Optical Properties and Antioxidant Activity of Water-Ethanolic Extracts from Sempervivum tectorum L. from Bulgaria

Abstract

Sempervivum tectorum L. is an evergreen plant belonging to a large family of the Crassulaceae. The fresh juice of the plant is used as a folk remedy almost exclusively for external purposes. The combination of several instrumental methods—transmission and fluorescence spectroscopy, ICP-MS spectrometry, and assays for the assessment of antioxidant activities were used for the characterization of water-ethanolic extracts from the leaves of Sempervivum tectorum L. with different polarities (ethanol content in the extracts varied between 10% and 95%). The highest total phenolic content was found in the 50% ethanol extract—0.84 ± 0.08 mg GAE/mL. Furthermore, this extract also possessed the highest antioxidant potential evaluated by DPPH and FRAP assays—7.02 ± 0.71 mM TE/mL and 6.15 ± 0.25 mM TE/mL, respectively. High correlation coefficients were found between the total phenolic contents and the antioxidant activities of water-ethanolic extracts from Sempervivum tectorum L. The same is true for the strong relationship between the phenolic contents and the concentrations of Na and K. Most likely, the bioavailable species of elements such as Fe, Zn, Ca, and Mg are mostly aqueous soluble. For all the studied extracts, the toxic element (As, Cd, Pb, Tl, Hg) contents are very much below the permissible limits for pharmaceutical products. On the contrary, the concentrations of compounds such as β-carotene and chlorophyll increase with the increase in ethanol in the extract. Results from this study may be used for the preliminary prognosis of pharmaceutical applications of extracts from Sempervivum tectorum L.

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 [2,3,4]. Aromatic and other biologically active substances are extracted from many herbs [5,6,7].

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 [8,9,10]. 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 yet 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 light-emitting 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 the test sample, and the second probe is fixed on top of the drop surface.

Phenolic contents.

Total phenolic contents (TPC) were determined using a Folin-Ciocalteu reagent (Sigma-Aldrich, Germany) [20] with some modifications. Briefly, Folin-Ciocalteu reagent (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 reagent 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 reagent 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.

3. Results

The results for the total phenolic contents and antioxidant activities of the obtained plant extracts are summarized in

Table 1. Total phenolic content (mg GAE/mL) and in vitro antioxidant activities (mM TE/mL) in extracts.

Sample	TPC, mg GAE/mL	DPPH, mM TE/mL	FRAP, mM TE/mL
10% ethanolic extracts	0.49 ± 0.05	4.06 ± 0.47	3.85 ± 0.30
50% ethanolic extracts	0.84 ± 0.08	7.02 ± 0.71	6.15 ± 0.25
95% ethanolic extracts	0.18 ± 0.04	1.86 ± 0.09	1.56 ± 0.47

. Two methods evaluated the antioxidant activities of extracts: DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging ability assay based on mixed hydrogen atom transfer (HAT) and FRAP (ferric reducing antioxidant power) assay based only on a single electron transfer mechanism.

The color coordinates were obtained in two different colorimetric systems XYZ and CIELab. The results are presented in

Table 2. Color parameters in two different colorimetric systems.

Sample	x ± 2.10−4	y ± 2.10−4	a	b
10% ethanolic extracts	0.3314	0.3381	10.25	0.08
50% ethanolic extracts	0.3426	0.3507	14.87	0.32
95% ethanolic extracts	0.3510	0.3762	18.82	−4.98

.

The chromaticity and color tone angle were determined by using the data in Table 2. The brightness L decreases and the chromaticity increases when the ethanol concentration is high. The values for the color tone angle of 10% and 50% water-ethanolic extracts are between 88–90 degrees, but the value is −75 degrees for the 95% ethanol concentration.

Transmission spectra for the investigated samples have been obtained (Figure 1). Chlorophyll and β-carotene are computed based on the transmission spectrum of the sample, and the color characteristics are obtained using the specially developed soft wear application Lovibond™ PFX/PFXi Software Upgrade for Chlorophyll and Beta-Carotene. The average results and standard deviations are shown in

Table 3. Content of pigments in ethanol extracts.

Sample	Chlorophyll Content, ppm	β-Carotene, ppm
10% ethanolic extracts	<DL *	3.51 ± 0.01
50% ethanolic extracts	0.31 ± 0.01	5.89 ± 0.03
95% ethanolic extracts	4.74 ± 0.03	11.12 ± 0.07

* Detection limit (DL) 0.01 ppm.

.

The linear dependencies between the color parameters, obtained in Table 2, and the contents of β-carotene from Table 3 were obtained. They are presented in Figure 2.

The fluorescence spectra of ethanolic extracts from Sempervivum tectorum L. with excitation light of 395 nm, 405 nm, 410 nm, 425 nm, 435 nm, and 450 nm were obtained. The results are presented in Figure 3. In addition, for better fluorescence visualization, excitation-emission matrices of ethanol extracts are shown in Figure 4.

For better visualization, in addition to the individual fluorescence spectra shown in Figure 3a–c, the three-dimensional emission matrices and two-dimensional contours with isolines are presented, representing a universal profile of the so-called fingerprints for selected samples extracts. The matrices presented were obtained after averaging the fluorescence spectra of the samples from a given concentration.

The contents of essential and toxic elements in three extracts were determined by ICP-MS. Results are presented in

Table 4. Concentrations of essential and toxic elements in water-ethanolic extracts (RSD for all samples varied between 2–7%).

Ethanol, %	K, g kg−1	Ca, g kg−1	Mg, g kg−1	Fe mg kg−1	Mn mg kg−1	Zn mg kg−1	Cu mg kg−1	Pb, mg kg−1
10%	10.8	20.0	1.06	123	7.18	9.09	0.83	0.13
50%	11.9	2.96	1.01	35.2	1.46	2.24	0.93	0.07
95%	2.63	0.09	0.07	1.63	0.24	1.35	1.54	0.09
Ethanol, %	Na, mg kg−1	Al, mg kg−1	Ba, mg kg−1	Cr, mg kg−1	Co, mg kg−1	Ni, mg kg−1
10%	13.2	4.53	16.4	0.16	0.19	0.68
50%	26.0	2.57	0.84	0.09	0.05	0.36
95%	10.9	1.26	0.05	<0.05	<0.02	0.04

Tl, As, Cd, and Hg concentrations are below detection limit (0.02 mg kg⁻¹) for all extracts.

.

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 agreement 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 (Figure 3 and Figure 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.

DPPH = 8.405 ∗ TPC, R² = 0.991

(1)

FRAP = 7.4975 ∗ TPC, R² = 0.991

(2)

β-carotene = 0.9026 ∗ b − 6.2895, R² = 0.923

(3)

T = −0.554 ∗ Chlorophyll + 59.527, R² = 0.961

(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⁻¹ Cd and for lead it is 10.0 mg kg⁻¹ 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⁻¹ 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.

5. Conclusions

The highest total phenolic content 0.84 ± 0.08 mg GAE/mL was found in 50% ethanol extract. This extract also has the highest antioxidant potential as assessed by DPPH and FRAP assays. High correlation coefficients were found between total phenolic contents and antioxidant activities of water-ethanolic extracts from Sempervivum tectorum L. The same is valid for the strong relationship between phenolic contents and concentrations of Na and K. Most likely bioavailable species of elements such as Fe, Zn, Ca, and Mg are mostly aqueous soluble. On the contrary, concentrations of compounds like β-carotene and chlorophyll increase with the increase in ethanol in the extract. The contents of toxic elements, As, Cd, Tl, and Pb, are far below the permissible limits for pharmaceutical products. Results from this study may be used for preliminary prognosis of pharmaceutical applications of extracts from Sempervivum tectorum L.