Determination of the health-protective effect of different Sempervivum and Jovibarba species

2.1 Chemicals

For the ICP-OES analysis nitric acid (lach: ner, Bratislava, Slovakia), and hydrogen peroxide (Molar Chemicals, Halásztelek, Hungary) were used for the destruction method. For calibration, mono-element standards for calcium, potassium, magnesium, and phosphorus, and multielement standards for microelements were purchased from CPAchem (Bogomilovo, Bulgaria). The argon gas required for the operation of the ICP-OES device was procured from Messer Hungarogáz Kft (Budapest, Hungary).

Chemicals for the determination of polyphenol, and antioxidant content were 97 % ethanol (Reanal, Budapest, Hungary), anhydrous sodium carbonate (Riedel-de Haen, Seelze, Germany), Folin-Ciocalteu reagent (Merck), 2–4-6-tripyridyl-s-triazine (TPTZ) (Sigma-Aldrich, Budapest, Hungary), acetic acid (Reanal, Budapest, Hungary), anhydrous iron chloride (Merck, Budapest, Hungary), ascorbic acid (Sigma-Aldrich, Budapest, Hungary), and gallic acid (Sigma-Aldrich, Budapest, Hungary).

2.2 Houseleek samples

During the experiments, the total antioxidant and polyphenol content of 22 houseleek varieties were examined using spectrophotometric methods, and their elemental composition was also determined using ICP-OES equipment. Each variety of stone rose was in 3 densely overgrown pots with 10.5 cm diameter. The leaves of the same varieties were collected before drying and then divided into 3 equal parts to determine the dry matter content. All samples were subjected to two types of drying - oven drying (Heraeus T6, Germany) at 40 °C, and lyophilization (Flexy-Dry MP, FTS Systems, USA)- until mass was constant. The dry matter content of each sample was determined gravimetrically. The antioxidant and polyphenol contents were examined for both drying methods in order to determine the effect of the drying process and temperature on the compounds. The composition of micro- and macroelements is not affected by the drying method, so they were analyzed only in the case of drying cabinet samples. The dried houseleek samples were chopped with a coffee grinder (Sencor, SCG 2050RD, FAST Hungary Kft., Hungary). The examined houseleek varieties were the following (Fig. 1):

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Fig. 1

Houseleek varieties in the experiment.

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2.3 Sample destruction

From the dried, and chopped samples, 0.4 g were weighed into a 90 mL tetrafluoromethoxyl (TFM) digestive vessel. To the weighed samples was added 5 mL of 65 % HNO₃, and 1 mL of 30 % H₂O₂. The samples were digested in MARS 6 iWave (CEM Corporation, Matthews, NC, USA) microwave digestion system according to the following program: After a 15-minute heating phase, the destruction was performed at a temperature of 210 °C for 15 min under continuous temperature control. The reaction vessels were cooled, and then the resulting solutions were diluted to a volume of 25 mL with a 0.1 mol/L nitric acid solution. Parallel to the experimental samples, we also prepared blind samples. After digestion, samples were diluted to 25 mL and assayed with ICP-OES.

2.4 ICP-OES analysis

For the determination of the micro-and macroelement composition of the samples an Agilent (Santa Clara, CA, USA) 5110 ICP-OES type equipment was used. We measured the elements shown in Table 2 at the described wavelengths with the settings in Table 1. In the case of the examined macroelements, for the quantitative determination, analytical measuring solutions were prepared in the concentration range of 1–500 mg/L. In the case of microelements, we calibrated in a lower concentration range of 2.5–1000 μg/L.

2.5 Sample preparation for determination of total antioxidant and polyphenol content

To determine the amount of antioxidants and polyphenols in dried houseleek samples, the content of the active ingredient had to be extracted from the matrix by solvent extraction. For extraction, 0.5–0.5 g of samples were weighed into 100 mL Erlenmeyer flasks on an analytical balance and 10 mL of an extraction mixture containing ethanol and water (50:50 V/V%) was added. The extraction was performed at room temperature with a laboratory shaker (Elpan 358 S, Poland) for 2 h at 120 rpm. The extracts were centrifugated at room temperature, 2500g, 20 min, and the filtrate was further analysed. We also examined the total polyphenol and, antioxidant content in the watery decoction (tea) of the plants. For this, 5 g of fresh houseleek leaves were scalded with 250 mL of boiling water. After it cooled down, it was filtered and the filtrate was examined.

2.6 FRAP assay

The FRAP assay procedure is based on the method described by Benzie and Strain (1996). 200 µL of extracted sample or tea, 3 mL of FRAP solution, and 100 µL of water were pipetted into a test tube. The finished solutions were placed in a dark place for 5 min and then their absorbance was measured with a Spectroquant Pharo 100 spectrophotometer (Merck, Germany) at a wavelength of 593 nm against the blank. Ascorbic acid (40–500 mg/L) was used as a standard and the results were expressed as ascorbic acid equivalent (AAE)/ g dry matter.

2.7 Folin-Ciocalteu assay

Determination of total polyphenol content based on the Folin-Ciocalteau method described by Singleton et al. (1999) with some modifications (Barba et al., 2013). To 200 µL of houseleek extract or tea, 1.5 mL of high-purity water was pipetted and the reagents were added. First 2.5 mL of 10 % Folin-Ciocalteu reagent, then 2 mL of 7,5 % Na₂CO₃. The tubes containing the mixture were placed in a dark place for 90 min, and then the absorbance was measured at 725 nm versus the blank. Gallic acid was used as a standard (25–1000 mg/L).

2.8 Data analysis

The total antioxidant, polyphenol, and micro-macroelement contents of houseleek samples and teas were determined in Microsoft Office Excel from the absorbance values measured for the samples using the equation of the second-order least squares analytical curve fitted to the measurement solutions using the nonlinear least-squares method. All the results are expressed as means (n = 3) + / - standard deviation. All measurements were performed in triplicate, and the results are expressed as the mean standard deviation (SD). Analyses of variance (ANOVA) followed by the Tukey post hoc test were used to compare thes ignificant differences in the data. Differences were considered statistically significant when p < 0.05. The statistical analyses were carried out with Microsoft Excel 2013 software.

3.1 Dry matter content of houseleek samples

Table 3 shown the dry matter contents of the houseleek samples after drying to constant mass. In many cases, we detected significant differences in the dry matter content of the examined varieties. The lowest dry matter content had the 'Sempervivum orostachys spinosa' variety with 3.02 m/m% value, in contrast, the 'Sempervivum granat' species had the largest dry matter content (16.79 m/m%). The average moisture content of the plants was 91.21 m/m%. A dry matter content of 10.5 m/m% for Sempervivum tectorum leaves was determined by Mladenović et al. (2021), but in the case of succulent plants, this value also depends on how much moisture they can absorb from the soil and store in their leaves. The examined houseleek varieties were cared for with the same irrigation procedure, so it can be assumed that the differences in the measured dry matter content resulted from morphological differences.

3.2 Microelement content of houseleek samples

Based on the results presented in Table 4, it can be said that the examined houseleek varieties have a high microelement content, which is influenced by the species. Among the microelements included in the study, the highest value is observed in manganese content. 'Sempervivum pilatus' has the lowest manganese content (20.67 mg/kg), while 'Sempervivum orostachys spinosa' contained the highest amount (140.00 mg/kg). Compared to the received concentrations, the recommended daily intake of manganese for an average, adult human body can be 5 mg (Kiss, 2009). The smallest concentration of boron determined in the variety of 'Sempervivum havendijker teufel' (10.19 mg/kg),while the 'Sempervivum calcareum' species (31.58 mg/kg), contained the largest amount. The least amount of copper measured in the variety 'Sempervivum mystic' (7.43 mg/kg), and the most in the 'Sempervivum arachnoideum webbianum aureum' (30.75 mg/kg) species. Regarding zinc, 'Sempervivum mystic' showed the least (16.03 mg/kg), and the variety of 'Sempervivum orostachys spinosa' had the most zinc concentration (85.74 mg/kg). The smallest iron content was obtained in the 'Sempervivum mystic' (13.57 mg/kg) variety, while the highest was in the species of 'Sempervivum havendijker teufel' (67.04 mg/kg). The recommended daily intake is 10–15 mg for iron, 1.1 mg for copper, and 9–10 mg for zinc (Forrai et al. 2020). Compared to the results of the studies presented by Szentmihályi et al. (2004), Turan et al. (2003), and Gentscheva et al., (2021a), we obtained similar results. The elements in the houseleek were better dissolved by the extractant with a higher water content, according to Gentscheva et al., (2021b), so it is assumed that these elements are water-soluble and easily accessed by living organisms.

3.3 Macroelement content of houseleek samples

Just like in the case of the microelement contents, the houseleek contains a large amount of macroelements (Table 5). The content of calcium and sodium should be emphasized. The smallest amount of calcium was detected in the case of 'Sempervivum havendijker teufel' (44.43 g/kg), and the lowest sodium concentration was in the 'Sempervivum gamma' (31.63 g/kg) species. The variety of 'Sempervivum orostachys spinosa' has the most calcium, and sodium content, with 95.27 g/kg and 128.5 g/kg values. Regarding potassium the lowest concentration measured in the variety of 'Sempervivum granat' (10.41 g/kg), and the highest in 'Sempervivum arachnoideum webbianum aureum' (39.43 g/kg). The least magnesium was detected in the species of 'Jovibarba arenaria' (5.82 g/kg), while the most were contained in the variety of 'Sempervivum orostachys spinosa' (13.51 g/kg). Sulfur and phosphorus, samples showed lower amounts. For sulfur, the 'Sempervivum mystic' (1.83 g/kg) variety contains the least, while 'Jovibarba arenaria ostirol' (9.89 g/kg) variety the most amount. We measured the least concentration in the variety of 'Jovibarba arenaria' (1.88 g/kg), while in the species of 'Sempervivum arachnoideum webbianum aureum' (6.05 g/kg) the most phosphorus content. The recommended daily intake is 3.5 g for potassium, 800–1000 mg for calcium, 2 g for sodium, 620 mg for phosphorus, and 300–350 mg for magnesium (Fritz et al. 2019; Forrai et al. 2020). The measured concentrations compared to the result of Szentmihalyi et al. (2004), Turan et al. (2003), Blázovics et al. (2003), and Gentscheva et al., (2021a), get almost similar quantities. The problem with using plants in the food industry is that they can accumulate toxic elements in addition to beneficial elements. Getcheva et al. (2021b) also examined the content of toxic elements in the houseleek extracts using the ICP-MS method, however, the content of titanium, arsenic, cadmium, and mercury was in all cases below the detection limit (0.02 mg/kg) and the highest measured lead concentration was 0.13 mg/kg. Comparing these values with WHO (2007) regulations, according to which the permissible limit value for cadmium in plant samples is 0.3 mg/kg, while for lead is 10 mg/kg, it can be concluded that the stone rose does not accumulate large amounts of toxic elements harmful to health.

3.4 Antioxidant content of houseleek samples

Based on the results of Table 6, it can be said that overall better values were obtained using the lyophilization method, but this is not typical for all varieties. The lowest antioxidant content was measured in the species of 'Sempervivum arachnoideum webbianum aureum' (2.22 mg AAE/g), this sample was dried in the drying cabinet. The most antioxidants were detected in 'Sempervivum manuel' (305.91 mg AAE/g), in the case of all drying methods. For lyophilized samples, ‘Sempervivum orostachys spinosa’ (41.45 mg AAE/g) contains the lowest concentration of antioxidant compounds, while the most was in 'Sempervivum manuel' (317.74 mg AAE/g). Drawing a parallel between the houseleek varieties and the results, it can be concluded that the antioxidant content is influenced by the variety of plants and the used drying method. To our knowledge, there is no comprehensive publication in the literature that compares the antioxidant content of such a large number of houseleek varieties.

The used different drying processes resulted in distinct antioxidant values for the same houseleek species. However in terms of the tested compound groups, in most cases, lyophilization eventuated more favorable results, because it is a gentler drying method, thus caused less damaging to the compounds with heat-sensitive antioxidant effects. However, there were samples where on the contrary, oven drying resulted in higher polyphenol and antioxidant content. The reason for these differences could be that the different drying processes caused different plant cell walls' permeability, thus influencing the extraction efficiency, but the applied drying temperatures could also lead to the formation or decomposition of other compounds. Jankov et al. (2023) investigated the free radical scavenging capacity of the Sempervivum tectorum leaf extract and determined that the most typical phenolic compounds with significant antioxidant effects in the houseleek leaves are kaempferol, kaempferol 3-O-glucoside, quercetin 3-O-glucoside, caffeic acid, and gallic acid.

3.5 Polyphenol content of houseleek samples

The results of the total polyphenol content of the dried samples are shown in Table 7. Just as in the case of antioxidant contents, clearly illustrates that the amount of polyphenolic compounds is influenced by the type of plant, as well as in most cases these values are more favorable for samples dried with a lyophilizer. Mihai et al. (2019) investigated the phytochemical composition of ‘Sempervivum ruthenicum’ and determined that the most typical polyphenolic acids in the plant are gallic acid, chlorogenic acid, caffeic acid, ferulic acid, cinnamic acid, p-coumaric acid, and ellagic acid. The sample with the least polyphenol content was 'Sempervivum arachnoideum webbianum aureum' (5.63 mg GAE/g), which was dried with a drying cabinet. For this drying method, the highest concentration of polyphenols contained 'Sempervivum tectorum var. pyreneum' (144.16 mg GAE/g). In the case of lyophilized samples 'Sempervivum orostachys spinosa' (19.75 mg GAE/g) contained the least amount of polyphenolic compounds, on the other hand, 'Sempervivum ronsdorfer hybride' contained in highest amount (126.45 mg GAE/g). Comparing the examined results with the article of Knez Marevci et al. (2021), similar polyphenol concentrations are seen.

3.6 Antioxidant and polyphenol content of houseleek decoctions

Regarding the total antioxidant and polyphenol content of teas made from different houseleek varieties, it can be clearly established that significantly fewer antioxidant and polyphenol compounds contain, like dried houseleek plants (Table 8). Plant decoctions containing antioxidant compounds in larger quantities, these amounts to each species are closely similar. The most antioxidants (4.61 mg GAE/g) were in the tea of 'Sempervivum arachnoideum webbianum aureum' variety, and the least (2.38 mg GAE/g) was in the 'Sempervivum noir' decoction. Polyphenolic compounds were only present in trace amounts in the teas, higher amounts were contained in the variety of 'Sempervivum tectorum' var. pyreneum' (1.11 mg GAE/g) and 'Sempervivum hey-hey' (0.9 mg GAE/g). Although houseleek tea contains antioxidants, water is not the best extractant to extract these compounds, with other solvents, for example alcohols we could probably achieve better efficiency (Gentscheva et al., 2021b).