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Influence of soil composition on the major, minor and trace metal content of Velebit biomedical plants
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Influence of soil composition on the major, minor and trace metal content of Velebit biomedical plants ˇ c´ c , Michaela Zeiner a,∗ , Iva Juranovic´ Cindric´ b , Martina Poˇzgaj b , Raimund Pirkl a , Tea Sili a Gerhard Stingeder a
Division of Analytical Chemistry, Department of Chemistry, BOKU – University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria Laboratory of Analytical Chemistry, Faculty of Science, Universty of Zagreb, Horvatovac 102a, 10000 Zagreb, Croatia c Velebit Botanic Garden, Northern Velebit National Park Public Institution, Krasno 96, 53274 Krasno, Croatia b
a r t i c l e
i n f o
Article history: Received 4 August 2014 Received in revised form 10 October 2014 Accepted 11 October 2014 Available online xxx Keywords: Medical plants Metals Soil composition Uptake Velebit
a b s t r a c t The use of medical herbs for the treatment of many human diseases is increasing nowadays due to their mild features and low side effects. Not only for their healing properties, but also for their nutritive value supplementation of diet with various herbs is recommended. Thus also their analysis is of rising importance. While total elemental compositions are published for many common herbs, the origin of toxic as well as beneficial elements is not yet well investigated. Thus different indigenous medicinal plants, namely Croatian spruce (Picea abies), savory (Satureja montana L.), mountain yarrow (Achillea clavennae), showy calamint (Calamintha grandiflora), micromeria (Micromeria croatica), yellow gentian (Gentiana lutea) and fir (Abies alba) together with soil samples were collected in the National Park Northern Velebit. The macro- and trace elements content, after microwave digestion, was determined by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and inductively coupled plasma mass spectroscopy (ICP-MS). The study focuses on the one hand on essential elements and on the other hand on non-essential elements which are considered as toxic for humans, covering in total Al, As, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, Sr and Zn. © 2014 Elsevier B.V. All rights reserved.
1. Introduction All over the world thousands of medical plants are nowadays applied. For healing properties and nutritive value supplementation of diet with various herbs is recommended among individual consumers [1]. This rising interest in the chemical composition of medical plants reflects the development in nutrition and in biochemical surveying and mineral prospecting [2,3]. The medical plants investigated in the presented study are indigenous plants, some species found only in small areas. Thus the knowledge on them is scarce. Their medical use is proven, but not entirely investigated by now. Micromeria croatica belonging to the Lamiaceae family is one of the six Micromeria species found in Croatia and used in folk medicine due to antimicrobial and antioxidant activities [4]. Mt Velebit was only recently described as new locality for M. croatica [5]. Micromeria species in general have many
∗ Corresponding author. Tel.: +43 1 47654 6543/6547; fax: +43 47654 6059. E-mail address: [email protected] (M. Zeiner).
pharmacological activities, e.g. sedative, anaesthetic, antiseptic, abortifacient, antioxidant, anti-inflammatory and antirheumatic [6]. Five species of genus savory (Satureja L., Lamiaceae), including Satureja montana, grow in Croatia [7]. Its medicinal application is based on the antioxidative and antimicrobial activity of the essential oil of this aromatic species, furthermore it is used as spice [8]. Calamintha grandiflora (Lamiaceae) is a wide spread medicinal herb, known mainly for its essential oil with antioxidative power [9]. Warmed resins from Abies alba as well as Picea abies, both wide spread representatives of the Pinaceae family, are mainly used in combination with other herbs, like Alchillea, as balms for skin and eye injuries [10]. Gentiana lutea (Gentianaceae) is used in traditional medicine in whole Europe, as herbal bitter it is consumed for digestives disorders [11], furthermore it exhibits hepatoprotective activity [12]. Achillea clavennae (Asteraceae) was described regarding medical use already in 1609 [13]. Cytotoxic and antioxidant activities as well as application as antifertility agent are reported [14].
http://dx.doi.org/10.1016/j.jpba.2014.10.012 0731-7085/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: M. Zeiner, et al., Influence of soil composition on the major, minor and trace metal content of Velebit biomedical plants, J. Pharm. Biomed. Anal. (2014), http://dx.doi.org/10.1016/j.jpba.2014.10.012
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Metal pollution in the environment is a serious problem. By the accumulation of heavy metals in soils, crops or plants, they can affect human physiological functions through the food chain. Since trace elements are involved in the formation processes of active chemical constituents present in medicinal plants, they impact their medicinal/beneficial as well as harmful/toxic properties [15,16]. For man zinc, iron, copper, chromium, molybdenum, selenium and cobalt are besides others considered as essential and become harmful only at high concentrations, in contrast to, e.g. lead and cadmium presenting a health risk even in low concentrations [3,17–20]. Not only humans suffer of deficiencies of certain elements, but similar problems are described also for agricultural crops [21]. Plants require essential elements for maintaining osmotic balance, as structural components in proteins and carbohydrates, as components of organic molecules crucial for regular metabolism [22]. Potassium plays an indispensable role in stress response [23], and calcium is an important constituent of plant cell wall and membranes [24]. Magnesium, being part of chlorophyll, is involved in plants’ photosynthesis and in consequence influencing their growth. Iron regulates photosynthesis, since it is required for chlorophyll synthesis. Not only in men, but also in plants zinc deficiency is wide spread resulting in stunted growth, chlorosis and smaller leaves, spikelet sterility [25]. Copper presents an important element for the plants’ root metabolism, and. Nickel is reported as growth regulator, exemplarily for wheat [26]. Aim of the present work was the determination of the element composition in different indigenous medicinal plants, namely Croatian spruce (Picea abies), savory (Satureja montana L.), mountain yarrow (Alchillea clavennae), showy calamint (Calamintha grandiflora), micromeria (Micromeria croatica), yellow gentian (Gentiana lutea) and silver fir (Abies alba) together with soil samples collected in a remote area, namely the Croatian National Park Northern Velebit. The macro- and trace elements content, after microwave digestion, was determined by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and inductively coupled plasma mass spectroscopy (ICP-MS). Additionally, the soil samples were characterized regarding pH-value, CEC (cation exchange capacity), and mineral composition.
2. Experimental 2.1. Chemicals and glassware For the experimental work performed nitric acid (HNO3 , 65%, w/w, p.a., sub-boiled), hydrochloric acid (HCl, 36%, w/w, p.a., subboiled), hydrogen peroxide (30%, w/w, p.a.) and ICP Multi-element Standard IV (Merck, Darmstadt, Germany) were used. The standard reference material of strawberry leaves (LGC7162) was purchased from LGC Standards (United Kingdom). All glass- and plastic-ware were cleaned with nitric acid and rinsed with supra-water prior to use. The ultrapure water used for the experiments was produced in-house.
2.2. Sampling and sample preparation All plant (leaves, blossoms, shoots, needles and roots) and soil samples (taken from A-horizon) were collected in July 2012 in the Croatian National Park Northern Velebit (N 44.46◦ –E 14.59◦ ; altitude 1400 m). At each sampling site plants and soil were taken. All plant parts were washed with supra-pure water, dried at 105 ◦ C, homogenized in a metal free mortar and stored in paper bags at room temperature prior to analysis. The soil samples were dried, homogenized and stored at ambient temperature in plastic bags before sample preparation.
For the microwave assisted digestion of the plant samples (in triplicate) a MWS-2 Microwave System Speedware BERGHOF was used applying the following procedure: approximately 0.15 g of the homogenized sample was weighed into a Teflon reaction vessel. The samples were digested with 4.0 mL HNO3 (7 mol/L) + 2.0 mL H2 O2 with a three-step programme (1 – 150 ◦ C/15 min, 2 – 175 ◦ C/15 min and 3 – 130 ◦ C/15 min). The final volume was 10.0 mL. Additionally, a strawberry leaves standard reference material (LGC7162) was digested in the same way. To determine the metal concentrations in the soils, the dried samples underwent an acidic microwave assisted digestion procedure. Approximately 100 mg dried matter were treated with 1 mL HNO3 (14 mol/L) + 1.0 mL H2 O2 + 3.0 mL HCl (12 mol/L). The power of 1400 W was reached after 10 min and then held for 20 min in the MLS-1200 MEGA digestion apparatus. The solutions were brought to 10 g final mass and diluted 1:10 prior to analysis. 2.3. Soil characterization All soil samples were characterized regarding pH-value, CEC, and mineral composition. The measurements of the pH-value were carried out in two ways, in a suspension of the dried soil in ultrapure-water and in a suspension of dried soil in calcium chloride solution (0.01 mol/L). The glass electrode used was calibrated with buffers pH 4 and pH 7 prior to analysis. The determination of CEC was performed according to a modified method of Bergaya and Vayer [27,28]. Dried soil (200 mg) was mixed with 8 mL 0.01 mol/L solution of Cu(EDA)2 2+ (copper ethylene diamine) + tris buffer (pH 7) and filled up with ultra-pure water to 25 mL final volume. After centrifugation for 30 min, the pH was checked and the absorbance of the supernatant of the suspension was measured at 548 nm. 2.4. ICP-AES measurements The metal concentrations of the all digests were determined using a Prodigy High Dispersive ICP-AES spectrometer (Teledyne Leeman, Hudson, NH, USA) working in a simultaneous mode. The instrument is equipped with a high resolution Echelle polychromator and a large format programmable array detector (L-PAD), a RF-Generator (40 MHz “free-running”, output power 1.1 kW), a peristaltic pump (sample uptake flow 1.0 mL/min), a pneumatic nebulizer, and a glass cyclonic spray chamber. The measurements were run with previously optimalized argon flows, namely coolant: 18 L/min, auxiliary: 0.8 L/min; axial plasma viewing, a sample uptake delay of 30 s and 3 replicates. The emission lines selected for the determination of the elements (wavelength in nm) were: Al (396.152), B (249.677), Ba (455.403), Ca (396.847), Cd (214.441), Co (228.615), Cr (267.716), Cu (324.754), Fe (259.940), K (766.491), Mg (285.213), Mn (259.372), Na (589.592), Ni (231.604), Pb (220.353), Sr (407.771), and Zn (213.856). The limits of detection (LOD) were calculated according to Boumans using 3 and limits of quantification (LOQ) using 9 for pure element standards and microwave digested samples by measuring an appropriate reagent blank solution ten times and spiked microwave assisted digested samples also ten times. For estimating the accuracy of the method, digest solutions of strawberry leaves standard reference material (LGC7162) were analyzed on the one hand, and for the elements not contained in the certified reference material (CRM) on the other hand spiking experiments of digests were carried out at two concentration levels (1.0 and 5.0 mg/L) by adding aqueous multielement standard solutions. The precision was evaluated by measuring the repeatability of the method for all analytes and sample types. The slope of the calibration curves was the basis for the evaluation of the sensitivity of
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the method. Good repeatability was obtained even without internal standard, thus only external calibration was used for quantification. All measurements were performed in triplicate. 2.5. ICP-MS measurements These measurements were carried out using an Element 2 ICP-SFMS (Inductively coupled plasma-sector field mass spectrometer; Thermo Fisher, Bremen, Germany) equipped with the self-aspirating PFA (perfluoroalkoxy) microflow nebulizer (ESI; Elemental Scientific Inc., Cuming, Omaha, USA) at a flow of 100 L/min a PC3 (Peltier cooled) cyclonic quartz chamber (ESI) operated at 4 ◦ C, a quartz injector pipe and torch (Thermo Fisher), aluminium sampler and skimmer cone (Thermo Fisher). The instrumental conditions applied were: RF power 1300 W and plasma gas flow 16 L/min, sample gas and auxiliary gas flows were set to 1.06 L/min and 0.86 L/min, respectively. The following isotopes were analyzed: at low resolution 88 Sr, 111 Cd, 208 Pb; at medium resolution 52 Cr, 55 Mn, 56 Fe, 59 Co, 60 Ni, 65 Cu, and 66 Zn at high resolution 75 As. 115 Indium (1.1 g/L) was used as internal standard at all resolution levels. Nominal mass resolutions of the Element 2 ICP-SFMS for low resolution (LR), medium resolution (MR) and high resolution (HR) were 350, 4500 and 10,000, respectively. For calibration quality control TM 27.2 (Certified Reference Waters for Trace Elements) was used, whereby the values registered agreed with the certified ones within measurement uncertainty. All samples and blank solutions were measured in triplicate. 2.6. Calibrations For both methods, measurements were accomplished by external calibration using aqueous mixed standards prepared by diluting a Merck VI multi-elemental standard with 2% (w/w) HNO3 . All calibration curves were based on eight standard solutions, including a blank. The calibration ranges were selected according to the expected concentrations of the respective analyte. 3. Results and discussion 3.1. Methods for elemental analysis The limits of detection (LOD) for all analytes determined in the digest solutions of soils and plant samples for ICP-AES as well as for ICP-MS are given along with the elemental concentration of the elements determined in Tables 2 and 3. The recoveries determined with the standard reference material of strawberry leaves (LGC7162) range from 94 up to 129% for the digested plant material by ICP-AES and from 89 to 109% for ICP-MS. The slopes of the calibration curves of all analytes, their correlation coefficients all being beyond 0.9995, showed good sensitivity. The precision expressed as RSD ranged from 0.05 up to 1.7%. Within day repeatability was found to be <4.2%. 3.2. Soil characteristics All investigated soil samples from the National Park Northern Velebit have the following mineral composition: quartz, illite, chlorite, chlorite-smectite and plagioclase. The soil pH-values measured in water suspension is between 4.78 and 7.59, and 4.14 and 7.09 in CaCl2 solution. pHdetermination in a weak electrolytic solution (0.01 mol/L CaCl2 ) offers the possibility to measure the acidic potential of a soil, including the acidic influence of protons, which are usually in H2 O
3
Table 1 pH values and CEC data of the investigated soils. CEC (pH 7) [mEq/100 g] Soil 1 Soil 2 Soil 3 Soil 4 Soil 5 Soil 6 Mean + sd
23.9 19.7 28.2 21.1 19.3 10.9 21 ± 6
pH (H2 O)
pH (CaCl2 )
7.59 4.78 6.63 7.16 5.08 7.90
7.09 4.14 6.34 6.73 4.27 7.13
6.5 ± 1.3
6.0 ± 1.4
solution ligated to ionic exchange structures of soil components like humic acids. The cation exchange capacity (CEC) results range from 10.3 to 28.3 mEq/100 g. CEC is defined as the maximum quantity of cations, which a soil is capable of holding at a certain pH-level [28] and provides information about soils’ potential to bind or to release cations (as nutrients or pollutants) as an interdependency between soil and its surrounding aqueous solution. The detailed results per soil are listed in Table 1. In addition to the single results, the mean as well as the standard deviations are given. The data obtained for all soil sample are in the common range for clay soils. 3.3. Metal content of soil samples Table 2 contains the obtained elemental concentrations in the soils from the six sampling sites in the National Park along with the LOD of the respective analyte and the method used for its determination. The relative standard deviations (r.s.d.) of the contents are mainly about 30%, laying in the acceptable range for inhomogeneous biological specimens. For each sampling site (soil samples 1–6) the r.s.d. values range from 2 to 5%. Higher variations were found for Cd, which is present only in very low concentrations and for Ca and Sr, elements strongly related to the local bedrock composition. Higher Ca and Mg levels were expected in dolomite dominated soils, like in Velika Goriza (Croatia) [29]. In an organic apple orchard about 36 g/kg Ca were found, in comparison to 1.5 up to 15 g/kg in the present study. Regarding the trace elements Co, Cr, Cu, Mn, Ni, Pb, Sr, and Zn and the major element Fe, similar elemental contents were found as reported for the apple orchard soil [29]. Arsenic was found in concentrations below the limit for agricultural soil in Chine (30 mg/kg) [30]. But even being located in a remote area the levels of certain elements exceed different limit concentrations given by different jurisdictions, e.g. soils 2 and 3 have Cd concentrations above the limits stated in Canada (1.4 mg/kg) [31] and France (2 mg/kg) [32]. Only soil 5 is below the maximum value for chromium in soil according to Canadian jurisdiction (64 mg/kg) [31], but none is beyond the French limit of 150 mg/kg [32]. Whereas Chinese and French regulations stipulate maximum copper level in soil of 100 mg/kg [32,33], the Canadian Ministry of Environment limits Cu in agricultural soil to 63 mg/kg [31]. All analyzed soils are lower than these values. Regarding Pb, only soil 3 is beyond the limit given by the Canadian Ministry (70 mg/kg) [31]. The maximum allowed Ni level in soils stated by French and Chinese regulations (50 mg/kg) [32,30], only soil 5 and 6 go below this limit. Zn was found far below the maximum regulated soil concentrations. Regulations limit Zn levels in soil to 300 g/g in France [32] and to 250 g/g in China [30]. 3.4. Metal content of plant parts All elemental concentrations obtained for the various plant parts are summarized in Table 3. For better clarity no standard deviations
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Table 2 Elemental concentrations in soil samples along with LOD. Content in mg/kg LOD Method
Al 45 MS
As 0.039 MS
Ba 0.16 AES
Ca 1.8 AES
Soil 1 Soil 2 Soil 3 Soil 4 Soil 5 Soil 6
2237 1397 1408 2238 3397 2956
26.5 29.7 12.0 22.5 16.4 27.5
136 12,469 76.3 6664 82.7 9934 118 4712 105 1476 153 15,629
Mean r.s.d.
2272 35
22 31
112 27
8481 61
Cd 0.0009 MS 0.89 2.00 3.76 0.67 0.469 0.384 1.4 97
Co 0.0070 MS
Cr 0.15 MS
Cu 0.092 MS
Fe 0.75 MS
K 0.058 AES
Mg 1.2 AES
Mn 0.025 MS
Na 17 AES
Ni 0.18 MS
Pb 0.026 MS
Sr 0.13 MS
Zn 4.7 MS
18.4 17.9 15.0 17.7 13.2 13.6
108 111 98 88 49.6 73
28.0 17.2 21.5 34.8 14.9 30.5
52,864 48,628 42,160 38,235 32,401 29,120
4420 2277 3237 3609 7053 3197
6373 5439 5335 3892 6102 4418
903 713 1239 693 1751 965
473 242 747 330 238 331
89 67 53.4 62 38.3 45.8
58.4 49.6 72.3 37.4 56.4 25.4
60.1 63.3 64 74 29.1 174
99 126 138 110 97 67
24 32
40,568 23
3966 42
5260 18
1044 38
394 49
59 30
50 33
77 64
106 23
16 15
88 27
are added, they range depending on plant species, organ and element from 1.7 to 10%. It can be clearly seen that the medical plants are rich in K, Ca, Mg, Na, Fe, Mn, Zn, and B, some also in Cu and Ni. These elements play irreplaceable roles in their efficacy as folk medicine, such as improving immune function and disease prevention. Comparing these plants with two medicinal herbs analyzed in a previous study, namely sage (Salvia officinalis L) and mountain germander (Teucrium montanum L) also sampled in the National Park Northern Velebit, it can be seen, that the amounts of trace elements are in the same order of magnitude [33]. K in contrast is about 10–15 times lower. The Ca levels found in different plant organs from the herbs are much higher than those determined for sage (approx. 4000 mg/kg), only the contents found in the needles and shoots of the trees Abies alba and Picea abies are in the same range. A study on the whole plant of Micromeria croatica showed completely different results, regarding macro elements [4]. Whereas in the single organs of this investigation Ca is present in highest concentration, the working group of Kremer found K as leading element in contents 30-fold higher than our results and Ca almost 1000-fold lower. Thus not only the genetic background determines the accumulation, but also the locality. Even if widely applied for healing reasons, no standards exist for medical raw plant materials, establishing a permissible level of certain metals. The World Health Organization WHO mentions maximum permissible levels in raw plant materials only for As, Cd and Pb, namely 1.0, 0.3 and 10 mg/kg, respectively [34]. Whereas no exceedings were registered for As, to high Cd levels were found in Abies alba in needles and shoots. Regarding Pb only the leaves of Alchillea clavennae contain more than the stipulated by WHO. Nevertheless the medical plants analyzed can be considered as safe for usage as alternative medicine, since they are not consumed in total, but as decoct, infusion or balm of oils. 3.5. Uptake and accumulation behaviour Due to the high variation of the soil composition the exact calculation of the plant uptake factors, the relation content in plant divided by content in soil, was not considered justifiable. Nevertheless general conclusions can be drawn regarding the metal uptake and accumulation behaviour comparing soil and plant elemental concentrations. In all investigated plant organs the As level was very low, even below the LOD (19 g/kg), whereas in the soil about 20 mg/kg were found. Thus the uptake is only approx. 1–6‰. A clear distinction between herbs and tree can be seen by the Ca contents. In needles and shoots of Abies alba and Picea abies less Ca is found than in soils, except for soil 5, whereas the other five plants studied contain more Ca than the soils. Furthermore the Ca concentration is always highest in the leaves, only in Calamintha grandiflora leaves and blossoms have the same level. Achillea clavennae is the herb with the highest B accumulation in leaves as well as blossoms compared with the other plants. Calamintha grandiflora is leading in Ba,
Fe, Mn and Sr uptake. Regarding Cd the behaviour is vice versa to Ca, whereas the trees accumulate less Ca than herbs, Cd is uptaken in highest concentration by Abies alba and Picea abies. Co shows this tendency only for Abies alba. 3.6. Possible contamination of environment, food commodities and workplace Medicinal plants are naturally grown and their content of metals, beneficial as well harmful to men, is determined by the surroundings, thus there is no risk of contaminating any environmental compartment. Moreover it is more likely that the herbs may accumulate certain elements at levels which in consequence can exhibit negative effects on human health from the environment, from soil via root uptake or from air due to precipitation. As shown before the investigated plants do not contain elements regulated by WHO, such as As, Cd or Pb in unacceptable high concentrations. Thus during industrial processing or preparing decocts and infusions of medical herbs collected under controlled conditions in private home, the risk of metal contamination of other food commodities or workplace is negligible for the normal population. Persons more susceptible to harmful effects of uptaken low doses of heavy metals by diet, like children, pregnant or elderly people might show adverse reactions. Plants from polluted areas however, may present a health risk when consumed and contaminate various processing utensils in private kitchen and industrial sites. Due to the growing interest in alternative medicine, the cultivation of selected herbs is forced and drastic use of plant protection products is necessary in order to meet the requirements of the market. Also less attention is paid to comply with regulations for safe processing and storage. In consequence the herbs brought on the market may be contaminated with pesticides, microbial contaminants, heavy metals, and chemical toxins, thus representing a risk for worker as well as consumers [35]. Furthermore the potential of adulteration rises with increasing interest in a product, e.g. with synthetic drugs, resulting in a risk for both consumers as workers handling these formulations [36,37] or with other herbs. As shown the mineral composition is characteristic for each medicinal plant, thus the metal determination may be used as method for discovering adulteration of synthetic drugs or the mixture with other cheaper plants. Besides the metals contained in the medical plants as discussed in detail in the previous subchapters, they contain organic compound, mainly responsible for their bioactivity, such as antioxidative compounds. Processed plant products contain often only a small percentage of the active substances present in the raw material. Thus in agriculture and industry waste and by-products still contain high quantities of valuable substances [9]. In order not to spoil these resources or to generate disorders in the environment by disposing them, more emphasis should be placed on the recycling of bioactive material.
Please cite this article in press as: M. Zeiner, et al., Influence of soil composition on the major, minor and trace metal content of Velebit biomedical plants, J. Pharm. Biomed. Anal. (2014), http://dx.doi.org/10.1016/j.jpba.2014.10.012
Al 4.2 AES
As 0.019 MS
B 0.45 AES
Ba 0.16 AES
Ca 1.8 AES
Cd 0.0005 MS
Gentiana lutea Leaves Roots
*
155
0.027 0.030
30.7 18.1
8.10 38.7
19,558 12,350
0.0072 0.11
Co 0.0035 MS *
0.0083
Cr 0.075 MS * *
32.3 87.8
0.060 0.047
26.3 31.4
19.6 14.9
29,741 23,453
*
Satureja montana Shoots Leaves Blossoms
30.2 76.1 86.7
*
29.5 23.7 16.8
18.4 12.4 8.31
16,022 27,940 22,887
*
0.088
24.9 15.2
54.8 43.3
12,164 12,492
0.037 0.047
0.044 0.060
0.368 0.639
Calamintha grandiflora 118 Leaves 61.5 Blossoms
*
0.022 *
*
* *
*
0.0094 *
0.0077 0.080
*
0.886 1.10
K 0.058 AES
Mg 1.2 AES
Mn 0.013 MS
Na 17 AES
20.9 25.7
269 116
3029 1070
6.73 2.02
573 302
*
*
*
*
29.9 36.0
279 251
2175 1792
5.62 5.91
652 154
*
*
*
*
*
0.437 0.801 5.54
25.0 28.1 55.6
488 385 304
1153 2767 2420
4.90 7.48 19.9
430 328 201
*
*
*
*
10.6 16.2
103 76.1
454 435
2730 3145
194 47.3
413 398
1726 1307
*
105 102
0.648 9.72
92.1 26.5
333 283
1.20 0.84
0.153 0.045
40.2 48.9
1.94 0.71
24,070 14,205
0.277 0.076
0.067 0.021
0.911 0.490
21.8 10.2
Picea abies Needles Shoots
20.5 28.1
*
16.4 18.4
4.57 7.91
2565 3168
0.269 0.158
0.066 0.037
0.251 0.266
10.7 9.72
28.0 18.5
356 455
1008 676
102 56.11
144 151
Abies alba Needles Shoots
67.2 62.6
0.024
14.2 18.1
5.56 6.92
7172 4585
0.471 0.717
0.380 0.204
0.672 0.291
10.0 13.6
24.2 28.5
497 425
1004 872
132 72.0
188 182
0.056 0.103 0.045
0.056
Salvia officinalis [33] Aerial parts Leaves Blossoms
102 134 57.2
n.d. n.d. n.d.
n.d. n.d. n.d.
n.d. n.d. n.d.
3890 3780 4760
Teucrium montanum [33] Aerial parts 133 85.6 Leaves 62.6 Blossoms
n.d. n.d. n.d.
n.d. n.d. n.d.
n.d. n.d. n.d.
58 66 81
* * *
*
*
0.069 *
0.231
*
*
257 253
98.4 44.4
*
Pb 0.013 MS
2.21
Achillea clavennae Leaves Blossoms
*
Ni 0.090 MS
*
*
*
Fe 0.38 MS
0.764 4.86 8.71
Sr 0.06 MS
Zn 2.4 MS
1.80 4.09
5.35 8.35
6.64 5.56
3.89 6.98
4.07 4.11 5.26
6.82 7.24 18.8
11.7 14.5
49.1 70.0
11.0 4.83
14.4 3.22
100 33.9
1.13 0.779
4.44 6.25
2.14 5.11
53.89 41.65
2.42 2.39
6.28 5.47
1.70 2.80
52.50 47.71
0.855 0.301
4.13 10.5 11.4
96.7 126 64.8
8420 9440 9020
3200 4010 3020
29.7 39.1 24.5
38 91 56
110 148 74.5
6.53 10.2 10.4
7.81 9.25 5.77
27.1 39.4 27.8
0.386 0.263 0.356
3.60 3.67 6.72
107 69.8 59.9
6470 5920 6750
1430 1320 1470
25.9 32.8 21.8
18 54 30
125 81.6 71.5
6.02 6.19 9.45
3.77 3.89 4.27
14.9 22.4 23.9
n.d. not determined. *
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Micromeria croatica Leaves Blossoms
Cu 0.046 MS
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Content in mg/kg LOD Method
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Table 3 Elemental concentrations in plant samples along with LOD.
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4. Conclusions Metal monitoring of medicinal herb is not only a tool for showing their nutritional value, but also assures the safe use of herbal supplements by proving the absence of harmful metals. Each plant exhibits its own metal uptake and accumulation behaviour, which is the basis for herb characterization by elemental pattern, which can be applied for adulteration detection. Due to the fact that medicinal herbs are natural products the risk of contamination of the environment, food commodities and workplaces is unlikely. In contrast herbs collected in polluted areas, not well stored or processed may turn into a harmful instead of beneficial product. Acknowledgement This project was financially supported by the Wissenschaftlichtechnische Zusammenarbeit Austria-Croatia (Austrian Exchange Service ÖAD) HR-23-2012. References [1] E. Ernst, Harmless herbs? A review of the recent literature, Am. J. Med. 104 (1998) 170–172. [2] I. Rodushkin, T. Ruth, A. Huhtasaari, Comparison of two digestion methods for elemental determinations in plant material by ICP techniques, Anal. Chim. Acta 378 (1999) 191–200. [3] T. Karak, R.M. Bhagat, Trace elements in tea leaves, made tea and tea infusion: review, Food Res. Int. 43 (2010) 2234–2252. [4] D. Kremer, E. Stabentheiner, V. Dunkic, I. Dragovic Müller, L. Vujic, I. Kosalec, D. Ballian, F. Bogunic, N. Bezic, Micromorphological and chemotaxonomical traits of Micromeria croatica (Pers.) Schott, Chem. Biodivers. 9 (2012) 755–768. [5] D. Kremer, M. Randic, I. Kosalec, A. Brklacic, G. Lukac, I. Krusic, D. Baillan, F. Bogunic, Ks. Karlovic, New localities of the subendemic species Berberis croatica, Teucrium arduini and Micromeria croatica in the Dinaric Alps, Acta Bot. Croat. 70 (2011) 289–300. [6] M. Öztürk, U. Kolak, G. Topcu, S. Öksüz, M. Iqbal Choudhary, Antioxidant and anticholinesterase active constituents from Micromeria cilicica by radicalscavenging activity-guided fractionation, Food Chem. 126 (2011) 31–38. [7] V. Dunkic, D. Kremer, I. Dragojevic Müller, E. Stabentheiner, S. Kuzmic, R. Jurisic Grubesic, L. Vujic, I. Kosalec, M. Randic, S. Srecec, N. Bezic, N. Bezic, Chemotaxonomical and micromorphological traits of Satureja montana L. and S. subspicata Vis. (Lamiaceae), Chem. Biodiv. 9 (2012) 2825–2842. [8] S. Cavar, M. Maksimovic, M.E. Solic, A. Jerkovic-Mujkic, R. Besta, Chemical composition and antioxidant antimicrobial activity of two Satureja essential oils, Food Chem. 111 (2008) 648–653. [9] D. Dobravalskyte, P.R. Venskutonis, T. Talou, Antioxidant properties and essential oil composition of Calamintha grandiflora L., Food Chem. 135 (8) (2012) 1539–1546. [10] B. Saric-Kundalic, C. Dobes, V. Klatte-Asselmeyer, J. Saukel, Ethnobotanical study on medicinal use of wild and cultivated plants in middle, south and west Bosnia and Herzegovina, J. Ethnopharmacol. 131 (2010) 33–55. [11] L. Cornara, A. La Rocca, L. Terrizzano, F. Dente, M.G. Mariotti, Ethnobotanical and phytomedical knowledge in the North-West Ligurian Alps, J. Ethnopharmacol. 155 (2014) 463–484.
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Influence of soil composition on the major, minor and trace metal content of Velebit biomedical plants ˇ c´ c , Michaela Zeiner a,∗ , Iva Juranovic´ Cindric´ b , Martina Poˇzgaj b , Raimund Pirkl a , Tea Sili a Gerhard Stingeder a
Division of Analytical Chemistry, Department of Chemistry, BOKU – University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria Laboratory of Analytical Chemistry, Faculty of Science, Universty of Zagreb, Horvatovac 102a, 10000 Zagreb, Croatia c Velebit Botanic Garden, Northern Velebit National Park Public Institution, Krasno 96, 53274 Krasno, Croatia b
a r t i c l e
i n f o
Article history: Received 4 August 2014 Received in revised form 10 October 2014 Accepted 11 October 2014 Available online xxx Keywords: Medical plants Metals Soil composition Uptake Velebit
a b s t r a c t The use of medical herbs for the treatment of many human diseases is increasing nowadays due to their mild features and low side effects. Not only for their healing properties, but also for their nutritive value supplementation of diet with various herbs is recommended. Thus also their analysis is of rising importance. While total elemental compositions are published for many common herbs, the origin of toxic as well as beneficial elements is not yet well investigated. Thus different indigenous medicinal plants, namely Croatian spruce (Picea abies), savory (Satureja montana L.), mountain yarrow (Achillea clavennae), showy calamint (Calamintha grandiflora), micromeria (Micromeria croatica), yellow gentian (Gentiana lutea) and fir (Abies alba) together with soil samples were collected in the National Park Northern Velebit. The macro- and trace elements content, after microwave digestion, was determined by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and inductively coupled plasma mass spectroscopy (ICP-MS). The study focuses on the one hand on essential elements and on the other hand on non-essential elements which are considered as toxic for humans, covering in total Al, As, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, Sr and Zn. © 2014 Elsevier B.V. All rights reserved.
1. Introduction All over the world thousands of medical plants are nowadays applied. For healing properties and nutritive value supplementation of diet with various herbs is recommended among individual consumers [1]. This rising interest in the chemical composition of medical plants reflects the development in nutrition and in biochemical surveying and mineral prospecting [2,3]. The medical plants investigated in the presented study are indigenous plants, some species found only in small areas. Thus the knowledge on them is scarce. Their medical use is proven, but not entirely investigated by now. Micromeria croatica belonging to the Lamiaceae family is one of the six Micromeria species found in Croatia and used in folk medicine due to antimicrobial and antioxidant activities [4]. Mt Velebit was only recently described as new locality for M. croatica [5]. Micromeria species in general have many
∗ Corresponding author. Tel.: +43 1 47654 6543/6547; fax: +43 47654 6059. E-mail address: [email protected] (M. Zeiner).
pharmacological activities, e.g. sedative, anaesthetic, antiseptic, abortifacient, antioxidant, anti-inflammatory and antirheumatic [6]. Five species of genus savory (Satureja L., Lamiaceae), including Satureja montana, grow in Croatia [7]. Its medicinal application is based on the antioxidative and antimicrobial activity of the essential oil of this aromatic species, furthermore it is used as spice [8]. Calamintha grandiflora (Lamiaceae) is a wide spread medicinal herb, known mainly for its essential oil with antioxidative power [9]. Warmed resins from Abies alba as well as Picea abies, both wide spread representatives of the Pinaceae family, are mainly used in combination with other herbs, like Alchillea, as balms for skin and eye injuries [10]. Gentiana lutea (Gentianaceae) is used in traditional medicine in whole Europe, as herbal bitter it is consumed for digestives disorders [11], furthermore it exhibits hepatoprotective activity [12]. Achillea clavennae (Asteraceae) was described regarding medical use already in 1609 [13]. Cytotoxic and antioxidant activities as well as application as antifertility agent are reported [14].
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Metal pollution in the environment is a serious problem. By the accumulation of heavy metals in soils, crops or plants, they can affect human physiological functions through the food chain. Since trace elements are involved in the formation processes of active chemical constituents present in medicinal plants, they impact their medicinal/beneficial as well as harmful/toxic properties [15,16]. For man zinc, iron, copper, chromium, molybdenum, selenium and cobalt are besides others considered as essential and become harmful only at high concentrations, in contrast to, e.g. lead and cadmium presenting a health risk even in low concentrations [3,17–20]. Not only humans suffer of deficiencies of certain elements, but similar problems are described also for agricultural crops [21]. Plants require essential elements for maintaining osmotic balance, as structural components in proteins and carbohydrates, as components of organic molecules crucial for regular metabolism [22]. Potassium plays an indispensable role in stress response [23], and calcium is an important constituent of plant cell wall and membranes [24]. Magnesium, being part of chlorophyll, is involved in plants’ photosynthesis and in consequence influencing their growth. Iron regulates photosynthesis, since it is required for chlorophyll synthesis. Not only in men, but also in plants zinc deficiency is wide spread resulting in stunted growth, chlorosis and smaller leaves, spikelet sterility [25]. Copper presents an important element for the plants’ root metabolism, and. Nickel is reported as growth regulator, exemplarily for wheat [26]. Aim of the present work was the determination of the element composition in different indigenous medicinal plants, namely Croatian spruce (Picea abies), savory (Satureja montana L.), mountain yarrow (Alchillea clavennae), showy calamint (Calamintha grandiflora), micromeria (Micromeria croatica), yellow gentian (Gentiana lutea) and silver fir (Abies alba) together with soil samples collected in a remote area, namely the Croatian National Park Northern Velebit. The macro- and trace elements content, after microwave digestion, was determined by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and inductively coupled plasma mass spectroscopy (ICP-MS). Additionally, the soil samples were characterized regarding pH-value, CEC (cation exchange capacity), and mineral composition.
2. Experimental 2.1. Chemicals and glassware For the experimental work performed nitric acid (HNO3 , 65%, w/w, p.a., sub-boiled), hydrochloric acid (HCl, 36%, w/w, p.a., subboiled), hydrogen peroxide (30%, w/w, p.a.) and ICP Multi-element Standard IV (Merck, Darmstadt, Germany) were used. The standard reference material of strawberry leaves (LGC7162) was purchased from LGC Standards (United Kingdom). All glass- and plastic-ware were cleaned with nitric acid and rinsed with supra-water prior to use. The ultrapure water used for the experiments was produced in-house.
2.2. Sampling and sample preparation All plant (leaves, blossoms, shoots, needles and roots) and soil samples (taken from A-horizon) were collected in July 2012 in the Croatian National Park Northern Velebit (N 44.46◦ –E 14.59◦ ; altitude 1400 m). At each sampling site plants and soil were taken. All plant parts were washed with supra-pure water, dried at 105 ◦ C, homogenized in a metal free mortar and stored in paper bags at room temperature prior to analysis. The soil samples were dried, homogenized and stored at ambient temperature in plastic bags before sample preparation.
For the microwave assisted digestion of the plant samples (in triplicate) a MWS-2 Microwave System Speedware BERGHOF was used applying the following procedure: approximately 0.15 g of the homogenized sample was weighed into a Teflon reaction vessel. The samples were digested with 4.0 mL HNO3 (7 mol/L) + 2.0 mL H2 O2 with a three-step programme (1 – 150 ◦ C/15 min, 2 – 175 ◦ C/15 min and 3 – 130 ◦ C/15 min). The final volume was 10.0 mL. Additionally, a strawberry leaves standard reference material (LGC7162) was digested in the same way. To determine the metal concentrations in the soils, the dried samples underwent an acidic microwave assisted digestion procedure. Approximately 100 mg dried matter were treated with 1 mL HNO3 (14 mol/L) + 1.0 mL H2 O2 + 3.0 mL HCl (12 mol/L). The power of 1400 W was reached after 10 min and then held for 20 min in the MLS-1200 MEGA digestion apparatus. The solutions were brought to 10 g final mass and diluted 1:10 prior to analysis. 2.3. Soil characterization All soil samples were characterized regarding pH-value, CEC, and mineral composition. The measurements of the pH-value were carried out in two ways, in a suspension of the dried soil in ultrapure-water and in a suspension of dried soil in calcium chloride solution (0.01 mol/L). The glass electrode used was calibrated with buffers pH 4 and pH 7 prior to analysis. The determination of CEC was performed according to a modified method of Bergaya and Vayer [27,28]. Dried soil (200 mg) was mixed with 8 mL 0.01 mol/L solution of Cu(EDA)2 2+ (copper ethylene diamine) + tris buffer (pH 7) and filled up with ultra-pure water to 25 mL final volume. After centrifugation for 30 min, the pH was checked and the absorbance of the supernatant of the suspension was measured at 548 nm. 2.4. ICP-AES measurements The metal concentrations of the all digests were determined using a Prodigy High Dispersive ICP-AES spectrometer (Teledyne Leeman, Hudson, NH, USA) working in a simultaneous mode. The instrument is equipped with a high resolution Echelle polychromator and a large format programmable array detector (L-PAD), a RF-Generator (40 MHz “free-running”, output power 1.1 kW), a peristaltic pump (sample uptake flow 1.0 mL/min), a pneumatic nebulizer, and a glass cyclonic spray chamber. The measurements were run with previously optimalized argon flows, namely coolant: 18 L/min, auxiliary: 0.8 L/min; axial plasma viewing, a sample uptake delay of 30 s and 3 replicates. The emission lines selected for the determination of the elements (wavelength in nm) were: Al (396.152), B (249.677), Ba (455.403), Ca (396.847), Cd (214.441), Co (228.615), Cr (267.716), Cu (324.754), Fe (259.940), K (766.491), Mg (285.213), Mn (259.372), Na (589.592), Ni (231.604), Pb (220.353), Sr (407.771), and Zn (213.856). The limits of detection (LOD) were calculated according to Boumans using 3 and limits of quantification (LOQ) using 9 for pure element standards and microwave digested samples by measuring an appropriate reagent blank solution ten times and spiked microwave assisted digested samples also ten times. For estimating the accuracy of the method, digest solutions of strawberry leaves standard reference material (LGC7162) were analyzed on the one hand, and for the elements not contained in the certified reference material (CRM) on the other hand spiking experiments of digests were carried out at two concentration levels (1.0 and 5.0 mg/L) by adding aqueous multielement standard solutions. The precision was evaluated by measuring the repeatability of the method for all analytes and sample types. The slope of the calibration curves was the basis for the evaluation of the sensitivity of
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the method. Good repeatability was obtained even without internal standard, thus only external calibration was used for quantification. All measurements were performed in triplicate. 2.5. ICP-MS measurements These measurements were carried out using an Element 2 ICP-SFMS (Inductively coupled plasma-sector field mass spectrometer; Thermo Fisher, Bremen, Germany) equipped with the self-aspirating PFA (perfluoroalkoxy) microflow nebulizer (ESI; Elemental Scientific Inc., Cuming, Omaha, USA) at a flow of 100 L/min a PC3 (Peltier cooled) cyclonic quartz chamber (ESI) operated at 4 ◦ C, a quartz injector pipe and torch (Thermo Fisher), aluminium sampler and skimmer cone (Thermo Fisher). The instrumental conditions applied were: RF power 1300 W and plasma gas flow 16 L/min, sample gas and auxiliary gas flows were set to 1.06 L/min and 0.86 L/min, respectively. The following isotopes were analyzed: at low resolution 88 Sr, 111 Cd, 208 Pb; at medium resolution 52 Cr, 55 Mn, 56 Fe, 59 Co, 60 Ni, 65 Cu, and 66 Zn at high resolution 75 As. 115 Indium (1.1 g/L) was used as internal standard at all resolution levels. Nominal mass resolutions of the Element 2 ICP-SFMS for low resolution (LR), medium resolution (MR) and high resolution (HR) were 350, 4500 and 10,000, respectively. For calibration quality control TM 27.2 (Certified Reference Waters for Trace Elements) was used, whereby the values registered agreed with the certified ones within measurement uncertainty. All samples and blank solutions were measured in triplicate. 2.6. Calibrations For both methods, measurements were accomplished by external calibration using aqueous mixed standards prepared by diluting a Merck VI multi-elemental standard with 2% (w/w) HNO3 . All calibration curves were based on eight standard solutions, including a blank. The calibration ranges were selected according to the expected concentrations of the respective analyte. 3. Results and discussion 3.1. Methods for elemental analysis The limits of detection (LOD) for all analytes determined in the digest solutions of soils and plant samples for ICP-AES as well as for ICP-MS are given along with the elemental concentration of the elements determined in Tables 2 and 3. The recoveries determined with the standard reference material of strawberry leaves (LGC7162) range from 94 up to 129% for the digested plant material by ICP-AES and from 89 to 109% for ICP-MS. The slopes of the calibration curves of all analytes, their correlation coefficients all being beyond 0.9995, showed good sensitivity. The precision expressed as RSD ranged from 0.05 up to 1.7%. Within day repeatability was found to be <4.2%. 3.2. Soil characteristics All investigated soil samples from the National Park Northern Velebit have the following mineral composition: quartz, illite, chlorite, chlorite-smectite and plagioclase. The soil pH-values measured in water suspension is between 4.78 and 7.59, and 4.14 and 7.09 in CaCl2 solution. pHdetermination in a weak electrolytic solution (0.01 mol/L CaCl2 ) offers the possibility to measure the acidic potential of a soil, including the acidic influence of protons, which are usually in H2 O
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Table 1 pH values and CEC data of the investigated soils. CEC (pH 7) [mEq/100 g] Soil 1 Soil 2 Soil 3 Soil 4 Soil 5 Soil 6 Mean + sd
23.9 19.7 28.2 21.1 19.3 10.9 21 ± 6
pH (H2 O)
pH (CaCl2 )
7.59 4.78 6.63 7.16 5.08 7.90
7.09 4.14 6.34 6.73 4.27 7.13
6.5 ± 1.3
6.0 ± 1.4
solution ligated to ionic exchange structures of soil components like humic acids. The cation exchange capacity (CEC) results range from 10.3 to 28.3 mEq/100 g. CEC is defined as the maximum quantity of cations, which a soil is capable of holding at a certain pH-level [28] and provides information about soils’ potential to bind or to release cations (as nutrients or pollutants) as an interdependency between soil and its surrounding aqueous solution. The detailed results per soil are listed in Table 1. In addition to the single results, the mean as well as the standard deviations are given. The data obtained for all soil sample are in the common range for clay soils. 3.3. Metal content of soil samples Table 2 contains the obtained elemental concentrations in the soils from the six sampling sites in the National Park along with the LOD of the respective analyte and the method used for its determination. The relative standard deviations (r.s.d.) of the contents are mainly about 30%, laying in the acceptable range for inhomogeneous biological specimens. For each sampling site (soil samples 1–6) the r.s.d. values range from 2 to 5%. Higher variations were found for Cd, which is present only in very low concentrations and for Ca and Sr, elements strongly related to the local bedrock composition. Higher Ca and Mg levels were expected in dolomite dominated soils, like in Velika Goriza (Croatia) [29]. In an organic apple orchard about 36 g/kg Ca were found, in comparison to 1.5 up to 15 g/kg in the present study. Regarding the trace elements Co, Cr, Cu, Mn, Ni, Pb, Sr, and Zn and the major element Fe, similar elemental contents were found as reported for the apple orchard soil [29]. Arsenic was found in concentrations below the limit for agricultural soil in Chine (30 mg/kg) [30]. But even being located in a remote area the levels of certain elements exceed different limit concentrations given by different jurisdictions, e.g. soils 2 and 3 have Cd concentrations above the limits stated in Canada (1.4 mg/kg) [31] and France (2 mg/kg) [32]. Only soil 5 is below the maximum value for chromium in soil according to Canadian jurisdiction (64 mg/kg) [31], but none is beyond the French limit of 150 mg/kg [32]. Whereas Chinese and French regulations stipulate maximum copper level in soil of 100 mg/kg [32,33], the Canadian Ministry of Environment limits Cu in agricultural soil to 63 mg/kg [31]. All analyzed soils are lower than these values. Regarding Pb, only soil 3 is beyond the limit given by the Canadian Ministry (70 mg/kg) [31]. The maximum allowed Ni level in soils stated by French and Chinese regulations (50 mg/kg) [32,30], only soil 5 and 6 go below this limit. Zn was found far below the maximum regulated soil concentrations. Regulations limit Zn levels in soil to 300 g/g in France [32] and to 250 g/g in China [30]. 3.4. Metal content of plant parts All elemental concentrations obtained for the various plant parts are summarized in Table 3. For better clarity no standard deviations
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Table 2 Elemental concentrations in soil samples along with LOD. Content in mg/kg LOD Method
Al 45 MS
As 0.039 MS
Ba 0.16 AES
Ca 1.8 AES
Soil 1 Soil 2 Soil 3 Soil 4 Soil 5 Soil 6
2237 1397 1408 2238 3397 2956
26.5 29.7 12.0 22.5 16.4 27.5
136 12,469 76.3 6664 82.7 9934 118 4712 105 1476 153 15,629
Mean r.s.d.
2272 35
22 31
112 27
8481 61
Cd 0.0009 MS 0.89 2.00 3.76 0.67 0.469 0.384 1.4 97
Co 0.0070 MS
Cr 0.15 MS
Cu 0.092 MS
Fe 0.75 MS
K 0.058 AES
Mg 1.2 AES
Mn 0.025 MS
Na 17 AES
Ni 0.18 MS
Pb 0.026 MS
Sr 0.13 MS
Zn 4.7 MS
18.4 17.9 15.0 17.7 13.2 13.6
108 111 98 88 49.6 73
28.0 17.2 21.5 34.8 14.9 30.5
52,864 48,628 42,160 38,235 32,401 29,120
4420 2277 3237 3609 7053 3197
6373 5439 5335 3892 6102 4418
903 713 1239 693 1751 965
473 242 747 330 238 331
89 67 53.4 62 38.3 45.8
58.4 49.6 72.3 37.4 56.4 25.4
60.1 63.3 64 74 29.1 174
99 126 138 110 97 67
24 32
40,568 23
3966 42
5260 18
1044 38
394 49
59 30
50 33
77 64
106 23
16 15
88 27
are added, they range depending on plant species, organ and element from 1.7 to 10%. It can be clearly seen that the medical plants are rich in K, Ca, Mg, Na, Fe, Mn, Zn, and B, some also in Cu and Ni. These elements play irreplaceable roles in their efficacy as folk medicine, such as improving immune function and disease prevention. Comparing these plants with two medicinal herbs analyzed in a previous study, namely sage (Salvia officinalis L) and mountain germander (Teucrium montanum L) also sampled in the National Park Northern Velebit, it can be seen, that the amounts of trace elements are in the same order of magnitude [33]. K in contrast is about 10–15 times lower. The Ca levels found in different plant organs from the herbs are much higher than those determined for sage (approx. 4000 mg/kg), only the contents found in the needles and shoots of the trees Abies alba and Picea abies are in the same range. A study on the whole plant of Micromeria croatica showed completely different results, regarding macro elements [4]. Whereas in the single organs of this investigation Ca is present in highest concentration, the working group of Kremer found K as leading element in contents 30-fold higher than our results and Ca almost 1000-fold lower. Thus not only the genetic background determines the accumulation, but also the locality. Even if widely applied for healing reasons, no standards exist for medical raw plant materials, establishing a permissible level of certain metals. The World Health Organization WHO mentions maximum permissible levels in raw plant materials only for As, Cd and Pb, namely 1.0, 0.3 and 10 mg/kg, respectively [34]. Whereas no exceedings were registered for As, to high Cd levels were found in Abies alba in needles and shoots. Regarding Pb only the leaves of Alchillea clavennae contain more than the stipulated by WHO. Nevertheless the medical plants analyzed can be considered as safe for usage as alternative medicine, since they are not consumed in total, but as decoct, infusion or balm of oils. 3.5. Uptake and accumulation behaviour Due to the high variation of the soil composition the exact calculation of the plant uptake factors, the relation content in plant divided by content in soil, was not considered justifiable. Nevertheless general conclusions can be drawn regarding the metal uptake and accumulation behaviour comparing soil and plant elemental concentrations. In all investigated plant organs the As level was very low, even below the LOD (19 g/kg), whereas in the soil about 20 mg/kg were found. Thus the uptake is only approx. 1–6‰. A clear distinction between herbs and tree can be seen by the Ca contents. In needles and shoots of Abies alba and Picea abies less Ca is found than in soils, except for soil 5, whereas the other five plants studied contain more Ca than the soils. Furthermore the Ca concentration is always highest in the leaves, only in Calamintha grandiflora leaves and blossoms have the same level. Achillea clavennae is the herb with the highest B accumulation in leaves as well as blossoms compared with the other plants. Calamintha grandiflora is leading in Ba,
Fe, Mn and Sr uptake. Regarding Cd the behaviour is vice versa to Ca, whereas the trees accumulate less Ca than herbs, Cd is uptaken in highest concentration by Abies alba and Picea abies. Co shows this tendency only for Abies alba. 3.6. Possible contamination of environment, food commodities and workplace Medicinal plants are naturally grown and their content of metals, beneficial as well harmful to men, is determined by the surroundings, thus there is no risk of contaminating any environmental compartment. Moreover it is more likely that the herbs may accumulate certain elements at levels which in consequence can exhibit negative effects on human health from the environment, from soil via root uptake or from air due to precipitation. As shown before the investigated plants do not contain elements regulated by WHO, such as As, Cd or Pb in unacceptable high concentrations. Thus during industrial processing or preparing decocts and infusions of medical herbs collected under controlled conditions in private home, the risk of metal contamination of other food commodities or workplace is negligible for the normal population. Persons more susceptible to harmful effects of uptaken low doses of heavy metals by diet, like children, pregnant or elderly people might show adverse reactions. Plants from polluted areas however, may present a health risk when consumed and contaminate various processing utensils in private kitchen and industrial sites. Due to the growing interest in alternative medicine, the cultivation of selected herbs is forced and drastic use of plant protection products is necessary in order to meet the requirements of the market. Also less attention is paid to comply with regulations for safe processing and storage. In consequence the herbs brought on the market may be contaminated with pesticides, microbial contaminants, heavy metals, and chemical toxins, thus representing a risk for worker as well as consumers [35]. Furthermore the potential of adulteration rises with increasing interest in a product, e.g. with synthetic drugs, resulting in a risk for both consumers as workers handling these formulations [36,37] or with other herbs. As shown the mineral composition is characteristic for each medicinal plant, thus the metal determination may be used as method for discovering adulteration of synthetic drugs or the mixture with other cheaper plants. Besides the metals contained in the medical plants as discussed in detail in the previous subchapters, they contain organic compound, mainly responsible for their bioactivity, such as antioxidative compounds. Processed plant products contain often only a small percentage of the active substances present in the raw material. Thus in agriculture and industry waste and by-products still contain high quantities of valuable substances [9]. In order not to spoil these resources or to generate disorders in the environment by disposing them, more emphasis should be placed on the recycling of bioactive material.
Please cite this article in press as: M. Zeiner, et al., Influence of soil composition on the major, minor and trace metal content of Velebit biomedical plants, J. Pharm. Biomed. Anal. (2014), http://dx.doi.org/10.1016/j.jpba.2014.10.012
Al 4.2 AES
As 0.019 MS
B 0.45 AES
Ba 0.16 AES
Ca 1.8 AES
Cd 0.0005 MS
Gentiana lutea Leaves Roots
*
155
0.027 0.030
30.7 18.1
8.10 38.7
19,558 12,350
0.0072 0.11
Co 0.0035 MS *
0.0083
Cr 0.075 MS * *
32.3 87.8
0.060 0.047
26.3 31.4
19.6 14.9
29,741 23,453
*
Satureja montana Shoots Leaves Blossoms
30.2 76.1 86.7
*
29.5 23.7 16.8
18.4 12.4 8.31
16,022 27,940 22,887
*
0.088
24.9 15.2
54.8 43.3
12,164 12,492
0.037 0.047
0.044 0.060
0.368 0.639
Calamintha grandiflora 118 Leaves 61.5 Blossoms
*
0.022 *
*
* *
*
0.0094 *
0.0077 0.080
*
0.886 1.10
K 0.058 AES
Mg 1.2 AES
Mn 0.013 MS
Na 17 AES
20.9 25.7
269 116
3029 1070
6.73 2.02
573 302
*
*
*
*
29.9 36.0
279 251
2175 1792
5.62 5.91
652 154
*
*
*
*
*
0.437 0.801 5.54
25.0 28.1 55.6
488 385 304
1153 2767 2420
4.90 7.48 19.9
430 328 201
*
*
*
*
10.6 16.2
103 76.1
454 435
2730 3145
194 47.3
413 398
1726 1307
*
105 102
0.648 9.72
92.1 26.5
333 283
1.20 0.84
0.153 0.045
40.2 48.9
1.94 0.71
24,070 14,205
0.277 0.076
0.067 0.021
0.911 0.490
21.8 10.2
Picea abies Needles Shoots
20.5 28.1
*
16.4 18.4
4.57 7.91
2565 3168
0.269 0.158
0.066 0.037
0.251 0.266
10.7 9.72
28.0 18.5
356 455
1008 676
102 56.11
144 151
Abies alba Needles Shoots
67.2 62.6
0.024
14.2 18.1
5.56 6.92
7172 4585
0.471 0.717
0.380 0.204
0.672 0.291
10.0 13.6
24.2 28.5
497 425
1004 872
132 72.0
188 182
0.056 0.103 0.045
0.056
Salvia officinalis [33] Aerial parts Leaves Blossoms
102 134 57.2
n.d. n.d. n.d.
n.d. n.d. n.d.
n.d. n.d. n.d.
3890 3780 4760
Teucrium montanum [33] Aerial parts 133 85.6 Leaves 62.6 Blossoms
n.d. n.d. n.d.
n.d. n.d. n.d.
n.d. n.d. n.d.
58 66 81
* * *
*
*
0.069 *
0.231
*
*
257 253
98.4 44.4
*
Pb 0.013 MS
2.21
Achillea clavennae Leaves Blossoms
*
Ni 0.090 MS
*
*
*
Fe 0.38 MS
0.764 4.86 8.71
Sr 0.06 MS
Zn 2.4 MS
1.80 4.09
5.35 8.35
6.64 5.56
3.89 6.98
4.07 4.11 5.26
6.82 7.24 18.8
11.7 14.5
49.1 70.0
11.0 4.83
14.4 3.22
100 33.9
1.13 0.779
4.44 6.25
2.14 5.11
53.89 41.65
2.42 2.39
6.28 5.47
1.70 2.80
52.50 47.71
0.855 0.301
4.13 10.5 11.4
96.7 126 64.8
8420 9440 9020
3200 4010 3020
29.7 39.1 24.5
38 91 56
110 148 74.5
6.53 10.2 10.4
7.81 9.25 5.77
27.1 39.4 27.8
0.386 0.263 0.356
3.60 3.67 6.72
107 69.8 59.9
6470 5920 6750
1430 1320 1470
25.9 32.8 21.8
18 54 30
125 81.6 71.5
6.02 6.19 9.45
3.77 3.89 4.27
14.9 22.4 23.9
n.d. not determined. *
ARTICLE IN PRESS
Micromeria croatica Leaves Blossoms
Cu 0.046 MS
G Model
PBA-9763; No. of Pages 6
Content in mg/kg LOD Method
M. Zeiner et al. / Journal of Pharmaceutical and Biomedical Analysis xxx (2014) xxx–xxx
Please cite this article in press as: M. Zeiner, et al., Influence of soil composition on the major, minor and trace metal content of Velebit biomedical plants, J. Pharm. Biomed. Anal. (2014), http://dx.doi.org/10.1016/j.jpba.2014.10.012
Table 3 Elemental concentrations in plant samples along with LOD.
5
G Model PBA-9763; No. of Pages 6
ARTICLE IN PRESS M. Zeiner et al. / Journal of Pharmaceutical and Biomedical Analysis xxx (2014) xxx–xxx
6
4. Conclusions Metal monitoring of medicinal herb is not only a tool for showing their nutritional value, but also assures the safe use of herbal supplements by proving the absence of harmful metals. Each plant exhibits its own metal uptake and accumulation behaviour, which is the basis for herb characterization by elemental pattern, which can be applied for adulteration detection. Due to the fact that medicinal herbs are natural products the risk of contamination of the environment, food commodities and workplaces is unlikely. In contrast herbs collected in polluted areas, not well stored or processed may turn into a harmful instead of beneficial product. Acknowledgement This project was financially supported by the Wissenschaftlichtechnische Zusammenarbeit Austria-Croatia (Austrian Exchange Service ÖAD) HR-23-2012. References [1] E. Ernst, Harmless herbs? A review of the recent literature, Am. J. Med. 104 (1998) 170–172. [2] I. Rodushkin, T. Ruth, A. Huhtasaari, Comparison of two digestion methods for elemental determinations in plant material by ICP techniques, Anal. Chim. Acta 378 (1999) 191–200. [3] T. Karak, R.M. Bhagat, Trace elements in tea leaves, made tea and tea infusion: review, Food Res. Int. 43 (2010) 2234–2252. [4] D. Kremer, E. Stabentheiner, V. Dunkic, I. Dragovic Müller, L. Vujic, I. Kosalec, D. Ballian, F. Bogunic, N. Bezic, Micromorphological and chemotaxonomical traits of Micromeria croatica (Pers.) Schott, Chem. Biodivers. 9 (2012) 755–768. [5] D. Kremer, M. Randic, I. Kosalec, A. Brklacic, G. Lukac, I. Krusic, D. Baillan, F. Bogunic, Ks. Karlovic, New localities of the subendemic species Berberis croatica, Teucrium arduini and Micromeria croatica in the Dinaric Alps, Acta Bot. Croat. 70 (2011) 289–300. [6] M. Öztürk, U. Kolak, G. Topcu, S. Öksüz, M. Iqbal Choudhary, Antioxidant and anticholinesterase active constituents from Micromeria cilicica by radicalscavenging activity-guided fractionation, Food Chem. 126 (2011) 31–38. [7] V. Dunkic, D. Kremer, I. Dragojevic Müller, E. Stabentheiner, S. Kuzmic, R. Jurisic Grubesic, L. Vujic, I. Kosalec, M. Randic, S. Srecec, N. Bezic, N. Bezic, Chemotaxonomical and micromorphological traits of Satureja montana L. and S. subspicata Vis. (Lamiaceae), Chem. Biodiv. 9 (2012) 2825–2842. [8] S. Cavar, M. Maksimovic, M.E. Solic, A. Jerkovic-Mujkic, R. Besta, Chemical composition and antioxidant antimicrobial activity of two Satureja essential oils, Food Chem. 111 (2008) 648–653. [9] D. Dobravalskyte, P.R. Venskutonis, T. Talou, Antioxidant properties and essential oil composition of Calamintha grandiflora L., Food Chem. 135 (8) (2012) 1539–1546. [10] B. Saric-Kundalic, C. Dobes, V. Klatte-Asselmeyer, J. Saukel, Ethnobotanical study on medicinal use of wild and cultivated plants in middle, south and west Bosnia and Herzegovina, J. Ethnopharmacol. 131 (2010) 33–55. [11] L. Cornara, A. La Rocca, L. Terrizzano, F. Dente, M.G. Mariotti, Ethnobotanical and phytomedical knowledge in the North-West Ligurian Alps, J. Ethnopharmacol. 155 (2014) 463–484.
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Please cite this article in press as: M. Zeiner, et al., Influence of soil composition on the major, minor and trace metal content of Velebit biomedical plants, J. Pharm. Biomed. Anal. (2014), http://dx.doi.org/10.1016/j.jpba.2014.10.012