Determination of macro, essential trace elements, toxic heavy metal concentrations, crude oil extracts and ash composition from Saudi Arabian fruits and vegetables having medicinal values

2.1.1 Food samples, sample collection, and sample preparation

Several fruit and vegetable samples—Common fig (F. carica), Annona seeds (Annonaceae seeds), Annona fruits (Annonaceae fruits), Butter 1 (C. munbayana), Butter 2 (C. hesperidum), Fennel (F. vulgare), Fennel flowers (N. sativa), and Vigna (V. unguiculata)— were collected in the summer of 2015 in Saudi Arabia. The fruit and vegetable samples were either sun or oven dried (Fisher Scientific Isotemp oven) at 55 °C to remove moisture in order to prevent food decay and microbial activity. The dried samples were subsequently grounded in a clean mortar and pestle and stored in pre-nitric acid washed and dried polyethylene bags prior to any further laboratory analysis.

2.1.2 Acid digestion of fruits and vegetables

The element concentrations in the food samples were determined using a standard analytical procedure (Latimer Jr., 2012; Watson, 1994). In brief, approximately 5 grams of the dried and ground sample was digested with 23 mL 6 M HNO₃ (trace element grade, Fisher, NY) in a digestion flask for approximately 8 h in order to ensure a complete sample digestion. The digested samples were cooled to room temperature, filtered with Whatman filter paper, and diluted to the mark with triply deionized water (Thermo Scientific, GenPure UV-TOC/UF, Hungary) in a 25-mL volumetric flask.

2.1.3 Analysis of digests using ICP-OES

Two multi-element stock solutions containing 100 μg/mL As, Cd, Co, Cr, Cu, Ni, Pb, Se, and Zn (SCP Science) and 5,000 μg/mL Ca, Mg, K, and Na (Environmental Express), along with individual single-element stock solutions containing Al, Fe, and Mn (SCP Science) were used to prepare calibration standards. The internal standard was prepared from 10,000 μg/mL Y (SCP Science). Calibration standards were diluted with 2% (v/v) HNO₃ prepared from PlasmaPure nitric acid (SCP Science) and water with a resistance of at least 18 ohms from a Barnstead Nanopure system. Calibration standards contained 0.01–5 mg/L Al, As, Cd, Cr, Cu, Ni, and Pb; 0.1–20 mg/L Fe and Mn; and 5–50 mg/L Ca, K, Mg, and Na. Samples that exceeded the concentration of the highest standard were diluted and re-run.

All digests were analyzed on a simultaneous Varian 710 ES axial ICP-OES with CCD detector. A Cetac autosampler with 15-mL sample tubes was connected to the peristaltic pump. A Burgener Teflon Mira Mist® nebulizer (SCP Science) and glass cyclonic spray chamber were used for sample introduction. The internal standard (2 mg/L Y) was added to all standards and samples via the sample introduction system. The plasma power was 1.2 kW, the argon flow rate was 15.0 L/min with an auxiliary flow of 1.50 L/min, the read time was 60 s, and the nebulizer pressure was 250 kPa. Instrument detection limits were determined by measuring the emission intensities of seven blanks. Each sample was analyzed three times (n = 3) for each element. For quality control, a laboratory fortified blank (LFB) containing 2.5 mg/L Al, As, Cd, Cr, Cu, Fe, Mn, Ni, Pb, Se, and Zn and 15 mg/L Ca, K, Mg, and Na was measured every ten samples.

2.1.4 Determination of crude oil and ash composition of food samples

The crude oil composition of the food samples was determined using a petroleum ether Soxhlet extraction procedure (Latimer Jr., 2012; Watson, 1994). Approximately 10 grams of the food samples was defatted using 200 mL of petroleum ether (60–80 °C) in a continuous Soxhlet extractor for 48 h until the solvent appeared colorless. The obtained petroleum ether extract was subsequently rotary evaporated in a Rotavapor R-205. The ash content of the food samples was determined gravimetrically using the standard muffle furnace dry ashing protocol (Latimer Jr., 2012; Watson, 1994). A known weight of the oven dried food sample was weighed into a pre-weighed crucible and placed in a programmable muffle furnace (Ney Tech Vulcan 3-550A) and ashed at 700 °C for 17 h. The crucible and the ash were allowed to cool to room temperature in a desiccator and reweighed. The weight of the ash was determined by differences in the weight of the crucible before and after ashing using Eq. (1).

3.1.1 Concentration of macro and essential trace elements in food samples

The average concentration of the macro and essential trace elements in each food samples is shown in Table 2. Each sample was analyzed three times (n = 3) for each element. The result shows the average ± standard deviation of the three replicate sample analyses. The concentrations of the essential elements varied widely in the food samples. As expected, Ca, K, Na, and Mg had the highest concentrations in each of the food samples. Ni and Se had the lowest concentrations. In general, the highest concentrations of Ca (24,749 μg/g), Mg (10,069 μg/g), Fe (565 μg/g), and Al (469 μg/g) were found in the Butter #1 fruit. The highest Zn concentration (9.3 μg/g) was found in fennel seeds and the fennel flower, which also contained a relatively higher Cu concentration (18.0 μg/g). The average concentration of each element obtained in fennel seeds and fennel flower was generally similar. The average Ca (5094 ± 53 μg/g), Mg (1844 ± 9 μg/g), Na (87.1 ± 2.9 μg/g), Fe (57.9 ± 4.0 μg/g), Al (137 ± 15 μg/g), Zn (14.8 ± 1.3 μg/g), Cu (4.71 ± 0.32 μg/g), Ni (2.79 ± 0.78 μg/g), and Se (0.58 ± 0.03 μg/g) were obtained in common fig fruit. The corresponding average Ca (1084 ± 20 μg/g), Mg (1423 ± 49 μg/g), Na (158 ± 9 μg/g), Fe (111 ± 5 μg/g), Al (83 ± 21 μg/g), Zn (6.31 ± 0.56 μg/g), Cu (5.38 ± 5.27 μg/g), Ni (1.99 ± 1.45 μg/g), and Se (0.40 ± 0.29 μg/g) concentrations were obtained in Annona fruit. The average Ca (4510 ± 504 μg/g), Mg (3355 ± 446 μg/g), Na (81.3 ± 8.3 μg/g), Fe (38.9 ± 12.4 μg/g), Al (29.9 ± 9.3 μg/g), Zn (27.1 ± 1.9 μg/g), Cu (11.5 ± 1.7 μg/g), Ni (1.76 ± 0.59 μg/g), and Se (0.43 ± 0.19 μg/g) concentrations were also found in Vigna samples.

3.1.2 Concentration of toxic heavy metals (Pb, Cd, As, Cr, and Mn) in food samples

The average Pb, Cd, As, Cr, and Mn concentration in each food sample is shown in Table 3. The common fig contained the following average concentrations: Pb (0.18 ± 0.05 μg/g), Cd (0.01 ± 0.00 μg/g), As (0.34 ± 0.03 μg/g), Cr (0.44 ± 0.02 μg/g), and Mn (14.1 ± 0.2 μg/g). The average concentrations in the Annona fruit were as follows: Pb (0.21 ± 0.02 μg/g), Cd (0.01 ± 0.00 μg/g), As (0.27 ± 0.15 μg/g), Cr (0.31 ± 0.02 μg/g), and Mn (6.27 ± 0.45 μg/g). The levels of Pb, Cd, As, Cr, and Mn detected in Annona seed were comparable to the levels of Pb, Cd, As, Cr, and Mn found in Annona fruit. The concentration of Pb (0.27 ± 0.06 μg/g), Cd (0.03 ± 0.00 μg/g), As (0.26 ± 0.16 μg/g), and Cr (0.89 ± 0.05 μg/g) found in Butter #1 was similar to the Pb (0.47 ± 0.23 μg/g), Cd (0.04 ± 0.00 μg/g), As (0.29 ± 0.05 μg/g), and Cr (1.03 ± 0.11 μg/g) concentrations detected in Butter 2. However, the concentration of Mn (149 ± 16 μg/g) found in Butter 2 was nearly twice the Mn concentration (78.7 ± 3.4 μg/g) detected in Butter 1 fruit. The levels of Pb (0.24 ± 0.12 μg/g), Cd (0.03 ± 0.01 μg/g), As (0.30 ± 0.04 μg/g), and Cr (0.33 ± 0.10 μg/g) found in fennel were also similar to the Pb (0.36 ± 0.07 μg/g), Cd (0.03 ± 0.01 μg/g), As (0.29 ± 0.13 μg/g), and Cr (0.27 ± 0.08 μg/g) concentrations obtained in fennel flower. However, the Mn concentration of 60.4 ± 4.4 μg/g found in fennel fruit was approximately three times the Mn concentration (20.3 ± 6.1 μg/g) in Fennel flower.

In contrast to macro and essential trace elements, heavy metals (Pb, Cd, As, Cr, and Mn) have no nutritional value. Detection of Pb, Cd, As, Cr, Mn, and other heavy metals in various food items has been widely reported elsewhere (Al-Ahmary, 2009; Davydova, 2005; Goldhabe, 2003; Hu et al., 2013; IRAC, 2006; Jarup, 2003; Mehari et al., 2015; Sharma et al., 2009; WHO, 1992; Zaidi et al., 2005). The need to effectively monitor the concentrations of heavy metals in foods and natural products is not only of environmental concern, but also of a considerable global public health safety interest because various health related issues including cancer diseases, cardiovascular problems, children low intelligent quotients, depression, hematic, gastrointestinal and renal failure, osteoporosis, tubular and glomerular dysfunctions, and other health issues have been directly linked to high levels of heavy metals in humans (ASTDR, 2005; EFSA, 2012; Fewtrell et al., 2003; Steenland and Boffeta, 2000; Schwartz, 1994; WHO, 2010). The concentrations of Pb, Cd, As, Cr, and Mn in food from this study are comparable with the concentrations of Pb, Cd, As, Cr, and Mn in food products reported in other studies (Al-Ahmary, 2009; Davydova, 2005; Hu et al., 2013; IRAC, 2006; Jarup, 2003; Mehari et al., 2015; WHO, 1992; Zaidi et al., 2005). The detected levels of Pb, Cd, As, Cr, and Mn in the investigated food products in this study may be attributed to the natural background of heavy metal concentrations in the soil geochemistry.

3.1.3 Daily dietary intake estimate of fruits and vegetable samples

The intake of essential elements must be closely monitored in the human diet, because both a deficiency and an excess can have negative health implications. In addition, elements such as Pb, Cd, and As have no nutritional value. The daily intake of the elements investigated in this study was calculated in μg/person/day from the average concentration of each food along with the daily consumption of each food, which was estimated through a survey of 95 families in southern Saudi Arabia. Estimated daily dietary intake of elements per person per day was calculated using Eq. (2).

Table 4 shows the results, where it can be seen that these foods will provide the recommended daily amounts of essential elements (Gupta and Gupta, 2014) but will not provide high concentrations of potentially toxic elements.

3.1.4 Inter-element association in fruit and vegetable samples

Linear regression analysis was used to evaluate the inter-element associations in the food samples, as shown in Table 5. The concentrations of most macro and essential trace elements were generally strongly correlated in the fruit and vegetable samples. For example, strong association between Mg and Ca (R² = 0.86), Mg and Na (R² = 0.95), and between Ca and Cr (R² = 0.88) was observed in the food samples. High correlations between the concentrations of Fe and Al (R² = 0.94), Cr and Mn (R² = 0.83), Cr and Fe (R² = 0.89), Mn and Al (R² = 0.88), Mn and Fe (R² = 0.93), and Cr and Al (R² = 0.93) were also found in the food samples. The concentrations of Fe and Ca (R² = 0.69), Fe and K (R² = 0.76), Al and Ca (R² = 0.72) and Al and K (R² = 0.67) were also found to be moderately correlated in the fruit and vegetable samples. On the contrary, the concentrations of potentially toxic heavy metals were found to be poorly correlated in the food samples. For instance, poor association between the concentration of Pb and Cd (R² = 0.21), Pb and As (R² = 0.00), Pb and Ni (R² = 0.23), Cd and As (R² = 0.01), Cd and Ni (R² = 0.08), As and Cr (R² = 0.02), As and Ni (R² = 0.19), As and Mn (R² = 0.01) was observed in the food samples.

The high correlations among the macro and essential trace elements indicate that these fruits and vegetables are sources of multiple elements. Consumers of these foods will be obtaining multiple macro and essential trace elements in their diet that will improve their overall health and well-being. The poor correlations among heavy metals indicate that there is no point source or a common source of these toxic elements in the food aside from the natural background levels.

3.1.5 Quality control, analytical method validation, and recovery study

Every necessary precaution was taken during sample collection, preparation, digestion, and analysis in order to preserve the sample integrity and to ensure accurate results. First, the food samples were collected in pre-nitric acid washed and dried polyethylene bags. The samples were immediately sun- or oven-dried to eliminate sample decomposition or microbial activity. The samples were prepared in a clean, dust free laboratory to eliminate contamination. Only trace element grade nitric acid (purity, 99.999%) was used for digestion and solution preparation. All glassware was pre-soaked in 6 M HNO₃ for three days and thoroughly rinsed with triply de-ionized water before use. Each food sample was analyzed in triplicate. The LFBs measured by ICP-OES demonstrated good accuracy, as most of the percent recoveries were 90–100%, with the exception of Ca and Mg, which had percent recoveries slightly greater than 100%. Specifically, percent recoveries of Pb (95.1%), Cd (94.6%), As (93.8%), Cr (95.2%), Ni (95.6%), Mn (100%), Se (94.0%), Al (98.3%), Zn (96.9%), Fe (90%), Mg (106%), Ca (114%), Na (99.0%), K (92.3%), and Cu (95.6%) were obtained for the LFB analysis.

3.1.6 Percent crude oil extract and ash compositions of fruits and vegetables

The determined percent crude oil compositions varied widely in the food samples and ranged between 0.8% in Vigna and 25% in Fennel Flower (Table 6). The highest crude oil extract compositions of 25% and 21% were found in Fennel Flower and Annona seed, respectively. Fats and oils are critical components of the human diet and may serve as sources of energy, glycerol and fatty acid. Several natural oils also contain pharmacologically active compounds, phenolics, and antioxidants that may reduce aging, lower high blood pressure, prevent cardiovascular diseases, and cancer diseases (Chen et al., 2012; Gajalakshmi et al., 2011; Veberic et al., 2008). Dietary intake of adequate amounts of oils in fruit and vegetables is therefore desirable to promote human health and well-being.

The percent ash compositions in the fruit and vegetable samples ranged from 1.7% in Annona seed to 9.5% in Butter 2 fruits. The ash composition of foods is often utilized as a measure of the level of inorganics, macro and essential elements, and other minerals in the fruit and vegetable samples.