanz., v. 649, n. 1, p. 105 - 149, 1975.

[10] KELMAN, D.; POSNER, E. K.; MCDERMID, K. J.; TABANDERA, N. K.; WRIGHT, P. R.; WRIGHT, A. D. Antioxidant activity of Hawaiian marine algae. Mar. Drugs, v. 10, n. 2, p. 403 - 16, 2012.

[11] KHACHIK, F. Analysis of carotenoids in nutritional studies. In BRITTON, G.; LIAAEN-JENSEN, S.; PFANDER, D. H. (Eds.). Nutrition and health Basel: Boston: Berlin: Birkhäuser, 2009. p. 7 - 44. (Carotenoids; v. 5).

[12] KOPEC, R. E.; COOPERSTONE, J. L.; CICHON, M. J.; SCHWARTZ, S. J. Analysis methods of carotenoids. In: XU, Z.; HOWARD, L. R. (Eds.). Analysis of antioxidant-rich phytochemicals Hoboken: Chichester: Wiley-Blackwell, 2012. p.105 - 149.

[13] KOPECKY, J.; SCHOEFS, B.; LOEST, K.; STYS, D.; PULZ, O. Microalgae as a source for secondary carotenoid production: a screening study. Arch. Hydrobiol., Suppl., v. 133, p. 153-168, 2000.

[14] LICHTENTHALER, H. K.; BUSCHMANN, C. Chlorophylls and carotenoids: measurement and characterization by UV-VIS. In: WROLSTAD, R. E.; ACREE, T. E.; DECKER, E. A.; PENNER, M. H.; REID, D. S.; SCHWARTZ, S. J.; SHOEMAKER, C. F.; SPORNS, P. Current protocols in food analytical chemistry New York: John Wiley, 2001. p. F4.3.1 - F4.3.8.

[15] MARINHO-SORIANO, E. Effect of depth on growth and pigment contents of the macroalgae Gracilaria bursa-pastoris. Rev. Bras. Farmacogn., v. 22, n. 4, p. 730 - 735, 2012.

[16] PINTO, E.; CARVALHO, A. P.; CARDOZO, K. H. M.; MALCATA, F. X.; ANJOS, F. M.; COLEPICOLO, P. Effects of heavy metals and light levels on the biosynthesis of carotenoids and fatty acids in the macroalgae Gracilaria tenuistipitata (var. liui Zhang & Xia). Rev. Bras. Farmacogn., v. 21, n. 2, p. 349 - 354, 2011.

[17] RAVEN, P. H.; EVERT, R. F.; EICHHORN, S. E. Biologia vegetal 7. ed. Rio de Janeiro: Guanabara Koogan, 2007. 830 p.

[18] ROSSO, V. V.; MERCADANTE, A. Z. Identification and quantification of carotenoids, by HPLC-PDA-MS/MS, from Amazonian fruits. J. Agric. Food Chem., v. 55, n. 13, p. 5062 - 5072, 2007.

[19] SCHMIDT, É. C.; MARASCHIN, M.; BOUZON, Z. L. Effects of UVB radiation on the carragenophyte Kappaphycus alvarezii (Rhodophyta, Gigartinales): changes in ultrastructure, growth, and photosynthetic pigments. Hydrobiologia, v. 649, n. 1, p. 171 - 182, 2010.

[20] VAN BREEMEN, R. B.; DONG, L.; PAJKOVIC, N. D. Atmospheric pressure chemical ionization tandem mass spectrometry of carotenoids. Int. J. Mass Spectrom., v. 312, p. 163 - 172, 2011.

[21] WELLBURN, A. R. The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J. Plant Physiol., v. 144, n. 3, p. 307-313, 1994.

[22] WRIGHT, S. W.; JEFFREY, S. W.; MANTOURA, R. F. C. Evaluation of methods and solvents for pigment extraction. In: JEFFREY, S. W.; MANTOURA, R. F. C.; WRIGHT, S. W. (Eds.). Phytoplankton pigments in oceanography: guidelines to modern methods. Paris: UNESCO, 1997. p. 261 - 282. (Monographs on oceanographic methodology; 10).

[23] URSI, S.; PLASTINO, E. M. Crescimento in vitro de linhagens de coloração vermelha e verde clara de Gracilaria birdiae (Gracilariales, Rhodophyta) em dois meios de cultura: análise de diferentes estádios reprodutivos. Rev. Bras. Bot., v. 24, n, 4, supl., p. 587 - 594, 2001.