Analytical MethodsUltra-performance liquid chromatographic separation of geometric isomers of carotenoids and antioxidant activities of 20 tomato cultivars and breeding lines
Highlights
► A new UPLC method for the separation of carotenoids (lycopene, β-carotene and lutein). ► Identify and quantify three all-trans and 22 cis-carotenoids of tomato by UPLC in 15 min. ► Carotenoid profiles and genetic variation of 20 accessions of tomato (many are unique). ► Antioxidant activities of 20 tomato cultivars and breeding lines. ► Correlation between the carotenoids and antioxidant activities.
Introduction
Fruits and vegetables, as important sources of natural antioxidants, play an important role in human nutrition. Tomato (Solanum lycopersicum) is one of the most widely consumed vegetables in the world for its essential nutrients and bioactive phytochemicals, including carotenoids, vitamins C and provitamin A, folic acid and other compounds, many of which contribute significantly to human health (Alshatwi et al., 2010). Epidemiological and experimental studies have suggested a strong association between the intake of carotenoid-rich foods and the reduced risk of chronic degenerative diseases, cardiovascular diseases and age-related macular degeneration (Rao & Rao, 2007).
Lycopene, β-carotene and lutein are the major carotenoids of tomato which are also considered nutritionally most valuable. There is a growing awareness of the health benefits of carotenoids in the human diet with individual carotenoids having different beneficial properties. Specifically, lycopene reduces several cancer types and the risk of heart attack (Rao, Ray, & Rao, 2006). β-Carotene is a provitamin A carotenoid and its deficiency can cause xerophthalmia, blindness and premature death (Krinsky & Johnson, 2005). Regular intake of lutein can reduce the risk of age-related macular degeneration, cataracts and atherosclerosis (Bone, Landrum, Dixon, Chen, & Llerena, 2000).
In nature, carotenoids mostly exist in all-trans-form and the double bonds can isomerise from the trans-form to mono- or poly-cis forms under the influence of heat, light, oxygen, or certain chemical reactions (Heredia, Peinado, Rosa, & Andrés, 2010). Usually, cis-isomers are thermodynamically less stable and have lower melting points than their all-trans counterparts, due to a decreased tendency to crystallise (Heredia et al., 2010). The presence and distribution of cis-carotenoids in biological tissues also varies. It has been well documented that cis-lycopene is more bioavailable than trans-lycopene in vitro and in vivo probably because cis-isomers are more soluble in bile acid micelles and may be preferentially incorporated into chylomicrons (Boileau, Boileau, & Erdman, 2002).
Objective measurements of colour and chromaticity values can be used to estimate the concentration of lycopene, the predominant red pigment in tomato. Improving the colour and lycopene content of tomatoes can be accomplished using classically defined genes, quantitative trait loci from wild species and cultivated tomatoes (Yuan, Chen, Shen, & Yang, 2008). For example, the old gold crimson (ogc) gene can increase the level of lycopene through a biochemical mechanism at the expense of β-carotene (Blanco, 1990).
Factors affecting the total carotenoid content and its relationship with the total antioxidant activity of tomato have been studied widely; genetics, environmental conditions, production techniques used and post-harvest storage conditions have been found to impact the most (Javanmardi & Kubota, 2006). However, limited information is available on how the trans- or cis-conformations affect the antioxidant activity of carotenoids.
At present, the technique of choice for the determination of carotenoids is high performance liquid chromatography (HPLC). However, most HPLC methods with a C18 column are not technically satisfactory, and they often failed to resolve carotenoids and their geometrical isomers. Methods with a C30 stationary phase provide excellent resolution of all-trans-carotenoids and their cis-isomers, but they require a complex elution gradient program which is time and solvent consuming (about 50 min or longer) (Lin & Chen, 2003). Serial connection of several columns has been proposed as an alternative to the single column LC for the analysis of carotenoids, however the increased physical length also lead to extended run time and column pressure. Recently, ultra-high performance liquid chromatography (UPLC or UHPLC) has emerged as a liquid chromatographic technique offering much rapid and efficient separation. Advantages reported for UPLC include increased signal-to-noise (S/N) ratio, enhanced peak resolution, and a significantly reduced analysis time and mobile phase solvent consumption (Welch et al., 2010). A good UPLC method has not been developed for separating carotenoids in tomato. On the other hand, in a conventional LC method, the detection of carotenoids is often based on the retention time and UV–Vis absorption pattern. The λmax of the different carotenoids are near 450 nm, and a unique feature of cis-isomers is the additional absorbance near 330 or 360 nm. The absorption pattern and the Q-ratio value have been used in the identification of different cis-isomers of carotenoids (Chen et al., 2004, Lee and Chen, 2001). The latter, i.e. the Q-ratio, is defined as the height ratio of the cis peak to the main absorption peak of a spectrum (Liu, Lin, & Yang, 2009).
Photochemiluminescence (PCL) assay is a highly sensitive and quantitative method that measures the antioxidant activity by its capability in scavenging superoxide anion, which is easy to perform, not subject to pH variations and useful to analyse both hydrophilic and lipophilic compounds (Wang et al., 2006). In this assay, luminol is used as a photosensitiser which generates superoxide radicals and a chemiluminogenic probe for free radicals. The antioxidant activities are quantified based on the inhibitory effect on luminescence generation and are expresses as trolox equivalents (Besco et al., 2007). Meanwhile, the DPPH assay is based on the purple chromogen radical, which is reduced by antioxidant/reducing compounds to the corresponding pale-yellow hydrazine. The scavenging capacity is generally evaluated in organic media by monitoring the absorbance decrease at 515–528 nm until the absorbance remains constant (Magalhães, Ribeiro, Segundo, Reis, & Lima, 2009). The stable DPPH assay is also recommended as fast, easy and accurate, for measuring the antioxidant activity of plant foods (Sánchez-Moreno, Plaza, de Ancos, & Cano, 2006).
The objectives of the present investigation are to: (a) develop a rapid and sensitive method using UPLC with a C18 column to analyse all-trans and cis-isomers of lutein, lycopene and β-carotene in tomato; (b) examine the antioxidant activities of carotenoid extracts of 20 tomato cultivars and breeding lines using the photochemiluminescence (PCL) assay and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging capacity assay; and (c) assess the relationships between the antioxidant activity and carotenoid concentration and composition of tomatoes.
Section snippets
Plant materials
The 20 tomato cultivars and breeding lines used for this study were grown in University of Guelph processing tomato breeding plots, on a Brookston silt loam soil near Ridgetown, Ontario, Canada. These accessions were a collection of one of the authors (S.L.). Transplants were set in the field in May 2008, in single rows, 45 cm plant spacing within the row, and rows spaced 150 cm apart. The plots were maintained according to a standard processing tomato production management system with the
UPLC analysis of carotenoids
Different solvent systems have been reported for the extraction of carotenoids in plants (Guil-Guerrero and Rebolloso-Fuentes, 2009, Hu et al., 2008, Lin and Chen, 2003). We have compared four typical solvent systems used in the literature for their efficiency in extracting total carotenoids in tomato samples. A mixture of ethanol–hexane (4:3, v/v) was found to yield the highest total carotenoid content as compared to the other three systems, i.e. ethanol–acetone–hexane (2:1:1, v/v/v),
Acknowledgements
This project was funded by the A-Base research (Project 205) of Agriculture & Agri-Food Canada, and the Sustainable Productions Program of the University of Guelph/Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA). Original seed of LA3006, LA1016 and LA1593 was provided by The Tomato Genetics Resource Center, University of California, Davis, CA.
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