Antimicrobial and Antioxidant Efficacy of the Lipophilic Extract of Cirsium vulgare

The aim of this study was to investigate the compounds in the hexane extract of Cirsium vulgare (Savi.) Ten. and to determine the antibacterial, antifungal, and antioxidant activities of different extracts. The Cirsium vulgare (NGBB 7229) plant was collected from Turkey’s Trakya region. Crude extracts were obtained using different solvents. The chemical composition of Cirsium vulgare was determined in hexane extract using gas chromatography mass spectrometry. The antioxidant activities of the extracts were evaluated by Trolox equivalent antioxidant activity (TEAC), ferric-reducing antioxidant power (FRAP), cupric-reducing antioxidant capacity (CUPRAC), the β-carotene bleaching method, and the determination of superoxide anion scavenging activities. The antibacterial activity was tested against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, and Salmonella typhimurium, whereas the antifungal activity was tested against Candida albicans, Candida glabrata, Candida parapsilosis, Candida krusei, Penicillium chrysogenum, and Aspergillus fumigatus by applying microdilution methods. A total of 41 bioactive compounds were identified using the GC–MS library. Terpenoids were found to be dominant (52.89%), and lup-20(29)-en-3-yl-acetate and lupeol were the most abundant terpenoids. The highest total flavonoid content (25.73 mg catechin/g) and antioxidant capacity were found in the methanolic extract. The highest antibacterial activity was detected against Bacillus subtilis in the ethyl acetate extract, and the highest antifungal activity was found against Candida krusei and Aspergillus fumigatus in the hexane extract. The observed antioxidant characteristics of the C. vulgare extracts could be attributed to the presence of flavonoids. The high antifungal activity of the hexane extract against all fungal strains can be attributed to its constituents, i.e., terpenoids. This study discloses the potential antioxidant and antimicrobial activities, including some bioactive components, of Cirsium vulgare and implies that Cirsium vulgare holds possible applications in the food and pharmaceutical industries as an antioxidant, antibacterial, and antifungal agent.


Introduction
Bioactive compounds obtained from different sources exhibit great potential for preventing free radical damage when used as functional food components and antioxidant agents in the food or pharmaceutical industries.On the other hand, safer alternative bactericides and fungicides are needed to reduce resistance to synthetic antimicrobials.Therefore, drugs derived from natural sources play a significant role in the prevention and treatment of human diseases.
Molecules 2023, 28, 7177 2 of 14 The Asteraceae family is one of the largest families of flowering plants, with about 1600 genera and over 23,000 species; the bioactivities of a number of the Asteraceae species have not yet been investigated [1].The Cirsium genus belongs to the Asteraceae family and is believed to be harmful in agricultural areas, as it can exhibit uncontrollable reproduction and growth.But the stem and roots of different Cirsium genus have been used as a food source and food additive in rural areas in Turkey for years [2].In different countries, different species of Cirsium have been used in hepatoprotective folk medicine [2][3][4].Some species have been used traditionally for the treatment of gastritis, diabetes, hemorrhoids, and cough [5].The leaves and stems of many species are also edible and can be used in tea, soup, and salads [6,7].There are several reports regarding the antioxidant, antimicrobial [2,[8][9][10], antidiabetic [11], and anti-tumor activities [12] of some Cirsium species.More than ten species of Cirsium have been used as folk medicines, and modern pharmacological studies have shown that Cirsium exhibits liver protection, along with antioxidant, anti-tumorigenesis, anti-inflammation, antibacterial, and other beneficial effects [13].
C. vulgare is present in a wide variety of habitats, mostly with a high degree of disturbance [14].Our recent study revealed that the extracts from C. vulgare showed DPPH radical scavenging activity and antibacterial activity, according to zone diameters [15].Previous phytochemical studies have reported that C. vulgare contains flavonoids and phenolic compounds [16,17].In addition to these polar compounds, non-polar compounds, such as terpenes and fatty acids, have been discovered from Cirsium species [18].In a recent study, Fernández-Martínez et al. [19] suggested that despite their non-polar constituents, the hexane extracts are not free radical scavengers, as is the case for the flavonoids of the Cirsium polar extracts.
Recently, attention has been focused on natural plant products, used alone or in combination with synthetic fungicides, for use in the food and pharmaceutical industries.Moreover, recent literature screened the most promising examples of dual-active antimicrobial-antioxidant sources, and C. vulgare was determined to be promising.The phytochemical studies of different Cirsium species and their renowned pharmacological activities could be exploited for pharmaceutic product development [20].Shahrajabian [21] reported that spear thistle (C.vulgare) can promote good health and serve as a primary defense mechanism against diseases.The aim of the present study was to analyze the composition of volatile/semi-volatile compounds of hexane extract and to determine the total flavonoid content; the antibacterial, antifungal, and antioxidant efficacy of lipophilic extracts (hexane and diethyl ether); and the ethyl acetate and methanol extracts obtained using different extraction methods of wild C. vulgare.There is no study in the current literature regarding the composition of hexane extracts of C. vulgare.The present work is the first report on the antibacterial and antifungal activities of C. vulgare in terms of minimum inhibitory concentration (MIC) values and various antioxidant properties of different extracts.

Total Flavonoids Content
The highest TFC was found in methanol extract, determined as 25.7 mg catechin and 44.6 mg RE in the gram extract (Table 3), while the highest total flavonoid content was found in the methanol extract, followed by ethyl acetate and then diethyl ether extract.TFC was not found in the hexane extract.Our findings are lower than those obtained by Nazaruk et al., who determined a TFC between 170-209 mg catechin equivalent in one g extract of C. vulgare [35].According to antioxidant activity assays, methanol extract showed the highest antioxidant activity in all tested antioxidant assays.Recently reported by Griškevičien ė et al. [36], the highest amounts of flavonoids obtained by heating with reflux from C. vulgare leaves were rutin, hyperoside, isoquercitrin, chlorogenic acid, and apigenin-7-O-glucoside, respectively.

Antioxidant Activity
The highest TEAC capacity was determined in methanol extract (0.86 mmol Trolox/g).The hexane fraction exhibited the lowest activity, with a 0.34 Trolox/g value.According to the results, the methanol extract exhibited 2.53 times more activity than did the hexane extracts (Table 3).The ABTS •+ radical reducing ability results of the C. vulgare extracts are in agreement with the findings of Malejko et al., in which they determined 3.23 times higher ABTS activity in the refluxed methanolic extracts than in ethyl acetate for the C. palustre extracts (Table 3) [37].Zhao et al. [38] found that the various parts of seven Cirsium species in Taiwan showed varying degrees of antioxidant activities against free radicals in regards to the 16 methanolic standards, according to the ABTS and DPPH methods.
The highest FRAP capacity was determined for methanol extract (1436.6 µmol Fe 2+/ g extract), and the lowest value was obtained for hexane extract (49.7 µmol Fe 2+/ g extract).
Based on the EC 50 value of the FRAP assay, the strength of FRAP power was in the order of: methanol (14.7 µg/mL) > ethyl acetate (80.5 µg/mL) > diethyl ether (127.5 µg/mL) > hexane (651.8 µg/mL).The differences between FRAP activities in different solvent extractions could be explained by solvent polarity, as the use of different solvents of varying polarities may lead to higher and lower mass transfers of different plant phenolics.
The CUPRAC assay is a redox potential-based method, and the results of the CUPRAC assay determined that the highest activity is obtained from methanol extract (2.14 mmol Trolox/g; EC 50 of 18.5 µg/mL) and ethyl acetate extract, followed by values of 1.62 mmol Trolox/g and EC 50 of 33.5 µg/mL.The hexane fraction exhibited the lowest activity (0.38 mmol Trolox/g extract; EC 50 of 140.6 µg/mL) (Table 3).It was shown here that the potency of methanol extract was around 1.81, 3.27, and 7.6 times as high as the potency of the ethyl acetate, diethyl ether, and hexane extracts, respectively.The values are higher than those reported by Karasakal et al., who reported lower CUPRAC values (0.18 mmol/g) after 80% methanol extraction in the C. vulgare varieties [39].On the contrary, Boga et al. reported that acetone and methanol extracts and isolated compounds from two endemic Cirsium species and C. eriophorum grown in Turkey [40] did not show CUPRAC activity.Higher CUPRAC activities in the methanol, ethyl acetate, and diethyl ether extracts compared to those of the hexane extract could be explained by differences in solvent polarity, similar to the results found for the TEAC and FRAP capacities of the previously mentioned extracts.
In the β-carotene-linoleic acid emulsion model, the hexane extracts showed the lowest inhibition effect.The highest efficient antioxidant activity in the lipid system seems to be related to the compounds extracted by methanol, similar to the ABTS and FRAP activity results.According to the results, 28.47% of β-carotene in the methanol extracts remained non-oxidized at the end of the oxidation reaction time (180 min).The inhibition of βcarotene was determined as 25.32% for diethyl ether, 12.13% for hexane, and 7.72% for ethyl acetate extract.According to the results, methanol extract presented the highest flavonoid content and the highest effect against the oxidation of β-carotene in the linoleic acid emulsion system (Figure 1).Nazaruk et al. [35] found that ethyl acetate extract from the C. vulgare flower exerted a 38.5% inhibition effect on β-carotene after one hour, and in this study, we determined higher inhibition effects for methanol and diethyl ether extracts after one hour (49.33% and 42.11%, respectively).However, different extraction methods were used.
FRAP activity results.According to the results, 28.47% of β-carotene in the methanol extracts remained non-oxidized at the end of the oxidation reaction time (180 min).The inhibition of β-carotene was determined as 25.32% for diethyl ether, 12.13% for hexane, and 7.72% for ethyl acetate extract.According to the results, methanol extract presented the highest flavonoid content and the highest effect against the oxidation of β-carotene in the linoleic acid emulsion system (Figure 1).Nazaruk et al. [35] found that ethyl acetate extract from the C. vulgare flower exerted a 38.5% inhibition effect on β-carotene after one hour, and in this study, we determined higher inhibition effects for methanol and diethyl ether extracts after one hour (49.33% and 42.11%, respectively).However, different extraction methods were used.The superoxide anion radical scavenging activity of the extracts at a concentration of 1 mg/mL are given in Table 3, and the results are compared to those for L-ascorbic acid.According to our results, methanol extract exhibited strong superoxide radical scavenging activity comparable to that of L-ascorbic acid.The inhibition of superoxide anion was found to be 74.85%,whereas that of L-ascorbic acid was found to be 99.08%.Demirtas et al. also reported the occurrence of higher superoxide anion radical scavenging activity in the C. arvense methanol-chloroform extracts compared to that of standard compounds, namely a-tocopherol, BHT, and BHA [2].

Antibacterial Activity
The antibacterial MIC levels of C. vulgare extracts against S. aureus were in the range of 15.62-250 mg/mL (Table 4).Diethyl ether and ethyl acetate extracts exhibited the highest inhibition effect on S. aureus, with MIC values of 15.62 mg/mL.All extracts had an effect on B. subtilis, and the MIC values ranged from 3.9 to 250 mg/mL.The highest inhibition effect was found in ethyl acetate extract, with an MIC level of 3.9 mg/mL.The MIC levels of four different extracts for E. coli were in the range of 15.62-125 mg/mL.The hexane extract showed highest inhibition effect on E. coli.The MIC levels of extracts for P. aeruginosa and P. mirabilis were in the ranges of 15.62-250 mg/mL and 31.25-250mg/mL, respectively, and the diethyl ether extract exhibited the highest inhibition effect.The MIC levels of extracts for S. typhimurium were in the range of 31.25-250mg/mL, and the di- The superoxide anion radical scavenging activity of the extracts at a concentration of 1 mg/mL are given in Table 3, and the results are compared to those for L-ascorbic acid.According to our results, methanol extract exhibited strong superoxide radical scavenging activity comparable to that of L-ascorbic acid.The inhibition of superoxide anion was found to be 74.85%,whereas that of L-ascorbic acid was found to be 99.08%.Demirtas et al. also reported the occurrence of higher superoxide anion radical scavenging activity in the C. arvense methanol-chloroform extracts compared to that of standard compounds, namely a-tocopherol, BHT, and BHA [2].

Antibacterial Activity
The antibacterial MIC levels of C. vulgare extracts against S. aureus were in the range of 15.62-250 mg/mL (Table 4).Diethyl ether and ethyl acetate extracts exhibited the highest inhibition effect on S. aureus, with MIC values of 15.62 mg/mL.All extracts had an effect on B. subtilis, and the MIC values ranged from 3.9 to 250 mg/mL.The highest inhibition effect was found in ethyl acetate extract, with an MIC level of 3.9 mg/mL.The MIC levels of four different extracts for E. coli were in the range of 15.62-125 mg/mL.The hexane extract showed highest inhibition effect on E. coli.The MIC levels of extracts for P. aeruginosa and P. mirabilis were in the ranges of 15.62-250 mg/mL and 31.25-250mg/mL, respectively, and the diethyl ether extract exhibited the highest inhibition effect.The MIC levels of extracts for S. typhimurium were in the range of 31.25-250mg/mL, and the diethyl ether extract showed the highest inhibition effect.According to antimicrobial activity results, the current study revealed that the highest antibacterial activity was found against B. subtilis in the ethyl acetate extract.Kenny et al. reported that neither the ethanol nor water extracts generated from C. arvense and C. vulgare exhibited any activity against S. aureus, MRSA, B. cereus, E. coli, or S. typhimirium [1].Conversely, the water and ethanol extracts of C. palustre were active against S. aureus, while the ethanol extract showed further inhibition against strains of MRSA (MIC = 375 µg/mL), B. cereus (MIC = 500 µg/mL), and E. coli (MIC = 375 µg/mL).A study by Karasakal et  observed that the herbal acetate extract from C. tenoreanum inhibited the growth of S. aureus (MIC = 0.5 mg/mL) and E. coli (MIC = 1 mg/mL) [42].Nazaruk and Jakoniuk proved that aqueous, methanol, and 70% ethanolic extracts from C. rivulare flowers and leaves also showed some antimicrobial activity, in which the aqueous leaf extract exhibited high activity, especially against Gram-positive bacteria.These extracts demonstrated antimicrobial activity against S. aureus (MIC = 6.2-25 mg/mL), B. subtilis (MIC = 6.2-25 mg/mL), E. coli (MIC = 6.2-50 mg/mL), and P. aeruginosa (MIC = 6.2-50 mg/mL) [43].Kozyra et al. reported that extracts from C. canum had no influence on the growth of the reference strains of Gram-negative bacteria and of yeasts belonging to Candida spp.However, the fractions possessed the highest activity against Gram-positive bacteria, especially S. aureus (MIC = 125-1000 µg/mL) and S. pneumonia (MIC = 125-1000 µg/mL), which are pathogens; and S. epidermidis (MIC = 125-1000 µg/mL), B. cereus (MIC = 62.5-1000 µg/mL), and B. subtilis (MIC = 125-1000 µg/mL) which are opportunistic microorganisms [18].Kozyra et al. isolated essential oils from the herb of C. vulgare, proving antimicrobial activity for Gram-positive and Gram-negative bacteria (concentration: 20 mg/mL) [6].A study conducted by Gadisa and Tadesse [44] showed that methanol extract of C. englerianum showed antibacterial activity against S. aureus (MIC = 16 µg/mL), E. faecalis (MIC = 1 µg/mL), E. coli (MIC = 64 µg/mL), and K. pneumoniae (MIC = 2 µg/mL).Another study using C. englerianum extract showed that the plant possessed inhibitory potential in regards to multidrug-resistant and the reference strains.This methanol extract demonstrated inhibitory activity against S. aureus (MRSA and MSSA) (MIC = 16 µg/mL), S. pyogenes (MIC = 1 µg/mL), E. coli (MIC = 64 µg/mL), and K. pneumoniae (MIC = 2 µg/mL) [45].Shahid et al. [46] reported that methanol extract from C. swaticum Petr.showed antimicrobial activity against S. aureus, S. typhi, B. megaterium, B. subtilis, P. mirabilis, and E. coli.In this study, extracts from C. vulgare possessed the highest antibacterial activity, especially against B. subtilis, S. aureus, E. coli, and P. aeruginosa.

Antifungal Activity
Antifungal MIC levels of extracts against C. albicans were in the range of 1.95-250 mg/mL (Table 5), and the highest inhibition effect was determined in the hexane extract.The MIC levels against C. glabrata, C. parapsilosis, and C. krusei were in the ranges of 1.95-125 mg/mL, 1.95-15.62mg/mL, and 0.97-31.25 mg/mL, respectively, and the highest inhibition effect was determined for the hexane extract.The MIC levels of four different extracts against P. chrysogenum and A. fumigatus were in the ranges of 3.9-31.25mg/mL and 0.97-250 mg/mL, respectively.Similarly, the highest inhibition effects were found in the hexane extract (Table 5).To the best of our knowledge, the present work is the first report on the antibacterial and antifungal activities of C. vulgare in different extracts in terms of MIC values and various antioxidant properties.According to our study results, the highest antifungal activity was found against C. krusei and A. fumigatus in the hexane extract.Current information regarding the antifungal efficacy of extracts from the Cirsium species is scarce.In some studies [6,18], no antifungal activity was detected against fungal strains of extracts from the Cirsium species.A study by Nazaruk and Jakoniuk proved that aqueous, methanol, and 70% ethanol extracts from C. rivulare flowers and leaves showed inhibitory activity against C. albicans (MIC = 25-50 mg/mL) [43].Ozcelik et al. reported that various extracts from C. hypoleucum showed antifungal activity against C. albicans (MIC = 64 µg/mL) and C. parapsilosis (MIC = 64 µg/mL) [47].In other studies, methanol extracts of C. englerianum showed antifungal effects on C. albicans, with MIC values of 128 µg/mL [43,44].In our study, hexane extract exhibited the highest inhibition effects against all tested fungal strains.However, no previous literature study reported the antifungal activity of the extracts of C. vulgare, as demonstrated in this study.

Plant Material
In this study, the whole plant parts of C. vulgare (root, stem, leaf, and flower) were collected from a natural habitat in the Trakya region of Turkey in June 2016.These plants were identified by Prof. Dr. E. Cabi of the Faculty of Science, Department of Biology, at Tekirdag Namik Kemal University, and a voucher specimen was deposited with the voucher number NGBB 7229.

Extractions
The whole plant parts (228 g) were ground and homogenized after being dried at room temperature.The extractions were carried out for 3 days, and a total of two macerations were performed in each solvent.The ground plants were macerated at room temperature using pure hexane (5.819 g), diethyl ether (0.973 g), ethyl acetate (0.905 g), and methanol (4.228 g) as solvents.The solvents were evaporated under vacuum using a rotary evaporator (Büchi Labortechnik, Flawil, Switzerland, Model: R-210 Rotavapor).The extraction yields were calculated as 2.55% for hexane extract, 0.42% for diethyl ether extract, 0.39% for ethyl acetate extract, and 1.58% for methanol extract.Then, the compositions of volatile compounds for the hexane extract of C. vulgare were investigated using GC, and the antibacterial, antifungal, and antioxidant activities were determined for all four extracts.

GC-MS Analysis
Chromatographic analyses were conducted using a Hewlett-Packard HP 6890 series GC/MS device.HP-5MS (5% phenyl methyl siloxane, 30 m × 250 µm × 0.25 µm) was used as the capillary column.Helium was used as the carrier gas, at a flow rate of 1.0 mL/min.The column's initial temperature was 180 • C 1 min after injection.The temperature was increased to 250 • C with an 8 • C/min heating ramp and a 1 min hold time, and the temperature was increased to 300 • C, with a 2 • C/min heating ramp over 10 min.The injection was performed in split mode (split ratio: 10:1).For analysis, the interface temperature was 250 • C, the injector temperature was 280 • C, and the running time was 49 min.The MS scan range was m/z 20-1000 using electron impact (EI) ionization (70 eV) and an ion source temperature of 250 • C. The components were identified by the comparison of their mass spectra with those of Wiley 9 and the NIST library.The relative percentages of the separated compounds were calculated with total ion chromatography using the computerized integrator.The retention indices (RI) were recognized externally using a series of n-alkanes (C6-C22), under the same chromatographic conditions [24].

Total Flavonoids Content (TFC)
Total flavonoid content was determined according to the suggestions of Zhishen et al. [48] by using the AlCl 3 -NaNO 2 method at a wavelength of 510 nm.A 0.25 mL aliquot of extract was mixed with 1.25 mL of distilled water in a test tube, followed by the addition of 75 µL of 5% sodium nitrite solution.After an incubation time of 6 min, 150 µL of 10% aluminium chloride was added.After 5 min, 0.5 mL of 1 M sodium hydroxide solution was added to the mixture.The mixture was immediately diluted to 2.5 mL by adding distilled water, mixing thoroughly.The absorbance of the mixture, which is pink in color, was determined at 510 nm against a blank containing all reagents except the extract samples.The total flavonoid content of the C. vulgare was calculated as mg catechin (CAT) and rutin (RE) equivalents per gram of the extracts (mg/g).The total flavonoid content was calculated with the help of the the standard curve equation: y = 21,782x + 0.0349, where R 2 = 0.9938 for catechin, and y = 12,714x + 0.0017, where R 2 = 0.9941 for rutin.The Trolox equivalent antioxidant capacity (TEAC) was estimated by using the method of Re et al. [49].For this assay, 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) cation radical (ABTS •+ ) solution was prepared by dissolving 96 mg of ABTS in 2.45 mmol/L Na 2 S 2 O 8 .This solution was shaken for 16 h at room temperature in the dark until a stable oxidative state was achieved.The ABTS •+ stock solution was diluted with methanol to an absorbance of 0.70 ± 0.02 at 734 nm before analysis.For the spectrophotometric assay, 2 mL of the ABTS •+ solution and 20 µL of C. vulgare extracts were mixed, and the absorbance was recorded at 734 nm (Hitachi spectrophotometer, 121-002.IR) after incubating the samples at 30 • C for 6 min.The calibration curve was plotted by using 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) as a standard.The results were expressed as mmol Trolox equivalents per g of extract, with the help of the the standard curve equation, which was y = 50,152 Trolox (mmol) + 93,842 (R 2 : 0.993).

Ferric-Reducing Antioxidant Power
The ferric-reducing antioxidant power (FRAP) assay was performed as previously described by Benzie and Strain [50].The working FRAP reagent was prepared by mixing 10 volumes of 300 mmol/L acetate buffer, pH 3.6, with 1 volume of 10 mmol/L 2,4,6-tris(2pyridyl)-S-triazine (TPTZ) in 40 mol/L HCl and with 1 volume of 20 mmol/L FeCl 3 × 6H 2 O.A volume of 2.25 mL of a working FRAP reagent was warmed to 37 • C.Then, 75 µL of the sample and 225 µL of deionized water were added to the FRAP reagent, and the absorbance was measured at 593 nm against reagent blank after 30 min of incubation.The results were expressed as µmol Fe 2+ equivalents per g of extract with the help of the calibration curve prepared in the concentration range of 0.1-1.0µmol/mL FeSO 4 × 7H 2 O (y = 0.6655 Fe 2+ (µmol) + 0.0021 (R 2 : 0.9978).The extract concentration providing 0.5 absorbance (EC 50 ) was calculated from the graph of absorbance at 593 nm against the extract concentration range of 0.0292-0.0882mg/mL.

β-Carotene-Linoleic Acid Emulsion Oxidation
The β-carotene bleaching test in the β-carotene-linoleic acid emulsion system was determined according to the methods suggested by Miller [51].A total of 1.6 mg of βcarotene was dissolved in 2 mL of chloroform, and then 400 mg of Tween 40 and 40 µL of linoleic acid were added to prepare the emulsion.The chloroform was evaporated, and 2 mL of methanol and 50 mL of water were added to the residue.A total of 250 µL of the emulsion mixture was vortexed with 10 µL of the extract solution (1 mg/mL).Methanol was added to the control sample.The absorbance of the samples was measured at 470 nm at 30 min intervals throughout the 180 min oxidation process.The reaction temperature was 42 • C. The results were expressed as the percentage of non-oxidized β-carotene after 180 min of reaction.The absorbance of the extracts and the control were measured immediately (t = 0).The tubes were incubated at 42 • C, and the absorbance was measured using a spectrophotometer at 30 min intervals at 470 nm for 180 min (t = 180).The antioxidant activity (AA) of the extracts was evaluated in terms of bleaching of the β-carotene using the following formula: where A 0 -absorbance value of the extract at zero time of incubation; A t -absorbance value of the extract at t minutes of incubation; A 0 0 -absorbance value of the control at zero time of incubation; A t 0 -absorbance value of control at t minutes of incubation; BHT (1 mg mL −1 ) was taken as the positive control sample.

Cupric-Reducing Antioxidant Capacity (CUPRAC)
For determination of the CUPRAC activity, CuCl 2 solution (1.0 × 10 −2 M), neocuproine alcoholic solution (7.5 × 10 −3 M) and NH 4 Ac buffer solution (pH = 7) were used for the analyses [52], and absorbance readings were obtained at 450 nm.The CUPRAC activity of the extracts (mM trolox/g) was calculated from the calibration curve obtained using Trolox as standard.The extract concentration providing 0.5 absorbance (EC 50 ) was calculated from the graph of absorbance at 450 nm against the µg/mL extract concentration.

Superoxide-Radical Scavenging Activity
Superoxide anion scavenging activities were determined according to the method described previously by Robak and Gryglewski [53].The reaction mixtures were arranged in 0.1 M phosphate buffer at pH 7.4.A total of 1 mL (156 µM) of nitrobluetetrazolium (NBT), 1 mL (468 µM) of reduced nicotinamide adenine dinucleotide (NADH), and 1 mL of the extracts were mixed at 100 µg/mL concentrations.A total of 100 µL of phenazine methosulphate (PMS, 60 µM) was added for reaction initiation.Incubation was performed at 25 • C for 5 min.Absorbance was measured at 560 nm using L-ascorbic acid as a control.The percentage of inhibition was determined using the following formula: superoxide inhibition percentage = [(A 0 − A 1 )/A 0 ] × 100 where A 0 -absorbance of the control, and A 1 -absorbance of the extracts.

Minimum Inhibitory Concentration (MIC)
The plant extracts were dissolved in H 2 O and DMSO at 1000 mg/mL of stock concentration to determine their antibacterial and antifungal activities.Penicillin G, gentamycin, and fluconazole were used as the standard antibacterial and antifungal drugs.These tests were conducted by applying the microdilution method in a liquid medium, according to CLSI standards [54][55][56].The procedure involves preparing two-fold dilutions of the plant extracts (e.g., 0.48, 0.97, 1.95, 3.90, 7.81, 15.62, 31.25, 62.5, 125, and 250 mg/mL) in a liquid growth medium (Mueller-Hinton Broth and RPMI 1640).The MIC values were determined as the lowest concentration of extracts inhibiting the visible growth of each organism on the plate.

Conclusions
In addition to the determination of some phytocompounds in the hexane extract, this study reports the antioxidant, antibacterial, and antifungal activity of different organic solvent extracts of C. vulgare.The highest antibacterial activity was found against B. subtilis in the ethyl acetate extract, while the highest antifungal activity was found against C. krusei and A. fumigatus in the hexane extract.According to the results of the antioxidant assays, the highest observed antioxidant activity of the C. vulgare extracts in the methanol extract could be attributed to the presence of extractable flavonoid compounds and a high flavonoid content.The highest levels of total flavonoids were found in the polar methanol extract, followed by the ethyl acetate because the flavonoid class compounds tend to be semipolar-polar, so that more flavonoid compounds could be extractable in semipolarpolar solvents, such as methanol, ethyl acetate, and diethyl ether, compared to that obtained from hexane.C. vulgare may provide even higher antioxidant activities to the methanol extracts than to the hexane extract.
On the other hand, the hexane extract exhibited a high antifungal activity against all fungal strains; therefore, we can conclude that constituents such as terpenoids, esters, and hydrocarbons found in lipophilic extract could be responsible of their high bioactivity and the reinforcement of these actions.The GC-MS analysis of the hexane extract of C. vulgare showed the existence of some important chemical compounds with different chemical structures.The present work discloses the potential antioxidant and antimicrobial activities of C. vulgare, along with some bioactive components, indicating that C. vulgare might hold potential for use as an antioxidant, antibacterial, and antifungal agent in food and pharmaceutical industries.

Table 1 .
The composition (%) of volatile compounds of the hexane extract of C. vulgare.

Table 2 .
The chemical class distribution of the C. vulgare hexane extract.

Table 3 .
The total flavonoid content (TFC) and antioxidant activities of C. vulgare extracts.
* As standard agents, penicillin G was used only for S. aureus, and gentamicin was used for the other bacteria.
* Fluconazole was used as a standard agent.