Comparative Study on the Essential Oils from Five Wild Egyptian Centaurea Species: Effective Extraction Techniques, Antimicrobial Activity and In-Silico Analyses

The genus Centaurea is recognized in folk medicine for anti-inflammatory, anti-itch, antitussive, purgative, astringent, and tonic activities. To study the chemical determinant for antimicrobial activity essential oils (EOs), five Centaurea species were analyzed including: C. scoparia, C. calcitrapa, C. glomerata, C. lipii and C. alexandrina. Conventional hydro-distillation (HD) and microwave-assisted extraction (MAE), as new green technologies, were compared for the extraction of essential oils. GC/MS analysis identified 120 EOs including mostly terpenoid except from C. lipii and C. alexandrina in which nonterpenoids were the major constituents. Major terpenoids included spathulenol, caryophyllene oxide and alloaromadendrene oxide-2. To probe antibacterial activity, potential EO inhibitors of a bacterial type II DNA topoisomerase, DNA gyrase B were screened via an in silico molecular docking approach. Spathulenol and alloaromadendrene oxide-2 possessed the best binding affinity in the ATP- binding pocket of Gyrase B enzyme. Principal component analysis and agglomerative hierarchical clustering were used for sample classification and revealed that sesquiterpenes contributed the most for accessions classification. In vitro antimicrobial activity against Staphylococcus aureus, Escherichia coli and Aspergillus niger for all EOs were also evaluated. EOs from C. lipii, C. glomerata and C. calcitrapa exhibited significant MIC against S. aureus with an MIC value of 31.25 µg/mL.


EOs Chemical Profiles of Centaurea Species
EOs from five Centaurea species were extracted via HD and MAE. The yield of the extracted EOs by HD were found to be 0.03, 0.054, 0.043, 0.055 and 0.037% v/w, whereas by MAE they were 0.023, 0.031, 0.037, 0.044, and 0.024% v/w from C. scoparia, C. calcitrapa, C. glomerata, C. lipii and C. alexandrina, respectively. Table 1 summarized the compounds, retention time (Rt), Kovat's index (KI), and percentage of each metabolite. EO yield variations are attributed to differences in the extraction method. Differences in environmental conditions may contribute to differences in EOs between the plant species [37].
Terpenoids are the main components in EOs from C. scoparia via HD and MAE (73.47 and 76.06%, respectively); in addition, EOs contain nonterpenoids (10.64 and 14.46%) as well as carotenoid-derived compounds (8.41 and 6.86%), respectively ( Figure 1). Among the identified terpenes, sesquiterpenes comprised the major component with a concentration of (72.68 and 76.06%), while diterpenes were detected in trace amounts (0.79 and 0%) and monoterpenes were not detected.

EOs Chemical Profiles of Centaurea Species
EOs from five Centaurea species were extracted via HD and MAE. The yield of the extracted EOs by HD were found to be 0.03, 0.054, 0.043, 0.055 and 0.037% v/w, whereas by MAE they were 0.023, 0.031, 0.037, 0.044, and 0.024% v/w from C. scoparia, C. calcitrapa, C. glomerata, C. lipii and C. alexandrina, respectively. Table 1 summarized the compounds, retention time (Rt), Kovat's index (KI), and percentage of each metabolite. EO yield variations are attributed to differences in the extraction method. Differences in environmental conditions may contribute to differences in EOs between the plant species [37].
Terpenoids are the main components in EOs from C. scoparia via HD and MAE (73.47 and 76.06%, respectively); in addition, EOs contain nonterpenoids (10.64 and 14.46%) as well as carotenoid-derived compounds (8.41 and 6.86%), respectively ( Figure 1). Among the identified terpenes, sesquiterpenes comprised the major component with a concentration of (72.68 and 76.06%), while diterpenes were detected in trace amounts (0.79 and 0%) and monoterpenes were not detected. Two EOs derived from C. calcitrapa revealed the abundance of terpenoids with concentrations of 72.34 and 82.87% alongside with nonterpenoids (21.65 and 9.23%) ( Figure  1). An amount of 4.48% of EO extracted by MW were characterized as carotenoid-derived compounds, while there was a complete absence of these compounds in HD-EO ( Figure  1).
The EOs from C. glomerata displayed a preponderance of terpenoids (60.03 and 79.75%) as well as a high concentration of nonterpenoids (32.07 and 18.68%) ( Figure 1). The data analysis exhibited a total absence of carotenoid-derived compounds in both methods, which agrees with a previous EO extraction of chemical components from C. glomerata [38].
The chemical profile of the HD-EO from C. lipii revealed a majority of nonterpenoids (82.37%) and a low concentration of terpenoids (14.96%) with complete absence of carotenoids. In contrast, extracted EO by MW terpenoids were 79.56%, comprising the main constituents, while a low concentration of nonterpenoids (16.11%) and traces of carotenoid-derived compounds (1.87%) was detected ( Figure 1).
The obtained HD-EO of C. alexandrina was characterized by the abundance of nonterpenoids (64.25%), a significant concentration of terpenes (30.17%) and traces of carotenoid-derived compounds (0.46%). On the other hand, the terpenes were characterized as Two EOs derived from C. calcitrapa revealed the abundance of terpenoids with concentrations of 72.34 and 82.87% alongside with nonterpenoids (21.65 and 9.23%) ( Figure 1). An amount of 4.48% of EO extracted by MW were characterized as carotenoid-derived compounds, while there was a complete absence of these compounds in HD-EO ( Figure 1).
The EOs from C. glomerata displayed a preponderance of terpenoids (60.03 and 79.75%) as well as a high concentration of nonterpenoids (32.07 and 18.68%) ( Figure 1). The data analysis exhibited a total absence of carotenoid-derived compounds in both methods, which agrees with a previous EO extraction of chemical components from C. glomerata [38].
The chemical profile of the HD-EO from C. lipii revealed a majority of nonterpenoids (82.37%) and a low concentration of terpenoids (14.96%) with complete absence of carotenoids. In contrast, extracted EO by MW terpenoids were 79.56%, comprising the main constituents, while a low concentration of nonterpenoids (16.11%) and traces of carotenoid-derived compounds (1.87%) was detected ( Figure 1).
The obtained HD-EO of C. alexandrina was characterized by the abundance of nonterpenoids (64.25%), a significant concentration of terpenes (30.17%) and traces of carotenoidderived compounds (0.46%). On the other hand, the terpenes were characterized as the main components of EO extracted via MW with concentration of 58.17% including mono-(1.24%), sesqui-(39.31%) and di-(7.63%) terpenes with the presence of a high concentration of nonterpenoids (33.53%) and an absence of carotenoid-derived compounds ( Figure 1).
Sesquiterpenes (54.24 and 74.07%) and diterpenes (5.79 and 5.68%) represented the overall categories of terpenoids in both EOs derived from C. glomerata. The disappearance and/or minority of mononterpenes in EOs of C. scoparia and C. Calcitrapa, as well as their total absence in the EOs for C. glomerata, is in agreement with the described analysis of EOs of different species of Centaurea [16,27].
The abundance of hydrocarbons in hydro-distilled EO from C. lipii, especially that of fatty acids, is in agreement with the two Centaurea plants, C. calcitrapa and C. spaerocephala. Furthermore, previous studies of C. pannonica essential oil extracted by HD revealed that oil was rich in fatty acids (43.7%), with 9-octadecenoic acid (34.0%) and (Z,Z)-9,12octadecadienoic acid (8.6%) as the major compounds [39]. However, 79.56% from total mass of EO extracted via MW were characterized as terpenoids, including 65.14% of sesquiterpenes in addition to 6.59% diterpenes and 7.83% monoterpenes. These findings were totally in agreement with the observation of EOs of the above three Centaurea plants (C. scoparaia, C. calcitrapa, C. glomerata), as well as the previous described data of EOs of the other Centaurea ecoplants [14,17,28]. Alloaromadendrene oxide-2 (31.55%) was found to be the main sesquiterpenoid of EO extracted via MW, as well as spathulenol (8.14%), 6,10,14trimethylpentadecan-2-one (8.34%) and torreyol (7.11%). Most of these major compounds were characterized from all EOs of the above plants in addition to others such as C. iberica, C. virgate [18], C. kilaea [27] and C. helenioides [16].
Sesquiterpenoids (39.30%) were identified as the main class of EO extracted from C. alexandrina via MW in addition to a high concentration of diterpenes (17.63%) and minors of monoterpenes (1.56%). Caryophyllene oxide, (17.01%), 6, 10, 14-trimethyl pentadecan-2-one (14.23%) and aromadendrene oxide-1 (3.12%) were proven to be the major characterized sesquiterpenes. Thunbergol (9.05%), 13-epi-manool (6.53%) and phytol (1.13) were assigned as the main diterpene constituents. Only two monterpenes, 1, 8-cineole and boronal, were assigned as overall identified monoterpenes. All these findings were in complete agreement with the above four Centaurea plants in addition to the documented data of others such as C. iberica, C. virgate [18], C. kilaea [27] and C. helenioides [16].             Monoterpenes, basically constructed from an isoprene unit, were recognized as the main components in most of the EOs derived from the plant kingdom [40]. However, several Centaurea species were described to include traces and/or absence of monoterpenes such as C. appendicigera, C. helenioides [16] and C. kilaea [27]. These data agree with our results of C. scoparia. At the same time, diterpenes are described as rare components in EOs with some exceptions, such as Lactuca serriola [41] and the Indian leaves of Araucaria heterophylla [42]. Our results with C. scoparia are also in line with diterpenes found in trace amounts in EO extracted by hydro-distillation, with one compound (phytol, 0.79%), while the was a complete absence in EOs extracted via microwave.
Similar to the present data of EOs of C. scoparia and the reported EO constituents of the EOs of C. appendicigera, C. helenioides [16] and C. kilaea [27], the non-existence of monoterpenes were recorded in EOs of C. calcitrapa. In contrast with the data of EOs of C. scoparia and most investigated Centaurea species, this revealed the presence of substantial concentrations of diterpenoids with abundance of phytol in EOs of C. scoparia (7.90 and 16.89%, respectively). The abundance of phytol was already reported for Centaurea species such as C. aggregata ssp. aggregate, C. behen [24], C. stenolepis [25] and others. In complete agreement with the above two Centaurea plants and previous published data of others [16,27], the nonexistence of monoterpenes were observed from the EOs of C. glomerata. On the other side, low concentrations of diterpenes (5.79 and 5.68%) were identified in the two EOs of this plant with only one identified compound, phytol, that is already characterized from numerous Centaurea plants [24,27].
Like the above three plant (C. glomerata, C. scoparia, C. calcitrapa) and others [16,27], the presence of traces of monoterpenes were noticed only in extracted EO from C. lipii, by MAE, in a concentration of 7.83% with d-isothujone (3.12%) as the main component. From the extracted EO by HD, only two diterpenoids, dehydroabietane and trans-geranyl geraniol (0.70%, each), were identified from EO extracted via hydrodistillation, while only one compound, phytol (6.59%), was characterized from extracted EO by MW.
The nonterpenoid compounds of EOs of C. scoparia were categorized with considerable concentrations in the two EO samples (10.64% and 14.46%). Methyl palmitate was the main nonterpenoidal compound with concentrations of 2.71% and 6.69%. Both EO samples of C. scoparia were found to contain carotenoid-derived compounds with respective concentrations of 8.41% and 6.86%. Theaspirane A (3.75 and 3.16%) and theaspirane B (2.00 and 1.91%) were characterized as the main compounds. The high concentration of the nonterpenoids in EOs of C. scoparia was in accordance with the previous described EOs of the Centaurea species, such as C. kilaea [27], C. amanicola [17], C. armena [43], C. cadmea and C. calolepis [44].
The two extracted EOs from C. glomerata nonterpenoids were characterized by high concentrations (32.07 and 18.68%), in which n-tricosane (10.51 and 1.64%) and phenylethyl stearate (4.41 and 3.35%) were found as major components.
The nonterpenoids (82.37%) represent the main components of the extracted EOs of C. lipii via hydrodistillation, in which n-heneicosane (61.07%) and n-eicosane (12.68%) were found as the main constituents. This result is completely in contrast with the above EOs of the three Centaurea plants, C. scoparia, C. calcitrapa and C. glomerata, and also with the extracted EO by MAE from the same plant. These results strongly elucidate the effect of the extraction method on the chemical composition of EOs. In the other side, 16.11% from the total mass of EOs derived via MAE were characterized by the hydrocarbon n-heneicosane (6.71%) and phenylethyl stearate (2.67%), which constitute the main entities.
The nonterpenoids of the extracted EO from C. alexandrina via hydro-distillation were assigned as (Z)-9-octadecenamide (19.13%), methyl arachidonate (14.25%), arachidic acid (9.81%) and phenylethyl stearate (6.82%). These results were in complete uniformity with the published data of the EOs of C. lycopifolia, in which arachidic acid (5.0 %) was considered as one of the major components [30]. In EOs extracted by MAE from C. alexandrina, the nonterpenoids represented by high concentrations with an abundance of methyl arachidonate (12.52%), (Z)-9-octadecenamide (3.92%) and phenylethyl stearate (3.70%). These results were in complete agreement with the results of the EOs of C. lipii, especially that extracted via hydrodistillation and also with the published data of the EOs of C. calcitrapa and C. spaerocephala. This significant un-symmetry strongly supports the theory of the effects of the extraction techniques on the EOs chemical components.

Unsupervised PCA and HCA Data Analyses
The correlation between the five Centaurea species was established based upon the main compounds of the EOs via PCA and AHC (Figure 2A  from the total mass of EOs derived via MAE were characterized by the hydrocarbon nheneicosane (6.71%) and phenylethyl stearate (2.67%), which constitute the main entities. The nonterpenoids of the extracted EO from C. alexandrina via hydro-distillation were assigned as (Z)-9-octadecenamide (19.13%), methyl arachidonate (14.25%), arachidic acid (9.81%) and phenylethyl stearate (6.82%). These results were in complete uniformity with the published data of the EOs of C. lycopifolia, in which arachidic acid (5.0 %) was considered as one of the major components [30]. In EOs extracted by MAE from C. alexandrina, the nonterpenoids represented by high concentrations with an abundance of methyl arachidonate (12.52%), (Z)-9-octadecenamide (3.92%) and phenylethyl stearate (3.70%). These results were in complete agreement with the results of the EOs of C. lipii, especially that extracted via hydrodistillation and also with the published data of the EOs of C. calcitrapa and C. spaerocephala. This significant un-symmetry strongly supports the theory of the effects of the extraction techniques on the EOs chemical components.

Unsupervised PCA and HCA Data Analyses
The correlation between the five Centaurea species was established based upon the main compounds of the EOs via PCA and AHC (Figure 2A  The PCA and AHC data revealed a strong correlation between the five plants. Firstly, C. calcitrapa exhibited strong correlation with C. scoparia and moderate correlation with C. alexandrina. Similarly, C. scoparia and C. calcitrapa exhibited a weak correlation with C. alexandrina. Furthermore, C. glomerata and C. lipii exhibited strong correlations, especially via the EO derived by microwave. The significant variations between the five plants might be attributed to the microclimatic and environmental condition variations [41,45].
C. scoparia and C. calcitrapa exhibited a strong correlation depending on the main components in which spathulenol, caryophyllene oxide, trans-caryophyllene and 6, 10, 14trimethyl pentadecan-2-one were found as the main constituents. Caryophyllene oxide and spathulenol were characterized as major components in the EOs of the three plants C. scoparia, C. calcitrapa and C. alexandrina. Otherwise, the correlations of the two plants C. glomerata and C. lipii were deduced via the main compounds of the EOs of the plants, including spathulenol, alloaromadendrene oxide-2,6,10,14-trimethyl pentadecan-2-one and phytol. The correlations of the five plants were also observed via the main compounds of their EOs, such as spathulenol, 6,10,14-trimethyl pentadecan-2-one and phytol. The PCA and AHC data revealed a strong correlation between the five plants. Firstly, C. calcitrapa exhibited strong correlation with C. scoparia and moderate correlation with C. alexandrina. Similarly, C. scoparia and C. calcitrapa exhibited a weak correlation with C. alexandrina. Furthermore, C. glomerata and C. lipii exhibited strong correlations, especially via the EO derived by microwave. The significant variations between the five plants might be attributed to the microclimatic and environmental condition variations [41,45].
C. scoparia and C. calcitrapa exhibited a strong correlation depending on the main components in which spathulenol, caryophyllene oxide, trans-caryophyllene and 6, 10, 14-trimethyl pentadecan-2-one were found as the main constituents. Caryophyllene oxide and spathulenol were characterized as major components in the EOs of the three plants C. scoparia, C. calcitrapa and C. alexandrina. Otherwise, the correlations of the two plants C. glomerata and C. lipii were deduced via the main compounds of the EOs of the plants, including spathulenol, alloaromadendrene oxide-2,6,10,14-trimethyl pentadecan-2-one and phytol. The correlations of the five plants were also observed via the main compounds of their EOs, such as spathulenol, 6,10,14-trimethyl pentadecan-2-one and phytol.
In conclusion, the results of chemometrics analysis deduced that there are significant variations between the five Centaurea species in EO composition depending upon the variations of the plant species and/or extraction method. Moreover, the significant variations might be attributed to the variations of the plant species more than the variations of the extraction method. Therefore, our results confirmed the effects of variations in plant species and extraction methods on the quantity and quality of EO composition.

Antimicrobial Activity
The microdilution assay was used to determine MIC values for EOs of the five Centaurea species against S. aureus, E. coli and A. niger. Strikingly, MAE-EOs of C. glomerata, C. lipii and C. calcitrapa showed a potent antibacterial activity against S. aureus, with MIC 31.25 µg/mL ( Table 2). Our results suggest that MAE is the method of choice for extraction of Centaurea EOs for the purpose of antimicrobial activity. The major constituents of the EOs may play a basic role as antimicrobial agents, whether as a singular role or synergistically with the other compounds. For example, alloaromadendrene oxides, as major components, were described to have an antimicrobial role in EOs in several plants such as Lippia alb [46], Aloysia citriodora [47], Curcuma aeruginosa [48] and others. According to the literature, spathulenol was found to be effective against several bacterial and fungal pathogens [49]; 6, 10, 14-Trimethyl pentadecan-2-one is one of the most common compounds from EOs of plants. This compound was described to have an important role in the antimicrobial activities of EOs derived from the flowers of several plants such as Citrus aurantium L [50]. Based on these studies, these major compounds might have a principal role in the antimicrobial activities of EOs. In addition to these main components, there are other major and minor compounds that are reported to have antimicrobial potentialities. Finally, the synergistic effects of the main and minor constituents of the EOs might be a possible pathway into the bioactivity of the oils.

Molecular Docking Inhibitory Effect of EOs Major Metabolites on ATP-Binding Pocket of Gyrase B Enzyme and N-Myristoyltransferase
The advantages of computer-aided drug discovery via structural studying against targeted enzymes as well as drug repurposing have led them to become an essential program for major pharmaceutical companies, owing to the speed and lower costs of their processes.
A specific lipidic modification for the -N-terminal glycine residue mainly sign to N-myristoylation protein in many viral and eukaryotic proteins [52].

Plant Material
The aerial parts of C. scoparia, C. calcitrapa, C. glomerata, C. lipii and C. alexandrina were collected during the flowering stage in April 2017 from Saint-Catherine-Southern Sinai Governorate, Ismalia City-Ismalia Governorate, Rashid centre-Beheira Governorate, Mediterranean coastal belt at Alexandria City and Borg El-Arab City-Alexandria Governorate, Egypt respectively. All Centaurea species under study were deposited in National Research Centre herbarium and were identified by the taxonomist Dr. I. El-Garf, (Professor of Botany, Faculty of Science, Cairo University, Cairo, Egypt) with and voucher specimens as the following:

Extraction of EO by Hydrodistillation
The dried aerial parts (250 g) of investigated Centaurea species were extracted for 3 h over a Clevenger-type apparatus using our previous protocol [53]. The EO samples kept were stored in sealed air-tight glass vials at 4 • C for analyses.

Microwave-Assisted Extraction of EOs
A microwave apparatus working at 2450 MHz and 1600 W maximum power (CEM Corporation, Matthews, NC, USA) was used to examine the plant species (250 g) under study using our previously reported protocol [53]. Extraction parameters: power, time and temperature were 800 W, 60 min and 100 • C respectively. All extracted EOs were dried using anhydrous sodium sulfate and kept directly in 4 • C till analysis.

GC-MS Analysis
Gas Chromatography-Mass Spectrometry (GC-MS) (THERMO Scientific Corp., Waltham, MA, USA), connected with a thermo mass spectrometer detector (ISQ Single Quadrupole Mass Spectrometer; Model ISQ spectrometer, electron ionization (EI) at 70 eV, m/z 40-450 a spectral range of), was used for plants EO analyses. A TR-5 MS (30 m × 0.32 mm i.d., 0.25 µm) column and helium as carrier gas (flow rate (one mL/min; split ratio (1:10); temperature program (60 • C for one min; rising at 4.0 • C/min to 240 • C and held for one min) were used and both the injector and detector were held at 210 • C. One µL of the mixtures diluted by n-hexane (1:10, v/v) were injected.

Identification of EO Constituents
The EO's main constituents were recognized via AMDIS software (www.amdis.net), according to its retention indices (relative to n-alkanes C 8 -C 22 ), mass spectrum corresponding to authentic standards, Wiley spectral library collection and NIST library databases as well.

MS Data Processing for Multivariate Analysis: PCA and HCA
The data of the major compounds of extracted EOs of the five samples (C. scoparia, C. calcitrapa, C. glomerata, C. lipii and C. alexandrina) were subjected to an agglomerative hierarchical cluster (AHC) using XLSTAT statistical computer software package (version 2018, Addinsoft, New York, NY, USA, www.xlstat.com (accessed on 29 January 2021)). Moreover, the matrix of data was also submitted to correlation by a principal component analysis (PCA) to identify whether a significant correlation exists between different samples using XLSTAT also.

Determination of Minimum Inhibitory Concentrations
The MIC values were determined by the broth microdilution assay (NCCLS, 2008) with slight modification [55]. The assay was carried out in nutrient broth medium for bacteria and potato dextrose broth medium for fungus. The assay was performed according to the reported procedure [56], with slight modifications. Briefly, 3 µL of the essential oil of concentration 1 mg/mL DMSO were prepared as an initial concentration in the first column of the sterile polystyrene 96 well plates. Then 197 µL of the tested microbial suspension adjusted to 5 × 10 5 CFU/mL was added. Serial dilutions were done by addition of 100 µL of the first column to the second one and so on. The final volume was adjusted to 200 µL on each well by addition of the microbial suspension to get final concentrations of tested compounds from 31.25 to 1000 µg. Negative control was made by adding an equal volume of DMSO instead of tested extracts. Blank control was prepared using broth medium. The plates were incubated in sterile conditions at a suitable temperature for the microbial growth. The turbidity of the culture media was taken as an indicator for microbial growth. The MIC value was taken as the lowest concentration of the test agent that caused complete inhibition (100%) of microbial growth [57]. Thiophenicol (Thiamphenicol, Sanofi-Aventis, France) and triflucan (Fluconazole, Egyptian International Pharmaceutical Industries Company (EIPICO), Naser City, Cairo, Egypt) were used as antibacterial and antifungal positive control drugs, respectively.

Conclusions
In the present study, GC/MS analysis was used to investigate the chemical composition of five Egyptian Centaurea species of EOs obtained by HD and MAE techniques, and the impact of an extraction method on the composition of metabolites of each EO was further assessed. The qualitative and quantitative variations in the composition of essential oils prepared by MAE and HD is probably due to climatic and environmental factors as well as to the possible degradation of products by hydrolysis, oxidation and trans-esterification, due to prolonged extraction time of HD when compared to MAE. These results could be useful in designing the best extraction method. Interestingly, it is the first report on the analysis of essential oils of C. lippi, C. glomerata and C. scoparia. Sesquiterpenes constitute the major classes in MAE-and HD-EOs of C. scoparia, C. calcitrapa and C. glomerata. In general, the amount of total terpenoids in MAE-EOs were higher than the HD-EOs. In contrast, the percentage of nonterpenoids for all studied species was higher in the HD-EOs than the MAE-EOs except for C. scoparia. The possible reason for this contradictory result is that the content of nonterpenoids of the oil was dependent on the species instead of the extraction method. The EOs of C. lipii, C. glomerata and C. calcitrapa, isolated by MAE in this study, exhibited significant inhibitory responses against the S. aureus, likely due to their enrichment in sesquiterpene compounds. In silico exploration, alloaromadendrene oxide-2 and spathulenol exhibited a significant inhibition of the ATP-binding pocket of the Gyrase B enzyme. These phytoconstituents could serve as potential candidates for the discovery of antibacterial and antifungal drugs, however their therapeutic potential is yet to be validated using in vitro and in vivo studies.