Introduction

The Salvia genus, with about 900 different species all around the world, belongs to the very important plant family Lamiaceae1,2. The morphology of Salvia pratensis (Meadow Clary or Meadow Sage) presents an erect herb which forms rosettes. This perennial herb has a wide distribution through Europe, mainly in central Europe. Sage prefers warm temperature and dry environments3,4. The species is capable of adapting to different conditions, changing its characteristics accordingly. However, the Flora Europaea does not recognize infraspecific taxa5,6. Salvia pratensis is categorized as a Temperate Element of the European flora7. As an ornamental species, Salvia is commonly found in gardens4. The species occurs in dry, calcareous grasslands8. Based on its preferred ecological niche, S. pratensis can be found along roadsides, in unmanaged meadows or in pastures5,9. Although it is not considered an alpine species, it has been found at altitudes of up to 1,920 m3. Salvia pratensis is one of the most favored herbs for grazing animals. Different animal species as sheep, deer or cattle feed on the aerial parts of S. pratensis, which aids in the recovery of the grass and supports grow of new shoots10,11. Terpenoids and polyphenols are specific phytochemicals produced by Salvia species, serving as their main secondary metabolites12. The essential oils of “sister” taxa S. officinalis is very popular in traditional medicines4. Research has been conducted on the essential oil hydrodistillated from different samples of S. pratensis to identify the main components which could be used as potential substitutes for S. officinalis in several treatments13. In another study, the quantity of essential oil extracted from S. pratensis was found to be quite low, approximately 0.073% in air-dried inflorescences. A total of 42 components were identified in this essential oil: the sesquiterpene hydrocarbons represented major chemical group (50%). The dominant compound was thymol (30%) following by caryophyllene (25–28%) and p-cymene (9%)14. The S. officinalis extract was found to contain flavonoids, specifically luteolin, quercetin, hispidulin, and apigenin15,16,17. Additionally, phenolic acids such as salvianolic, rosmarinic, sagerinic, caffeic acids, and sagecoumarin were identified in this extract17. In contrast to S. officinalis, S. pratensis remains relatively underexplored in scientific inquiry. However, recent investigations into the biological potential of essential oils derived from S. pratensis have shown promising results particularly, noting a greater antimicrobial efficacy exhibited by the essential oil of S. pratensis compared to that of S. officinalis1,18. Commercial herbicides are widely used for weed management in agricultural systems due to their cost-effectivness and long-standing tradition. Their primary objective is the eradication of unwanted plant species. The effectiveness of herbicides is influenced by various factors including plant species, environmental conditions, weather patterns, and the specific type of herbicide employed19,20,21. Sustainable agricultural practices encompass a variety of approaches adopted by farmers. Advances in biotechnology have facilitated the development of climate-resilient plant varieties high-quality seeds, and other reproductive plant parts, leading to reduced reliance on synthetic pesticides and promoting greater sustainability. Recent attention has been directed towards eco-herbicides, natural products derived from plant secondary metabolites, reflecting a growing interest in environmentally friendly weed control methods22,23. Exploring the chemical composition of lesser-known plants offers a valuable opportunity to discover novel components with herbicidal properties. Within the Lamiaceae family, certain plant species are distinguished by their potent essential oils (EOs) which exhibit a wide range of biological activities, including antioxidant, anti-inflammatory, antimicrobial, fungicidal, insecticidal, and notably, herbicidal properties24,25,26,27,28. Extracts from Lamiaceae species contribute to almost 50% of bioherbicidal activity25. Both water and alcohol extracts from these plants also contain secondary metabolites, with polyphenols and flavonoids being the predominant groups29. Various classes of secondary metabolites such as terpenoids, alkaloids, and flavonoids play a crucial role in bioherbicidal activity25. Phenolic compounds, including anthocyanins, flavonoids, and phenolic acids, among others, are biologically active substances29, making them a focus of research for new commercial bioherbicides. Our research revealed that S. pratensis possesses a relatively low amount of essential oil. Consequently, alternative extracts were employed for biological assays to evaluate potential phytotoxic and antioxidant activities. Furthermore, pioneering analyses utilizing Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) were conducted on S. pratensis extracts for the first time to compare and correlate them with observed biological activities.

Results

Dry mass

Various extraction methods applied to Salvia pratensis L. plant material yielded different amounts of dry mass. The highest dry mass (10.1 mg/mL) was registered in sample W60. Approximately half of this amount (5.6 mg/mL) was obtained from sample S3, which underwent extraction using an alcohol solution via Soxhlet extraction. Sample S1 and S2 produced similar dry mass of 4.4 mg/mL and 4.0 mg/mL; the lowest amount was obtained in sample S4 (2.7 mg/mL). The extracts mutually differed significantly in dry mass content (Table 1).

Table 1 Content of dry mass in different types of extracts from Salvia pratensis.
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Phytochemical parameters of extracts

All extracts were tested for their content of phenolic (TPC) and flavonoids (TFC) compounds. The results are shown in Table 2. The levels of rosmarinic acid, the predominant phenolic component in all extracts, were determined through HPLC analysis (Fig. 1), as also detailed in Table 2. The choice of extraction method significantly influenced the concentration of phenolic compounds in the extracts. The extracts obtained using the Soxhlet apparatus, particularly those utilizing higher ethanol concentration in the extraction solvent (30% and 70%), exhibited elevated levels of TPC, TFC, and rosmarinic acid. Additionally, the W60 extract displayed a notable phenolic compound content, likely influenced by the ratio of plant material weight to extract volume rather than the extraction method itself (water extraction at 60 °C for one hour in a water bath). When considering the different dry masses of the extracts, extract S3 demonstrated higher levels of total phenols, total flavonoids, and rosmarinic acid compared to extract W60.

Fig. 1
figure 1

HPLC-DAD chromatogram of Salvia pratensis flowering shoot extract.

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Table 2 Content (mean ± standard deviation) of phenols, flavonoids, and rosmarinic acid in Salvia pratensis extracts.
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DPPH· radical scavenging activity

In the free DPPH• radical scavenging assay, extract S3 demonstrated the highest effectiveness, with its IC50 value closely resembling that of reference antioxidant ascorbic acid. Conversely, extract W60 exhibited the lowest antioxidant activity against DPPH• radicals (Table 3). The phenolic constituents present in the extracts likely contribute to their DPPH• radical scavenging ability. Among all the extracts analyzed, extract S3 stood out for its elevated levels of total phenols, total flavonoids, and rosmarinic acid in dry matter. Notably, rosmarinic acid emerged as a potent antioxidant, displaying superior DPPH• scavenging activity compared to ascorbic acid.

Table 3 DPPH• radical scavenging activity (mean ± standard deviation) of Salvia pratensis extracts and reference standard compounds, ascorbic acid and rosmarinic acid.
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Phytotoxic activity

Among all the Salvia extracts tested, the 10% alcoholic extract (S2) demonstrated the highest efficacy, as no germination was observed in any of the four model organisms after applying the highest doses of 50 mL and 100 mL, corresponding to 2.2 mg/mL and 4.0 mg/mL dry mass (DM), respectively (Table 4). Complete inhibition of germination was also observed in R. sativum and S. alba with a lower dose of 25 mL corresponding to 1.1 mg/mL DM. At the dose of 10 mL (0.6 mg/mL DM), the germination of R. sativum and S. alba was significantly lower in comparison to the control group. Among the model organisms, R. sativum exhibited the highest sensitivity, displaying markedly reduced germination rates with all concentrations of the 10% salvia alcoholic extract (S2). In contrast, the germination of H. vulgare and T. aestivum remained unaffected by the Salvia extract S3, except for a slight impact on H. vulgare at a dose of 100 mL (5.6 mg/mL DM).

Both R. sativum and S. alba showed significantly lower germination in comparison to the control group when exposed to extract doses with a DM content exceeding 0.7 mg/mL, a trend also observed with the S4 extract. This extract affected the germination of H. vulgare and T. aestivum compared to the control only at the highest dose of 100 mL (2.7 mg/mL DM). The W60 extract did not show any antigerminative effect on either H. vulgare seeds or T. aestivum seeds compared to the control; only the 100 mL dose (corresponding to 10.1 mg/mL DM) significantly reduced germination of the seeds of S. alba and R. sativum. This same dose significantly affected root growth across all model organisms except for H. vulgare. The extract S1 displayed effectiveness solely on R. sativus: the dose of 100 mL, corresponding to 4.4 mg/mL DM, caused a significantly lower % of germinated seeds in comparison to the control (Table 5). Concerning root growth, the extract S2 was the most effective of all the Salvia extracts used. Generally, water-based extracts (W60 and S1) exhibited lower phytotoxic effects compared to alcohol-based extracts (S2, S3, S4), with the water extract obtained at 60 °C (W60) showing greater efficacy than that obtained at 100 °C (S1) on H. vulgare and T. aestivum seeds. A similar pattern was observed in S. alba and R. sativum, where the extract S1 was significantly more effective than the W60 extract (Table 5).

Table 4 Seed germination (%) of model plants after exposure to different doses of Salvia pratensis extracts prepared in different ways.
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Table 5 Roots length (cm) after exposure to different doses of Salvia pratensis extracts prepared in different ways.
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PCA and HCA analysis

The different effects of water vs. alcoholic extracts were also confirmed by PCA and HCA analyses. Thirteen original variables were compared using PCA, which were then condensed into two principal components, collectively representing 84.1% of the total variability. PC1 accounts for the primary component, explaining 53.9% of the total variability, while PC2 represents the secondary component and explains 30.1% of the total variability (Table 6).

Table 6 Loadings of the significant variables on two first principal components from data analysis.
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PC1 (53.95%) was represented mainly by almost all variables related to phytotoxic activity in the positive scores, with a minor contribution from DPPH• IC50 and dry biomass in the negative scores. On the other hand, PC2 (30.10%) was primarily characterized by positive scores for most variables, except for Sinapis alba % inhibition of root length and Raphanus sativus % inhibition of root length, which were positioned in the lower right quadrant on the negative side of PC2. The scores of the extracts exhibited a strong correlation, with samples S2, S3, and S4 clustered on the positive side of PC1, indicating higher inhibition percentages of germination and root length in R. sativus and S. alba. Conversely, the similarity between S1 and W60 was also evident because these two extracts displayed the highest scores on the negative side of PC1 (Fig. 2).

Fig. 2
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Biplot (loading and scores plots), with 95% confidence ellipse, obtained by principal component analysis (PCA) of five extracts of Salvia pratensis based on the thirteen different variables in the two-dimensional space. The vectors shown are the eigenvectors of the covariance matrix.

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In the Hierarchical Cluster Analysis (HCA) (Fig. 3), the Euclidean distance between groups revealed two distinct clusters with varying levels of similarity. The first cluster, with a similarity measure of less than 3.73, comprised extracts S2, S3, and S4, distinguished by their pronounced effects on seed germination and root length. The second cluster, with a similarity level greater than 3.75, consisted of extracts W60 and S1, characterized by their lesser impact on seed germination and root length.

Fig. 3
figure 3

Dendrogram obtained by HCA based on the Euclidian distances between 5 different.

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Discussion

Recent studies have corroborated the potential utilization of aqueous herbal extracts, particularly those sourced from S. officinalis leaves, as natural herbicides against weeds30. Prior evaluations of the phytotoxicity of various extracts from Calamintha nepeta L. (Savi), also belonging to the Lamiaceae family, have indicated that different classes of molecules, including phenolic compounds, may be involved in plant-plant allelopathy, suggesting their possible use as promising herbicides31. In the water extract of Eucalyptus globulus several phenolic compounds (such as ellagic, two ρ-coumaric derivatives, hyperoxide, chlorogenic, quercitrin, rutin and kaempferol 3-O-glucoside) along with five low weight organic acids (succinic, fumaric, citric, shikimic and malic acids) were identified by HPLC analyses and were tested for the herbicidal activity23. The antioxidant activity of extracts (water/methanol solution) and bioactive compounds determination in S. pratensis were previously investigated29. The content of polyphenols was about 400 mg GAE/100 g FW, and the DPPH• value was about 10 µmol TE/g FW, values that align closely with those reported in this study. For related species such as Salvia tesquicola and Salvia verticillata, phenolic levels in water-bath extracts ranged from 35.66 to 43.9 mg/g dry mass (DM)32.

In the current study, phenolic content varied depending on the extraction method: water extracts yielded lower phenolic amounts (W60 = 91.1 ± 1.9 GAE mg/g DM; S1 = 88.1 ± 1.3 GAE mg/g DM) compared to alcohol extracts (S2-S4 ranging from 95.7 ± 1.9 GAE mg/g DM to 148.4 ± 2.6 GAE mg/g DM). The DPPH· IC50 values in this study ranged from 15.6 ± 0.4 µg/mL DM (in S3) to 37.1 ± 0.2 µg/mL DM (in W60). Similar studies on other Salvia species, such as Salvia officinalis, reported high polyphenol content and significant antioxidant capacity16. The antioxidant capacity determined in Salvia extracts varied significantly based on growth locality, with total phenols ranging from 5502.0 to 7787.5 mg RA/100 g DM and relatively consistent DPPH• values between 170.5 and 175.5 µmol TE/100 g. The main polyphenols identified in S. officinalis leaves included rosmarinic acid (2460–3844 mg/100 g) and lutolin-3-glucuronide (634–840 mg/100 g)16.

In a previous study, the primary compounds identified in the methanol extract of Salvia officinalis were 12-methoxy carnosic acid (21,918.3 µg/g) and rosmarinic acid (17,678.7 µg/g), determined using UHPLC coupled with QTOF-MS17. In our study, rosmarinic acid was also a dominant compound, with values ranging from 15.9 ± 0.2 mg/g DM in the W60 extract to 43.2 ± 0.3 mg/g DM in the S3 extract. These findings suggest that the extraction method significantly impacts the composition of the extract. Also, biological properties were linked to extraction techniques: both antioxidant and antifungal properties were positively correlated to the phenolic composition of S. officinalis, as previously reported15. The phenolic compounds were identified in plant water extracts and various studies noted their phytotoxic effect against model species33,34.

The bioassay involved using one monocot species (Agrostis stolonifera L.) and one dicot species (Lactuca sativa L.) as model plants to assess the herbicidal activity of the water extract from Eucalyptus globulus Labill. Secondary metabolites produced by E. globulus can be naturally released from leaf into the soil and influence the germination and development of neighboring plant species through allelopathic interactions23. Plant-plant interactions are driven by the production and release of secondary metabolites, which can exert positive or negative impacts on each other35. These secondary metabolites can inhibit the growth and development of neighboring plants when released from the mother plants into the environment36. The variations in the allelopathic mechanisms among different plant species are based on their phytotoxic compounds37,38. The modern analytical chromatographic techniques have significantly advanced in recent years, enhancing the chemical identification of phenolic compounds39. A study involving 81 medicinal plant species from Pakistan across 39 families revealed that 66 extracts exhibited growth inhibitory effects on model plants, while 15 extracts stimulated lettuce growth40. Similarly, a separate study conducted in Iran assessed the herbicidal effects of extracts from 68 medicinal plants, with 57 showing herbicidal impacts on model plants and 11 notably stimulating the growth of lettuce seedlings41.

In Bangladesh, water extracts from 55 medicinal plants were tested on Raphanus sativus at four different concentrations. Six plant species demonstrated complete inhibition (100%) of R. sativus growth at a concentration of 1:5 (w/v), while another 15 species showed 95% of inhibition at the same concentration42. Many studies, investigating the impact of plant extract activity on different weeds, reported diverse levels of effectiveness, with monocot plant species generally being more resistant than dicots43,44,45. Additionally, various crops were often affected to a lesser extent than weeds44. The dosage of potential herbicides applied to crops plays a crucial role in their impact; however, existing studies have not provided a clear explanation for the varying sensitivities observed among different crops. The current research endeavors to address this gap by elucidating the mechanisms of action associated with various phytochemicals. Understanding these mechanisms is vital for advancing knowledge in this field and developing effective natural herbicides25.

Conclusions

Our study focused on the potential biological activity of the less investigated species S. pratensis compared to very well-studied S. officinalis. While the biological activities of essential oils from various plants are well documented, S. pratensis contains less than 0.1% of EO, making it challenging to work with this EO. Therefore, different types of extracts prepared using water and alcohol were compared. An elementary study of composition, of antioxidant activity and of potential herbicidal activity was carried out. Active components extracted by using 10% ethanol were most active against the dicot plant species in the highest doses while the influence on monocot species was not significant. The mean identified amount of dry mass extracted from plant material as well as the mean content of phenols or flavonoids were in correlation of herbicidal activity. We can conclude that the phenolic compounds and potential of antioxidant activity do not play a crucial role in phytotoxicity of extracts from S. pratensis. Another chemical compound, or currently not well understood mode of action was responsible for positive biological activity. Finally, S. pratensis is still a little-studied plant species, overshadowed by its relative S. officinalis, but it is certainly a rich source of phytochemicals that deserve to be studied for different biological activities. The PCA and HCA analyses, also conducted for the first time on S. pratensis extracts, corroborated the results obtained by biological assays.

Methods

Plant materials

The fresh aboveground parts of Salvia pratensis L. were collected during the flowering period from a meadow along the Torysa river in Prešov, Eastern Slovakia (48° 59’ 22.6855183” N, 21° 13’ 56.8595123” E) in August 2019. The freshly collected plant material was laid out on filter paper in the laboratory and stored at room temperature until it dried. Once dried, the plant material was ground into a powder using an electric mill. This prepared sample was utilized for the preparation of various extracts and subsequent analysis. The plant material is stored in the herbarium collection at the Department of Ecology, University of Prešov under the number: Eko_2019/8_SalviaPratensis. The identification of plant species was conducted identified by Dr. Daniela Grulova. The material deposited is available to the public upon request. The experiment compiled with relevant institutional, national, and international guidelines and legislation.

Extracts preparation

One extract (W60) was prepared using a water bath. Twenty grams of plant powder were mixed with 200 mL of distilled water in an Erlenmeyer bank (EB). The EB was placed in water bath maintained at 60 °C, with a cooler placed on top of the flask to avoid water evaporation. The extraction lasted for 1 h. After cooling, the extract was filtrated by using a Büchner funnel connected to a vacuum pump (V-700, Vacuum Pump, Büchi). This filtration process was repeated twice. After filtration, the extracts were also centrifuged for 30 min at 6000 rpm. The water extract was stored in a freezer until analysis. Four different extracts (S1 – S4) were prepared using the Soxhlet apparatus. A paper cartridge was filled with 10 g of plant powder. The following solutions were used as extraction agents: distilled water (S1), a 10% ethanol solution in water (S2) (v/v), a 30% ethanol solution in water (S3) (v/v), and a 70% ethanol solution in water (S4) (v/v). Three hundred mL of each solution was used for seven cycles of plant extraction. After that, each extract was filtered using the same method described above, and then centrifugated. The extracts prepared with different alcohol solutions were subsequently placed in a vacuum evaporator to remove any remaining alcohol, ensuring it would not impact on the biological assays.

Dry mass evaluation

One mL of each extract was put into a glass Petri dish and placed into the oven at 105 °C until the constant weight was achieved, indicating complete water evaporation. This process was carried out to determine the dry mass of the extracts. Each determination was repeated four times.

Table 7 Preparation of the extract solution for the evaluation of phytotoxic activity.
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Determination of total phenolic content

The phenolic components in the extracts were determined using the Folin-Ciocalteu method46, with some modifications. Specifically, 0.1 mL of appropriately diluted extract was mixed with 0.2 mL of Folin-Ciocalteu phenol reagent, 2 mL of double distilled water (DDW), and 1 mL of a 20% (w/v) Na2CO3 solution. The resulting reaction mixture was kept in the dark at room temperature for 90 min. Absorbance was measured at a wavelength of 765 nm using a Shimadzu UV-1800 spectrophotometer, with DDW serving as the blank. A calibration curve of gallic acid (Merck) was also established for quantification. The results were expressed as gallic acid equivalents (GAE) per mL of extract, and each measurement was repeated four times for accuracy. Fresh extracts were prepared on the day of measurement to ensure the reliability of the results.

Determination of total flavonoid content

Total flavonoid content was determined by aluminum chloride (AlCl2) colorimetric method47, with slight modifications. Briefly, 0.2 mL of appropriately diluted extract, 1.8 mL of DDW, 0.1 mL of AlCl2 water solution (10%, w/v), 0.1 mL of 1 M CH3COOK, and 2.8 mL of DDW were kept in the resulting reaction mixture for 30 min at room temperature. The absorbance of the mixture, at a wavelength of 415 nm, was measured on a Shimadzu UV-1800 spectrophotometer using a corresponding blank in which AlCl2 solution was replaced by DDW. A calibration curve for quercetin (Sigma-Aldrich) was prepared for quantification, and the results were expressed as quercetin equivalents (QE) per mL of extract. Each measurement was repeated four times for accuracy, and extracts were prepared freshly on the day of measurement.

HPLC-DAD analysis of rosmarinic acid

The extracts were firstly filtrated through a syringe filter (Whatman, Puradisc 130, nylon membrane, 45 μm) before being analyzed by gradient reversed-phase High-Performance Liquid Chromatography (HPLC) using a Dionex UltiMate 3000 Quaternary Analytical System equipped with a diode array detector and a Dionex Acclaim 120 C18 column (5 μm, 250 × 4.6 mm), maintained at 25 °C. The mobile phase A (HPLC gradient grade water with 0.1% formic acid, v/v) and mobile phase B (HPLC gradient grade acetonitrile with 0.1% formic acid, v/v) were used at a 1.0 mL/min according to the gradient program: 0 min, 5% B; 0–50 min, 5–30% B; 50–53 min, 30–100% B; 53–58 min, 100% B; 58–60 min, 100–5% B; 60–65 min, 5% B. The peak of rosmarinic acid was identified based by comparison on the retention time and UV-VIS spectra of the corresponding standard compound (Sigma-Aldrich). The peak area values, obtained at a wavelength of 320 nm, were used for quantitative evaluation. The content of rosmarinic acid was calculated by means of a five-point calibration curve (30–500 µg/mL, R2 > 0.999).

DPPH· (2,2-Diphenyl-1-picrylhydrazyl) radical scavenging assay

The DPPH• assay was used to determine the free radical scavenging ability of the extracts48. The details can also be found in our previous publication49. Ascorbic acid (Sigma-Aldrich) was used as a reference antioxidant. Rosmarinic acid (Sigma-Aldrich), the major phenolic compound found in the extracts, was also assayed. The half maximal inhibitory concentration (IC50) value was calculated (g DW/mL). The assay was repeated four times. All solutions were used on the day of preparation.

Biological assay to evaluate phytotoxic effect of extracts

Model plants

Four model plant seeds were used for the evaluation of phytotoxic activity: two monocot species, Hordeum vulgare L. (barley) and Triticum aestivum L. (winter wheat), as well as two dicot plant species, Sinapis alba L. (white mustard) and Raphanus sativus L. (radish). The seeds were obtained from the Breeding Research Center in east Slovakia (Malý Šariš).

Phytotoxic activity

The experimental design was established based on previously reported methods50, with some modifications. The factors considered in the experiment included: (i) model plants: [dicots: radish, white mustard, and monocots: winter wheat and barley]; (ii) different extracts of Salvia pratensis; and (iii) different extract concentrations of these extracts. Extracts were prepared with a primary determined dry mass (DM) and mixed with distilled water to create different concentrations (Table 7). Distilled water was used as control. Before the bioassay, seeds were surface sterilized with 95% EtOH for 15 s and then rinsed three times with distilled water. Ten seeds were placed into each Petri dish (90 mm diameter), containing 5 layers of Whatman filter paper, to which 7 milliliters of extracts of different concentration were added. Each variation was replicated three times. The Petri dishes were then placed in the phytochamber (Sanyo, MLR-351 H) under controlled conditions (20 ± 1 °C, 16 h light, 8 h dark). The evaluation of germination and the radicle length (cm) were measured after 5 days. The influence of extracts was declared as inhibitory (I) or stimulatory (S) effect in comparison to control.

Statistical analysis

General differences in the germination percentages and roots length (i) between control and experiments, (ii) between experiments mutually and (iii) between different type of extracts were statistically processed by using Student´s T-Test and the significance was determined in three different levels (p < 0.05; p < 0.01; p < 0.001). Student´s T-Test was provided in Excel, for all other statistical analyses were used software PAST, Version 4.0351. Principal Components Analysis (PCA) and Hierarchical Cluster Analysis (HCA) were provided by using Matlab software and were used to assess the similarity between different extracts of Salvia pratensis based on the following original variables: total phenols (GAE mg/mL), total flavonoids (QE mg/mL), rosmarinic acid (mg/mL), DPPH IC50 (mg/mL DM), dry biomass mg/mL, and percentage inhibitions of germination and root length for four model plants (Hordeum vulgare, Triticum aestivum, Sinapis alba, Raphanus sativus. The results of the MTS-assay analysis express the mean value with addition of standard deviation (SD) from two independent experiments. Significant differences were determined using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test in GraphPad Prism 9.0.0 software. The level of significance was determined in three different levels: significant (*) for p-value ≤ 0.05, very significant (**) for p-value ≤ 0.01, extremely significant for p-value ≤ 0.001 (***), and not significant (ns).