**4. Discussion**

As seen from Table 1, *Sempervivum tectorum* L. contains an extremely high calcium content, exceeding by between 3–10 times the concentrations of the second highest content element, K. No statistically significant differences were found for the total content of essential elements (except Ca and Mn) in plants from different regions, confirming the biouptake ability of plant toward essential elements [14]. Unexpectedly a higher total content of Ca was observed in rural and urban plants in comparison with plants from fertilized and mountain regions. Significantly higher concentrations of Mn were determined in the plants grown on fertilized soils, which might be explained by the high bioavailable Mn content in these soils, as the same concentrations were measured in other herbs from the same region. The total content of essential elements presented at lower concentration levels in *Sempervivum tectorum* L. is close to the content of these elements in other herbs from these regions [15,16].

Table 2 lists the results obtained for some nonessential elements. As can be seen, the plants grown on agricultural (fertilized) soils differed from the others with their higher concentrations of Al, Co, and Ba. However, only the concentrations of cobalt were surprising, as Al of such and higher concentrations is found in herbs from this region [15].

Another critical aspect is the good quality control of medicinal herbs, to protect consumers from contamination, as many medicinal herbs and their mixtures can present a health risk due to toxic elements [17].

Toxic element levels in raw plant material or prepared products/extracts/infusions is regulated by documents at global, national, or regional level. Strict control of contaminant levels and their minimization is required by the World Health Organization (WHO) through guidelines such as the good agricultural and collection practices (GACP) for medicinal plants and good manufacturing practices (GMP) for herbal medicines. Maximal values for toxic elements in herbal drugs and extracts have been discussed and compared by several authors [18–20].

According to the World Health Organization, cadmium concentrations and lead in herbal medicines and products are regulated at 0.3 mg kg−<sup>1</sup> Cd and 10.0 mg kg−<sup>1</sup> Pb [18]. In different countries, the law sets lower limits, and a very good comparison of the various permissible limits is presented by Luo et al. [20]. As shown in Table 3, the concentrations of toxic elements meet the requirements of the WHO, and only in one single case was the cadmium concentration exceeded, for urban soil. Expectedly, the results for elements such as As, Cd, and Pb are highest in plants grown on urban soils. Most probably, in this case both soil pollution and aerosol deposition are responsible for the high toxic element content. Although, it is clearly important to harvest medicinal plants from clean sites

without anthropogenic influences such as mountain regions. A relatively high content was determined for Ni and Pb in plants grown on fertilized soils, most likely connected with Ni and Pb contamination by the phosphate fertilizers applied.

In this study, sampling for all studied plants and sampling sites was performed in the summer season, with some efforts to use plants in the same vegetation period. Taking into account that *Sempervivum tectorum* L. is a perennial plant, additional research is required to elucidate any correlation between plant age and chemical element content.

In Table 4, the results found for the operationally defined bioavailable content (see Section 2.3) of Ca, Mg, Zn, Mn, and Fe in fresh leaves of *Sempervivum tectorum* L. (after two hours of treatment in pH 1.0–1.5) are depicted. Plants growing on mountains, villages, and fertilized soils were used. The percentage content of bioavailable fraction varied between 4–14% for all studied essential elements (Table 7). It should be pointed out that the content of Ca and Fe, which might be accepted as being most responsible for the health functions of *Sempervivum tectorum* L., is almost constant in the bioavailable fractions from all samples. High concentrations of Ca in this fraction justify the use of *Sempervivum tectorum* L. as a national remedy for the treatment of gastric ulcers, possibly because of the beneficial calcification effect.

**Table 7.** Bioavailable concentrations of Ca, Mg, Zn, Mn, and Fe as a percentage of total content.


Although the concentration of Mn is still highest as a bioavailable concentration, the degree of extraction was significantly lower, most probably depending on the different Mn species present in the leaves. Therefore, it might be assumed that the Mn bio-uptake would be highest from agricultural (fertilized) soils, most probably because of suitable pH values.

Determination of elements in juice from fresh leaves. Fresh juice obtained by squeezing leaves from *Sempervivum tectorum* L. was widely used as folk medicine against ear pain. As shown in Table 6, this effect can most probably be explained by the high Mg concentrations, analogous to the pharmaceuticals used for external application (Mg-gels or Mg-oils) with anti-inflammatory and regenerative actions and improved blood circulation [21–23]
