Phytochemical Characterization, and Antioxidant and Antimicrobial Properties of Agitated Cultures of Three Rue Species: Ruta chalepensis, Ruta corsica, and Ruta graveolens

The in vitro cultures of the following three species of the genus Ruta were investigated: R. chalepensis, R. corsica, and R. graveolens. The dynamics of biomass growth and accumulation of secondary metabolites in the 3-, 4-, 5-, 6-, and 7-week growth cycle were analysed. The antioxidant capacity of the methanol extracts obtained from the biomass of the in vitro cultures was also assessed by different in vitro assays: 1,1-diphenyl-2-picrylhydrazil (DPPH), reducing power, and Fe2+ chelating activity assays. Moreover, a preliminary screening of the antimicrobial potential of the extracts was performed. The extracts were phytochemically characterized by high-performance liquid chromatography (HPLC), which highlighted the presence of linear furanocoumarins (bergapten, isoimperatorin, isopimpinellin, psoralen, and xanthotoxin) and furoquinoline alkaloids (γ-fagarine, 7-isopentenyloxy-γ-fagarine, and skimmianine). The dominant group of compounds in all the cultures was coumarins (maximum content 1031.5 mg/100 g DW (dry weight), R. chalepensis, 5-week growth cycle). The results of the antioxidant tests showed that the extracts of the three species had varied antioxidant capacity: in particular, the R. chalepensis extract exhibited the best radical scavenging activity (IC50 = 1.665 ± 0.009 mg/mL), while the R. graveolens extract displayed the highest chelating property (IC50 = 0.671 ± 0.013 mg/mL). Finally, all the extracts showed good activity against Staphylococcus aureus with MIC values of 250 μg/mL for the R. corsica extract and 500 μg/mL for both R. graveolens and R. chalepensis extracts.


Introduction
The genus Ruta belongs to the Rutaceae family [1]. The best-known species of the genus are Ruta graveolens L. and Ruta chalepensis L. Other rue species are less known and may occur, for example, as endemic species such as Ruta corsica D. C. [2]. The individual rue species differ slightly in terms of morphology.
R. graveolens is a plant whose natural habitats were originally located in south-eastern and eastern Europe, and the species then spread to the area of southern Europe and the African coast of the Mediterranean Sea. R. chalepensis is a common species on the Mediterranean coast. It grows on the Italian, Albanian, Greek, Spanish and French coasts, as well as in North Africa (e.g., in Algeria and Libya). It also grows in the areas of India, Latin America, and Western Asia, including countries such as Syria, Iran, Turkey, and Israel. R. corsica is a species endemic to the Corsican mountains [3,4].
The chemical composition of the investigated rue species has been studied to varying extents. The chemical composition of R. graveolens is well known, while that of R. corsica is least known.
R. graveolens exhibits a broad spectrum of activity, such as antioxidant, anti-inflammatory, spasmolytic, sedative, antibacterial, antifungal, and antidiabetic effects [9,10]. R. graveolens is a valuable source of linear furanocoumarins (derivatives of psoralen) that have been used for treating skin diseases such as vitiligo and psoriasis in the so called PUVA therapy (psoralens and UVA irradiation) [11,12].
In vitro plant cultures can provide high-quality plant material for pharmaceutical or cosmetic purposes. In addition, the use of natural products as screening libraries has recently come of interest and may contribute to the advancement of drug discovery [19]. In vitro cultures of R. graveolens are a convenient material for conducting multidirectional research because they exhibit a significant increase in biomass within a short time and show a high capacity for organogenesis and accumulation of many secondary metabolites. Therefore, numerous studies are conducted to determine the quantitative and qualitative composition of the biomass from in vitro cultures of this plant species and subspecies R. graveolens ssp. divaricata [20][21][22][23][24][25].
The cultures of other Ruta species have not been thoroughly investigated. There are few reports on the chemical constituents in R. chalepensis in vitro cultures [26,27]. However, there are no studies on the in vitro cultures of R. corsica in the available literature. Therefore, the present study aimed to investigate the biosynthetic, antioxidant, and antimicrobial potential of the in vitro cultures of R. chalepensis and R. corsica and to compare them with the potential of cultures of the most known species, R. graveolens.

In Vitro Cultures
The shoot cultures of Ruta ssp. were initiated in the Department of Pharmaceutical Botany UJ CM in Cracow: R. graveolens-from hypocotyl segments of sterile seedlings derived from the Botanical Garden at the Purkyny University in Brno (Czech Republic); and R. chalepensis and R. corsica-from seeds derived from the Botanical Garden at the Maria Skłodowska-Curie University in Lublin (Poland).
The cultures were maintained in Erlenmeyer flasks (500 mL), containing 150 mL medium. Initial biomass was 1 g. Cultures were performed on a shaker (Altel) with a rotation frequency of 140/min. The cultures were maintained under artificial light with an intensity of 4 W/m 2 at 25 ± 2 • C. The cultures were performed in Linsmaier and Skoog (LS) medium with the following content of plant growth regulators: NAA-0.1 mg/L; and BAP-0.1 mg/L. The culturing period was 3, 4, 5, 6, and 7 weeks (3 replicates were performed for each species in each week). After this period the biomass was separated from the medium and dried at 38 • C.

Free Radical Scavenging Activity
The free radical scavenging activity of the Ruta spp. extracts was determined using the DPPH (1,1-diphenyl-2-picrylhydrazyl) method [30]. The extracts were tested at different concentrations (0.0625-2 mg/mL). An aliquot (0.5 mL) of the methanol solution containing different amounts of sample solution was added to 3 mL of daily prepared methanol DPPH solution (0.1 mM). The change in optical density at 517 nm was measured 20 min after the initial mixing by using a model UV-1601 spectrophotometer (Shimadzu). Butylated hydroxytoluene (BHT) was used as a reference. The scavenging activity was measured as the decrease in the absorbance of the samples versus the DPPH standard solution. Results were expressed as radical scavenging activity percentage (%) of the DPPH, defined by the formula ((A o −A c )/A o ) × 100, where A o is the absorbance of the control and A c is the absorbance in the presence of the sample or standard.
The results obtained from the average of three independent experiments are reported as the mean radical scavenging activity percentage (%) ± standard deviation (SD) and mean 50% inhibitory concentration (IC 50 ) ± SD.

Reducing Power Assay
The reducing power of the Ruta spp. extracts was evaluated by spectrophotometric detection of Fe 3+ -Fe 2+ transformation [31]. The extracts were tested at different concentrations (0.0625-2 mg/mL). Varying amounts of samples in 1 mL solvent were mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of 1% potassium ferricyanide (K 3 Fe(CN) 6 ). The mixture was incubated at 50 • C for 20 min. The resulting solution was cooled rapidly, mixed with 2.5 mL of 10% trichloroacetic acid, and centrifuged at 3000× g rpm for 10 min. The resulting supernatant (2.5 mL) was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% fresh ferric chloride (FeCl 3 ), and the absorbance was measured at 700 nm after 10 min. The increased absorbance of the reaction mixture indicates an increase in reducing power. As a blank, an equal volume (1 mL) of water was mixed with a solution prepared as described above. Ascorbic acid and BHT were used as a reference. The results, obtained from the average of three independent experiments, are expressed as the mean absorbance values ± SD. The reducing power was also expressed as the ascorbic acid equivalent (ASE/mL).

Ferrous Ions (Fe 2+ ) Chelating Activity
The ferrous (Fe 2+ ) ion chelating activity of Ruta spp. extracts was estimated by measuring the formation of the Fe 2+ -ferrozine complex, according to the method described previously [32]. The extracts were tested at different concentrations (0.0625-2 mg/mL). Briefly, varying concentrations of each sample in 1 mL solvent were mixed with 0.5 mL of methanol and 0.05 mL of 2 mM FeCl 2 . The reaction was initiated by adding 0.1 mL of 5 mM ferrozine. The mixture was then shaken vigorously and left to stand at room temperature for 10 min. The absorbance of the solution was measured spectrophotometrically at 562 nm. The control contained the FeCl 2 and ferrozine complex. Ethylenediaminetetraacetic acid (EDTA) was used as a reference. The percentage of inhibition of the ferrozine-(Fe 2+ ) complex formation was calculated by the formula ((A o − A c )/A o ) × 100, where A o is the absorbance of the control and A c is the absorbance in the presence of the sample or standard. The results obtained from the average of three independent experiments are reported as mean inhibition of the ferrozine-(Fe 2+ ) complex formation (%) ± SD and IC 50 ± SD.

Antimicrobial Activity
The antimicrobial properties of the Ruta spp. extracts were tested against the following microbial strains: Staphylococcus aureus ATCC 65381, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, and the yeast Candida albicans ATCC 10231; these strains were obtained from the in-house culture collection of the Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina (Italy). Overnight cultures of the bacterial strains and C. albicans were grown at 37 • C in Mueller-Hinton Broth (MHB, Oxoid) and Roswell Park Memorial Institute (RPMI) 1640 medium, respectively. The Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal/Fungicidal Concentration (MBC/MFC) of the extracts were established according to the guidelines of the Clinical and Laboratory Standards Institute [33,34], with some modifications. The methanol extracts were dissolved in dimethyl sulfoxide (DMSO) and further diluted using MHB or RPMI 1640 to obtain a final concentration of 1 mg/mL. Two-fold serial dilutions were prepared in a 96-well plate. The tested concentrations ranged from 500 to 0.49 µg/mL. Working cultures were adjusted to the required inoculum of 1 × 10 5 CFU/mL for bacteria and 1 × 10 3 CFU/mL for yeast. Growth controls (medium with the inoculum but without the extracts) and vehicle controls (medium with the inoculum and DMSO) were also included. The solvent (DMSO) did not exceed 1% concentration. Tetracycline (TC), as a positive control, was tested against all the bacterial strains at concentrations ranging from 32 to 0.016 µg/mL, while itraconazole (ITC) was tested against the yeast. The MIC of TC was considered as the lowest concentration of the extracts at which there was no bacterial growth. The MIC of ITC was defined as the lowest drug concentration that inhibited ≥50% growth as compared to the control [35]. To determine MBC/MFC values, bacterial or yeast aliquots (10 µL) were taken from each well and cultured on Mueller-Hinton Agar (MHA, Oxoid) or Sabouraud Dextrose Agar (SDA, Oxoid), respectively. The cultures were incubated for 24-48 h at 37 • C.

Statistical Analysis
Statistical comparison of data was performed using one-way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparisons test (GraphPAD Prism Software for Science LLC, San Diego, CA, USA). p-values less than 0.05 were considered statistically significant.

Biomass Increments
The in vitro cultures of R. graveolens, R. corsica, and R. chalepensis were maintained as agitated shoot cultures in a 7-week growth cycle (inoculum mass: 1 g). The cultures were characterized by a similar increase in biomass. The cultures showed a good biomass growth up to the fifth week of cultivation, except for R. graveolens cultures, which reached their maximum growth after four weeks of breeding. In the sixth week the growth gradually stopped, and after 7 weeks of cultivation, the shoots started to show signs of dieback. The best (33.7-fold) increase in biomass was observed in R. chalepensis cultures after 5 weeks of cultivation. For R. graveolens and R. corsica, the following biomass increase was recorded: 33.3-fold (after 4 weeks) and 31.3-fold (after 5 weeks), respectively ( Figure 1). fold serial dilutions were prepared in a 96-well plate. The tested concentration from 500 to 0.49 μg/mL. Working cultures were adjusted to the required inoculu 10 5 CFU/mL for bacteria and 1 × 10 3 CFU/mL for yeast. Growth controls (medium inoculum but without the extracts) and vehicle controls (medium with the inocu DMSO) were also included. The solvent (DMSO) did not exceed 1% concentratio cycline (TC), as a positive control, was tested against all the bacterial strains at co tions ranging from 32 to 0.016 μg/mL, while itraconazole (ITC) was tested against The MIC of TC was considered as the lowest concentration of the extracts at wh was no bacterial growth. The MIC of ITC was defined as the lowest drug conce that inhibited ≥50% growth as compared to the control [35]. To determine MBC/M ues, bacterial or yeast aliquots (10 μL) were taken from each well and cultured on Hinton Agar (MHA, Oxoid) or Sabouraud Dextrose Agar (SDA, Oxoid), respectiv cultures were incubated for 24-48 h at 37 °C.

Statistical Analysis
Statistical comparison of data was performed using one-way analysis of (ANOVA) followed by Tukey-Kramer multiple comparisons test (GraphPAD Pr ware for Science LLC, San Diego, USA). p-values less than 0.05 were considered cally significant.

Biomass Increments
The in vitro cultures of R. graveolens, R. corsica, and R. chalepensis were main agitated shoot cultures in a 7-week growth cycle (inoculum mass: 1 g). The cultu characterized by a similar increase in biomass. The cultures showed a good growth up to the fifth week of cultivation, except for R. graveolens cultures, which their maximum growth after four weeks of breeding. In the sixth week the grow ually stopped, and after 7 weeks of cultivation, the shoots started to show signs of The best (33.7-fold) increase in biomass was observed in R. chalepensis culture weeks of cultivation. For R. graveolens and R. corsica, the following biomass incr recorded: 33.3-fold (after 4 weeks) and 31.3-fold (after 5 weeks), respectively (Fig
No flavonoids were detected. Phenolic acids were accumulated in very small amounts. The dynamics of accumulation of individual metabolites varied according to the time of the culture and species. Generally, the production of most compounds increased between the third and sixth week and decreased in the seventh week. Table 1 shows the mean contents (mg/100 g DW) of individual metabolites depending on the rue species and the growth cycle. Among the coumarins, xanthotoxin was accumulated in the greatest amount (maximum content: R. chalepensis-509.8 mg/100 g DW; R. corsica-375.9 mg/100 g DW, 5-week growth cycle; R. graveolens-428.3 mg/100 g DW, 6-week growth cycle). Bergapten was the next coumarin that was accumulated in high amounts with the maximum content obtained after the 6-week growth cycle in R. chalepensis and R. graveolens cultures and after the 7-week growth cycle in R. corsica cultures (281.4; 186.6; 174.7 mg/100 g DW, respectively). The quantity of psoralen was also high, with the maximum content of 268.8 mg/100 g DW in R. chalepensis microshoots (5-week growth cycle) and 278.7 and 129.3 mg/100 g DW in R. corsica and R. graveolens microshoots (6-week growth cycle). The amounts of isopimpinellin were lower and reached the maximum content of 145.25 and 57.45 mg/100 g DW in R. graveolens and R. corsica extracts of biomass (5-week growth cycle), respectively, and 77.1 mg/100 g DW in the R. chalepensis extract (6-week growth cycle). Isoimperatorin was accumulated in the lowest amount of 46.8, 43.1, and 23.9 mg/100 g DW in R. corsica, R. chalepensis, and R. graveolens microshoots, respectively (6-week growth cycle). Table 1. Average contents of metabolites (mg/100 g DW) in methanol extracts obtained from biomass of cultures of three rue species depending on the duration of the culture (3, 4, 5, 6, and 7 weeks). Means of three measurements ± SD. Different letters indicate significant differences (p < 0.05). Among the confirmed furoquinoline alkaloids, γ-fagarine was accumulated in the highest amount (293.7 mg/100 g DW in R. corsica cultures after the 4-week growth cycle, while its content was 155.9 and 124.3 mg/100 g DW in R. graveolens and R. chalepensis cultures after the 5-week growth cycle, respectively). The maximum skimmianine content was 133.4 mg/100 g DW in R. corsica cultures (4-week growth cycle) 94.6 mg/100 g DW in R. graveolens cultures (5-week growth cycle) and 55.5 mg/100 g DW in R. chalepensis cultures (7-week growth cycle). Isopentenyloxy-γ-fagarine was accumulated in the lowest amount: 26.5 mg/100 g DW in R. corsica microshoots (4-week growth cycle) and 6.8 and 4.4 mg/100 g DW in R. graveolens and R. chalepensis microshoots, respectively (5-week growth cycle) ( Table 1).

Accumulated Compounds
The highest total content (1031.5 mg/100 g DW) of the estimated coumarins was detected in the cultures of R. chalepensis (5-week growth cycle). Very high amounts of coumarins were also confirmed in the cultures of R. graveolens (917.23 mg/100 g DW, 5week growth cycle) and R. corsica (878.05 mg/100 g DW, 6-week growth cycle) ( Table 1). During the breeding cycle, the total content of coumarins in the biomass of R. chalepensis cultures increased from 617.5 mg/100 g DW (3-week growth cycle) to 1031.5 mg/100 g DW (5-week growth cycle) (1.38-fold), in the biomass of R. graveolens cultures from 461.4 mg/100 g DW (3-week growth cycle) to 917.2 mg/100 g DW (5-week growth cycle) (1.99-fold), and in the biomass of R. corsica cultures from 533.5 mg/100 g DW (3-week growth cycle) to 878.05 mg/100 g DW (6-week growth cycle) (1.65-fold) (Table 1, Figure 2).  Figure 2). Shoot cultures of R. corsica showed the highest ability to accumulate alkaloids. The highest total content (293.7 mg/100 g DW) of the estimated alkaloids was detected after the 4-week growth cycle and was 1.88-fold higher than the maximal content of alkaloids in R. graveolens cultures (155.9 mg/100 g DW, 5-week growth cycle) and 2.36-fold higher than the maximal content of alkaloids in R. chalepensis cultures (124.3 mg/100 g DW, 5week growth cycle) ( Table 1). During the breeding cycle the total content of alkaloids in the biomass of R. corsica cultures increased from 175.4 mg/100 g DW (3-week growth cycle) to 293.7 mg/100 g DW (5-week growth cycle) (1.67-fold), in the biomass of R. graveolens cultures from 105.1 mg/100 g DW (3-week growth cycle) to 155.9 mg/100 g DW (5-week growth cycle) (1.48-fold), and in the biomass of R. chalepensis cultures from 61.25 mg/100 g DW (3-week growth cycle) to 124.3 mg/100 g DW (5-week growth cycle) (2.03-fold) (Table 1, Figure 3). Shoot cultures of R. corsica showed the highest ability to accumulate alkaloids. The highest total content (293.7 mg/100 g DW) of the estimated alkaloids was detected after the 4-week growth cycle and was 1.88-fold higher than the maximal content of alkaloids in R. graveolens cultures (155.9 mg/100 g DW, 5-week growth cycle) and 2.36-fold higher than the maximal content of alkaloids in R. chalepensis cultures (124.3 mg/100 g DW, 5-week growth cycle) ( Table 1). During the breeding cycle the total content of alkaloids in the biomass of R. corsica cultures increased from 175.4 mg/100 g DW (3-week growth cycle) to 293.7 mg/100 g DW (5-week growth cycle) (1.67-fold), in the biomass of R. graveolens cultures from 105.1 mg/100 g DW (3-week growth cycle) to 155.9 mg/100 g DW (5-week growth cycle) (1.48-fold), and in the biomass of R. chalepensis cultures from 61.25 mg/100 g DW (3-week growth cycle) to 124.3 mg/100 g DW (5-week growth cycle) (2.03-fold) (Table 1, Figure 3).
The production of secondary metabolites from the group of linear furanocoumarins and some alkaloids was observed in the in vitro cultures of the studied rue species. A characteristic of in vitro cultures is that their metabolic profile may differ from that of their parent plants. Our previous studies on in vitro cultures of R. graveolens confirmed that the shoot cultures of this plant species accumulated mainly linear furanocoumarins and alkaloids, and significantly lower amounts of phenolic acids [21,23]. The results of current studies also confirmed this ability of the R. graveolens shoot culture. The same biosynthetic potential was now documented by us for two other rue species-R. chalepensis and R. corsica. The shoots of these species also produced mainly linear furanocoumarins and some alkaloids. However, some differences were noted between the in vitro cultures of the three rue species. R. corsica shoots produced higher amounts of alkaloids than R. chalepensis and R. graveolens shoot cultures. the biomass of R. corsica cultures increased from 175.4 mg/100 g DW (3-week growth cycle) to 293.7 mg/100 g DW (5-week growth cycle) (1.67-fold), in the biomass of R. graveolens cultures from 105.1 mg/100 g DW (3-week growth cycle) to 155.9 mg/100 g DW (5-week growth cycle) (1.48-fold), and in the biomass of R. chalepensis cultures from 61.25 mg/100 g DW (3-week growth cycle) to 124.3 mg/100 g DW (5-week growth cycle) (2.03-fold) (Table 1, Figure 3).  Previous studies [36] analysed the selected secondary metabolites (coumarins and alkaloids) in R. graveolens shoot stationary liquid cultures (LS medium containing 2 mg/L each of NAA and BAP). Seven secondary metabolites were identified. These included linear furanocoumarins (psoralen, bergapten, xanthotoxin, isopimpinellin), linear dihydrofuranocoumarin (rutamarine), and furoquinoline alkaloids (cocusaginine and skimmianine). Five of these were confirmed in the current HPLC analysis of R. graveolens biomass extracts. Additionally, we confirmed two furoquinoline alkaloids (γ-fagarine, isopentenyloxy-γfagarine) and isoimperatorin (furanocoumarin).
The results of dynamics of metabolites accumulation during the growth cycles showed that the maximal total amounts of furanocoumarins were obtained after the 5-week growth cycle. In our previous studies on agitated shoot cultures of R. graveolens, which were grown in a 6-week growth cycle, the maximal content (520.8 mg/g DW) of linear furanocoumarin was confirmed after 6 weeks (LS medium NAA/BAP 0.1/0.1 mg/L) [21].
In the study of Fischer et al., the authors isolated and identified three metabolites from R. chalepensis suspension cultures: isorutarin (dihydrofuranocoumarin), rutarensinan unknown ester of daphnorin (coumarin dimer) and 3-hydroxy-3-methylglutaric acid. The authors, however, did not isolate the linear furanocoumarins detected in our present study [27].
As part of this work, the R. chalepensis shoot cultures were conducted for the first time. The abovementioned authors investigated other types of cultures (callus and suspension cultures). A comparison of the results of previous studies with those obtained in the present study revealed significant qualitative differences in the accumulated compounds from the coumarin group between different types of cultures. The shoot cultures studied in the present research accumulated high amounts of xanthotoxin and psoralen. However, they were not found in callus and suspension cultures.
There are no studies on in vitro cultures of the endemic species R. corsica in the available literature. In the present study, the metabolic profile of the R. corsica shoot cultures was investigated for the first time.
On the basis of the obtained results, all the studied rue cultures can be proposed as an alternative, rich, in vitro-controlled source of linear furanocoumarins. Xanthotoxin and bergapten in particular are objects of interest in dermatology. These compounds have photosensitizing effects on human skin and are used for their pigmentation stimulating and antiproliferative properties in the symptomatic treatment of vitiligo, psoriasis, and mycosis fungoides [37]. The obtained results showed very high levels of xanthotoxin in R. chalepensis and R. graveolens cultures (above 500 and 420 mg/100 g DW) and approximately 400 mg/100 g DW in R. corsica cultures. The obtained maximal amounts of bergapten were also high (above 280 mg/100 g DW in R. chalepensis cultures, above 180 mg/100 g DW in R. graveolens cultures, and above 170 mg/100 g DW in R. corsica cultures). For comparison, the content of xanthotoxin and bergapten in plants cultivated in the field is as follows: for R. graveolens-xanthotoxin 100 mg/100 g DW, bergapten 160 mg/100 g DW; for R. chalepensis-xanthotoxin 10 mg/100 g DW, bergapten 260 mg/100 g DW [29]. The biosynthetic potential for the production of furoquinoline alkaloids is the highest for R. corsica cultures (above 130 mg/100 g DW). These cultures can therefore be considered as a source of furoquinoline alkaloids: γ-fagarine and skimmianine. Furoquinoline alkaloids show a number of biological activities, including antifungal and antibacterial properties, inhibitory activity towards AchE (acetylcholinesterase), and 5-HT2 receptor-inhibiting properties [38].

Antioxidant Activity
Antioxidants compounds neutralize free radicals and their negative effects by preventing their production, intercepting them, and repairing the damage caused by these molecules; these compounds use various mechanisms, such as functioning as reducing agents by trapping free radicals, donating hydrogen, acting as chelators, and quenching singlet oxygen. Antioxidants can be divided into primary (or chain-breaking) and secondary (or preventive), and the primary antioxidant reactions can be classified as hydrogen-atom transfer (HAT) and single-electron transfer (SET). The HAT mechanism occurs when an antioxidant scavenges free radicals by donating hydrogen atoms; while in the SET mechanism, an antioxidant acts by transferring a single electron to reduce any compound.
Antioxidants can act through various mechanisms, such as binding of transition metal ion catalysts, reducing capacity and radical scavenging, prevention of chain initiation, decomposition of peroxides, and prevention of continued hydrogen abstraction. Therefore, it is important to use methodologies with distinct mechanisms to evaluate the antioxidant capacity of plant-derived phytocomplexes or isolated compounds [39].
To determine the in vitro antioxidant capacity of methanolic extracts of Ruta spp., three in vitro tests based on different mechanisms of determination of the antioxidant capacity were used: the DPPH (1,1-diphenyl-2-picrylhydrazyl) method, (involving both HAT and SET mechanisms); the reducing power assay (based on SET mechanism); and the ferrous ion chelating activity assay, to assess secondary antioxidant ability.
The results of the DPPH assay showed that all Ruta spp. extracts exhibited the radical scavenging effect that increased with the increasing concentrations ( Figure 4). The R. chalepensis extract showed the best radical scavenging properties, reaching 60% of the activity at the higher concentration tested (Figure 4). As indicated by the IC 50 values, the activity of the extracts was found to be lower than that of butylated hydroxytoluene (BHT) used as a standard drug and it decreased in the following order: BHT > R. chalepensis > R. graveolens > R. corsica (Table 2).    [42,43]. Kacem et al. (2015) highlighted a decreasing DPPH radical scavenging potential in different extracts of R. chalepensis from Tunisia: ethanol extract > methanol/water (1/1) extract > methanol extract > ethyl acetate extract > water extract > hexane extract [14]. In contrast, other authors found a higher IC50 value for the methanolic extract of the aerial part of R. chalepensis from Saudi Arabia (320.7 μg/mL) [44]. Interestingly, the IC50 values in the DPPH test were found for decoction and ethanol extracts of stem and leaves of R. chalepensis from El Hamma (Tunisia) (IC50 value of 2.26 μg/mL for the leaf decoction) [45].
Regarding the antioxidant activity of R. graveolens, Molnar et al. (2017) reported that the ethanolic extracts obtained from commercial samples of the plant showed a higher DPPH scavenging activity (% inhibition = 59.3%) than the hexane extract (% inhibition = 16.8%) at the same concentration (250 μg/mL) [46]. Furthermore, methanolic and ethanolic extracts of wild-growing and cultivated Serbian R. graveolens at the beginning and end of the flowering season exhibited significant antioxidant potential. The methanolic extract of wild plants collected at the end of the flowering season showed the strongest antioxidant activity in DPPH tests (IC50 = 36.36 μg/mL) [47].  The antiradical activity of the extracts obtained from R. chalepensis grown in different countries has been extensively studied. Fakhfakh et al. (2012) found IC 50 values of 0.12 and 0.22 mg/mL for R. chalepensis ethanol and aqueous extracts, respectively [40]. Ereifej et al. (2015) reported a DPPH radical scavenging ability of Tunisian leaf extract with an IC 50 value of 70.01 µg/mL [41]. Similar results were obtained by Rached et al. (2010) who reported an IC 50 value of 61.41 µg/mL for an Algerian R. chalepensis leaf ethanol extract and by Loizzo et al. (2018) who reported an IC 50 value of 60.2 µg/mL for the methanol leaf extract obtained from Tunisian samples of this species [42,43]. Kacem et al. (2015) highlighted a decreasing DPPH radical scavenging potential in different extracts of R. chalepensis from Tunisia: ethanol extract > methanol/water (1/1) extract > methanol extract > ethyl acetate extract > water extract > hexane extract [14]. In contrast, other authors found a higher IC 50 value for the methanolic extract of the aerial part of R. chalepensis from Saudi Arabia (320.7 µg/mL) [44]. Interestingly, the IC 50 values in the DPPH test were found for decoction and ethanol extracts of stem and leaves of R. chalepensis from El Hamma (Tunisia) (IC 50 value of 2.26 µg/mL for the leaf decoction) [45].
Regarding the antioxidant activity of R. graveolens, Molnar et al. (2017) reported that the ethanolic extracts obtained from commercial samples of the plant showed a higher DPPH scavenging activity (% inhibition = 59.3%) than the hexane extract (% inhibition = 16.8%) at the same concentration (250 µg/mL) [46]. Furthermore, methanolic and ethanolic extracts of wild-growing and cultivated Serbian R. graveolens at the beginning and end of the flowering season exhibited significant antioxidant potential. The methanolic extract of wild plants collected at the end of the flowering season showed the strongest antioxidant activity in DPPH tests (IC 50 = 36.36 µg/mL) [47].
As shown in Figure 5, the Ruta spp. extracts showed a weak, dose-dependent reducing power. A comparison of the ASE/mL values showed that the reducing power of the Ruta spp. extracts and the standard decreased in the following order: BHT > R. chalepensis > R. corsica > R. graveolens (Table 2).
Antioxidants 2022, 11, x FOR PEER REVIEW 12 of 16 As shown in Figure 5, the Ruta spp. extracts showed a weak, dose-dependent reducing power. A comparison of the ASE/mL values showed that the reducing power of the Ruta spp. extracts and the standard decreased in the following order: BHT > R. chalepensis > R. corsica > R. graveolens (Table 2). In the Fe 2+ chelating activity assay, the Ruta spp. extracts exhibited good, dose-dependent chelating properties. R. graveolens showed the best activity, reaching approximately 80% at the highest tested concentration ( Figure 6). Based on the IC50 values, the activity of the Ruta spp. extracts and the standard decreased in the following order EDTA > R. graveolens > R. corsica > R. chalepensis (Table 2). Loizzo et al. (2018) conducted a study on the methanolic leaf extract of Tunisian R. graveolens and found that this extract showed low metal chelating activity (14.6%) as compared to EDTA (97.8%) [43].  In the Fe 2+ chelating activity assay, the Ruta spp. extracts exhibited good, dosedependent chelating properties. R. graveolens showed the best activity, reaching approximately 80% at the highest tested concentration ( Figure 6). Based on the IC 50 values, the activity of the Ruta spp. extracts and the standard decreased in the following order EDTA > R. graveolens > R. corsica > R. chalepensis (Table 2). Loizzo et al. (2018) conducted a study on the methanolic leaf extract of Tunisian R. graveolens and found that this extract showed low metal chelating activity (14.6%) as compared to EDTA (97.8%) [43].
Antioxidants 2022, 11, x FOR PEER REVIEW 12 of 16 As shown in Figure 5, the Ruta spp. extracts showed a weak, dose-dependent reducing power. A comparison of the ASE/mL values showed that the reducing power of the Ruta spp. extracts and the standard decreased in the following order: BHT > R. chalepensis > R. corsica > R. graveolens (Table 2). In the Fe 2+ chelating activity assay, the Ruta spp. extracts exhibited good, dose-dependent chelating properties. R. graveolens showed the best activity, reaching approximately 80% at the highest tested concentration ( Figure 6). Based on the IC50 values, the activity of the Ruta spp. extracts and the standard decreased in the following order EDTA > R. graveolens > R. corsica > R. chalepensis (Table 2). Loizzo et al. (2018) conducted a study on the methanolic leaf extract of Tunisian R. graveolens and found that this extract showed low metal chelating activity (14.6%) as compared to EDTA (97.8%) [43].  On the basis of the obtained results, it is evident that the methanolic extracts of the Ruta spp. cultures act as weak primary antioxidants and possess good secondary antioxidant properties.
The genus Ruta is rich in bioactive compounds. Phytochemical investigations conducted on extracts obtained from Ruta spp. grown in fields have detected the presence of compounds belonging to different chemical classes, such as coumarins, phenolic acids, flavonoids, alkaloids, and tannins [48].
In our HPLC study of in vitro cultures of Ruta spp., a different metabolic profile with respect to that of the parent plant was highlighted. In all the extracts, flavonoids were undetectable and phenolic acids were present only in small amounts. The dominant group of compounds in all cultures were coumarins followed by alkaloids. Among the coumarins, xanthotoxin was accumulated in the highest amount. It was reported that coumarins imperatorin, xanthotoxin, and bergapten did not exhibit anti-radical activity against DPPH, while they showed a moderate level of reducing power, moreover, they were effective in the ferrous ion-chelation test [49]. Coumarins can reduce the level of oxidative stress through chelation of redox-active Cu and Fe, thus suppressing the formation of ROS through the Fenton reaction [50].
Thus, the good secondary antioxidant properties observed for R. graveolens, R. Corsica, and R. chalepensis methanol extracts could be mainly related to the presence of coumarins, but it cannot be excluded that other compounds present in the phytocomplex could also contribute to the observed effects.

Antimicrobial Activity
The development of antimicrobial resistance in pathogens has prompted extensive research to find alternative therapeutics. Staphylococcus aureus is the first pathogen that has become resistant to all known antibiotics and thus it is essential that new antimicrobial agents are discovered to combat this problem [51]. Plants rich in secondary metabolites are one of the go-to reservoirs for the discovery of potential resources to alleviate this problem [52,53].
The preliminary antimicrobial screening of the Ruta spp. extracts obtained from in vitro cultured biomass was performed against a small representative set of Gram-positive and Gram-negative bacterial strains and the yeast Candida albicans according to the protocols recommended by the Clinical and Laboratory Standards Institute [33,34]. The obtained results showed that all the methanolic extracts had a good bacteriostatic activity against the Gram-positive S. aureus at the tested concentrations. Among the extracts, the R. corsica extract showed the highest activity (MIC = 250 µg/mL) followed by R. graveolens and R. chalepensis extracts (MIC = 500 µg/mL). None of the tested strains were inhibited by DMSO (maximum 0.5% v/v), used as a negative control. The MIC of tetracycline, used as positive control, was 1 µg/mL.
Several studies have reported the antibacterial and antifungal potential of Ruta spp., with R. chalepensis and R. graveolens being the most frequently investigated [48].
Our results showed good antibacterial activity of the methanolic extract obtained from Ruta spp. in vitro cultured biomass against S. aureus at concentrations lower (MIC range: 250-500 µg/mL) than those reported by Alzoreky and Nakahara (2003) for wild or cultivated R. chalepensis and R. graveolens (MIC = 2640 mg/mL) [54]. There are no data on R. corsica.
The in vitro cultures studied in the present research accumulated mainly metabolites from the group of coumarins and alkaloids. Alkaloids exhibit antimicrobial activity by enzyme activity inhibition, intracellular molecular imbalance, and cell membrane disturbances, which can cause cell death [55]. All the extracts obtained from Ruta spp. in vitro cultured biomass contained furoquinoline alkaloids, namely, γ-fagarine, 7-isopentenyloxyγ-fagarine, and skimmianine, whose strong antibacterial activity against S. aureus has been widely demonstrated [56][57][58].
The highest total content of the identified alkaloids was found in the R. corsica extract, which verified the greater activity shown by this extract than the other two extracts.
Coumarins possess a wide variety of biological activities [59]; however, only a limited number of coumarin structures have been assessed for antimicrobial activity [48]. Among these, the furanocoumarin xanthotoxin proved to be effective in inhibiting the growth of S. aureus while bergapten was not active [53].
Thus, it can be hypothesized that the good antibacterial activity observed for all Ruta spp. extracts against S. aureus could depend mainly on the presence of the alkaloids γ-fagarine and skimmianine and the coumarin xanthotoxin contained in the phytocomplex.

Conclusions
The present research proved a similar biosynthetic potential of shoot cultures of three rue species: R. chalepensis, R. corsica, and R. graveolens. The metabolism of the in vitro cultures of these species differs from that of the parent plants and is mainly directed towards the production of linear furanocoumarin compounds and furoquinoline alkaloids. The antioxidant and antibacterial activities of all rue cultures were confirmed. These activities are derived from the presence of the dominant metabolites from the furanocoumarin group and alkaloids, but they may also be related to the action of other compounds present in the phytocomplex. The obtained contents of the described groups of metabolites are higher than those in plants grown in the field. Therefore, in vitro cultures of Ruta spp. can be proposed as an alternative source of obtaining these metabolites: all rue cultures tested can be used to obtain furanocoumarins, while the most effective source for obtaining alkaloids is an R. corsica culture.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/antiox11030592/s1, Figure S1: Sample chromatogram of the extract from Ruta corsica in vitro cultures; Figure S2: Sample chromatogram of the extract from Ruta chalepensis in vitro cultures; Figure S3: Sample chromatogram of the extract from Ruta graveolens in vitro cultures.

Conflicts of Interest:
The authors declare no conflict of interest.