Spectrophotometric Determinations of Chloroplastidic Pigments in Physalis angulata L . Leaves Using Different Methodologies

The aim of this study was to compare different protocols for extraction and quantification of photosynthetic pigments, considering different solvents, extraction time and temperature, to determine the best methodology for Physalis angulata L. leaves. The efficiency of immersion of leaf discs in 95% ethanol, 80% acetone and dimethyl sulfoxide (DMSO) was evaluated for 24, 48 and 72 hours. The effect of temperature on extraction with DMSO was also evaluated, with the samples incubated at room temperature and in a water bath at 65 °C. These protocols were compared with the classical methods of plant tissue maceration in 80% acetone followed by filtration, which proved to be the most efficient for Physalis angulata plants and indicated for use in the laboratory. The use of high temperatures (65 °C) is not necessary for the chlorophyll extraction process with DMSO, the room temperature is most efficient in the extraction of carotenoids. Considering the total chlorophyll content, the leaf discs immersion technique in a solvent is acceptable, with 95% ethanol and 24 hours incubation and the most economically viable alternative.


Introduction
Chlorophylls are the most abundant natural pigments found in plants.Studies show that chlorophyllian pigments are the same in a wide variety of plants, and the apparent differences in plant color due to the presence and distribution of other associated pigments (Streit, Canterle, Canto, & Hecktheuer, 2005;Von Elbe, 2000).The chlorophyll α is the pigment used to perform the photochemical step, corresponding to 75% of the total green pigments in plants.The other pigments, known as accessory pigments such as chlorophyll β and carotenoids, aid in the light absorption and in the transfer of radiant energy to the reaction centers (Taiz & Zeiger, 2004).
The photosynthetic pigments found in chloroplasts and are responsible for the conversion of light radiation into energy, such as ATP and NADPH, because of that, closely related to the photosynthetic efficiency of plants and primary productivity (Taiz & Zeiger, 2004).The determination of the leaf pigment content is an important tool for studies of plant physiology, whether for characterization of the material, for distinguishing treatments or the interaction between plants and environmental factors (Lambers, Chapin, & Pons, 1998).How nitrogen is a constituent of the chlorophyll molecule, the content of this pigment in the leaf also used to predict the nutritional level of nitrogen in plants (Silveira, Braz, & Didonet, 2003).
The extraction and quantification of the photosynthetic pigments based on the absorbance and reflectance properties of the leaf constituents, which can be a destructive method or not (Lichtenthaler, 1987).Because the fragile (non-covalent) bonds between the chlorophyll molecules, these can be disrupted by the use of organic solvents, and polar once such as acetone, ethanol and dimethylformamide the most effective (Streit et al., 2005).The use of dimethyl sulfoxide (DMSO) is efficient, as well, for complete extraction of the pigments, easier to use, since it only requires immersion of the foliar material in a known volume of this solvent (Barnes, Balaguer, Manrique, Elvira, & Davison, 1992).The choice of the ideal method for chlorophyll extraction will depend on the nature, polarity and purity of the solvent, the temperature and the incubation time, and the correct equations used to calculate chlorophyll concentrations (Ritchie, 2008;Tait & Hik, 2003).
The morphological and anatomical characteristics of the leaves of different species also influence the extraction process of photosynthetic pigments, which requires evaluating the solvent efficiency.In some fruits, such as Ficus carica, Diospyros kaki and Prunus persica, it has been reported higher efficiency for extraction using 80% acetone (Barbosa, Scopel, & Vieira, 2008), In Vittis vinifera the DMSO was more effective when compared to 80% acetone (Santos et al., 2007).In other cases, there is no difference in the use of different solvents, such as pineapple leaves (Ananas comosus) (Barboza, Portes, & Teixeira, 2008).
The Physalis angulata L. is a fruit tree, belonging to the Solanaceae family, characterized by sweet taste fruits with a high nutraceutical value, and which stands out for the production of secondary metabolites with wide medicinal potential (Tomassini, Barbi, Ribeiro, & Xavier, 2000;Lorenzi & Matos, 2008).Despite the importance of the species, it lacks basic studies related to physiology, such as the extraction and quantification of photosynthetic pigments.Some studies that use chlorophyll content to distinguish between treatments in species of Physalis commonly use 80% acetone (Bertoncelli, Alamino, Oliveira, Marchesan, & Loss, 2015;Passos, 2013), however, there is no evaluation about the efficiency of different solvents for any species of the genus.Arruda et al. (2014) found higher extraction efficiency with 80% acetone for Solanum paniculatum, a species of the same P. angulata family.
The quantity of these pigments varies with the plant species, which demands methodological adjustments for the extraction and conservation of these pigments become efficiently.Thus, this work aims to compare the different protocols for extraction and quantification of photosynthetic pigments, considering different solvents, extraction time and temperature to determine the best methodology for use in Physalis angulata leaves.

Method
This work was performed at the Experimental Unit Horto Florestal of State University of Feira de Santana (UEFS), Brazil, in March 2017.
The Physalis angulata leaf samples obtained from cultivated plants in a hydroponic system with Sarruge's (1975) nutrient solution modified for the Physalis growth (Leite, Tanan, Nascimento, Oliveira, & Abreu, 2017).Eight randomly selected plants used, from which the first pair of fully expanded leaves collected to measure chloroplastidic pigment contents.Twelve 10 mm diameter discs were cut from each leaf and immediately transferred to test tubes wrapped with aluminum foil to exclude light, containing 5 mL of the extractors evaluated.
The effect of three different solvents: 80% acetone, 95% ethanol and dimethylsulfoxide (DMSO) and, three incubation periods: 24, 48 and 72 hours were analyzed.The tubes were sealed and kept in the dark at room temperature (±25 °C).At the same time, the effect of the temperature on extraction with DMSO evaluated, with the tubes placed in a water bath at 65 °C for 1 hour and the readings performed immediately and after 24 hours of incubation at room temperature.For comparison, chloroplastidic pigments extracted from the same leaves by vegetal tissue maceration and subsequent filtration using 80% acetone as solvent (Arnon, 1949).
After each incubation period, the absorbance of the samples was determined using quartz cuvettes in a double beam spectrophotometer (FEMTO 800XI).The wavelengths varied with the solvent used and the contents of chlorophyll α, β, total (α + β) and carotenoids were calculated according to the equations proposed by Lichtenthaler and Wellburn (1983) for 80% acetone, by Lichtenthaler (1987) for ethanol and by Wellburn (1994) for DMSO.The experimental design was completely randomized, with three replicates, taking one qualitative factor (extraction method) and another quantitative one (extraction time).The quantitative factor was analyzed per non-linear regression using SigmaPlot software v.11.0 (Systat Software Inc., Chicago, USA), while the qualitative one was submitted to analysis of variance.The Tukey test was applied to compare means with 5% significance using the software Sisvar v.5.3 (Ferreira, 2008).

Results and Discussion
The concentrations of the chloroplastidic pigments over the extraction time in P. angulata leaves are shown in Figure 1.In relation to the time required for chlorophyll extraction, 95% ethanol and DMSO were faster, being efficient with 24 hours of incubation.For 80% acetone the time required was 48 hours, and after this period, there was no difference in the concentrations, evidencing the complete extraction of the pigments.With both solvents used, the data were set for the same model, corresponding to a rectangular hyperbole; the adjustments were highly efficient, as demonstrated by R² values above 0.95 obtained for chlorophylls, except carotenoids.Note.Means followed by the same letter in the column did not differ significantly by the Tukey's test (P < 0.05).
For extraction of chlorophyll β, several methods were efficient, with the highest values observed with the 80% acetone maceration, followed by 95% ethanol immersion leaf disks and DMSO at 65 °C after 1 h and 24 h of incubation, with no statistical difference between them.The opposite was observed for the carotenoid content, where the disks immersed in 80% acetone and DMSO at room temperature had the highest concentrations (Table 1).The total carotenoid content can be easily determined by measuring the maximum absorption of chlorophyll α and β, and additionally the maximum absorption at the wavelength of 470-480 nm.Thus, a correct determination of carotenoids requires the prior and correct determination of the concentration of chlorophyll β.If the chlorophyll β level is overestimated, the resulting values for the total carotenoids will be very low and vice versa (Lichtenthaler, 1987).
The differences between solvent efficiency depend on the kind of plant and the structure of the cell wall (Dere, Günes, & Sivaci, 1998).Different plant tissues react to a different kind of extractor.Some of them are inefficient, some are highly efficient, but still require maceration and centrifugation or filtration, and some require immersion of the tissue in aqueous solvents from which the pigments are extracted (Wellburn, 1994).For P. angulata plants, considering the total chlorophyll content (α + β), the highest extraction efficiency was obtained using the 80% acetone maceration technique, followed by 95% ethanol leaf discs immersion (Table 1), however without statistical differences between them.
In order to determine the best method other factors should be considered.Acetone is not suitable for field work due to volatility, propensity to leak from containers, flammability and despite the best results observed, it is permissible to lose a certain precision and use another method under field conditions, when it involves the comparison of a high number of treatments and repetitions (Ritchie, 2008;Barbieri Junior et al., 2010).Protocols, where part of the plant tissue is immersed in a solvent, are recommended due to the practicality, the possibility of preparing a large number of samples in a short time.In addition, the spectrophotometric readings can be made after a few days in extracts stored in a dark and cool place (Hiscox & Israelstam, 1979;Barnes et al., 1992).
Several authors recommend the use of DMSO (Cruz et al., 2007;Santos et al., 2007;Pompelli et al., 2013).Has high diffusion capacity through semipermeable membranes, also recognized for its efficiency as proteins carrier that provides agility in the process, the extracts prepared with DMSO being more stable than those are prepared whit acetone (Barnes et al., 1992;Cruz et al., 2007).However, some authors report the inefficiency of DMSO to extract in vivo plants chlorophyll β (Barnes et al., 1992;Shinano et al., 1996).Ehanol is more practical, easier to transport and handle, and cheaper than other solvents evaluated.However, the absorption peaks of chlorophyll α are usually smaller and wider and may interfere with the absorbance of chlorophyll β, underestimating the pigments concentration in leaf extracts (Ritchie, 2008;Pompelli et al., 2013).

Conclusion
Several factors should be considered at moment to choose the chloroplastidic pigments extraction method.The use of the 80% acetone tissue maceration protocol followed by filtration was the most efficient for Physalis angulata plants, indicated for use in the laboratory.In field conditions, the immersion technique of foliar disks in 95% ethanol and 24 hours incubation is acceptable and the most practicable economically.

Table 1 .
Mean content of photosynthetic pigments extracted from leaves of Physalis angulata by different solvents and methodologies