Investigation of composition, antioxidant, antimicrobial and cytotoxic characteristics from Juniperus sabina and Ferula communis extracts

Plants have been one the most valuable sources of biologically active compounds. This study investigates the chemical composition, as well as the antioxidant, antimicrobial, and cytotoxic activities of methanolic and ethanolic extracts from Juniperus sabina and Ferula communis leaves, grown in Cyprus. Total phenolic and flavonoids content of methanol and ethanol extracts were quantified. Chemical constituents of the leaf extracts were analysed using gas chromatography/mass spectrometry (GC/MS). Mome inositol was the predominant component in the J. Sabina’s extracts. The most dominant component in F. communis ethanolic extract was phytol, while in FCL methanolic extract 1,3,4,5 tetrahydroxycyclohexanecarboxylic acid. Antioxidant activities were evaluated by 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging ability. Antioxidant activity results revealed concentration dependent activity for methanolic and ethanolic extracts from the plant leaves. Antibacterial activity of plant extracts was tested against Gram-negative and Gram-positive bacteria using disk diffusion and minimal inhibitory concentration methods. Cytotoxic activity of plant extracts were evaluated on MCF-7 and MDA-MB-231 breast cancer cell lines, where they demonstrated their potential on the viability of both cell lines. The biological activity revealed by plants is due to the bioactive compounds found in the extracts. These bioactive components could be used as anticancer drug candidates.


Preparation of methanolic and ethanolic extracts of JSL and FCL.
Juniperus sabina and F. communis (50 g each) were extracted separately using 250 ml of 95% methanol or 95% ethanol at a temperature below the boiling point of the solvents. Obtained mixtures were filtered using Whatman filter paper No. 1 and concentrated under reduced pressure at 45 °C using the rotary evaporator (Heidolph, Germany), leaving a viscous residue, dark green with an aromatic odor. These samples were stored in the refrigerator at 4 °C for further analysis.
Total phenolic content. The total phenolic content was determined with some slight modifications 20 .
Appropriately diluted extract (100 µl) was mixed with 200 µl of an undiluted Folin-Ciocalteu reagent for 5 min. 1 ml of 20% Na 2 CO 3 (Sodium carbonate) was added to the mixtures and the total volume was adjusted to 10 ml with distilled water. This was incubated at room temperature in darkness for 2 h, and the absorbance measured at 765 nm using a UV Visible Spectrophotometer (UV-2450). The results were estimated as gallic acid equivalent (GAE), expressed as mg GAE/g. The calibration curve range was 1.25-20 mg/ml (R 2 = 0.99). The data are presented as means ± standard deviation (SD) from three biological repeats.
Total flavonoid content. Total flavonoid content was calculated by the aluminum chloride (AlCl 3 ) colorimetric method with some modifications 21 . Briefly, 1 ml of aliquots, 4 ml of distilled water and 0.3 ml of 5% NaNO 2 (sodium chloride) were placed in separate test tubes. After 5 min, 0.3 ml of 10% AlCl 3 and 2 ml of 1 M NaOH (sodium hydroxide) were added to the mixture. The mixture was adjusted to 10  www.nature.com/scientificreports/ and mixed well, developing an orange/yellow color. Using the spectrophotometer (UV-2450), the absorbance was measured at 510 nm wavelength versus a prepared blank. The total flavonoid contents of each JSL and FCL plant extract were expressed as milligrams of quercetin equivalents per gram of dry matter (mg QE/g) through the calibration curve with quercetin. All results are presented as means (± SD) from three biological repeats.
GC-MS analysis. The components of methanolic and ethanolic extracts of both JSL and FCL were identified using gas chromatography-mass spectrometry (GC-MS) analysis. The GC-MS apparatus used was Shimadzu QP-2010 equipped with a fused-silica column. The operating conditions were: flow rate of helium gas 1 ml/min; oven temperature 50-310 °C with a rate of 8 °C/min; injector temperature 275 °C; split injection mode of ratio 1:10; pressure 112.2 kPa; Ionization energy 70 eV. The extract components were identified from the retention indices (RI) obtained by computer matching with the MSdata bank (Wiley Library), and relative percentages were calculated.
DPPH radical scavenging assay. The total antioxidant activity of each sample was determined using the 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity assay 22 . The DPPH was freshly made using pure ethanol of 0.004% w/v. A volume of 1 ml of diluted samples (concentrations 100, 50, 25, 12.5, 6.25 µg/ ml) were mixed with 1 ml of DPPH. These were incubated in darkness for 30 min, at 25 °C, before the absorbance of each sample was measured at 517 nm. A blank was prepared by mixing 0.5 ml of DPPH solution with 0.5 ml of ethanol. A positive control of ascorbic acid was prepared to compare the results of decreased absorption induced by the samples. The capability to scavenge 50% of DPPH was calculated using Eq. (1). All results presented are means (± SD) from three biological repeats.
Antimicrobial activity. Microorganisms. The antimicrobial activity of methanolic and ethanolic extracts obtained from both JSL and FCL were determined using four strains of microorganisms: E. coli O157:H7 (932), S. typhimurium (B-4420), B. cereus (ATCC 7064) and S. aureus (6538 P). "Minimum inhibitory concentrations (MIC)" and "Disk diffusion method" were used to determine antimicrobial activity. Nutrient agar and nutrient broth were prepared according to the manufacturer and sterilized in the autoclave for 20 min at 121 °C. The final microorganism concentration was adjusted to 0.5 McFarland Standard (1.5 × 108 CFU/ml).

Minimum inhibitory concentrations (MIC).
MIC of methanolic and ethanolic extracts of JSL and FCL were estimated using the broth dilution method. The diluted sections of five concentrations (6.25, 12.5, 25, 50 and 100 mg/ml) were prepared using 30% dimethyl sulfoxide (DMSO) 23 . The MIC was estimated using 3 ml of sterile nutrient broth cultured with 1 ml of the bacterial suspension. 1 ml of each extract concentration was added to the mixture and incubated for 24 h at 37 °C before the results were collected. Each assay was performed in triplicate.
Disc diffusion method. Nutrient agar plates were inoculated with 100 µl of prepared bacterial suspension using a sterile wire loop swabbed on the surface of the agar plates. Sterile 6 mm discs were soaked with 15 µl of one of the five concentrations of methanolic and ethanolic extracts of JSL and FCL. The diluted extracts of five different concentrations (6.25, 12.5, 25, 50 and 100 mg/ml) were prepared by using 30% DMSO 24 . On each plate, five 6 mm discs were placed on the surface of the nutrient agar. Plates were incubated for 24 h at 37 °C. DMSO was used as a negative control. The diameter of the zones of inhibition (ZI) around the discs determined bacterial growth inhibition. The zones of inhibition were measured using a ruler and recorded in millimeters. Each assay was performed in triplicate with DMSO as a negative control. The effects of each concentration of extracts tested on the bacteria were compared with sensitivity to antibiotics chloramphenicol and amoxicillin.
Cytotoxic activities. Cell lines and cell culture conditions. Human breast cancer cell lines MDA-MB-231 and MCF-7 were obtained from Imperial College London, UK (courtesy of Prof. Dr. Mustafa Djamgoz). Both cancer cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 4 mM lglutamine and 10% fetal bovine serum. The cells were harvested at a sub-confluence of 80-100% using 0.05% trypsin/EDTA. The cells were incubated in 5% CO 2 and 100% relative humidity at 37 °C.
In vitro cytotoxicity profile. Antiproliferative activities of methanolic extracts of JSL and FCL were determined against MDA-MB-231 and MCF-7 using Trypan blue exclusion assay 25 . The antiproliferative activities of the extracts and the controls were investigated at five concentrations (10, 20, 50, 150 µg/ml). 35 mm sterile dishes (3 dishes per each condition) were used to grow cultured cells at a density of 3 × 104 cells per dish, incubated overnight to settle. Cells were treated with extracts for 24 h in a humidified incubator of 5% CO 2 at 37 °C. The medium was removed, and 0.4% Trypan blue solution was added. After 10 min, the diluted Trypan blue was removed. Around 15 fields of view were counted randomly using an inverted microscope (Leica, Germany) to estimate the percentage of cell viability. Control dishes contained cells with DMSO 24 . Each assay was performed at least in triplicate.
Statistical analysis. All experiments were performed at least in triplicate. The results obtained were expressed as the mean values ± standard deviation (SD) for all experiments. Student's t-test determined significant differ-

Results and discussion
Total phenolic content. Table 1 shows the quantitative analysis of the total phenolic content of methanolic and ethanolic extracts from JSL and FCL. The total phenolic content for both plants was found to be higher in ethanolic extracts compared to methanolic extracts. For methanolic extracts, the phenolic content of JSL (77.02 ± 3.3 mg GAE/g) was higher than FCL (17.97 ± 0.45 mg GAE/g). For ethanolic extracts, the phenolic content of JSL (124.73 ± 1.9 mg GAE/g) was also higher than FCL (91.5 ± 3.01 mg GAE/g). The quantitative analysis of the total phenolic content of both extracts from JSL showed a higher content (methanolic 77.02 and ethanolic 124.73 mg GAE/g), than those revealed by FCL extracts (methanolic 17.97 and ethanolic 91.5 mg GAE/g). Ethanolic extracts of both plants provided a higher extraction of phytochemicals than methanolic extracts. F. communis from Jordan was previously shown to have a similar total phenolic content in the methanol extract (18.4 mg GAE/g) 26 . According to Öztürk et al., J. sabina showed lower total phenolic content of methanolic extracts from plants collected in the Karabük province in Turkey (31.58 mg GAE/g), as compared to this study 27 . Zengin et al., studied the difference in total phenolic content of F. halophila extracted from three solvents, with the highest phenolic content observed from the acetone extract, followed by methanol, then chloroform 28 .
Total flavonoid content. Table 1 shows the quantitative analysis of total flavonoids of both methanolic and ethanolic extracts from JSL and FCL. Higher total flavonoid content was found for both JSL and FCL in ethanolic extracts than methanolic extracts. For methanolic extracts, the flavonoid content of JSL (77.02 ± 3.3 mg QE/g) was higher than FCL (17.97 ± 0.45 mg QE/g). Among the ethanolic extracts, the flavonoid content of JSL (124.73 ± 1.9 mg QE/g) was also higher than FCL (91.5 ± 3.01 mg QE/g). The quantitative analysis of total flavonoids in JSL methanolic and ethanolic extracts revealed higher total flavonoid content, 13.83 mg QE/g and 15.66 mg QE/g, respectively, compared to those seen in FCL extracts, 7.37 and 8.37 mg QE/g. Ethanolic extracts of both plants showed a higher extraction of phytochemicals than methanolic extracts. Öztürk et al. showed the total flavonoid content of J. sabina methanolic plant extracts was lower, at 8.83 mg QE/g 27 . Zengin et al. showed that, for F. halophila, the highest flavonoid content was obtained from the acetone extract (34.52 mg RE/g extract), followed by the methanolic extract (24.13 mg RE/g extract), and then the chloroform extract (8.61 mg RE/g extract) 28 .
In a previous study, the DPPH assay percentage of inhibition for the methanolic extract of J. Sabina was 24.66%, 34.94%, and 55.49% for concentrations 25, 50, and 100 µg/ml respectively 27 . At the same concentrations, our research showed JSL had a higher percentage of inhibition at 84.4%, 84.7% and 85%, respectively. In another study, Jordanian F. Communis showed 86.7 µmol TE g −1 DW antioxidant activity 26 . Zengin et al. methanolic extracts from F. halophila also showed a high antioxidant activity of 95.28 ± 3.80% due to the polarity of methanol, enabling the extract to scavenge the DPPH radicals more actively 28 . Results from Rahali et al. revealed that the different organs of the F. communis plant had various antioxidant activities. Thus, flower extracts showed the highest DPPH-scavenging compared to stems and fruits, indicating the highest antioxidant capability was in this organ. This might be because it also contains the highest total phenolic content 30 . Natural plant antioxidants, including phenolic compounds, can prove beneficial by removing harmful free radicals. Therefore, phenolic compounds help to protect cells from oxidative damage. Chanjirakul et al. reported that plant extracts rich in phenolic compounds also provide antibacterial activity 33 . Results from another study confirm this result. According to Onyebuchi et al., extraction temperature significantly affected the total phenolic content and radical-scavenging properties of the different extracts 22 .

Microbiological inhibition analysis. MIC antibacterial activity.
In this study, the antimicrobial activity of methanolic and ethanolic extracts of JSL and FCL growing on the island of Cyprus was tested against two strains of gram negative bacteria, Escherichia coli O157:H7 (932) and Salmonella typhimurium (B-4420), and gram positive bacteria Bacillus cereus (ATCC 7064) and Staphylococcus aureus (6538 P). Both methanolic and ethanolic extracts of JSL and FCL showed effective antimicrobial results in minimal inhibitory concentration (MIC) assays. As shown in Table 4, the best MIC values were detected against S. typhimurium with all extracts, except for the FCL ethanolic extract. FCL methanolic extract had the best MIC against E. coli. The weakest anti-   Disk diffusion method for antibacterial activity. The FCL ethanol extract (100 mg/ml) had the greatest antibacterial zone of inhibition (ZI) (16.5 mm) against E. coli, and the JSL ethanol extract (100 mg/ml) had the greatest ZI (12 mm) against S. aureus. Ethanolic extracts of both JSL and FCL were ineffective against B. cereus, with no activity seen. Chloramphenicol (10 μg/disc) and Amoxicillin (10 μg/disc) were used as positive controls to determine the sensitivity of microbial strains. Disks containing DMSO were used as a negative control. The highest ZI for methanolic extracts was from JSL (16.5 mm) against S. aureus, followed by the FCL methanolic extract against E. coli (12 mm), both at a concentration 100 mg/ml. FCL methanolic extract against S. typhimurium (Table 5) detected no antimicrobial activity.
Öztürk et al. showed the antibacterial effect of the J. sabina methanolic extract against several bacterial strains, with no ZI detected against E. coli, and an 8.5 mm ZI detected against S. aureus 27 . The best results for inhibiting bacterial growth were seen on multiple-antibiotic-resistant Staphylococci and some strains of multiple-antibioticresistant Stenotrophomonas maltophilia. In this research, JSL ethanolic and methanolic extracts had an effective antibacterial activity against E. coli (ZI = 8.5 mm) and S. aureus (ZI = 16.5 mm), respectively. In another study, the methanolic extract of J. phoenicea was effective for inhibiting the growth of E. coli and S. aureus, with ZI size increasing with extract concentration; as the concentration increased from 20, 30, to 40%, the ZI measured 11, 12 and 13 mm for E. Coli and 15, 17 and 20 mm for S. aureus, respectively 23 . Sitotaw et al. showed that the in vivo antibacterial activity of root and stem methanolic and ethanolic extracts of F. communis varied by concentration (100 mg/ml and 200 mg/ml) and that the methanolic extract showed higher antibacterial activity against S. aureus and E. coli than ethanolic extracts. Significantly higher antibacterial activity was detected using 200 mg/ ml of extracts. However, they also showed that F. communis ethanolic stem extracts could inhibit the growth of E. coli at 100 mg/ml concentration with a minimal ZI (8.00 ± 0.00 mm) 37 . These findings show that extracts from different parts of the plant have other antibacterial activity. This might be due to the distribution of active ingredients between various plant parts, whether in leaves, stems or roots. 38 and thus have anticancer properties by inhibiting cell proliferation 39,40 which brings about various biological effects. To test the anticancer properties of the methanolic and ethanolic extracts from JSL and FCL, we applied them in multiple concentrations to two breast cancer cell lines.
Methanolic extracts from FCL showed a more significant reduction in cellular viability on MDA-MB-231 than that of JSL, as shown in Fig. 4. Contrastingly, the JSL methanolic extract caused the highest reduction of cellular viability for MCF-7, shown in Fig. 5.
FCL ethanolic extract showed a lower cellular viability percentage than JSL ethanolic extract against MDA-MB-231 (Fig. 6). However, for ethanolic extracts, JSL showed better cytotoxic results overall at higher concentrations (Fig. 7). The most significant reduction was observed for all experiments with the 150 µg/ml concentration of the JSL methanolic and ethanolic extracts applied to MCF-7.
Our findings showed methanolic and ethanolic extracts from JSL and FCL had significant antiproliferative activity against breast cancer cell lines MDA-MB-231 and MCF-7. No other data has been recorded against these specific cell lines with extracts from our selected plants. Another study showed cytotoxic activities against Hela and MDA-MB-468 cells from fruit extracts and branchlets of male and female Iranian J. sabina 32 . Also, total extracts from the aerial parts of J. sabina had promising hepatoprotective activity against CCl 4 -induced toxicity in rats 41 . In another study, the cytotoxicity of ferulenol, isolated from F. communis, on human breast cancer (MCF-7), ovarian cancer (SKOV-3), leukemic cancer (HL-60), and colon cancer (Caco-2) cells was measured,   44 .
Only a few studies have compared the cytotoxic effects of methanolic and ethanolic extracts for J. Sabina. In Sadeghi-Aliabadi et al., ethanolic extracts of J. Sabina branchlets were tested for cytotoxic effect against MDA-MB-468 cells using 3 different concentrations, 5, 10, and 20 μg/ml. Still, they did not show any cytotoxic activity 45 . One study on Juniper species J. foetidissima reported the cytotoxic effects of nardosinen extracted in the acetone extract from leaves and branchlets tested against various cancer cells, showing the cytotoxic effect against MCF-7 was dose-dependent 46 .

Data availability
All data is included in the submitted manuscript file.