61 SPECTROPHOTOMETRIC DETERMINATION OF CHLOROPHYLLS AND CAROTENOIDS. AN EFFECT OF SONICATION AND SAMPLE PROCESSING Jana Braniša 1 , Zita Jenisová 1 , Mária Porubská 1 , Klaudia Jomová* 1 , Marián Valko 2 Address(es): RNDr. PhD. Klaudia Jomová, 1 Constantine the Philosopher University, Faculty of Natural Sciences, Department of Chemistry, Tr. A. Hlinku 1, 949 74 Nitra, Slovak Republic,+421 37 6408656. 2 Slovak Technical University, Faculty of Chemical and Food Technology, Department of Physical Chemistry, Radlinského 9, 812 37 Bratislava, Slovak Republic. *Corresponding author: kjomova@ukf.sk ABSTRACT Keywords: Carotenoids, chlorophylls, sonication INTRODUCTION Many of epidemiological studies have shown that the consumption of diets rich in fruit and vegetables helps to prevent a wide range of diseases associated with long-term oxidative stress, such as cardio-vascular diseases, cancer and illnesses related to the aging (Kohlmeier and Hastings, 1995; Halliwell, 1997; Nishino et al., 2009; Bystrická et al., 2011; Jomová and Valko, 2011; Papaioannou et al., 2011; Tanaka et al., 2012). Well-known biologically beneficial natural substances are carotenoids endowed either with antioxidant activity or with pro- vitamin A activity (Bohm et al., 2012). Carotenoids are one of the most important groups of natural pigments abundant in many fruits and vegetables. Dietary carotenoid intake from vegetable and fruit sources has been correlated with a reduced cancer risk (Sporn and Suh 2002; Tanaka et al., 2012). However, recent studies have shown that also chlorophyll (Chl) consumption might be linked to a chemoprotective effect (Ferruzzi and Blakeslee, 2007). Beneficial effects of natural pigments on human health have led to increased interest in the study of these substances. Current research is oriented on identification and quantification of components in plant material and determination of their activity. The various sets of experimental data concerning the abundance as well as biological activity of effective substances in natural plant resources are required to develop the useful food and nutraceutical supplements. Identification and quantification of plant bioactive substances is influenced by various factors, the most significant being the sample preparation procedure, the type of extraction reagent and the method of their determination. The most commonly used techniques for identification and quantification of natural substances are either spectrophotometry or high-performance liquid chromatography (HPLC) (Jeffrey et al., 1997). Each of the methods has different advantages and limitations (Mantoura et al., 1997). In our work we verified applicability of two developed spectrophotometric methods for simultaneous determination of chlorophylls (a, b) and carotenoids in strawberry, apricot and raspberry fruits. The pigments were extracted in two organic mixtures according to the used method and ultrasonication was applied with the aim to enhance the extraction yields. MATERIAL AND METHODS Chemicals The used solvents (acetone and hexane) were purchased from Sigma Aldrich and were of analytical grade purity. Processing of raw fruits The fruits of strawberry, apricot and raspberry were obtained from market in a stage of full ripeness. The clean and dry fruit (free of stems or pits) was homogenized in household food blender fitted with a sieve with 2 mm perforations. The waste fraction was returned to blender one more time to assure complete homogenization. The whole isolation procedure was performed under dark conditions to avoid light degradation of the pigments. Assay for chlorophylls, β-carotene and lycopene In general, the samples prepared from raw fruits intended for pigment extraction were initially processed by two methods. In the first approach the fruit was processed only mechanically, the second approach involved mechanical processing plus sonication of the samples. The pigments were determined according to the method of Nagata and Yamashita (1992). 1g of fresh weight of each fruit slurry was separately homogenized (IKA-WERKE T10 Basic) with 10 mL of an acetone–hexane mixture (2:3) for 2 minutes to uniform mass. In parallel experiments under the same conditions, in order to ascertain the effect of sonication on extraction yield, the samples were sonicated (sonicator Bandelin HD3100 Sonopuls) for 3 minutes (5 cycles: puls 30 s, pause 10 s). Samples were maintained in an ice-water bath to prevent over-heating of the samples. Homogenates were centrifuged (Eppendorf) at 5000 rpm for 10 minutes at 20 °C. The absorbance spectrum of each supernatant was measured and the absorption maxima were read at 453, 505, 645 and 663 nm (UV/VIS spectrophotometer Cary 50 Scan). Chl a, Chl b, β-carotene and lycopene content was calculated from the following equations: Chlorophylls and carotenoids are abundant pigments in plants, algae and cyanobacteria. In this study we verified the applicability of two previously developed UV-vis spectrophotometric methods for simultaneous quantitative determination of chlorophylls (a, b) and carotenoids (lycopene, β-carotene or total carotenoids). The pigments were extracted from the strawberries, apricots and raspberries in both the acetone-water and acetone-hexane mixtures. Based on the statistical evaluation of the results the combination of mechanical disruption and sonication of fruit samples seems to be a suitable way to improve the pigment extraction efficiency from fruits in both types of solvents. In the case of apricot and raspberry fruit extracts the amount of chlorophylls and carotenoids calculated from the proposed equations was comparable to those published by other authors. However, the spectrophotometric determination of β-carotene content in strawberry acetone-hexane extract appeared to be problematic mainly due to the fact that carotenoids exhibited overlapping chlorophyll absorption bands. Overlap of bands leads to the negative values calculated from the proposed equation for the β-carotene content. The results indicate the limitations in use of the proposed set of equations for plant samples with comparable amounts of studied pigments. ARTICLE INFO Received 23. 10. 2013 Revised 28. 11. 2013 Accepted 16. 12. 2013 Published 1. 2. 2014 Regular article