**1. Introduction**

Egypt, Iran, China, and India have known about and applied the healing properties of some plants for more than 3000 years. Ancient scientists (Hippocrates, Theophrastus, Avicenna, and many others) have described the herbs used in their time. The first records of the use of regional herbs date back to the time of Theophrastus. In his work, "On Medicines", Dioscorides, the most famous pharmacologist of antiquity, describes the herbs used by the Thracians [1]. Even today, it is known that medicinal plants are a valuable source for making medicines. Many of the medicinal plants are the basis for obtaining nutritional supplements to reduce the action of free radicals and reduce oxidative stress in living cells [2–4]. Aromatic and other biologically active substances are extracted from many herbs [5–7].

**Citation:** Gentscheva, G.; Karadjova, I.; Minkova, S.; Nikolova, K.; Andonova, V.; Petkova, N.; Milkova-Tomova, I. Optical Properties and Antioxidant Activity of Water-Ethanolic Extracts from *Sempervivum tectorum* L. from Bulgaria. *Horticulturae* **2021**, *7*, 520. https://doi.org/10.3390/ horticulturae7120520

Academic Editor: Alessandra Durazzo

Received: 26 October 2021 Accepted: 22 November 2021 Published: 25 November 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

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In recent years, studies have been focused on the concentrations of potentially bioactive compounds in various plants and their antioxidant activities, detoxifying properties, and other essential properties for human health.

Plants offer a wide range of natural antioxidants. Many herbal decoctions and extracts have been widely used in folk medicine for centuries. *Sempervivum tectorum* is a plant known in folk medicine with fleshy, succulent leaves; the juice is used to treat inflammation of the ears, mild burns and wounds on the skin, warts, ulcers, skin rashes, and calluses [8–10]. The potent antioxidant activity and the ability to inhibit lipid peroxidation have been established [11,12], as well as the membrane-stabilizing and protective effects on the liver [13]. Drinking tea prepared from the leaves of the plant is recommended for stomach ulcers [14]. Chromatographically purified fractions of *S. tectorum* L. in which rare natural components have been identified—monosaccharide sedoheptulose and polyalcohol 2-C-methyl-D-erythritol, as well as known organic acids and flavonoids, demonstrate the therapeutic effects of the plant in the healing of wounds [15]. *Sempervivum tectorum* extracts have been used to normalize oxidative stress biomarkers in the blood of rats [16].

*Sempervivum tectorum* L., although often used in folk medicine, has been poorly studied. The available data in the scientific literature are still few [8,15,17]. Although several pharmacological properties have been reported for the leaves of *Sempervivum tectorum* L., a complete characterization of the extracts is not ye<sup>t</sup> available. Therefore, only the polyphenolic contents and, in part, the antioxidant activities have been studied [16,18].

The present study aims to: (i) determine the optical characteristics, antioxidant activities, and elemental composition of water-ethanolic extracts of *Sempervivum tectorum* L. with different polarities; (ii) elucidate the correlation between antioxidant activities and the contents of bioactive compounds such as polyphenols and beta carotene, and (iii) demonstrate the possible relationship between the essential element contents and polyphenols.

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

Plant materials.

The above ground parts of the *Sempervivum tectorum* L (leaves) were sampled randomly from the parks of Sofia in April 2021. Samples were botanically identified by Assoc. Prof. Iliya Slavov (Department of Botany, Faculty of Pharmacy, MU-Varna). *Sempervivum tectorum* L. is a cultivated ornamental plant and was not present in the herbarium until now.

Samples were washed with distilled water, dried at 40 ◦C to constant weight, and further ground in a coffee grinder.

Preparation of water-ethanolic extracts.

The dried and ground leaves of the plants were treated with different concentrations of ethanol (C2H5OH 96%, Sigma-Aldrich, Darmstadt, Germany)—10%, 50%, and 95% *v/v*, respectively. In all cases, the sample to alcohol ratio was 1:10 w/v. The extraction was performed for 48 h at room temperature by constantly shaking the sample at 50 rpm (Digital orbital shaker, SHO-2D, witeg Labortechnik GmbH, Wertheim, Germany). Finally, the resulting extracts were separated by filtration—0.20 μm filters (CHROMAFIL® CA-20/25, Düren, Germany) and kept in glass vessels in dark places.

Elemental composition.

To determine the elements in the water-ethanolic extracts, 10 mL was carefully dried under an IR lamp (until complete alcohol removal) and treated with nitric acid (65%, Suprapur®, Merck, Darmstadt, Germany) for the mineralization of the extracted organic components. The drying of the ethanol extracts and their further digestion was performed at a temperature below 40 ◦C, ensuring loss free determination of As and Hg. After digestion, the solutions were diluted with doubly distilled water to a final volume of 25 mL.

The quantitative determination of chemical elements was carried out using ICP-MS ("X SERIES 2"—Thermo Scientific, (Thermo Fisher Scientific, Bremen, Germany) under optimal instrumental parameters [19]. Multielement standard solution 5 for ICP (TraceCERT®, Sigma-Aldrich Production GmbH, Industriestrasse 25, 9471 Buchs, Switzerland, subsidiary of Merck) and standard solutions of Hg and As (TraceCERT ®, 1000 mg/L, Merck) were used for the preparation of diluted working standard solutions for the calibration of ICP-MS.

Optical measurements.

The test samples' color coordinates, color parameters, and brightness were measured with a Lovibond PFX 880 (UK) colorimeter in 10 mm cells at room temperature. Using the spectrum in the visible range and the values for the color parameters utilizing a software program developed especially for Lovibond PFX 880 by the manufacturer, chlorophyll and β-carotene were calculated.

The CIELab (1976) colorimetric system characterizing the colors of the extracts was used. In this colorimetric system, the color components a and b characterize the predominance of the red or green component and the yellow or blue component in the respective samples. The parameter L is called luminosity, and the smaller its value, the darker the sample.

The fluorescence of the ethanol extracts was studied by exciting them with lightemitting diodes (LEDs), emitting at 370 nm, 395 nm, 405 nm, 410 nm, 425 nm, 435 nm, and 450 nm. A 90-degree geometry for light detection in a 10 × 10 mm cuvette was used. Samples were studied without any preliminary solution. Fluorescence and scattering spectra were recorded using the fiber optic spectrometer Avantes 2048 with a spectral sensitivity within the 250–1100 nm range.

The resolution of the spectrometer is 8 nm for an input slit of 200 μm. An optical fiber with a diameter of 200 μm is used to bring the light to the probe and to measure the scattered and fluorescent light. A collimator with a lens with an aperture of D = 5 mm is used to collect more light and send it to the receiver. In general, in classical fluorescence spectroscopy, measurements are performed in dilute solutions where the absorbance is below 0.1. At higher optical densities, the fluorescence intensity decreases due to the effect of the internal filter. In this case, frontal-face fluorescence spectroscopy is more suitable for use. In the present study, to measure the fluorescence spectra of extracts without dilution, the cuvette holder is modified as follows: the first probe (optical fiber) is placed directly in thetestsample,andthesecondprobeisfixedontopofthedropsurface.

 Phenolic contents.

Total phenolic contents (TPC) were determined using a Folin-Ciocalteu reagen<sup>t</sup> (Sigma-Aldrich, Germany) [20] with some modifications. Briefly, Folin-Ciocalteu reagen<sup>t</sup> (1 mL) diluted five times was mixed with 0.2 mL sample and 0.8 mL 7.5% Na2CO3 (Sigma-Aldrich, Germany). The reaction was performed for 20 min at room temperature in darkness. Then the absorbance was measured at 765 nm by a UV/Vis spectrophotometer Camspec M107 (Spectronic-Camspec Ltd., Leeds, UK) against a blank, prepared with 70% methanol (Sigma-Aldrich, Germany). The calibration curve was linear in the range of 0.02–0.10 mg gallic acid (Sigma-Aldrich, Germany) and was used as a standard [21].

Antioxidant activities.

Two methods were used to evaluate the antioxidant activities: DPPH (1,1-diphenyl-2- picrylhydrazyl) radical based on mixed hydrogen atom transfer (HAT) and single electron transfer mechanisms and FRAP (ferric reducing antioxidant power) based only on a single electron transfer mechanism.

For the DPPH radical-scavenging ability, the analyzed sample (0.15 mL) was mixed with 2.85 mL freshly prepared 0.1 mM solution of DPPH (Sigma-Aldrich, Germany) in methanol (Merck). The sample was incubated for 15 min at 37 ◦C in darkness. The reduction of absorbance at 517 nm was measured by spectrophotometer a Vis spectrophotometer Camspec M107 (Spectronic-Camspec Ltd., UK) compared to the blank containing methanol, and the percentage of inhibition was calculated [22]. A standard curve was built with 6- hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (Sigma-Aldrich, Germany) in concentrations of between 0.005 and 1.0 mM.

The Ferric reducing antioxidant power (FRAP) assay was performed according to Benzie and Strain [23] with a slight modification. The reagen<sup>t</sup> was freshly prepared by mixing 10 parts 0.3 M acetate buffer (pH 3.6) (Sigma-Aldrich, Germany), 1 part 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) (Fluka) in 40 mM HCl (Merck, Germany) and 1 part 20 mM FeCl3.6H2O (Merck, Germany) in distilled H2O. The reaction was started by mixing 3.0 mL FRAP reagen<sup>t</sup> with 0.1 mL of investigated extract. The reaction time was 10 min at 37 ◦C in darkness, and the absorbance was measured at 593 nm by a Vis spectrophotometer Camspec M107 (Spectronic-Camspec Ltd., UK) against a blank prepared with methanol (Merck, Germany).

Statistical Analysis.

Data for the contents of pigments, phenols, antioxidant activity, and color characteristics were processed to obtain the mean and standard deviation of the mean (SD). One-way analysis of variance followed by a Student's *t*-test was used to compare the mean values. A value of *p* < 0.05 was considered to be statistically significant.

The linear dependences between the studied parameters were obtained by performing a one-way analysis of variance. To estimate the parameters of the regression model, the least squares method was applied. The coefficient of determination was determined and the adequacy of the model was checked using IBM SPS software.
