Determination of Selected Metals in Leaf and Root Bark of Malva Parviflora

In Ethiopia, Malva parviflora (family: Malvaceae) leaf is used as a vegetable and its root bark is used to treat furuncles, carbuncles, wound infections and other related ailments. However, no research has been done to analyze essential and toxic metals in leaf part and the metals having antibacterial and wound healing activities in root bark of this plant. Thus, the present study was aimed to determine concentration of K, Na, Mg, Ca, Fe, Zn, Cu, Cd, Pb, and Ni in leaf par; and Mg, Ca, Fe, Zn, Cu, Cd, Pb, Ni, and Co in root bark of Malva parviflora. The results revealed that the leaf part contains high amounts of Na, K, Mg, and Ca. Similarly, high concentrations of Mg and Ca were detected in the root bark. Appreciable amounts of Fe and Zn, and low amount of Cu were detected in both leaf part and root bark. Concentrations of Co, Ni, Cd, and Pb were below the method detection limit. Thus, leaf of Malva parviflora is a good source of essential nutrients (Na, K, Mg, Ca, Fe, and Zn) and the presence of Mg, Ca, Fe, Zn and Cu might contribute to the therapeutic action of root bark of Malva parviflora.


Pretreatment of Glasswares
To avoid contamination, all glasswares required for experiments were first washed with cold tap water using a detergent and then soaked in 20% (v/v) HNO3 for 48 hours, and finally rinsed with deionized water.

Collection of the Samples
Fresh plant materials were randomly collected from agricultural farmlands in the month of October, 2012 towards the end of the rainy season. Both leaf and root samples collected were kept separately in polyethylene plastic bags.

Sample Pretreatment, Drying and Grinding
The root materials were washed with tap water followed by deionized water to eliminate attached soil particles. The bark was the separated from the root and chopped into small pieces using a stainless steel knife. After drying in air, the sample was dried in an oven at 60 o C for 12 hr to assure complete removal of moisture. It was then grinded using mortar and pestle, sieved (2 mm) to obtain uniform particle size. Both leaf and root bark powders were weighed and then stored in clean dried plastic bottles at room temperature for further analysis.

Wet Digestion of Samples
The wet sample digestion was carried out using Kjeldahl digestion apparatus with a reflux condenser for both leaf and root bark samples. Three parameters: temperature (275°C), time (3 hrs) and reagent volume (15 mL HNO3) were optimized. Applying the optimized conditions, 1 g of each sample was digested for analysis. The digestion was carried out in triplicate for each sample. The digested solutions were allowed to cool at room temperature for 30 min. To the cooled clear yellow solutions, 20 mL of deionized water was added, and solutions were then shaken to dissolve precipitates remaining on the wall of the digestion apparatus. Then, solutions were filtered into 100 mL volumetric flasks through a Whatman No. 42 filter paper with a 0.45-μm to remove the impurities for metals analysis. The solutions were finally diluted to the mark with deionized water and kept in room temperature until analysis.

Constructing Calibration curves
To construct calibration curves, diluted standard solutions were prepared from their respective stock standard solutions (1000 ppm) which were already-made by dissolving appropriate amounts of the their respective metal salts of analytical grade (purity 99.9%) in HNO3 and diluting with deionized water. Preparation of the diluted standard solutions for each metal was depending upon the linear working range (Table 1). The absorbance of each solution was measured and calibration curves were constructed separately for selected metals to determine correlation coefficient (r 2 ).

Recovery Test
The optimized procedure was validated by spiking each of triplicate leaf and root bark samples with the known concentration of standard metal solutions using a pipette ( Table 2). The spiked samples were then digested using optimized procedure and analyzed to calculate percent recoveries.

Method Detection Limit
Method detection limits (3SD, where SD= Standard Deviation) for metals such as Fe, Co, Ni, Cu, Zn, Cd, and Pb were determined by digesting ten analytical reagent blank (n=10).

Analysis of the Selected Metals
Concentrations of Ca, Mg, Fe, Cd, Cu, Pb, Zn, Ni, and Co were analyzed using flame atomic absorption spectrometry; whereas analysis of Na and K was performed in flame photometer. Triplicate determinations were carried out for each metal and the mean values were reported. The instrumental conditions during the analysis of each the metal is present in Table 3.

Method Validation Parameters
The results of correlation coefficients are given in Table 4. According to (Mitra, 2003), the minimum correlation coefficient (r 2 ) should be 0.995. Thus, all calibration curves in this study showed good correlation between concentration and absorbance. The method detection limits determined for leaf and root bark samples were low enough to detect the presence of interested metals at trace levels ( Table 4). The reproducibility of the analytical procedure was checked by carrying out triplicate analysis and calculating the relative standard deviation (RSD) for each metal. The recommended value of RSD is less than 10% (Mitra, 2003). In this study, RSD values were below 8% (Table 4) of the mean which revealed that the analytical method used was precise and reliable. As shown in Table 4, the percentage recovery varied from 83.3 to 112.9 %, which are in the acceptable range according to Clesceri et al. (1999).

Concentration Level of Selected Metals in Leaf and Root bark of Malva parviflora
This study revealed that the leaf part contains high amounts of Na, K, Mg, and Ca. Similarly, high amounts of Mg and Ca were detected in the root bark. Appreciable amounts of Fe and Zn, and low amount of Cu were detected in both leaf part and root bark. Amounts of Co, Ni, Cd, and Pb were below the method detection limit (Table 5). The concentration level (38125.0±3011.9 ppm) of K indicates that leaf part of Malva parviflora rich in this element. K is the most abundant cation in the human body and its presence in edible part at high level is important (Ashraf et al., 2010). A daily recommended amount of K for adult is 4,700 mg/day (Ismail et al., 2011). K is a major component of many soils and derived from the weathering of soil parent materials such as potassiumaluminium-silicates. Other factors that increase amount of K in soils are application of fertilizers (Kirmani et al., 2011) and plants and other organisms holding K as free ions in their cells, once they die; it quickly reenters the