**1. Introduction**

There are over 3000 plant species in Bulgaria, of which more than 600 are used for medicinal purposes. Bulgarian herbs contain a high percentage of biologically active substances [1]. They are rich in various chemical compounds: alkaloids, glycosides, saponins, polysaccharides, tannins, flavonoids, coumarins, essential oils, vitamins, and trace elements. The pharmacological and medicinal action and application of Bulgarian herbs has been an important topic and subject of many studies. One of the most outstanding achievements of the Bulgarian pharmaceutical industry was the creation of the medicinal preparation "Nivalin" by Prof. D. Paskov. The active substance of which is the alkaloid galantamine, extracted from *Leucojum aestivum* L. [2]. Most of the achievements of contemporary medical science are based on bioactive compounds extracted from medicinal plants [3].

*Sempervivum tectorum* L. (synonym: Sempervivum tectorum var. arvernense (Lecoq & Lamotte) Zonn., Sempervivum tectorum var. andreanum (Wale) O.Bolòs & Vigo) belongs to a large family of Crassulaceae with crassulacean acid metabolism, native to the mountains of southern Europe and cultivated in the whole of Europe.

**Citation:** Gentscheva, G.; Karadjova, I.; Radusheva, P.; Minkova, S.; Nikolova, K.; Sotirova, Y.; Yotkovska, I.; Andonova, V. Determination of the Elements Composition in *Sempervivum tectorum* L. from Bulgaria. *Horticulturae* **2021**, *7*, 306. https://doi.org/10.3390/ horticulturae7090306

Academic Editors: Dasha Mihaylova and Aneta Popova

Received: 12 August 2021 Accepted: 9 September 2021 Published: 12 September 2021

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*Sempervivum tectorum* L. (houseleek) is an evergreen plant with fleshy blue-green leaves forming a rosette, which grows on dry to fresh sandy soils and in sunny to semisunny places. The plant is well-known in folk medicine and has been used for thousands of years.

In recent years, researchers have focused on studies of the characterization of the bioactive ingredients of this plant and their ability to restore liver function [4], their antioxidant properties [5], the potential for wound healing [6], anti-inflammatory action, and analgesic and detoxicating properties [7–9]. Most of these properties of *Sempervivum tectorum* L. are connected with phenolic compounds identified in fresh juices squeezed from plant leaves [10,11]. However, according to the author's knowledge, despite of the numerous uses of *Sempervivum tectorum* L. in folk medicine, it remains poorly known from the viewpoint of systematic investigations into trace element content, element bioavailability, and correlation between essential element content and antioxidant activity.

The trace element content is an essential characteristic of any plant. However, there are no such data for *Sempervivum tectorum* L., neither for environmental safety, nor the effect on human health. The objective of this study was to investigate the level of the elements Ca, K, Na, Mg, Mn, Fe, Zn, Cu, Co, Al, V, Cr, Ni, Mo, Ba. Pb, Cd, Hg, and As as a total content in plant samples of *Sempervivum tectorum* L. obtained from different sampling sites; natural and affected at different levels by human activities. The urban soil (A) is from an urban park close to center of the city, and the fertilized soil (C) is from land used for agriculture purposes for many years. The rural soil (B) and mountain soil (D) might be accepted as natural; however, with varying composition. The bioavailable fraction of essential elements Ca, Mg, Fe, Mn, and Zn, defined according to a standard procedure for element migration in hydrochloric acid that mimics food digestion processes in the stomach, was quantified. Additionally, K, Ca, Mg, Na, Fe, Mn, Zn, Al, Cu, and Cr were determined in the freshly squeezed juice from plant leaves, as directly used in folk medicine against ear pain.

### **2. Materials and Methods**

*Plant Material*. *S. tectorum* plants were from different habitats, grown on city soils (A), village soils (B), fertilized soils (C), and mountain soils (D). The leaves of the plants were removed, thoroughly washed with deionized water to remove all possible external contaminants, and used immediately for:


Sample preparation before analysis:

*Reagents*: 67% HNO3 (supra pure, Merck, Darmstadt Germany); 30% H2O2 (supra pure, Merck, Darmstadt Germany); 37% HCl (p.a. Sigma-Aldrich, Darmstadt Germany).

### *2.1. Determination of Total Content of Elements*

A dry sample of around 0.5 g was weighed in Teflon vessels of a microwave digestion system, 6 mL 67% HNO3 and 2 mL 30% H2O2 were added, and samples were left overnight. Microwave digestion was performed for 20 min: 10 min to reach 180 ◦C and 10 min maintained at this temperature. After cooling, samples were transferred to a 50 mL volumetric flask and diluted up to the mark with deionized water. A blank sample was passed through the whole analytical procedure.

#### *2.2. Determination of Bioavailable Fraction in Fresh Leaves*

A sample of 2.0 g of fresh leaves was milled with 50 mL deionized water in a plastic container. After that, 50 mL 0.14 mol L−<sup>1</sup> HCl were added, and the mixture was shaken for at least 1 min. The suspension was left for several minutes to settle, and the pH of the clear supernatant was measured. If the pH was above 1.5, 2 mol L−<sup>1</sup> HCl solution was added drop-wise while mixing until the pH reached values between 1.0 and 1.5. The container was closed and agitated at 37 ± 2 ◦C for 1 h. After that, the suspension was left for a further 1 h at 37 ± 2 ◦C. The mixture was protected from daylight. The solid matter was separated by centrifugation and, if necessary, filtrated through a membrane filter (0.22 μm) to remove all solid particles. The separation should be completed as soon as possible after completing the standing time; centrifuging should take no longer than 10 min. Next, the obtained solution was evaporated on a hot plate to 2–3 mL, 3 mL of conc. HNO3 was added for digestion of the organic components, and, finally, the sample was quantitatively transferred to a 25 mL flask and made up with deionized water [12].

#### *2.3. Determination of Elements in Juice Obtained by Squeezing of Fresh Leaves*

A sample of 2.0 g juice (obtained after filtration of fresh juice through a 0.22 μm membrane filter) was transferred in a glass beaker and treated with 1 mL 67% HNO3 on a hot plate. After 1 h, the solution was cooled and diluted in a 10 mL volumetric flask with distilled water.

Apparatus for quantitative measurement of chemical elements:

Flame atomic absorption spectrometry: The content of Fe, K, Mn, Mg, Na, and Zn was measured by flame atomic absorption spectrometry (Thermo Electron—SOLAAR Mkll M5 series, UK) in an air/acetylene flame under optimized instrumental parameters. The content of Ca was measured in N2O/acetylene flame, using the same instrument. Stock standard solutions of Ca, Fe, K, Mn, Mg, Na, and Zn (1.000 g <sup>L</sup>−1(Merck)) were used for the preparation of diluted working standards.

Inductively coupled plasma mass spectrometry: The content of As, Al, Ba, Cd, Co, Cr, Cu, Hg, Mo, Ni, Pb, and V was measured by ICP-MS using an inductively coupled plasma mass spectrometer "X SERIES 2"—Thermo Scientific, USA with a 3 channel peristaltic pump; concentric nebulizer; Peltier-cooled spray chamber (4 ◦C); Xt interface option; Ni cones. Optimized instrumental parameters: forward plasma power of 1400 W; plasma gas flow 13 L min/L; nebulizer flow 0.85 L/min; dwell time 30 ms; measurements 3 × 30 scans. Stock standard solutions: multielement standard solution 5 for ICP (TraceCERT ®, Merck), 1000 mg/L As (Fluka, Sigma-Aldrich) and 1000 mg/L Hg (Fluka, Sigma-Aldrich) were used for the preparation of diluted working standard solutions for calibration of ICP-MS.

The accuracy of the analytical procedure used was validated by the analysis of certified reference material NIST SRM 1573a Tomato leaves. The very good agreemen<sup>t</sup> with the certified values and the recoveries above 95% achieved for all certified elements confirmed the reliability of the results obtained for total element contents (see Table 1). Limit of detection and limit of quantification was calculated for each element based on standard deviation of blanks sample for the respective procedures using 3 σ criterium (LOD) and 10 σ criterium (LOQ). Calculated values for LOD and LOQ are presented in Table 1.
