Effect of organic and integrated farming on carotenoid and tocopherol content of apricot fruits

Apricot is among the fruits having special importance from nutritional point of view. The edible part of the fruit accumulate high amounts of biologically active carotenoids other macroand micro-nutrients (Mangels et al., 1993; Kita et al., 2007). Carotenoids and tocopherols, as naturally occurring bioactive nutrients, have received an increasing interest during the last decades. The reactive oxygen speciesscavenging activity is associated with reduced risk of serious diseases including cancer (Keleman et al., 2006; Larsson et al., 2010), cardiovascular disease (Arab and Steck, 2000) and age-related degeneration and disorder caused by deficiency of vital carotenoids and other bio-antioxidants (Bone et al., 2000; Mozaffariech et al., 2003; Frankel and German, 2003; Schneidr, 2005). At post-harvest of plant crops, particularly during processing, carotenoids undergo some chemical alteration, such as isomerisation and oxidation on their molecules (Niizu and Rodriguez-Amaya, 2005). Such a chemical alteration can change, to a considerable extent, their chemical and biological properties (Granado, 1992). Under certain biological conditions all-trans to cis geometrical isomerisation occur not only in processed foods but also in human serum (Molnár, 2009) The organic production of certain horticultural crops is increasing from year to year due to the increasing demand for fertilizerand pesticide-free products. In conventional production fertilizer and pesticide residues threaten the health of the consumers since they increase the risk of serious diseases such as cancer. Due to the increasing level of environmental pollution and food contamination there is a great need to raise the level of endogenous antioxidants such as carotenoids, vitamin C and vitamin E in fruits and vegetables to neutralize the oxidizing effect of many dangerous pollutants. Stracke and co-workers (2009) found slight difference between organically and conventionally produced ‘Golden Delicious’ apple fruits in their polyphenol contents and antioxidant capacities. The authors concluded that the impact of season climate is more effective than the cultivation technology. Carbonaro and Mattera (2001) found significantly higher polyphenol level in organic peaches, and more polyphenol oxidase activity in organic peaches and pears. The objective of the present work was to study the carotenoids and tocopherol composition and content in apricot fruits cultivated under organic farming and integrated conditions, by newly developed HPLC methods.


Introduction
Apricot is among the fruits having special importance from nutritional point of view.The edible part of the fruit accumulate high amounts of biologically active carotenoids other macro-and micro-nutrients (Mangels et al., 1993;Kita et al., 2007).Carotenoids and tocopherols, as naturally occurring bioactive nutrients, have received an increasing interest during the last decades.The reactive oxygen speciesscavenging activity is associated with reduced risk of serious diseases including cancer (Keleman et al., 2006;Larsson et al., 2010), cardiovascular disease (Arab and Steck, 2000) and age-related degeneration and disorder caused by deficiency of vital carotenoids and other bio-antioxidants (Bone et al., 2000;Mozaffariech et al., 2003;Frankel and German, 2003;Schneidr, 2005).
At post-harvest of plant crops, particularly during processing, carotenoids undergo some chemical alteration, such as isomerisation and oxidation on their molecules (Niizu and Rodriguez-Amaya, 2005).Such a chemical alteration can change, to a considerable extent, their chemical and biological properties (Granado, 1992).Under certain biological conditions all-trans to cis geometrical isomerisation occur not only in processed foods but also in human serum (Molnár, 2009) The organic production of certain horticultural crops is increasing from year to year due to the increasing demand for fertilizer-and pesticide-free products.In conventional production fertilizer and pesticide residues threaten the health of the consumers since they increase the risk of serious diseases such as cancer.Due to the increasing level of environmental pollution and food contamination there is a great need to raise the level of endogenous antioxidants such as carotenoids, vitamin C and vitamin E in fruits and vegetables to neutralize the oxidizing effect of many dangerous pollutants.Stracke and co-workers (2009) found slight difference between organically and conventionally produced 'Golden Delicious' apple fruits in their polyphenol contents and antioxidant capacities.The authors concluded that the impact of season climate is more effective than the cultivation technology.Carbonaro and Mattera (2001) found significantly higher polyphenol level in organic peaches, and more polyphenol oxidase activity in organic peaches and pears.
The objective of the present work was to study the carotenoids and tocopherol composition and content in apricot fruits cultivated under organic farming and integrated conditions, by newly developed HPLC methods.

Materials and methods
Freshly harvested fruits were obtained from the experimental fields of the University of Debrecen in Penedics and Boldogkôváralja.Standard all-trans bcarotene and lutein were purchased from Sigma-Aldricht (Budapest, Hungary).b-cryptoxanthin and lycopene were obtained from the Institute of Bio-and Medical Chemistry of the University of Péch (Hungary).HPLC-and analytical grade organic solvents were from Merck (Darmstat, Germany).A cross-linked Nucleodur ISIS, C-18 UPLC column was from Symetron Ltd (Budapest).

Extraction of carotenoids and tocopherols:
Five grams of fresh edible part ofthe fruit were crushed in a crucible mortar in presence of 1 g quartz sand and 0,5g ascorbic acid.The extraction procedure started with macerating with methanol to bind the water followed by addition of 1,2dichloroethan and initiation of liquid-liquid phase extraction of carotenoids according to a previously described procedure (Biacs and Daood (1994)) After pooling, dehydration on anhydrous Na 2 SO 4 of the pigment-containing phase, the solvent was evaporated under vacuum and the residues were redissolved in pure acetone.To saponify the extract it was redissolved in 20 ml petroleum ether and subjected to alkaline hydrolysis with 20 ml of 30%KOH in methanol for 1 h at dark place.Carotenoids were then extracted with ethyl acetate, which was pooled and washed with double distilled water.The organic layer was then dried on anhydrous Na 2 SO 4 and evaporated to dryness under vacuum at 40 °C.The pigment was re-dissolved in HPLC acetone before injection on reversedphase column.For tocopherol analysis the residues were redissolved in HPLC grade n-hexane before injection onto normal-phase silica column.

HPLC conditions and instrument:
A Waters Alliance liquid chromatographic instrument consisting of a Model 2695 Separation Module (Gradient pump, auto-sampler and column heater), a Model 2975 Photodiode-Array Detector and a Model 2475 fluorescence detector was used.Operation and data processing were performed by Empower software.
Separation of carotenoids was performed on Nucleodur ISIS, 3µm, 15 cm x 4,6mm, column (Macherey Nagel, Düren, Germany) with gradient elution of (A) water and (B) acetone.The elution started with 20% A in B, which changed to 10% A in B in 7 min then to 5% A in B in 5 min and stayed isocratic for 5 min and turned to 20% A in B in 3 min.The flow rate was 0.7 ml min -1 .Detection of carotenoids was between 200 and 700 nm.For quantification, each compound was detected at the maximum wavelength of its light absorbance.Identification of peaks from sample extract was based on comparison of their retention time and spectral characteristics with those of standard materials available in our laboratories and with data from literature.
As for tocopherol analysis, the separation was performed with isocratic elution on Nucleosil 100 Silica gel, 5 m, 250 x 4,6 mm column using mobile phase consisting of 99.6:0.4nhexane-absolute ethanol.The separated compounds were detected at EX: 295 nm and EM: 320 nm.Peak identification and quantification was based on use of external standard materials for γ-, b-, γ-, δtocopherol analogues.

Results and Discussion
HPLC profiles of carotenoids from un-hydrolysed extract of ripe apricot fruit is shown in Figure1.In such a crop, carotenoids occur un-esterified with fatty acids.According to the HPLC profile b-carotene and its cis-isomers were dominant carotenoids followed by all trans-lycopene, its cis isomer, neo-lycopene, rubixanthin and γ-carotene.As minor individuals, b-cryptoxanthin, a-carotene, and some unidentified carotenoids could be detected.This composition agrees, to a high extent, with that reported by Baurenfiend (1981) and Mangels et al., (1993) with more compounds could be separated and detected in the HPLC analysis developed recently by our group.Mono-and di-esters of zeaxanthin could also be detected.It is to be mention that esterification of bioactive carotenoids such as zeaxanthin and b-cryp toxanthin with fatty acid does not reduce their biological activity because mono-and di-esters can be hydrolysed by de-esterase enzyme in the human intestine, furthermore, antioxidant activity of carotenoid esters containing saturated fatty acids is almost the same of the correspondent free forms (Grenado et al., 1992) Table1 shows the data obtained for different carotenoids in ripe fruits of different apricot varieties cultivated under integrated and organic farming conditions.The highest level of carotenoids was recorded in Tomcot fruits, which look pink in colour due to relatively high concentration of most carotenoids particularly all-trans lycopene and its isomers.The fruits of Tomcot variety contained also the highest level of b-carotene, the biologically most active and the major vitamin A precursor carotenoid in plant kingdom, revealing the high nutritive and biological values of such variety.Goldstrike and kajszi ranked 2 nd , while ceglédi óriás and Bergarouge ranked 3 rd when grown under integrated cultivation conditions.
Figure 3 shows the content of the major carotenoids in fruits harvested from trees of two apricot varieties grown under organic farming and integrated cultivation conditions.It is evident that organic/bio farming increases significantly the content of the dominant carotenoids such lycopene, rubixanthin an b-carotene in both examined varietie.
This change accompanied by inverse change in the content of γ-carotene and cis-b-carotene indicating that organic farming has an effect on the enzyme system (isomerise enzymes) responsible for the formation such carotenoid derivatives.From nutritional point of view this change is favourable since all-trans carotenoids are more biologically active than γand cis-isomers.Hussein, G.D., Bánáti, D., Nyéki, J. & Szabó, Z.The HPLC analysis of vitamin E components in the fatsoluble extract of apricot fruit after alkaline hydrolysis revealed that the a-tocopherol, the biologically active analogue of vitamin E, is dominant in the edible portion of apricot fruit (Table 2).The different varieties differed significantly in their content of vitamin E with that in Begarouge being the lowest followed by the content in Goldstrike among examined varieties.As for the effect of organic and integrated cultivation the results showed that the slight difference or change is varietydependent since vitamin E content in Hungarian Kajszi is significantly higher than that found in fruits from harvested from trees of integrated cultivation, while a slight decrease was observed in the content of Ceglédi óriás apricot from bio farms.It is interesting and worthy to mention that band γ-tocopherol tended to significantly increase in organic/bio fruits.From chemical point of view the later analogue are more reactive, particularly as antioxidant, than a-tocophero.

Figure 1 .
Figure 1.HPLC profile of apricot fruit carotenoids separated on cross-liked ISIS column with gradient elution of water in acetone and detected at 450 nm.For details see text.
Acknowledgement: This work was financially supported by the Hungarian Ministry of Agriculture and Rural Development and National Office for Research and Technology (NFÜ) under grant no.Biodeb07 and NFÜ TECH_08-A3/2-2008-0373 grant.

Figure 3 .
Figure 3. Carotenoid content of apricot fruits from Kajszi and Ceglédi óriás varieties grown under organic and integrated cultivation conditions.

Table 1 .
Carotenoid composition and content of different apricot varieties grown under organic and integrated farming conditions