EFFECT OF FOLIAR CALCIUM CHLORIDE TREATMENT ON THE LEVEL OF CHLOROGENIC ACID ,-CAROTENE , LUTEIN AND TOCOPHEROLS IN LETTUCE ( Lactuca sativa L . )

The aim of the presented study was to determine the effect of foliar application of CaCl 2 on the level of -carotene, lutein, tocopherols and phenolic compounds in the leaves of lettuce cv. Omega (Lactuca sativa L.). The experiments were conducted in the greenhouse of the Faculty of Cultivation and Fertilisation of Horticultural Plants, University of Life Sciences in Lublin. During the growing period, 7 and 14 days before harvest, the plants were treated with a solution of CaCl2 with concentrations of 0.1 M and 0.2 M. Plants sprayed with water were the control treatment in the experiment. Analyses were performed on whole leaves and the blade without midrib. The obtained results indicate that the leaves of lettuce cv. Omega are a good source of phenolic compounds, carotenoids and tocopherols. The dominant carotenoid in lettuce cv. Omega was -carotene. Foliar application of CaCl2 on the plants did not have any significant effect on the level of -carotene and lutein in the whole leaves. The contents of tocopherols, total phenolic compounds and chlorogenic acid were dependent on the concentration of CaCl2. The application of 0.1 M CaCl2 solution in the plants resulted in a decrease in the level of total phenolic compounds and chlorogenic acid, and an increase in tocopherol content. However, the treatment of the plants with 0.2M CaCl2 solution caused a lowering of the concentration of tocopherols and an increase in the content of chlorogenic acid and total phe-


INTRODUCTION
In recent years, there has been a continuous increase in the consumption of leafy vegetables, which is a result of changes in the lifestyle of consumers (R a g a e r t et al. 2004; F e r r a n t e and M a g g i or e , 2007; M a r t i n -D i a n a et al. 2007).Diets rich in vegetables and fruits reduce the risk of occurrence of chronic diseases (S t e i n m e t z and P o t t e r , 1991; B l o c k et al. 1992), because they are sources of bioactive phytochemical compounds (M o e l l e r et al. 2000; N i i z u and R o d r i g u e z -A m a y a , 2005; C a l d w e l l and B r i t z , 2006).Lettuce is one of the important world-wide dietary products available the year round which include phenolic compounds and carotenoids such as lutein and -carotene as well as of vitamin E (N i c o l l e et al. 2004).
The antioxidant activity in lettuce arises from phenolic compounds such as flavonoids and derivatives of caffeic acid.One of them is chlorogenic acid, which is located mainly in the green parts of plants (O l t h o f et al. 2001).This acid is characterised by not only antioxidant activity but also anticancerogenic properties (G o n t i e r et al. 2003); it also decreased the level of choresterol and glucose in the blood of experimental animals (R o d r i q u e s d e S o t i l l o and H a d l e y , 2002).
Carotenoids are a group of natural pigments whose importance is related to reducing the risk of occurrence of degenerative diseases, such as cancers, cardiovascular disorders, cataract, and macular degeneration.They are sensitive to the effect of numerous factors, such as light and heat (H a r t and S c o t t , 1995) as well as growth conditions (K i m u r a and R o d r i g u e z -A m a y a , 2003; C a l d w e l l and B r i t z , 2006).
Another group of components that are important in terms of dietary properties are tocopherols.They are a group of lyophilic antioxidants, synthesised by photosynthetic organisms, occurring primarily in leaves and seeds.Tocopherols are considered to be antioxidants taking part in the protection of stability of cell membranes.Their antioxidant role is attributed to inhibition of peroxidation of membrane lipids and to scavenging of reactive forms of oxygen (F r y e r , 1992; T r e b s t et al. 2002; K r u k et al. 2005).Tocopherols also perform other functions, among which we can include the transport of sugars from the leaves to other parts of plants (R u p e r e z et al. 2001; M u n n e -B o s c h and A l e g r e , 2002; T r e b s t et al. 2002; H o f i u s et al. 2004; K r u k et al. 2005; S z y m a ń s k a and K r u k , 2008).
Lettuce, and especially butter lettuce, is prone to damage.To ensure high quality of freshly cut vegetables preserving the correct colouring and preventing the browning of tissues, a variety of processes are employed.The plants are subjected to various treatments during their growth or after harvest.Such processes comprise mainly biochemical transformations related with the occurrence of plant browning (D e g l ' I n n o c e n t i et al. 2005, 2007).One of the causes for leaf browning is a deficit of calcium ions.The most effective method is foliar supplementation of this element.Among the calcium compounds that are used for plant spraying, calcium chloride is absorbed the best (M i c h a ł o j ć and H o r o d k o , 2006).It has been found that a high content of calcium in fruits reduces the rate of respiration, delays ageing (F e rg u s o n , 1984).Our earlier studies showed that foliar application of CaCl 2 solution on plants of sweet and hot peppers at the seedling stage caused accelerated ripening of the fruits but did not have any significant effect on the level of vitamins.A tendency towards an increase in the content of tocopherols in the fruits of plants treated with CaCl 2 was only observed (P e r uc k a and M a t e r s k a , 2004).
The objective of the study presented here was to determine the effect of CaCl 2 applied on lettuce at the phase of growth on its quality expressed by the contents of -carotene, lutein, tocopherols and phenolic compounds and their antioxidant activities.

MATERIAL AND METHODS
The experimental material was lettuce cv.Omega.The study was performed in the form of a pot experiment, in the greenhouse of the Faculty of Cultivation and Fertilisation of Horticultural Plants, University of Life Sciences in Lublin, in 2006.The greenhouse temperature was 18 o C during the night and 23 o C during the day.2L pots were filled with transitional peat with pH = 5.4, limed to pH = 6.4 with calcium carbonate.The addition of nutrients was in conformance with the requirements for greenhouse cultivation of lettuce and contained 0.7 g N, 1.5 g K, and 0.45 g Mg per plant (P e r u c k a et al. 2007).After two and three weeks from planting in the pots, the plants were sprayed with a solution of CaCl 2 in various amounts.The plants were divided into three experimental series.One series was given foliar feeding in the form of spraying with CaCl 2 solution at a concentration of 0.1 M (Ca1), another one with a solution with a concentration of 0.2 M (Ca2), which corresponded to 55 mg and 110 mg per plant, respectively, while the third series comprised control plants sprayed with water.
The chemical analysis included the assay of phenolic levels in whole leaves of lettuce cv Omega, whereas carotenoid and tocopherol contents in whole leaves and in the blades after removal of the midrib.
Total phenolic compounds and chlorogenic acid were analysed using freeze-dried material, while the carotenoids and tocopherols were determined on fresh material immediately after harvest.

Isolation and quantification of total phenolic compounds
The fraction containing phenolic compounds was isolated from plant material according to the method given by Chen et al. 2001 andby Nicolle et al. 2004, after certain modifications.Freeze-dried samples of lettuce (250 mg) were homogenised with 50 ml of a 70% aqueous solution of methanol for 30 min.The extract obtained was filtered and evaporated under reduced pressure (220-150 mbar) till dry at a temperature of 47 o C. Next, the dry residue was dissolved in water (5 ml) and purified on a Sep-Pak cartridge with RP-18 stationary phase which was preconditioned with methanol (5 ml) and 0,03M HCl (10 ml).The obtained extract was loaded onto the cartridge and washed with 10 ml of 0.03M HCl.The adsorbed fraction of phenolic compounds was eluted with 5 ml of 60% methanol.The determination of total phenolic compounds was made using the Folin-Ciocalteau reagent, following the method given by Nicolle et al. 2004 with modifications.The Folin reagent was diluted with water at 1:10, and a 3.2 ml solution of Na 2 CO 3 (75 g x l -1 ).Absorbance of the mixture obtained was measured after 1h, at a wavelength of 760 nm, on a Shimadzu UV -160A spectrophotometer.Total phenolic compounds were determined on the basis of the standard chlorogenic acid solution (within the range of 0.0015 -0.1 mg x ml -1 ) and their contents were expressed as chlorogenic acid equivalent.

Determination of chlorogenic acid
The determination of chlorogenic acid was made with the use of the method proposed by Llorach et al. 2004.The splitting of the fraction containing phenolic compounds was conducted by HPLC method using a Knauer WellChrom chromatograph with UV-Vis detection.The separation was made on a RP -18 Eurosil Bioselect 300 column, with dimensions of 300 x 4 mm, and particle diameter of 5 μm.The following system of solvents was used as the mobile phase: A -5% water solution of HCOOH, B -acetonitrile, with gradient elution, starting with 90%A and 10%B, in 25 min 82%A and 18%B, in 40 min.55%A and 45%B in isocrate to 50 min.The flow rate of the mobile phase was 1 ml/min and the detection was made at a wavelength 330 nm.Identification and quantification of chlorogenic acid were performed through comparison with retention time and plotting of a standard curve (within the range of 0.00156 -0.025 mg x ml -1 ), which was carried out under the same conditions as those for the analysed samples (Figs 1-2).

Antioxidant activity assay
Antioxidant activity was determined by the spectrophotometric method according to Nicolle et al. 2004.The free radical scavenging activity using the free radical of DPPH (1,1-diphenyl-2-picrylhydrazyl) was evaluated by measuring the variation in absorbance at 515 nm after 30 min. of reaction in a glass cuvette at room temperature.The reaction was started by adding the sample extract (0.5 ml) to the cuvette containing 2 ml of methanol solution of free radical DPPH (1 x 10 -4 M).The reaction was followed with a UV -Vis Varian spectrophotometer.The percentage inhibition was evaluated according to equation:

Isolation of carotenoids and tocopherols
The fraction containing carotenoids and tocopherols was obtained after homogenization of the plant material with a mortar and silicon dioxide in acetone (70 ml) that was cooled to -4 o C. Petroleum ether (70 ml) was added to the acetone extract obtained, and next it was washed continuously with water to remove the acetone.The ether extract obtained was dehydrated with anhydrous sodium sulphate (VI) and concentrated to 10 ml.The samples prepared as above were used for the assay of the contents of carotenoids and tocopherols.

Evaluation of carotenoids
The content of carotenoids was determined with the method of column chromatography, using calcium oxide as the adsorbent.-Carotene was eluted as the first carotenoid with petroleum ether used as the mobile phase, and the second one was lutein eluted with a 5% (v/v) solution of acetone in petroleum ether (P er u c k a , 1996).The carotenoid contents were analysed spectrophotometrically by absorption measurements at 450 nm and 470 nm for -carotene and lutein fractions, respectively, using the spectrophotometer UV-Vis Shimadzu -160A.

Vitamin E determination
The content of vitamin E was determined using the modified method of Müller-Mulot 1968.The petroleum ether extract obtained as described above was evaporated till dryness at 35 o C under reduced pressure, and the dry residue was dissolved in ethanol.The extract obtained was purified on a silica gel column by washing with hexane to remove chlorophylls and carotenoids.The fraction containing tocopherols was eluted with a mixture of hexane with acetone (4:1).The extract obtained was evaporated at 35 o C, under reduced pressure and the dry residue was dissolved in 2 ml of ethanol.An acetate buffer with pH = 3.5, ethanolic solutions of 0.2% FeCl 3 and 0.5% , '-dipyridyl were added to the ethanol extract.Absorbance of the mixture obtained in this way was measured after 20 min, at a wavelength of 525 nm.The concentration of tocopherols was determined based on a standard solution of -tocopherol (Sigma) within the range of 0.001 -0.018 mg x ml -1 .
Statistical analysis was made using Statgraphic v 3.1 for Windows.Tuckey's test (P<0.05)was used to evaluate significant differences among the means from three replicates.

RESULTS AND DISCUSSION
The results obtained in this study showed that the level of total phenolic compounds depended on the concentration of calcium chloride (Fig. 3).The application of CaCl 2 at a concentration of 0.1M caused a decrease in the level of total phenolic compounds, while an increase of this concentration to 0.2M had no significant effect on the accumulation of these compounds.The results obtained partially support the results obtained by R u i z et al. 2003 who, using much lower concentrations than those used in this study, showed a decrease in the level of phenolic compounds in tobacco leaves with increasing calcium ion concentrations (from 0.25 to 3 mM CaCl 2 ).There are diverse reports on the effect of Ca 2+ on the level of phenolic compounds in other plants.According to Castenada and Perez 1996, Ca 2+ plays a direct role in the synthesis of phenols.Those authors observed that foliar application of 10 μM CaCl 2 increased the accumulation of phenols in lemon seedlings.
The analysis of the results on the content of chlorogenic acid initially revealed a lower content of this acid following the application of 0.1M CaCl 2 , while a significant increase in its content was noted after the application of 0.2M CaCl 2 .The increase in the content of chlorogenic acid with an increase in the concentration of CaCl 2 may be attributed to stimulation of the activity of L-phenylalanine ammonia-lyase by Ca 2+ ions.According to a study by R u i z et al. 2003, the highest concentration of Ca 2+ caused the highest activity of L-phenylalanine ammonia-lyase that is the key enzyme in the synthesis of cinnamic acid derivatives, which also include chlorogenic acid.
The results of the effect of CaCl 2 treatment on antioxidant activity of phenolic fractions isolated from the leaves of lettuce are presented in Figure 5.The application of CaCl 2 solution on the plants 7 and 14 days before harvest caused a ca.9% decrease in antiradical abilities of the phenolic fraction obtained from the plants after 0.1 M CaCl 2 treatment and a slight decrease (3%) after using 0.2 M CaCl 2 compared to the control.
Lettuce cv.Omega is also a good source of lipophilic vitamins such as provitamin A ( -carotene), lutein and vitamin E (tocopherols) (Table 1).The dominant carotenoid in the leaves of the lettuce cultivar studied was -carotene.Its content varied depending on the part of the leaf and CaCl 2 concentration.The highest levels of -carotene characterized the leaf blades without veins, about 2 times higher than in the whole leaves in the control plants.Spraying of lettuce with CaCl 2 solution before harvest influenced only on the -Carotene levels in the leaf blades without midrib.We noticed that -carotene concentration decreased in the leaf blades about 20% compared to the control, but we did not notice any statistically significant differences in the concentration of this compound in the whole leaves of the plants sprayed with CaCl 2 solution and in the control plants.
The next very important carotenoid in lettuce was lutein.The level of lutein in the control plants was 2.5 to 3-fold lower than that of -carotene.The level of lutein in this study was similar to the values given by other authors for butter lettuce (H a r t and S c o t t , 1995; N i c o l e et al. 2004; H a m u ł k a et al. 2005).
The results of the study of the effect of CaCl 2 foliar treatment on lettuce indicated that in whole leaves only a slight increase in the levels of lutein was noticed after using CaCl 2 solution at 0.1 M and a decrease in the plants after treatment with 0.2 M CaCl 2 (Table 1).These changes were not significant statistically.Higher differences were noticed in the concentration of lutein in the leaves without midrib.After treatment of the lettuce plants with CaCl 2 at 0.1 M, the level of this carotenoid increased about 12%, while at 0.2 M it decreased about 43.5% compared to the control.
The content of tocopherols in lettuce leaves depends on the leaf type.It is the highest in the outer leaves (22.2 μg x g -1 f.m.), and lower in the central leaves -9.2 g x g -1 f.m. (S z y m a ń s k a and K r u k , 2008).These values are higher than the ones obtained in this study, as they are for leaves of whole lettuce heads (Table 1).This may be also due to inter-variety differences and the techniques applied for the determination of the tocopherol level.
The analysis of the results of the study on the effect of CaCl 2 on the level of tocopherols in lettuce leaves revealed greater differences between the control plants and those treated with calcium chloride, compared to the differences observed in the changes in lutein content.The whole leaves of the plants treated with CaCl 2 at a concentration of 0.1M were characterised by an 18% higher concentration of tocopherols and about 15% lower levels of these compounds after spraying with 0.2 M CaCl 2 compared to the control plants.
In the literature, there is a lack of studies on the effect of CaCl 2 , applied in the form of foliar feeding, on the quality of lettuce 7 and 14 days before harvest, expressed in terms of vitamin value, and it does not permit a comparison of the results obtained with the results of studies by other authors.
The results of our earlier study on the application of CaCl 2 on plants of sweet and semi-hot pepper cultivars during the vegetative growth stage showed a correlation between the content of -carotene and tocopherols in control plants and in plants subjected to the effect of CaCl 2 similar to that obtained in this study (P e r u c k a and M a t e r s k a , 2004).
In summary, the results obtained in the present study demonstrated that foliar application of CaCl 2 on lettuce during the growing period had no significant effect on the level of -carotene and lutein in whole leaves in terms of statistical significance.However, in the leaves of lettuce cv.Omega, after the application of CaCl 2 at a concentration of 0.1M, an increased level of vitamin E, -carotene and lutein was observed compared to the control.The greatest increase under the effect of CaCl 2 application was noted in the level of tocopherols.Foliar CaCl 2 treatment with 0.2 M solution slowly decreased the content of vitamin E and carotenoid but enhanced the level of chlorogenic acid in lettuce leaves.Thus, the results indicated that controlled foliar treatment with CaCl 2 can be used to enhance the level of antioxidants in lettuce.Wpływ dolistnego stosowania chlorku wapnia na poziom kwasu chlorogenowego, -karotenu, luteiny i tokoferoli w liściach sałaty (Lactuca sativa L.)