Determination of Major-to-Trace Minerals and Polyphenols in Different Apple Cultivars

The aim of this study was to investigate the concentration level of some major-to-trace elements and toxic minerals and polyphenols (catechin, epicatechin and quercetin) in fruits belonging to different apple cultivars (‘Auriu de Cluj’, ‘Florina’, ‘Generos’, ‘Golden Delicious’, ‘Prima’, ‘Productiv de Cluj’ and ‘Starkrimson’), under specific climate conditions to northwestern Romania. The apple minerals were determined by the inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma optical emission spectrometry (ICP-OES) after a preliminary, microwave-assisted, acid digestion. The polyphenols were determined by high performance liquid chromatography (HPLC) with MS/MS detection. According to the obtained results, the edible part of the apple contains many minerals with high relative nutritional value (RNV). The major minerals (Na, K, Ca, Mg and P), minor elements (Cu, Fe, Mn, Sr and Zn) and toxic trace elements (Cd, Cr and Pb) were comparatively identified and quantified. It was found that K (31976 mg/kg) was predominant among the major elements, while Fe (3.68 mg/kg) and Cu (3.57 mg/kg) were comparable. The toxic trace metals (Cd, Cr and Pb) were below the limits of detection (0.01, 0.10 and 0.01 mg/kg, respectively) and did not determine any health risk to consumers. The main polyphenols composition (catechin, epicathechin, quercetin) differs slightly among different cultivars. In ‘Idared’ and ‘Jonathan’ cultivars, the investigated polyphenols are in higher quantities comparing to the others. In all investigated apple cultivars, quercetin was proved to be the major polyphenol, followed by epicathechin and catechin. These results suggest that, regarding only the investigated polyphenols, ‘Idared’ and ‘Jonathan’ cultivars have the highest antioxidant character in all investigated genotypes.


Introduction
Apples are the second most produced tree fruits (apple crop is estimated at 69 million metric tonnes/year) in the world, after bananas (FAO, 2011).Apples represent an important part of the human diet because they are a good source of fiber, including the soluble fiber pectin, Vitamin A, Vitamin C, minerals and polyphenols (Wu et al., 2007).Besides 75-95% water, the edible portion of apple contains an important quantity of phenolic compounds, which contribute to the flavor and can protect the human body from free radicals and reactive oxygen species (Cindrić et al., 2012;Hyson, 2011;Pandey et al., 2009).
Numerous studies on apples have been carried due to their nutritional importance, mainly regarding their acid, amino acid, mineral, vitamin and polyphenol contents (Auclair et al., 2008;Vieira et al., 2009;Ko et al., 2005).Approximately 30 elements are considered essential to life.Essential nutrients which are needed in trace and ultratrace quantities (Cu, Fe, Ni, Zn, Mn, Co, Cr, and Se), are as important for life as macronutrients (Ca, K, Mg and Na) (Cindric et al., 2011).However, some essential elements may become toxic when their concentration increases.The polyphenol family (phenolic acids, stilbenes, chalcones, coumarins, cromones, lignans, flavonoids, isoflavonoids, neoflavonoids and tannins) has been shown to possess significant antioxidant capacities, while they are maintaining low toxicities (Escudero et al., 2008).Apple polyphenols have been registered among Japanese Standard Food Additives as antioxidants due to their strong antioxidant activity (Enomoto et al., 2006).Between the apple polyphenols, flavonoids such as epicatechin, catechin and quercetin (Tab. 1) are the most important being widely distributed in natural products and medicinal herbs (Chang and Wu, 2011).Catechin and epicatechin have antioxidant, commercialization conditions.In order to avoid the influence of these factors, the samples were collected from only one farm, during one harvest/year and not representative for the market.In Central Transylvania, Romania, the most famous and the most consumed apple cultivars were: 'Golden Delicious', 'Gustav', 'Jonathan', 'Kaltherer', 'Starkrimsom'.These old apple cultivars have become well adapted to the soil and climate conditions producing good yields and high-quality fruits.The renewal process of apple variety seems to be slow and difficult both due to high costs of a new apple orchard and the attitude of farmers/consumers.The most well-known and spreaded cultivars of apples created at Fruit Research Station Cluj-Napoca were 'Aromat de vara', 'Ardelean', 'Ancuta', 'Feleac", 'Rosu de Cluj', 'Auriu de Cluj', 'Estival' and 'Productiv de Cluj' (Sestras et al., 2006;Militaru et al., 2013).
The aim of this study was to determine the content of macro-(Na, K, Ca, Mg and P), micro-(Cu, Fe, Mn, Sr and Zn) and toxic trace elements (Cd, Cr and Pb) and polyphenols (catechin, epicatechin and quercetin) of ten cultivars of apple fruits, under the climate conditions of Cluj-Napoca area, Romania.
In order to obtain good quality data, several aspects such as: sample preparation, techniques used to identify the targeted compounds and the variables which influence the quality of the apple, must be considered.
Sample preparation is an important step in elemental analysis.Microwave-assisted digestion is characterized by short digestion time, small volume of reagent consumption, lower losses of volatile metals, good recoveries, accuracy, reproductibility and enhanced operator safety (Krushevska et al., 1993;Lamble et al., 1998;Bocca et al., 2007).ICP-AES is a rapid and accurate technique for the simultaneous determination of the minor and major element contents in apples and apple juices as well.ICP-MS technique has become to be considered as the most powerful analytical tool for simultaneous trace elemental analysis of food samples due to its extreme sensitivity, selectivity and last but not the least due to its multielemental and isotopic capability (Barnes, 1999;Davidowski et al., 2009;Froes et al., 2009;Zeiner et al., 2010;Cindrić et al., 2012).
Liquid chromatography tandem mass spectrometry (LC/MS/MS) in the multiple reaction monitoring (MRM) mode has been recognized as a powerful analytical tool due to its high sensitivity and short run time (Hossain et al., 2010).Through this technique, less toxic organic solvents are used as mobile phase and is the most free of interferences compared to other chromatographic techniques (Hossain et al., 2010;Weingerl et al., 2009).
The chemical composition of the apples is a good indicator of their quality, consumer acceptability and the health status of consumers.There are many factors that influence the chemical composition and the nutritional value of apples, such as: apple variety, soil quality, production area, farming practices, the quality of irrigation water, local climate conditions, storage and

Major, minor and toxic trace elements
The quality of the analytical procedure was assured using a Standard reference material (NIST-SRM 1515-Apple Leaves).The obtained values (Cd, Cr, Cu, Fe, Pb, Zn, Ca, K, Mg and Na) of CRM draw against certified values indicating that the measured values correlate well with certified values.Limits of detection (LOD) of every studied element were experimentally calculated as three times the standard deviation of ten measurements of independent reagent blank solutions.The obtained values (Cd, Cu, Fe, Mn, Pb, Zn, Ca, K and Mg) of CRM draw against certified values indicate that the measured values correlate well with certified values (Tab.4).
Apples are considered a good source of dietary minerals.The range of concentration (mean ± SD, mg/kg d.w.) of major and minor elements in 10 apple cultivars is given in Tabs. 5 and 6.According to the obtained results, in the edible LGC Standards GmbH, Wessel, Germany.Catechin, epicatechin and quercetin were purchased from AppliChem (USA).Methanol LC-MS Optigrade (≥99.8%),Ethanol LC-MS Optigrade (≥99.8%) and Ortophosphoric acid ULC-MS Optigrade (99-100%) were acquired from LGC Standards.All glassware was cleaned with nitric acid prior to use.For all dilutions ultrapure water (18.2MΩ/cm) obtained from a Millipore Direct-Q3 UV system (Millipore, Molsheim, France) was used.

Sample preparation
Fruits were picked at harvest maturity as it is recommended for fruits that are marked out for storage.Fruit samples consisted of five individual apple fruits picked from the same tree of the investigated orchard.The fruit samples were washed first, several times with deionized water to remove the dust particles and then dried at 105 °C for 24 h and homogenized.The dried samples were grounded, homogenized using a metalfree mortar and stored in polyethylene bags until acid digestion.
Per sample, 0.5 g was weighted accurately in a dry, clean PTFE vessel; 6 mL of HNO3 65% and 3 mL of H2O2 30% were added to each sample and the vessel content was kept 4 h at room temperature before digestion.After this, the samples were digested using a four-step digestion program (time [min] / power [W] / T [°C]: 10/700/170; 15/1000/200; 10/1000/160; 10/1000/100; 10 min ventilation).After mineralization, clear solutions were cooled at room temperature, and then quantitatively transferred to 25 mL volumetric flasks and diluted to the mark with deionized water.Certified reference material NIST-SRM 1515 apple leaves and blank, consisting of deionized water and reagents, were prepared in the same way as the samples.Three replicate measurements were made for each sample.
In order to determine the polyphenol content, the edible portion of the fruit was sliced into small pieces with a ceramic knife and lyophilized to avoid the degradation of the samples.100 g of each dried vegetable product were extracted for 5 hours with EtOH:H2O (90/10, v/v) using a Soxhlet.The extract was dried under constant flow of compressed nitrogen gas, and reconstituted in mobile phase before injection.The recovery degree for all three polyphenols was situated in 77-94% range and was measured using internal standard addition.

Instrumentation
For the microwave digestion of samples, a closed vessel microwave system Berghof MWS-3+ (Eningen, Germany) was used.The contents of Ca, Mg, Na, K, Fe and P were determined by ICP-OES (OPTIMA 5300 DV, Perkin Elmer, Norwalk, USA), while those of Cd, Cu, Mn, Pb, Sr and Zn by ICP-MS (ELAN DRC II, SCIEX, Perkin Elmer, Toronto, Canada).The calibration standards were prepared by diluting the stock multi-elemental standard solution (1000 mg/L) in 2% HNO3 and the calibration ranges were modified according to the expected concentration values of investigated elements.
The Regarding the annual variation on minor and major element concentrations, the obtained mean concentration was similar to those from both years.'Golden Delicious' apple had the highest average content of major elements.
One apple ( ∼ 300 g fresh weight or 40 g dry matter) can offer up to 1279 mg K (27.2% RDA), 0.38 mg Na (0.03% of RDA), 12.5 mg Ca (1.25% of RDA), 13.3 mg Mg (3.31% of RDA) and 26.3 mg P (3.75% of RDA).As a result, major elements were found to have good nutritional contribution in accordance to RDA.

Minor elements
The minor-elements also support important functions of the human body.Iron is necessary to produce hemoglobin and myoglobin, proteins that carry oxygen in the human body.Manganese, one of the vital important elements, enhances the absorption of calcium and plays an important role in the production of bones and connective tissues.Chromium enhances the action of insulin, making it important in blood sugar regulation processes.The human body needs copper and zinc as well, to produce enzymes involved in protection against oxidative processes.Strontium can improve the cellular make-up of bones and teeth by preventing tooth decay of soft bones (WHO, 2006;Vicente et al., 2014).

Toxic trace elements
In all samples, the contents of heavy metals (Cd, Cr and Pb) were below the limit of detection (0.01, 0.10 and 0.01 mg/kg, respectively).European legislation (1881/2006/EC setting maximum levels for certain contaminants in foodstuffs) regulates the maximum admitted concentration of Pb (0.10 mg/kg wet weight) and Cd (0.05 mg/kg wet weight) in fruits.The obtained concentrations of Pb and Cd part of the apple, the concentration for the major-elements varies in the following way: K > Ca > P > Mg > Na and Fe > Cu > Zn > Mn > Sr for the micro-elements, respectively.

Major elements
Among the major elements examined in apple fruits, K was the most abundant with an overall mean concentration of 31976 ± 5366 mg/kg.This result was comparable with the data reported for apple fruits by Cindric et al. (2012) (38600 mg/kg) and higher than those determined by Hamurcu et al. (2010) -863 mg/kg, Horsley et al. (2014) -10226 mg/kg, and Ozcan et al. (2012) -7000 mg/kg.The obtained values for Na (9.62 ± 1.63 mg/kg) were similar to those reported by Cindric et al. (2012) -9.92 mg/kg, and lower than those determined by Humurcu (37.0 mg/kg) and Ozcan (600 mg/kg).Adults should consume 4700 mg K/day and 1500 mg Na/day (deMan, 1999).Apple fruits may be a good source of potassium, which plays an important role in the human body, especially in the transmission process of nerve signals, but also in fluid balance and proper function of heart, muscles, kidney and hormones.Sodium plays a key role in muscle and nerve function and work with potassium to coregulate ATP and fluids.
Calcium and magnesium also play an essential role in muscle function, nerve transmission, bone and teeth formation and hormone secretion; furthermore, magnesium is required for processing ATP.The Recommended dietary allowance, RDA (average daily dietary intake level sufficient to meet the nutrient requirements of nearly all, healthy individuals in a particular life-stage or gender group) for adults is 1000 mg Ca/day, and 400 mg Mg/day (deMan, 1999).It should also have to be considered the role of calcium to the maintenance of fruit ripening and optimum quality during postharvest storage.This role is enhanced directly in the prevention of specific disorders (bitter pit) and in relationships between calcium and general quality properties (flesh firmness) (Ferguson and Watkins, 1982).The content of Ca (312 ± 102 mg/kg) and Mg (331 ± 66.4 mg/kg) were ten times lower than the reported results for apples (4410 mg/kg-Ca and 3140 mg/kg) by Cindric et al. (2012).
Phosphorous is a very important component of the bones and cells having an important role in protein production to satisfy the needs of the human body.The RDA for daily phosphorous for adults is 700 mg P/day (deMan, 1999).The obtained values for P (657 ± 113 mg/kg) were similar to those obtained by Hamurcu et al. (2010)

Polyphenols content
Three apple polyphenols, namely catechin, epicatechin and quercetin, were determined (Tab.7).The concentrations of polyphenols were slightly different among cultivars, with some increases for 'Idared' and 'Jonathan'.Several other studies confirmed earlier that the content of polyphenols differs by variety and is affected by different factors such as: apple variety, fruit development, conditions during long-term storage, superficial scald development in apple fruit and solar radiation (Lachman et al., 2006).Taking into consideration the fact that the investigated cultivars were selected from the same orchard, were grown under similar conditions and were sampled of their positions, it can be concluded that the differences in polyphenols quantity depends only on the apple variety.
In all investigated apple cultivars, quercetin was proved to be the major polyphenol, with concentrations from 8.99 to 12.21 mg/100 g d.w.The concentrations decreased to epicatechin (from 5.88 to 9.87 mg/100 g d.w) and to catechin (from 0.49 to 1.04 mg/100 g d.w).Similar results were obtained in other studies (Manach et al., 2005).
A recent study (Randall, 2012) suggest that the daily dose of 300 mg to 600 mg of apple polyphenols may be all that is needed to obtain good health benefits.The present results suggest that catechin, epicatechin and quercetin from 100 g dried apples cover only 0.666% from the necessary dose.Manach et al. (2005) support the idea that the most abundant polyphenols in our diet, are not necessarily leading to the highest concentration of active metabolites in target tissues, the three investigated polyphenols being of great interest for our health.

Conclusion
The results of this study provide valuable data regarding major-to-trace elements and polyphenols in different apple cultivars, under climate conditions of Cluj-Napoca area, Romania.The investigated apple samples contain significant amounts of several essential elements and polyphenolic compunds as well.Apple consumption represents a valuable and a very important source of minerals and antioxidants.Toxic element concentrations (Cd, Cr and Pb) were below the limits of detection showing no harmful effects caused by these elements through the high consumption of apples.Regarding the annual variation on minor and major element concentrations, the obtained mean concentration were similar in both years.The LC/MS/MS method was proved to be a robust and useful technique for polyphenols' determination.The results obtained are of interest especially for the medical world due to the aminoacid characteristics of the investigated polyphenols.These data should prove useful especially for the medical world when designing anti-cancer, antiangiogenic, anti-mutagenic, hypocholesterolemic, antiageing, anti-diabetic, anti-bacterial, anti-HIV and antiinflammatory treatments.