Studies concerning the extraction of chlorophyll and total carotenoids from vegetables

Romanian Biotechnological Letters Vol. 17, No.5, 2012 Copyright 2012 University of Bucharest Printed in Romania. All rights reserved ORIGINAL PAPER Studies concerning the extraction of chlorophyll and total carotenoids from vegetables Received for publication, June 13, 2012 Accepted, September 12, 2012 Abstract MANUELA ADRIANA COSTACHE 1), GHEORGHE CAMPEANU 2), GABRIELA NEATA 3) 1) Faculty of Horticulture, USAMV, Bd. Marasti No. 59, Sect. 1, Bucharest, Romania; E-mail: manuela_bth@yahoo.com 2 ) Faculty of Horticulture, USAMV, Bd. Marasti No. 59, Sect. 1, Bucharest, Romania; 3 ) Faculty of Horticulture, USAMV, Bd. Marasti No. 59, Sect. 1, Bucharest, Romania; Corresponding author: E-mail: manuela_bth@yahoo.com The main pigments found in vegetables are chlorophyll a, b and c. Chlorophyll a converts light energy into chemical energy through photosynthesis process. The content of chlorophyll pigments varies by species. The aim of the research was to determine simultaneously chlorophyll and carotene content from different vegetables by spectrophotometric method, testing three organic solvents to obtain the best extraction solution. 17 samples of cherry tomatoes, peppers and cucumbers, collected from different greenhouses located in southern part of Romania were analyzed. The carotenoids and chlorophyll pigments extracted in methanol, diethyl ether and acetone, were determined based on the absorbance of three wavelengths: 470 nm, 645 nm-653 nm and 662 nm-666 nm. The results were calculated according to the three "trichromatic equations" of Lichtentaler and Wellburn. The best extraction solvents for simultaneously determination of chlorophyll a and b and carotene were methanol and acetone. For cherry tomatoes and tomatoes the best extraction solvent was methanol for chlorophyll a and acetone for carotene. For peppers the best extraction solvent was acetone and for cucumbers it was methanol. Variation coefficients were high, both between vegetable species and between tested varieties or hybrids. Keywords: a and b chlorophylls, total carotenoids, extraction, vegetables Introduction The presence of pigments in plant tissues gives color to vegetables and fruit, which is different depending on variety and species. Pigments are substances with very different chemical structure; they are present in the form of porphyrin pigments, carotenoids, anthocyanins and flavones. The main porphyrin pigments found in vegetables are chlorophyll a, b and c. Chlorophyll a, the main pigment in plants, converts light energy into chemical energy through photosynthesis process. The content of chlorophyll pigments varies by species. Several studies concerning chlorophyll pigments determination using phytoplankton (ARAR [1, 2], BARLOW et al. [3], DON SANTOS et al. [7], FLANDER et al. [9], and HALLENGRAEFF [10]) and some species of algae (DERE et al. [8]) are reported. The change of chlorophyll pigment content from fruit and vegetables have a great importance for their color variation during the maturation phase. CIOFU [5] states that the chlorophyll content of tomatoes during ripening period varied from 0.95 mg/100 g fresh product to 0.12 mg/100 g fresh product after 7 day of storage (CȂMPEANU et al. [4], COSTACHE et al. [6]). Carotenoid pigments can be located in chromoplasts, contributing to the color of vegetables and fruits, or in chloroplasts, where, together with chlorophylls, are involved in the 7702 Romanian Biotechnological Letters, Vol. 17, No. 5, 2012

Studies concerning the extraction of chlorophyll and total carotenoids from vegetables two phytosystems. Carotenoids group and their derivatives, include about 70 compounds that are present in most vegetables and fruits. Among carotenoid pigments with 40 carbon atoms identified in vegetables and fruits, β-carotene is the most popular and widespread. In horticultural products lycopene has been extensively identified. It is found mostly in tomato and other 70 plant species. Total carotenoid content of tomato pigments varies between 6.55 mg/100 g and 12.0 mg/100 g fresh product. In pepper, the reported range is between 8.0 and 25.0 mg/100 g fresh product (CIOFU [5]). The aim of the research was to determine the chlorophyll and carotene pigments simultaneously from different vegetables, using three organic solvents, in order to obtain the best extraction solution. Absorptive properties of the pigments were followed up in quantitative analysis using a spectrophotometric method. The literature presents two methods to determine these pigments - the trichromatic method and monochromatic method, the two methods differing by the used wavelengths. It is known that chlorophyll pigments have broad absorption band from blue to red. Also the coextracted carotenoids have maximum absorption in blue band so that the chosen determination method is based on measuring the absorbance at three wavelengths for each type of matrix and then calculate chlorophyll and carotene using the three "trichromatic equations" of LICHTENTALER and WELLBURN [11, 12, 13]. Materials and methods The research was conducted on 17 samples of several vegetable species collected from different greenhouses located in southern part of Romania. Investigated species were cherry tomatoes, common tomatoes, peppers and cucumbers, in several varieties and hybrids existing in culture. Extraction of pigments was carried out in stoppered tubes. Vegetables samples were prepared with a laboratory homogenizer using about 1 g fresh material. Three extraction solutions were used for each sample: 95% aqueous diethyl ether solution (petroleum ether), 90% aqueous methanol solution and 100% acetone. The extraction ratio was 1:50. Homogenized mixture is separated by centrifugation at 3000 rpm, for 10 minutes. The analytical determination was performed with Helios α spectrophotometer at the following wavelengths: 645, 653, 662 and 664 nm, for chlorophyll a and b (according to each extraction solvent) and 470 nm for carotene. Equations used for calculation are presented below. Diethyl ether Chlorophyll a = 10.05 A662-0.766 A644 Chlorophyll b = 16.37 A664 3.140 A662 Carotene = 1000 A470 1.280 Chl a 56.7 Chl b/230 Methanol Chlorophyll a = 15.65 A666 7.340 A653 Chlorophyll b = 27.05 A653 11.21 A666 Carotene = 1000 A470 2.860 Chl a 129.2 Chl b/245 Acetone Chlorophyll a = 11.75 A662 2.350 A645 Chlorophyll b = 18.61 A645 3.960 A662 Carotene = 1000 A470 2.270 Chl a 81.4 Chl b/227 Romanian Biotechnological Letters, Vol. 17, No. 5, 2012 7703

Studies concerning the extraction of chlorophyll and total carotenoids from vegetables Measurements were performed in triplicates. Statistical interpretation of results was done using mean, standard deviation and coefficient of variation values (ANOVA programme). Results and discussions Analyses performed on several vegetables showed a large variety of chlorophyll a and b and also of carotene content. When performing diethyl ether extraction, the content of chlorophyll a was small, difficult to detect, except the values obtained at cucumber determinations that had ranged from 7.21 mg/100 g fresh product to 33.76 mg/100 g fresh product (Table 1). The averaged was 23.14 mg/100 g, the standard deviation was 10.31 and the coefficient of variation was 44.55% (Table 2). In the case of methanol extraction, the contents were higher, varying from 10.49 mg/100 g fresh product to 626.15 mg/100 g fresh product. In the case of cherry tomatoes, chlorophyll a content varied between 10.49 mg/100 g to 28 mg/100 g fresh product and in common tomatoes, range values were between 10.49 mg/100 and 45.89 mg/100 g fresh product. In pepper, chlorophyll a results obtained by methanol extraction ranged from 45.89 mg/100 g and 127.52 mg/100 g fresh product and in cucumber the values ranged from 354.06 mg/100 g to 626.15 mg/100 g fresh product. The acetone extraction of chlorophyll a, detected in tomatoes was low, ranging from 3.98 mg/100 g to 5.54 mg/100 g fresh product and in pepper the quantities of pigment had values between 181.06 mg/100 g and 416.07 mg/100 g fresh product. The highest values of chlorophyll extracted in acetone ranged between 259.89 mg/100 g and 466.27 mg/100 g fresh product. Table 1. Chlorophyll a and b and total carotenoids content, mg/100 g fresh product Species Cherry tomatoes Cultivar Diethyl ether 1:50 Mehtanol 1:50 Acetone 1:50 Chlorophyll a Chlorophyll b Carotene Chlorophyll a Chlorophyll b Carotene Chlorophyll a Chlorophyll bcarotene Belle F1 7.21 4.50 16.93 28.19 3.51 14.34 4.50 7.21 52.79 Yellow pears 7.41 4.65 1.39 10.49 4.50 3.39 4.12 6.23 6.37 Gold nugget 7.11 4.20 1.20 28.19 3.51 5.98 4.79 6.89 2.99 Muscato F1 6.85 4.45 5.78 10.49 4.50 11.95 5.54 6.58 54.78 Diamont F1 7.32 4.58 21.91 28.19 3.51 20.52 4.50 7.11 48.80 Tomatoes Carabella F1 7.21 4.12 15.54 10.49 4.50 10.76 4.50 7.21 32.67 7704 Romanian Biotechnological Letters, Vol. 17, No. 5, 2012

MANUELA ADRIANA COSTACHE, GHEORGHE CAMPEANU, GABRIELA NEATA Peppers Katerina F1 16.06 4.01 3.98 19.34 4.01 10.76 4.65 6.53 34.26 Ursula F1 7.11 4.50 11.95 19.34 4.01 7.77 3.98 6.89 37.65 Winona F1 7.31 4.62 4.98 45.89 2.52 12.55 4.56 7.48 26.89 Kaptur F1 7.11 4.15 1.59 45.89 2.52 4.78 48.02 105.86 11.95 Fat papper Bianca F1 7.21 4.50 1.20 47.87 63.45 2.59 181.06 357.56 35.46 Maradonna F1 7.02 4.32 8.96 127.52 59.00 42.83 416.07 714.78 74.70 Bettina F1 7.21 4.50 2.39 540.93 412.31 28.09 390.80 201.82 27.49 Asterix F1 16.06 4.01 1.00 478.64 359.84 25.10 440.80 272.28 21.31 Cucumbers Alibi F1 33.76 3.02 4.98 626.15 397.38 33.86 466.27 255.23 25.10 Fabrio 33.76 3.02 2.39 400.94 287.88 20.52 277.84 117.28 16.53 Mirabell F1 24.91 3.51 2.19 354.06 254.90 18.13 259.89 175.3 13.94 Table 2. Statistically interpretation of pigments content using different extraction solvents, mg/100 g fresh product Diethyl ether Methanol Acetone Species Cherry tomatoes Cultivars Chlorophyll a Chlorophyll b Carotene Chlorophyll a Chlorophyll b Carotene Chlorophyll a Chlorophyll b Carotene Average 7.18 4.4761 9.44 21.11 3.9071 11.23 4.69 6.804 33.15 Max 7.41 4.65 21.91 28.19 4.5 20.52 5.54 7.21 54.78 Min 6.85 4.2 1.2 10.49 3.51 3.39 4.12 6.23 2.99 Standard deviation 0.16 0.26 8.46 8.67 0.538 6.1 0.46 0.424 23.34 Romanian Biotechnological Letters, Vol. 17, No. 5, 2012 7705

Studies concerning the extraction of chlorophyll and total carotenoids from vegetables Cv% 2.22 5.81 2.75 41.07 13.79 54.31 9.8 6.23 70.4 Tomatoes Average 9.422 4.311 9.11 23.765 3.758 10.46 4.42 7.02 32.87 Max 16.06 4.62 15.54 45.89 4.5 12.55 4.65 7.48 37.65 Min 7.11 4.006 3.98 10.49 2.52 7.77 3.98 6.53 26.89 Standard deviation 3.83 0.27 4.82 13.3 0.734 1.7 0.28 0.479 3.87 Cv% 40.65 6.26 52.9 55.97 19.57 16.25 6.33 6.82 11.77 Peppers Average 7.113 4.323 3.92 73.76 41.658 16.73 215.05 392.73 40.7 Max 7.21 4.5 8.96 127.52 59 42.83 416.74 714.78 74.7 Min 7.02 4.15 1.2 45.89 2.52 2.59 48.02 105.86 11.95 Standard deviation 0.218 0.21 3.57 38.01 27.73 18.46 4.81 7.93 25.88 Cv% 3.06 4.86 91.07 51.53 66.57 90.27 22.39 20.23 91.27 Cucumbers Average 23.14 3.61 2.59 480.144 342.461 25.14 367.11 204.38 20.88 Max 23.76 4.5 4.98 540.93 412.31 33.86 466.26 272.28 27.49 Min 7.21 3.07 1 354.89 254.89 18.13 259.89 117.28 13.94 Standard deviation 10.31 0.574 1.3 97.85 61.39 4.16 84.2 57.3 5.05 Cv% 44.55 15.9 50.19 52.15 47.95 16.55 46.3 48.56 24.18 7706 Romanian Biotechnological Letters, Vol. 17, No. 5, 2012

MANUELA ADRIANA COSTACHE, GHEORGHE CAMPEANU, GABRIELA NEATA Comparing the values of the three extraction variants of chlorophyll a, best results were obtained with methanol and acetone. Chlorophyll b extraction with diethyl ether registered low results obtained in all the tested vegetables, being close to the limit of detection of the spectrophotometer. Extraction with methanol resulted in high values of chlorophyll b in peppers and cucumbers; the peppers values ranged between 2.52 mg/100 g and 63.45 mg/100 g fresh product and the cucumbers values ranged from 254.90 mg/100 g to 412.31 g/100 g fresh product. When using acetone, the extraction of chlorophyll b is decreased in tomatoes, near the limit of detection of the device and high in peppers and cucumbers. Results of the extraction of chlorophyll b showed that acetone solvent determined the best results followed by extraction with methanol. Statistical interpretation of the results for chlorophyll a determination showed that the best results were obtained from extraction with methanol where the coefficient of variation was 41.07% in cherry tomatoes, 55.97% in common tomatoes, 51.53% in peppers and 52.15% in cucumbers (Table 2). In the case of chlorophyll b extraction, the best coefficients of variation were obtained using extraction with methanol respectively 13.79% in cherry tomatoes 19.57% in common tomatoes, 66.57% in peppers and 47.95% in cucumbers (Table 2). Diethyl ether solvent determined a different extraction of carotene, so at tomatoes the values ranged between 1.20 mg/100 g and 21.91 mg/100 g in fresh product, while at peppers and cucumbers low extraction values were obtained (Table 1). Methanol extraction determined higher levels of carotene pigment in tomatoes, varying between 3.39 mg/100 g and 20.52 mg/100 g in fresh product, in pepper the concentrations of carotene were between 2.59 mg/100 g and 42.83 mg/100 g in fresh product, and in cucumbers the concentrations obtained ranged from 18.13 mg/100 g and 33.86 mg/100 g fresh product. In the third extracting solution acetone, the obtained carotene values were higher, thus in tomatoes were between 2.99 mg/100 g and 54.78 mg/100 g fresh product, in peppers between 11.95 mg/100 g and 74.70 mg/100 g fresh product, and in cucumbers the concentrations ranged from 13.94 mg/100 g fresh product and 27.49 mg/100 g fresh product (Table 1). Comparing the results of the three extractions of carotene, acetone extraction determined the best results in all analyzed vegetables: tomatoes, peppers and cucumbers. Statistical interpretation of results of carotene showed that acetone extraction resulted in the best variation coefficient respectively 70.4% at cherry tomatoes, 11.77% at common tomatoes, 91.27% at peppers and 24.18% at cucumbers. Methanol extraction also showed coefficient of variation of the results slightly lower than for acetone extraction, respectively 54.31% for cherry tomatoes, 16.25% for common tomatoes, 90.27% for peppers and 16.55% for cucumbers (Table 2). Conclusions The best extraction solvents for simultaneously determination of chlorophyll a and b and carotene were methanol and acetone. For cherry tomatoes and tomatoes the best extraction solvent was methanol for chlorophyll a and acetone for carotene. For peppers the best extraction solvent was acetone and for cucumbers is methanol; Variation coefficients were high, both between vegetable species and between tested varieties or hybrids. Romanian Biotechnological Letters, Vol. 17, No. 5, 2012 7707

Studies concerning the extraction of chlorophyll and total carotenoids from vegetables Determination of the three pigments from different vegetables can be provided with spectrophotometric method using methanol as extractant. Beacause the methanol is toxic, extraction with acetone is preferred, providing reliable results that must be corrected with a suitable coefficient according to the vegetable species. Aknowledgement These researches were supported by the POSDRU/88/1.5/S/52614 Programme. References 1. E.J. ARAR, Determination of Chlorophyll a, b, c1 and c2, and Pheophytin a in marine and freshwater phytoplankton by Spectrophotometry, EPA, (1997). 2. E.J. ARAR, Determination of Chlorophyll a,b, c1 and c2, and Pheophytin a in marine and freshwater phytoplankton by High Performance Liquid Cromatography, EPA, Cincinnati (1997). 3. R.G. BARLOW, R.F.C. MANTOURA, D.G.CUMMINGS, Phytoplankton pigments distribution and associated fluxes in the Bellinghauser Sea during the austral spring 1992, J. Mar. Sys. 17:97-113 (1998) 4. GH. CÂMPEANU,, M.A. COSTACHE,, G. NEAŢĂ, Research on the methodology of chlorophyll and carotene content extraction in cucumbers and peppers grown in the south of Romania area, Annals Of The University Of Craiova, XVI (LII), pp. 74-82 (2011). 5. R. CIOFU, N. STAN, V. POPESCU, P. CHILOM, S. APAHIDEAN, A.HORGOŞ, V.BERAR, K.F. LAUER, N. ATANASIU, Tratat de legumicultură, Ed. Ceres, București, (2003). 6. M. A. COSTACHE, GH. CÂMPEANU, G. NEAŢĂ, Research on the methodology of extraction of chlorophyll and carotene content of tomatoes grown in the south of Romania area, Scientifical Papers U.S.A.M.V. Bucharest, seria B, LV, pp. 069-073 (2011). 7. DON SANTOS, A.C.A., CALIJURI, M.C., MORAES, E.M., ADORNO, M.A.T., FLACO, P.B., CARVALHO, D.P., DEBERDT, G.L.B. & BENASSI, S.F., Comparison of three methods for Chlorophyll determination: Spectrophotometry and Fluorimetry in samples containing pigment mixtures and spectrophometry in samples with separate pigments through High Performance Liquid Chromatography, Acta Limnol. Bras., 15(3):7-18 (2003). 8. Ş.DERE, T.GUNES,R. SIVACI,, Spectrophotometric Determination of Chlorophyll - A, B and Total Carotenoid Contents of Some Algae Species Using Different Solvents, Tr. J. of Botany, 22, pp. 13-17 (1998). 9. P.V. FLANDER, T.SENKA, M. ALENKA, A. MARIJAN, Effect of mucilage event on the distribution of summer phytoplankton as reflected by phytoplankton pigments, Per. Biol.,102:169-177 (2000). 10. G.M. HALLENGRAEFF, Seasonal study of pkytoplankton pigments and species at a coastal station off Sidney: Importance of diatoms and the nanoplankton, Mar. Biol., 61:107-118 (1981). 11. H.K. LICHTENTHALER, A.R. WELLBURN, Determination of Total Carotenoids and Chlorophylls A and B of Leaf in Different Solvents, Biol. Soc. Trans., 11, pp. 591-592 (1985). 12. D.P. SATORY, Extraction of Chlorophyll A from Freshwater Phytoplankton for Spectrophotometric Analysis, M. Sc. Hesis. Univ. Orange free state, Bloemfontein, Republic of S. Africa (1982). 13. A.R. WELLBURN, The Spectral Determination of Chlorophylls A and B, as well as Total Carotenoids, Using Various Solvents with Spectrophotometers of Different Resolution, J. Plant Phys. 144, 307-313 (1994). 7708 Romanian Biotechnological Letters, Vol. 17, No. 5, 2012