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Article

Antimicrobial and Antioxidant Efficacy of the Lipophilic Extract of Cirsium vulgare

by
Mine Aydın Kurç
1,*,
Hakime Hülya Orak
2,
Dumrul Gülen
1,
Hilmican Caliskan
3,
Merve Argon
3 and
Temine Sabudak
3
1
Department of Medical Microbiology, Faculty of Medicine, Tekirdag Namik Kemal University, 59030 Tekirdag, Turkey
2
Department of Food Technology, Vocational School of Technical Sciences, Tekirdag Namik Kemal University, 59030 Tekirdag, Turkey
3
Department of Chemistry, Faculty of Science and Arts, Tekirdag Namik Kemal University, 59030 Tekirdag, Turkey
*
Author to whom correspondence should be addressed.
Molecules 2023, 28(20), 7177; https://doi.org/10.3390/molecules28207177
Submission received: 19 September 2023 / Revised: 13 October 2023 / Accepted: 13 October 2023 / Published: 19 October 2023
(This article belongs to the Section Medicinal Chemistry)
Browse Figure
Versions Notes

Abstract

:
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.

1. 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.
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.

2. Results and Discussion

2.1. GC–MS Analysis

The GC–MS chromatogram of hexane extract revealed the presence of 41 compounds. These compounds were characterized by their retention time (RT), retention index (RI), and their molecular formula and concentrations, according to the peak area (%), and are presented in Table 1. In this study, GC–MS results indicated that the hexane extract is a rich source of terpenoid compounds, and terpenoids exhibit antimicrobial, antiviral, antiallergic, and anti-inflammatory activities [22]. According to chemical class distribution, terpenoids (52.89%) were the most abundant compound, followed by esters (19.92%) and hydrocarbons (14.17%) (See Table 2). In the determination of the volatile components of the C. japonicum plant using GC–MS, Miyazawa et al. found terpenoids (45.22%) to be the most abundant compounds [23]. Lup-20(29)-en-3-yl-acetate (29.94%), lupeol (13.19%), linolenic acid ethyl ester (6.38%), norolean-12-en (5.15%), 1-nonadecene (4.23%), 9,12-octadecadien-1-ol (4.9%), hexadecanoic acid (palmitic acid) (3.21%), 1-tricosene (2.89%), and cycloeicosane (2.62%) were the major compounds found in the extract (Table 1). In the study conducted by Orhan et al. [24], palmitic acid was observed as the main component in hexane extract of C. hypoleucum. Additionally, Kozyra et al. [6] detected β-linalool (1.3%), β-cyclocitral (1.7%), and eugenol (1.7%) terpenoids in the essential oil analysis of the C. vulgare plant using the GC–MS method.
The most abundant terpenoids in the hexane extract were Lup-20(29)-en-3-yl-acetate, lupeol, norolean-12-ene, and methyl commate B (2.0%). Lupeol and lup-20(29)-en-3-yl-acetate are triterpenes that exhibit pharmacological activities, including anticancer, anti-inflammatory, and antimicrobial properties [25]. Lupeol also shows nephroprotective and hepatoprotective effects [26]. Methyl commate B is a pentacyclictriterpene glycoside which possesses antimicrobial and anti-inflammatory properties [27]. Hydrocarbons were also (14.17%) quite abundant, with significant amounts of 1-nonadecene (4.23%), 1-tricosene (2.89%), and cycloeicosane (2.62%). The n-alkanes are thought to be indigenous to plants and are formed as the result of the decarboxylation of long-chain fatty acids [28]. The long aliphatic hydrocarbons are found on the surfaces of the aerial organs of plants and are important for repelling water and controlling the gas balance within a plant [29]. Smaoui et al. [30] reported that 1-nonadecene from the Streptomyces sp. TN 256 strain exhibited antibacterial activity and strong antifungal activity against C. albicans [31]. Among the esters, hexadecanoic acid butyl ester (butyl palmitate) (9.89%) and linolenic acid ethyl ester (6.38%) were predominant. 9,12-Octadecadien-1-ol (linoleyl alcohol) (4.9%), which is a fatty alcohol produced by the reduction of linolenic acid, and palmitic acid (4.9%) were the major compounds found in the hexane extract. Leventhal et al. [32] reported that fatty acids can modulate immune responses, and Reifen et al. [33], suggested that α-linolenic acid has potential as an anti-inflammatory agent. A similar explanation was provided by Aparna et al., who reported that hexadecanoic acid may be an anti-inflammatory compound [34]. According to the literature; β-linalool (1.3%), β-cyclocitral (1.7%) and eugenole (1.7%) values were also revealed in the GC–MS analysis of C. vulgare plants [6]. Additionally, Orhan et al. [24] suggested that palmitic acid (26.35%) is the main fatty acid identified in the C. hypoleucum.

2.2. 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 Griškevičienė 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.

2.3. 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 Fe2+/g extract), and the lowest value was obtained for hexane extract (49.7 µmol Fe2+/g extract). Based on the EC50 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; EC50 of 18.5 µg/mL) and ethyl acetate extract, followed by values of 1.62 mmol Trolox/g and EC50 of 33.5 µg/mL. The hexane fraction exhibited the lowest activity (0.38 mmol Trolox/g extract; EC50of 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 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].

2.4. 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 31.25–250 mg/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 31.25–250 mg/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 al. indicated that C. bulgaricum demonstrated antimicrobial activity against a range of bacteria, including E. coli (MIC values = 250 and 500 µg/mL), S. enteritidis (MIC = 500 µg/mL), L. monocytogenes (MIC = 250 µg/mL), and S. aureus (MIC = 250 µg/mL) [39]. Borawska et al. also confirmed the antimicrobial activity of flower and leaf extracts from C. arvense, C. oleraceum, C. palustre, C. rivulare, or C. vulgare against S. aureus, B. subtilis, and P. aeruginosa [41]. Loizzo et al. 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.

2.5. 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, 1.95–15.62 mg/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 3.9–31.25 mg/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.

3. Materials and Methods

3.1. 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.

3.2. 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.

3.3. 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].

3.4. Total Flavonoids Content (TFC)

Total flavonoid content was determined according to the suggestions of Zhishen et al. [48] by using the AlCl3-NaNO2 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 R2 = 0.9938 for catechin, and y = 12,714x + 0.0017, where R2 = 0.9941 for rutin.

3.5. Antioxidant Activities

3.5.1. Trolox Equivalent Antioxidant Activity (TEAC)

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 Na2S2O8. 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 (R2: 0.993).

3.5.2. 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(2-pyridyl)-S-triazine (TPTZ) in 40 mol/L HCl and with 1 volume of 20 mmol/L FeCl3 × 6H2O. 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 Fe2+ equivalents per g of extract with the help of the calibration curve prepared in the concentration range of 0.1–1.0 μmol/mL FeSO4 × 7H2O (y = 0.6655 Fe2+ (μmol) + 0.0021 (R2: 0.9978). The extract concentration providing 0.5 absorbance (EC50) was calculated from the graph of absorbance at 593 nm against the extract concentration range of 0.0292–0.0882 mg/mL.

3.5.3. 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 β-carotene 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:
AA = [(1 − (A0 − At/A00 − At0)] × 100
where A0—absorbance value of the extract at zero time of incubation; At—absorbance value of the extract at t minutes of incubation; A00—absorbance value of the control at zero time of incubation; At0—absorbance value of control at t minutes of incubation; BHT (1 mg mL−1) was taken as the positive control sample.

3.5.4. Cupric-Reducing Antioxidant Capacity (CUPRAC)

For determination of the CUPRAC activity, CuCl2 solution (1.0 × 10−2 M), neocuproine alcoholic solution (7.5 × 10−3 M) and NH4Ac 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 (EC50) was calculated from the graph of absorbance at 450 nm against the µg/mL extract concentration.

3.5.5. 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 = [(A0 − A1)/A0] × 100
where A0—absorbance of the control, and A1—absorbance of the extracts.

3.6. Antimicrobial Activities

3.6.1. Microorganisms

The antibacterial activity was tested against two Gram-positive (Staphylococcus aureus ATCC 43300 and Bacillus subtilis NRRL NRS-744) and four Gram-negative bacterial strains (Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, Proteus mirabilis ATCC 12453, and Salmonella typhimurium ATCC 14028) that were grown in nutrient agar at 37 °C for 18 h. The antifungal activity was tested against six fungal strains (Candida albicans ATCC 90028, Candida glabrata ATCC 90030, Candida parapsilosis ATCC 22019, Candida krusei ATCC 6258, Penicillium chrysogenum ATCC 48271, and Aspergillus fumigatus ATCC 204305) that were grown in Sabouraud dextrose agar (SDA) at 27 °C.

3.6.2. Minimum Inhibitory Concentration (MIC)

The plant extracts were dissolved in H2O 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.

4. 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 semipolar–polar 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.

Author Contributions

Conceptualization, M.A.K., H.H.O. and T.S.; investigation, M.A.K., H.H.O., H.C. and M.A.; resources, H.C. and M.A.; writing—original draft, and writing—review and editing, H.H.O., D.G. and T.S.; supervision, T.S. All authors have read and agreed to the published version of the manuscript.

Funding

This study received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data used to support the findings of this study are included within the article.

Acknowledgments

The authors would like to thank Evren Cabi for plant identification.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Not applicable.

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Molecules 28 07177 g001
Figure 1. Inhibition of the oxidation of β-carotene-linoleic acid emulsion by C. vulgare extracts; BHT butyhlhidroxytoluene; (n = 3).
Figure 1. Inhibition of the oxidation of β-carotene-linoleic acid emulsion by C. vulgare extracts; BHT butyhlhidroxytoluene; (n = 3).
Molecules 28 07177 g001
Table 1. The composition (%) of volatile compounds of the hexane extract of C. vulgare.
Table 1. The composition (%) of volatile compounds of the hexane extract of C. vulgare.
No.RICompoundsPercentage [%]
18915-Eicosene1.11
2891Cycloeicosane2.62
3936Hexadecanoic acid (palmitic acid)3.21
49411-Nonadecene4.23
5975Hexadecadienoic acid, methyl ester0.74
69869,12-Octadecadien-1-ol4.90
7994cis-9-Hexadecenal1.15
81002Hexadecanoic acid, butyl ester9.89
91043Docosane0.50
101069Linolenic acid, ethyl ester6.38
1110801-Tricosene2.89
121129Pentacosane0.41
1311561,2-Benzenedicarboxylic acid, diisooctyl ester0.36
141165Tetrahydrogeraniol0.17
151183Pentafluoropropionic acid, heptadecyl ester1.99
16122817-Pentatriacontene0.18
17126310-Methylnonadecane0.24
1812731,3-Benzenedicarboxylic acid, bis(2-ethylhexyl) ester0.33
191292cis-9,10-Epoxyoctadecanamide0.31
201330Squalene0.11
2114072,2-dimehyl-3-(3,7,16,20-tetramethyl-heneicosa-3,7,11,15,19-pentaenyl) oxirane0.15
2214571-Hentetracontanol0.51
231470Acetic acid, octadecyl ester0.11
241492Octadecanal0.13
251590Solanesol0.14
261548Tetratetracontane1.99
271572Vitamin E0.16
2815831,10-dibromo-decane0.10
2916481-Triacontanol0.27
301682Octadecanal0.09
311684Stigmasterol0.35
321740Stigmast-5-en-3-ol1.17
331774Olean-12-ene1.82
3417861,54-dibromo-tetrapentacontane0.19
351813Methyl commate B2.00
361858Docosyl acetate 0.12
371860A′-Neogammacer-22(29)-ene
(Diploptene) 		0.37
381882Norolean-12-ene5.15
391936Lupeol13.19
402066Lup-20(29)-en-3-yl-acetate29.94
4123014′,5-dihydroxy-7-diglyocoside-flavone0.31
Table 2. The chemical class distribution of the C. vulgare hexane extract.
Table 2. The chemical class distribution of the C. vulgare hexane extract.
Volatile CompoundsPercentage [%]
Terpenoids52.89
Hydrocarbons (alkanes and alkenes)14.17
Esters19.92
Alcohols5.68
Fatty acids3.21
Sterols1.52
Carbonyl compounds (aldehyde-ketones)1.37
Other functional groups (amide, epoxide, alkyl halide, etc.)0.29 alkyl halide
0.31 amide
0.31 phenolics
0.15 epoxide
0.16 vitamin E
Total (%)99.98
Table 3. The total flavonoid content (TFC) and antioxidant activities of C. vulgare extracts.
Table 3. The total flavonoid content (TFC) and antioxidant activities of C. vulgare extracts.
Extracts1 Total Flavonoid ContentAntioxidant Activity Assays
TFC
(mg CAT/g) 		TFC
(mg RUT/g) 		TEAC
(mmol Trolox/g)		FRAP
(μmol Fe2+/g)		2 EC50 FRAP
(µg/mL)		CUPRAC
(mM Troloks/g)		3 EC50CUPRAC
(µg/mL)		4 Superoxide-Radical Scavenging Activity (%)
Methanol25.7 ± 2.244.6 ± 1.30.86 ± 0.041436.6 ± 11.114.7 ± 0.082.14 ± 0.00218.5 ± 0.00274.8 ± 0.8
Ethylacetate23.6 ± 0.940.3 ± 1.80.41 ± 0.01295.6 ± 4.180.5 ± 0.021.62 ± 0.00233.5 ± 0.03153.5 ± 1.2
Hexanend 5nd0.34 ± 0.0349.7 ± 3.7651.8 ± 0.010.38 ± 0.055140.6 ± 0.00324.9 ± 0.2
Diethyl ether16.3 ± 0.828.1 ± 0.8 0.40 ± 0.02115.3 ± 4.7127.5± 0.031.15 ± 0.01160.6 ± 0.00139.8 ± 2.5
1 The total flavonoid content was calculated as catechin and rutin equivalents in g extract, where the catechin equivalent was obtained according to y = 21.782 (CAT) + 0.0349 (R2: 0.9988), and the rutin equivalent was obtained according to the y = 12.714 (RUT) + 0.0017 (R2: 0.9941) equation. 2,3 The EC50 value (μg/mL) is the effective concentration giving an absorbance of 0.5, which indicates the concentration of the extract that yields a half-maximal response (efficient concentration = EC50). 4 ascorbic acid exhibited 99.09 ± 1.05% activity at the same concentration; 5 not detected.
Table 4. Antibacterial MIC results (mg/mL) for different extracts.
Table 4. Antibacterial MIC results (mg/mL) for different extracts.
BacteriaMethanolEthyl AcetateDiethyl EtherHexaneStandard (µg/mL)
S. aureus25015.6215.62≥2500.125 *
B. subtilis2503.915.622501
E. coli12562.531.2515.621
P. aeruginosa25012515.6231.251
P. mirabilis25012531.25≥2501
S. typhimurium25025031.25≥2502
* As standard agents, penicillin G was used only for S. aureus, and gentamicin was used for the other bacteria.
Table 5. Antifungal MIC results (mg/mL) for different extracts.
Table 5. Antifungal MIC results (mg/mL) for different extracts.
FungiMethanolEthyl AcetateDiethyl EtherHexaneStandard (µg/mL)
C. albicans25015.6215.621.952 *
C. glabrata12515.627.811.952
C. parapsilosis15.6215.6215.621.951
C. krusei31.253.97.810.974
P. chrysogenum7.8131.257.813.91
A. fumigatus25015.627.810.971
* Fluconazole was used as a standard agent.
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Aydın Kurç, M.; Orak, H.H.; Gülen, D.; Caliskan, H.; Argon, M.; Sabudak, T. Antimicrobial and Antioxidant Efficacy of the Lipophilic Extract of Cirsium vulgare. Molecules 2023, 28, 7177. https://doi.org/10.3390/molecules28207177

AMA Style

Aydın Kurç M, Orak HH, Gülen D, Caliskan H, Argon M, Sabudak T. Antimicrobial and Antioxidant Efficacy of the Lipophilic Extract of Cirsium vulgare. Molecules. 2023; 28(20):7177. https://doi.org/10.3390/molecules28207177

Chicago/Turabian Style

Aydın Kurç, Mine, Hakime Hülya Orak, Dumrul Gülen, Hilmican Caliskan, Merve Argon, and Temine Sabudak. 2023. "Antimicrobial and Antioxidant Efficacy of the Lipophilic Extract of Cirsium vulgare" Molecules 28, no. 20: 7177. https://doi.org/10.3390/molecules28207177

APA Style

Aydın Kurç, M., Orak, H. H., Gülen, D., Caliskan, H., Argon, M., & Sabudak, T. (2023). Antimicrobial and Antioxidant Efficacy of the Lipophilic Extract of Cirsium vulgare. Molecules, 28(20), 7177. https://doi.org/10.3390/molecules28207177

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