**4. Discussion**

The results from the total phenolic contents and antioxidant activities of the obtained plant extracts are summarized in Table 1. The highest total phenolic content was found in the 50% ethanolic extract about—0.84 ± 0.08 mg GAE/mL. This extract also possessed the highest antioxidant potential as evaluated by the DPPH and FRAP assays at about— 7.02 ± 0.71 mM TE/mL and 6.15 ± 0.25 mM TE/mL, respectively. The antioxidant activity and total phenolic content in the 10% ethanol extract showed approximately two times lower values than in the 50% ethanol extract. The lowest antioxidant activity was found in the 95% ethanol extract. Therefore, the extraction of bioactive compounds depended strongly on the water content in a solvent. A similar observation was reported by Taneva et al. for water and hydroethanolic extracts from rosehip (*Rosa canina* L.) fruits [24]. In agreemen<sup>t</sup> with their results, our study demonstrated that the 50% ethanol extract had the highest antioxidant potential. The better extraction of phenolic compounds from dried and lyophilized leaves of *Sempervivum tectorum* with a water to ethanol ratio of 1:1 was also reported. The total phenols reached 15-40 mg GA/g dry weight [25]. As might be expected, a strong correlation was found between the total phenolic content and the antioxidant activity. A correlation coefficient above 0.9 was calculated for both the total polyphenols and antioxidant activities of extracts: DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging ability and total polyphenols and FRAP (ferric reducing antioxidant power). Evidently, the antioxidant activity of the 50% ethanol extract was mainly due to the high level of total phenolic content in this extract. 50% ethanol extracts of *Sempervivum tectorum* could be successfully used for food and pharmaceutical purposes due to the safety of the solvent and as it is rich in polyphenols. As confirmation, Šentjurc et al. [26] explained that compounds in pure extracts of *Sempervivum tectorum* L. that possessed the highest antioxidant potential are oligomeric polyphenols. Knez Marevci et al. [25] showed the antioxidative potential of dried and lyophilized *S. tectorum* extracts only by the DPPH assay. Similar results for ABTS and DPPH radical scavenging assays used to evaluate the antioxidant potential of different extracts of *Sempervivum tectorum* leaves were observed by Alberti et al. [27]. In this study we demonstrated that the 50% ethanol extract showed better radical scavenging activity by the DPPH method than by the metal reducing activity (FRAP assay) (Table 1) *Sempervivum tectorum* 70 % (*v/v*) ethanol extract showed IC50 in concentration 74.5 ± 3.6 μM/mL (DPPH) [27], while in our case 50% ethanol extracts demonstrated much higher IC50 3.45 mM TE/mL (DPPH). The correlation of total polyphenol content and radical scavenging activity (DPPH), as well as total polyphenol content and metal reducing activity (FRAP) was considered as highly significant with r2 = 0.9988 and r2 = 0.9994, respectively.

The color coordinates were obtained in two different colorimetric systems (Table 2). Color parameters x and y, which are connected with the purity of the color of samples from *Sempervivum tectorum* L., have been increased with higher ethanol contents. The uniform color space CIELab has been used to better present the color characteristics of the investigated extracts. The increase in the color parameter b proves that the yellow component dominates in 10% and 50%, intensifying with increasing ethanol content. In 95% ethanol extract the blue component dominates—the parameter b < 0—in contrast to 10% and 50% extracts.

The results presented in Table 3 show that as the ethanol content increases, so does the amount of β-carotene. Chlorophyll exists in 50% and 95% ethanol extracts, but it is absent in 10% extract (Figure 1). The absorption band between 630 nm and 690 nm is absent in the 10% ethanol extract of *Sempervivum tectorum* L. because it does not contain chlorophyll. It has the greatest transmission in the visible range at about 60%–70%. For 95% of the ethanol extracts from *Sempervivum tectorum* L., the transmission spectrum in the visible region has two regions—the first is in the range of 440 nm–480 nm, and the second is between 610 nm–660 nm. There is a linear dependence between the transmission coefficient at 655 nm and the content of chlorophyll.

The main compounds associated with the antioxidant effect of *Sempervivum tectorum* L. extracts may also correlate with the fluorescence, absorption, or transmission spectra of the samples.

The fluorescence spectra of 50% and 95% ethanolic extracts of *Sempervivum tectorum* L. were similar (Figures 3 and 4). An intense fluorescent maximum in the range of 675–690 nm was observed, which was associated with the presence of chlorophyll. It is the most intense and clearly expressed for the 50% ethanol extract. The main compounds associated with the antioxidant activities were carotenoids and polyphenolic compounds (especially phenolic acids and flavonoids). In our case, the fluorescence spectra provide a fluorescence maximum in the range of 630–700 nm, which is associated with the presence of chlorophyll in the samples. For the sample with the 50% ethanol content, there is a low-intensity fluorescent band in the range of 500–580 nm, associated in the literature with the presence of β-carotene.

Analysis of the transmission spectra shows that intense absorption bands between 630 nm and 690 nm were observed only in 50% and 95% water-ethanolic extracts and were absent in the 10% ethanolic extract of *Sempervivum tectorum* L. (chlorophyll is not leached in this extract).

Several correlations have been established between the parameters of applied photonics and the contents of pigments and with antioxidant activities and total phenolic contents.

$$\text{LPPPH} = 8.405 \ast \text{TPC}, R^2 = 0.991 \tag{1}$$

$$\text{FRAP} = 7.4975 \,\text{\*}\,\text{TPC}, R^2 = 0.991 \,\tag{2}$$

$$\text{\\$-carotene} = 0.9026 \ast \text{b} - 6.2895, R^2 = 0.923 \tag{3}$$

$$T = -0.554 \ast \text{Chlorophyll} + 59.527, R^2 = 0.961 \tag{4}$$

Essential element content in studied extracts varied considerably. The concentrations of elements like K and Na showed relatively high correlation coefficients above 0.8 with total phenolic content showing most like the chemical compositions of phenols. Unexpectedly, content of elements like Fe, Cu, and Zn do not correlate with total phenols although it is well known that Fe, Zn, and Cu formed stable complexes with phenols. Most likely parts of these elements are bound to other constituents of investigated extracts. As can be seen from Table 4, the ethanol concentration significantly affects the extraction of elements such as Ca, Fe, Mn, Zn, Al, Co, Ba, and Cr. Their contents in the extracts decrease with the increase in ethanol content. Only the concentration of Cu increases with the increase in ethanol content.

Toxic element levels in prepared extracts/infusions are under regulation at the national or regional level. Permissible limits for toxic elements in extracts have been compared by several authors [28]. According to the World Health Organization, the permissible limit for cadmium concentrations in herbal products is 0.3 mg kg−<sup>1</sup> Cd and for lead it is 10.0 mg kg−<sup>1</sup> Pb [29]. Results obtained for all studied extracts showed very low concentrations for toxic elements, much below the permissible limits. Furthermore, the results for highly toxic elements like Cd, Tl, As, and Hg are below the detection limit of 0.02 mg kg−<sup>1</sup> for all studied extracts. As a general rule, the 95% ethanol extract has the lowest concentrations of elements, suggesting that chemical species of essential and toxic elements in *Sempervivum tectorum* L. are aqueous soluble and highly bioavailable.
