Determination of 17 metals and metalloids by ICP-MS and total antioxidant activity by Microwave-Assisted Extraction and DPPH Scavenging method of Salvia Fruticosa Ioannis N. Pasias , Nikolaos S. Thomaidis, Efrosini Piperaki Laboratory of Analytical Chemistry, Department of Chemistry, National and Kapodistrian University of Athens, 157 71 Athens, GREECE e-mail: piperaki@chem.uoa.gr A. Determination of metal and metalloid content in leaves, flowers and the infusion from Salvia Fruticosa Introduction This work describes the development of an Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) method for the determination of seventeen elements (V, Cr, Co, Se, Sr, Sn, Sb, Ba, Bi, Pb, Cd, As, Ni, Mn, Fe, Mg and Zn) in leaves, flowers and the infusions from Salvia Fruticosa, a sage cultivated in Crete island, Greece. The ICP-MS results were compared for some elements with the results obtained by Flame Atomic Absorption Spectrometry (Fe, Mg, Zn) and Electrothermal Atomic Absorption Spectrometry (V, Se, Pb, Cd, As, Ni, Mn). The elemental content was found to be in the range of 0.01-30.8 mg/Kg (leaves), 0.30-39.1 mg/Kg (flowers), 0.003-20.4 mg/Kg (infusions) for V, Cr, Co, Se, Sr, Sn, Sb, Ba, Bi, Pb, Cd, As, Ni, Mn and in the range of 0.07-3.21 g/kg (leaves) for Fe, Mg and Zn. 45 Sc, 72 Ge, 115 In and 232 Th (50 μg/L) were used as internal standards for elements in the mass range (m/z) 51-65, 75-82, 111-117, 206-209, respectively. The method LODs were determinated to be in the range of 1.6 μg/Kg (Cd) to 68.8 μg/Kg (Bi) for the leaves and the flowers and in the range of 2.0 μg/Kg (Cd) to 86.0 μg/Kg (Bi) for the infusion. The calculated recoveries were in the range of 57%( 82 Se) to109%( 53 Cr) for the leaves, 67%( 121 Sb) to 119%( 114 Cd) for the flowers and 58%( 209 Bi) to 119% for 114 Cd for the infusions. The precision was also checked and the relative standard deviation was ranged from 1.3% ( 52.9 mg/Kg 55 Mn ) to 26% (0.01 mg/Kg 111 Cd) for the leaves, 2.4% ( 61.9 mg/Kg 55 Mn ) to 25% (0.02 mg/Kg 114 Cd) for the flowers and 2.0% (2.5 mg/Kg 88 Sr ) to 38% (0.003 mg/Kg 59 Co) In addition, the antioxidant activity of the herbal was determined by measuring the DPPH scavenging activity. Microwave-assisted extraction (MAE) was used to extract total antioxidants. The effect of temperature, time and solvent in the extraction efficiency was investigated. The optimum extraction conditions were: 0.5 g of sample extracted by 10 mL of 100% ethanol at 90 0 C for 10 min. The determination of the antioxidant activity was based on the calculation of the % inhibition of the absorbance signal of the radical DPPH at 515 nm, after the addition of herbal’s extract. Samples from other areas of Greece were also examined for comparison reasons. The EC 50 values were found to be in the range of 10.6-32.2 mg/L. Figure 1: Sampling areas: a) Chania, Crete (Southern Greece), b) Thermopyles, Lamia (Central Greece), c) Amorgos, Kyklades (Eastern Greece), d) Lesvos (Eastern Greece). a b c d B. Investigation of the antioxidant activity by Microwave-Assisted extraction and DPPH scavenging method of Salvia Fruticosa Element C mean ±SD (mg/Kg) Range (mg/Kg) 60 Ni Leaves 3.46 ± 0.97 1.86-5.08 Flowers 1.40 ± 0.29 0.66-1.72 Infusion 0.58 ± 0.61 0.13-2.80 63 Cu Leaves 9.9 ± 2.9 5.3-17.9 Flowers 12.2 ± 1.9 9.5-16.4 Infusion 3.9 ± 2.6 0.8-13.2 75 As Leaves 0.32 ± 0.15 0.12-0.76 Flowers 0.22 ± 0.08 0.11-0.34 Infusion 0.050 ± 0.027 <0.006-0.106 82 Se Leaves 0.51 ± 0.37 0.09-1.46 Flowers 0.44 ± 0.16 0.11-0.67 Infusion 0.04 ± 0.04 <0.01-0.13 Element C mean ±SD (mg/Kg) Range (mg/Kg) 88 Sr Leaves 20.4 ± 7.9 7.5-33.7 Flowers 27.3 ± 6.3 14.6-36.5 Infusion 7.9 ± 3.1 3.1-15.5 114 Cd Leaves 0.04 ± 0.02 0.02-0.11 Flowers 0.10 ± 0.09 <0.005-0.31 Infusion <0.006 <0.006 118 Sn Leaves 0.15 ± 0.04 0.06-0.20 Flowers 1.12 ± 1.0 0.1-3.1 Infusion 0.003 ± 0.01 <0.002-0.05 121 Sb Leaves 0.05 ± 0.04 <0.01-0.15 Flowers 0.06 ± 0.02 0.03-0.10 Infusion 0.003 ± 0.004 <0.001-0.016 Element C mean ±SD (mg/Kg) Range (mg/Kg) 137 Ba Leaves 23 ± 12 9.5-61 Flowers 16.5 ± 9.0 6.8-43.8 Infusion 2.74 ± 0.99 1.43-5.40 208 Pb Leaves 2.94 ± 0.74 1.30-4.00 Flowers 1.12 ± 0.45 0.39-1.91 Infusion 0.19 ± 0.16 <0.01-0.65 209 Bi Leaves 0.01 ± 0.03 <0.01-0.12 Flowers 0.30 ± 0.63 <0.01-1.71 Infusion <0.08 <0.08 As referred, for validation of ICP-MS seven elements were measured by both ICP-MS and ETAAS. Linear least squares adjustment was applied. The regression line of the global results yielded the equation y = 1.18(±0.034)x + 0.31(±0.12), R 2 = 0.996 (n=7). The calculated recoveries were (91±14)% (Average recovery ± SD) (n = 14) for the leaves, (97±13)% for the flowers and (105±24)% for the infusions. Figure 2: Analytical procedure for elemental determination. Table 3: Ni, Cu, As and Se content. Table 4: Sr, Cd, Sn and Sb content. Table 5: Ba, Pb, and Bi content. Element C mean ±SD (g/Kg) Range (g/Kg) Zn 0,07 ± 0.04 0.02-0.18 Fe 0.50 ± 0.15 0.30-0.81 Mg 3.21 ± 0.65 2.32-4.20 Table 1: Zn, Fe and Mg content 60,0 65,0 70,0 75,0 80,0 85,0 90,0 95,0 100,0 40 50 60 70 80 90 110 % Relative Inhibition of DPPH T ( 0 C) MeOH EtOH Ethyl Acetate Figure 3: Effect of extraction’s temperature in different solvents. The optimum conditions were ethanol at 90 0 C 60,0 65,0 70,0 75,0 80,0 85,0 90,0 95,0 100,0 2 6 8 10 12 16 % Relative Inhibition of DPPH t (sec) MeOH EtOH Ethyl Acetate Figure 4: Effect of extraction’s time in different solvents. The optimum conditions were ethanol at 90 0 C for10 min. Plant Reference Compounds Sample Part EC 50 (mg/L) EC 50 (mg/L) I (Crete) Leaf 18.9 ± 5.0 Caffeic acid 3.3 Flower 39 ± 14 Rosmarinic acid 4.7 II (Crete) Leaf 19.2 ± 3.7 BHT 40.9 III (Crete) Leaf 14.1 ± 5.0 (+) Catechin 4.8 IV (Crete) Leaf 17.8 ± 2.2 Quercetin 3.2 V (Crete) Leaf 19.2 ± 8.3 3,4,5- Trihydroxybenzoic acid 0.71 VI (Crete) Leaf 27.4 ± 1.6 2-tert-buty-4- methoxyphenol 4.5 Thermopy les Leaf 27.9 Amorgos Leaf 40.1 Prigiani (Lesvos) Leaf 14.3 Oros (Lesvos) Leaf 32.2 White tea Leaf 9.4 Table 6: EC 50 values for ethanol extracts and some reference compounds. Figure 5: Analytical procedure for the investigation of antioxidant activity. EC 50 defined as the concentration of sample at which 50% of maximum scavenging activity was recorded. As lower as stronger the antioxidant activity is. CONCLUSIONS The results above indicated that Salvia Fruticosa contain large amounts of nutrients and is rich in Fe, Mg, and V. Salvia Fruticosa also contains Cr, Cu, Zn, and Mn which have an important role in the metabolism of cholesterol. Flowers and leaves of Salvia Fruticosa contain also some heavy metals in normal concentrations which don’t exist in the infusion According to this work strong antioxidant effects obtained from the herbal Salvia Fruticosa grown in all over Greece. On the other hand, we developed a fast (12 min) and quantitative method for the microwave-assisted extraction of antioxidants from sage. During the method development, several parameters were evaluated such as the extraction temperature, solvent and extraction time. The optimum extraction conditions were: 0.5 g of sample extracted by 10 mL of 100% ethanol at 90 0 C for 10 min (hold time). Element C mean ±SD (mg/Kg) Range (mg/Kg) 51 V Leaves 0.99 ± 0.40 0.11-1.78 Flowers 1.21 ± 0.32 0.76-1.90 Infusion <0.03 <0.03 53 Cr Leaves 4.7 ± 2.6 0.01-8.7 Flowers 2.60 ± 0.74 1.60-3.81 Infusion 0.04 ± 0.16 <0.04-3.80 55 Mn Leaves 30.8 ± 6.8 21.3-50.8 Flowers 39.1 ± 9.2 27.6-60.6 Infusion 20.4 ± 5.4 1.7-20.4 59 Co Leaves 0.30 ± 0.16 0.17-0.81 Flowers 0.36 ± 0.12 0.22-0.61 Infusion 0.020 ± 0.030 <0.01-0.11 Table 2: V, Cr, Mn and Co content. The results show that a) the leaves and the flowers have similar concentration in elements in contrast with the infusion, b) some essential elements, present in the leaves of sage, are not migrate into the infusion (e.g V) and c) the concentration of the heavy metals in samples conforms the E.U legislatives.