Phenolic compounds and vitamins in wild and cultivated apricot (Prunus armeniaca L.) fruits grown in irrigated and dry farming conditions

Phenolic profile

The study reports the effects of irrigation practices on human health related biochemical contents of cultivated (cvs. ‘Cataloglu’ , ‘Hacihaliloglu’ , ‘Kabaasi)’ and wild (Zerdali) apricot genotype. The findings in general indicated remarkably lower contents of phenolic compounds in the fruits of apricots grown in irrigated conditions compared to their dry farmed counterparts.

Vitamin content

Plant material

In the study 3 main commercial apricot cultivars (‘Cataloglu’ , ‘Hacihaliloglu’ and ‘Kabaasi’) and 1 wild apricot (Zerdali) genotype both grown in same dry and irrigated farming orchards were used. The trees of cultivars were grafted on seedlings of wild apricots. However wild apricot (Zerdali) was ungrafted that grown on their roots. All trees were 11 years old and found 1050 meter a.s.l. with 38°49′N, 37°56′E position. The study was conducted in Hekimhan district belongs to Malatya province which is the main apricot growing center in Turkey with its convenient climatic and geographic features. All cultivars and wild material were planted at 5×4 m row spacing and intra-row spacing in single crop orchards. Drip irrigation with 15 days intervals was employed for irrigated farming conditions. In dry farming conditions, no irrigation was performed and the examined cultivars only utilized rain water. A total 50 fruits of cultivars and wild material were harvested at commercial maturity period (fully orange-ripe stage) and quickly transferred to the laboratory. Then fruits were cut into small pieces, frozen in liquid nitrogen and stored at -80°C for subsequent analysis.

Extraction and determination of phenolic acids and flavonoids

The polyphenols in examined samples were extracted using a procedure described by Bengoecha et al. [24] Each apricot fruit puree (50 g) was mixed with 50 mL methanol/HCl (100:1, v/v) which contained 2% t BHQ, in inert atmosphere (N ) during 14 h at 35°C in dark. The extract was then centrifuged at 5000 rpm 15 min. Supernatant was evaporated to dryness under reduced pressure (35-40°C). The residue was dissolved in 25 mL of water/ethanol (80:20, v/v) and extracted four times with 25 mL of ethyl acetate. The organic fractions were combined, dried for 30-45 min with anhydrous sodium sulphate, filtered through the Whatman No. 40 filter paper (Whatman International Ltd., Kent, England) and evaporated to dryness under vacuum (35-40°C). The residue was dissolved in 1 mL of methanol/water (50:50, v/v) and filtered through 0.45 μm filter (Nylon Membranes, Supelco Inc., Bellefonte, PA, USA) before injected (20 μL) into the HPLC apparatus. Samples were extracted in triplicate.

Extractions were performed on a DionexASE 200 (Dionex Corp., Sunnyvale, CA, USA) system. Percentages of methanol and water in the solvent, temperature, pressure and static extraction time were the parameters under study. The pre-set default conditions were as follows: pre-heating period, 5 min; solvent flush volume, 6 0% of the extraction cell volume; number of extraction cycles, 3; purge, 90 s using pressurized nitrogen (99.995% of purity, 150 p.s.i.); and collection, in 60 ml glass vials with teflon coated rubber caps (I-CHEM,New Castle, DE, USA). The solvent used was previously degassed in order to avoid the oxidation of the analytes under the operating conditions. Optimum extraction conditions were determined as 1500 psi pressure, 40°C temperatures and an hour application time in PLE system. PEE (1 g) was mixed in gradient conditions with (methanol/water/hydrochloric acid; 75:20:5) which contained 2% tBHQ in 11-or 22-ml stainless steelextraction cells. Then, each one was filtered through a 0.45 μm nylon membrane (Lida, Kenosha, WI, USA) and transferred to a 50 ml volumetric flask. Solvents were evaporated to dryness in a Turbovap LV Evaporator (Zymark, Hopkinton, MA, USA) provided with a nitrogen stream in a water bath at 40°C. The residue was reconstituted in (2 ml) methanol-water-aqueous (50:50, v/v) and which was brought up to its volume with methanol-water filtered through a 0.45 μm PTFE filters (Waters, Milford, CA, USA) prior to injection into the HPLC system.

Polyphenol analysis were performed on a Agilent Series 1100 liquid chromatography, equipped with a vacuum degasser, a quaternary pump and a Agilent 1100, G 1315B DAD detector, connected to a HPChemStation software. A reversed-phase ACE 5 C18A11608 (250×4.6 mm, 4 μm) column was used. The content of solvents and used gradient elution conditions were previously described by Dragovic-Uzelac et al. [15]. For gradient elution mobile phase a contained 3% acetic acid in water; solution B contained a mixture of 3% acetic acid, 25% acetonitrile and 72% water. The following gradient was used: 0-40 min, from 100% A to 30% A, 70% B with flow rate 1 mL/min; 40-45 min, from 30% A, 70% B to 20%A, 80% B with flow rate 1 mL/min; 45-55 min, from 20% A, 80% B to 15% A, 85% B with flow rate 1.2 mL/min; 55-57 min, from 15% A, 85% B to 10% A, 90% B with flowrate 1.2 mL/min; and 57-75 min 10% A, 90% B with flow rate 1.2 mL/min. Operating conditions were as follows: column temperature, 30°C, injection volume, 20 μL, UV-VIS photo diode array detection at 280 nm. Detection was performed with UV-VIS photo diode array detector by scanning spectra from 210 to 360 nm. Stock standard solutions at a concentration of 1 mg ml, G1 were prepared in methanol/water (1:1) and stored at 4°C in darkness. Different range calibration curves for different polyphenol components in apricot samples were used.

Analytical quality control

Recoveries were measured by comparing retention times and spectral data with those of authentic standards. Quantitative determinations were carried out using calibration curves of the standards.

Extraction and determination of vitamins

Apricot samples (50 g) were mashed in a homogenizer and 4 g homogenate paste per sample were taken for extraction of vitamins A, E, β-carotene, lycopen and 1 g per sample was taken for extraction of vitamin C. To the above homogenates, 4 ml of ethanol were added, vortexed and the mixture centrifuged (Mistral#2000) at 2000 rpm for 3 min at 4 h. The supernatant was also filtered through a Whatman paper, and to the filtrate 0.15 ml n-hexane was added and mixed. Vitamins A, E and β-carotene were extracted twice in the hexane phase and the collected extract was dried under a stream of liquid nitrogen. Dried extract was solubilized in 0.2 ml methanol for HPLC. Injections were made in duplicate for each sample. The quantification [25] utilized absorption spectra of 326, 296 and 436 nm for vitamins A, E, and b-carotene, respectively.

HPLC separations were accomplished at room temperature consisting of a sample injection valve (Cotati 7125) with a 20 μl sample loop, an ultraviolet (UV) spectrophotometric detector (and a Techsphere ODS-2 packed (5 mm particle and 80 A pore size) column (250_4.6 i.d.) with a methanol: acetonitrile: chloroform (47:42:11, v/v) mobile phase at 1 ml per min flow rate. The extraction of vitamin C was according to the method of Cerhata et al. [26]. To 1 g homogenized apricot paste, 1 ml of 0.5 M perchloric acid was added, vortexed and the volume adjusted to 5 ml by adding ddH2O. The mixture was centrifuged at 5000 rpm for 8 min at 4°C, the supernatant was filtered, as earlier, and the vitamin C level was determined using the method of Tavazzi et al. [27] by HPLC, utilizing a column (250_3.9 i.d.) packed with Tecopak C18 reversed-phase material (10 mm particle size) with mobile phase (3.7 mM phosphate buffer, pH 4.0) at 1 ml min 1 flow rate.

Statistical analysis

Experimental data were evaluated using analysis of variance (ANOVA) and significant differences among the means of three replicates (p < 0.05) were determined by Duncan’s multiple range test, using the “SPSS 10.0 for Windows”.