ABSTRACT
This study evaluated how geographical origin (El Alamein vs. Red Sea regions in Egypt) and extraction methods saffect the chemical profile and antimicrobial efficacy of Cotula cinerea essential oils. Comparing conventional hydro‐distillation (HD) to microwave‐assisted extraction (MAE), researchers found that MAE delivered higher oil yields (0.35%–0.37%) than HD (0.31%–0.34%) in significantly less time. Gas chromatography‐mass spectrometry (GC/MS) identified 19 volatile compounds—predominantly oxygenated monoterpenes like pinocarvone, α‐thujone, and camphene—with statistical analyses confirming that both location and extraction technique distinctly altered this chemical composition. Furthermore, MAE‐extracted oils demonstrated superior antimicrobial activity, particularly those from El Alamein, which reached a notable 25 mm inhibition zone against Aspergillus niger. Ultimately, the results indicate that MAE is a highly efficient and reproducible method for harvesting bioactive C. cinerea oils for potential pharmaceutical and nutraceutical applications.
Keywords: antimicrobial activity, Cotula cinerea, essential oil, GC/MS analysis, microwave‐assisted extraction, principal component analysis
The present study investigated the effect of geographical origin and extraction technique on the chemical composition and antimicrobial activity of Cotula cinerea essential oils collected from two different locations in Egypt (El Alamein and Red Sea regions). Essential oils were extracted using conventional hydro‐distillation (HD) and microwave‐assisted extraction (MAE). With significantly less extraction time, MAE offered higher yields of oil (0.35%–0.37%) than did HD extraction (0.31%–0.34%). Nineteen volatile constituents (99.87%–99.99% of total oil composition) were identified by GC/MS. The major compounds were pinocarvone (43.88%–62.05%), α‐thujone (6.62%–31.67%), camphene (7.07%–13.49%), santolina triene (2.68%–8.08%), and α‐pinene (2.35%–4.86%). The major class of terpenes in all the samples was oxygenated monoterpenes. Antimicrobial studies showed that the antimicrobial properties of the oils were higher and the inhibition zone was larger in MAE‐derived oils, where the El Alamein sample oils showed higher antibacterial and antifungal properties, as the zone of inhibition reached 25 mm against Aspergillus niger. The results of PCA and HCA showed good separations of the samples based on geographical origin and extraction method, thus proving that they had definite effects on the composition and bioactivity of essential oils. The results indicate a potential application of MAE as an efficient method for the extraction of C. cinerea essential oil with reproducible chemical composition for pharmaceutical and nutraceutical uses, due to the biological activity of the essential oil.
1. Introduction
Medicinal and aromatic plants (MAPs) have been a subject of interest by many researchers in the last few decades, due to their potential value in the discovery and development of new therapeutic compounds used in the treatment of various diseases. In addition, MAPs are considered to be the backbone of traditional medicine [1, 2, 3]. Many problems arise from the over prescription and the misuse of antibiotics, and therefore, there is an urgent need to develop MAPs which possess antimicrobial activity [4, 5, 6].
The Egyptian flora comprises 2121 species and 153 infraspecific vascular plants [7]. Slightly more than 16% of these species have been repeatedly reported to be used in medical practices.
The genus Cotula (Asteraceae) comprises 80 species, and it is represented in Egypt by two species, Cotula cinerea Del (C. cinerea) and Cotula anthemoides L. C. cinerea (syn: Brocchiacinerea Del. and Tanacetumcinereum Del.). C. cinerea is a xerophytic plant, widely distributed in desert wadis and sandy plains [8, 9]. C. cinerea is used in folk medicine as anti‐inflammatory, analgesic, antipyretic, antiseptic, for rheumatism, and in the treatment of urinary and pulmonary infections; it is also used for the treatment of many digestive problems, including constipation and colic. Also, it's reported to treat headaches, migraines, and joint inflammation. In Egypt, C. cinerea is used to flavor tea as an alternative to peppermint; it is also used in popular medicine for its stomachic and broncho‐pulmonary properties [8].
Previous studies of C. cinerea revealed the presence of many constituents with many therapeutic benefits, including flavonoids, tannins, alkaloids, saponins, terpenoids, steroids, and cardenolides; also, the plant was found to be rich in essential oil [10].
An extensive review has been conducted on the chemical composition of essential oil of C. cinerea growing in Egypt, Algeria and Morocco [8]. The essential oil of C. cinerea fresh aerial parts collected from Egypt, in the desert area between Cairo and Ismailia, yielded 0.30% (V/ W) of oil, camphor (50%) (V/V) and alpha and beta‐thujone (15%) (V/V) were the major constituents identified by GC/MS of the Egyptian oil [11]. Another study revealed the presence of oxygenated monoterpenes, monoterpene hydrocarbons, and sesquiterpene hydrocarbons (87.38%, 11.39%, and 1.2%, respectively); camphor and thujone were the major constituents identified by GC/MS, representing 65.5% and 15.59%, respectively, of the total oil [12, 13]. GC/MS profiling of essential oil of C. cinerea growing in Algeria and Morocco revealed the presence of camphor, camphene, alpha‐pinene, 3‐carene, thujone, 4‐terpineol, (Z)‐betafarnesene and santolinatriene as the most abundant components of the extracted oil [14]. Studying the biological activities of the C. cinerea oil, C. cinerea essential oil was found to possess significant antibacterial, antifungal, and anticandidal activities, and also the oil has shown moderate cytotoxic activity against both colorectal adenocarcinoma and Hepatocellular carcinoma cell lines [8, 13]. However, its antioxidant potential was proved to be lower than expected. The essential oil of C. cinerea is characterized by changes in the chemo type due to various factors, including the part of the plant used, stage of plant development, genetic factors, environmental conditions, the harvest period, and the nature of the soil [13].
In the last few decades, great attention has been devoted by many researchers to the development of the extraction process of MAPs and replacing conventional methods with more efficient innovative techniques, owing to their advantages over conventional ones. Using microwave energy is considered one of the promising technologies for the extraction of phyto‐constituents from MAPs. Microwave energy was first introduced to laboratories and was applied to acid digestions for performing trace metal analysis by Abu‐Samra et al. [15]. In 1986 and 1987, microwave energy was first used in the development of an extraction process by Ganzler and coworkers [16, 17] for the extraction of vicine and convicine from faba beans. In addition, microwave (MW)‐assisted extraction has been used for the extraction of essential oils from different aromatic plants by many researchers, and this extraction method has been scaled up at pilot scale.
Chemometric techniques, specifically principal component analysis (PCA) and hierarchical clustering analysis (HCA), provide the necessary framework for interpreting complex GC‐MS datasets. These multivariate tools enable the reduction of numerous chemical variables into a few orthogonal factors (Principal Components, PCs) while maximizing the retained variance, thereby allowing for robust visual and statistical classification of samples. The primary objective of employing this combined chemometric approach is to mathematically decouple and quantify the effect of intrinsic biological factors (geographical origin, reflecting genetics and environment) from extrinsic methodological factors (extraction technique) on the essential oil's ultimate composition. The high degree of resolution reported in this analysis underscores the power of PCA/HCA in identifying and confirming stable chemical polymorphisms, or chemotypes, within the species [18].
Therefore, the present study investigated the influence of geographical origin and extraction methodology on the chemical composition and antimicrobial activity of C. cinerea essential oil collected from two ecologically distinct Egyptian regions. Additionally, the link between geographic location, extraction technique, and volatile chemical profile was evaluated using multivariate chemometric techniques, such as PCA and HCA. To the best of our knowledge, this is the first comparative study that examines Egyptian C. cinerea populations from two ecologically distinct regions using microwave‐assisted extraction, GC–MS profiling, antimicrobial evaluation, and multivariate chemometric analysis. It offers fresh perspectives on how processing and environmental factors affect the quality and bioactivity of essential oils, offering fresh perspectives on how processing and environmental factors affect the bioactivity and quality of essential oils.
2. Results and Discussion
2.1. Chemical Profiles of the EOs
There are several methods for extracting essential oils, based on the type of botanical material and the type of oil constituents. The extraction process is one of the key factors that determine the quality of the oil, whereas poorly executed extraction can damage the essential oil and alter its chemical constituents. The traditional methods are extraction by distillation, expression, and solvent extraction, while new methods include the use of liquid carbon dioxide or microwaves.
On the other hand, the production of essential oils depends on some external variations such as soil, climatic conditions, harvesting time, and the amount of water to which the herb is exposed. Apparently, these factors favor the formation of one isomer over the other [19, 20, 21]. This study evaluated the chemical constituents of the essential oil of C. cinerea collected from two different localities in Egypt using two different extraction methodologies (MW‐assisted hydro‐distillation and conventional hydro‐distillation) and studied the effect of both the location and the extraction method used on the antimicrobial activity of C. cinerea essential oil.
From the obtained results, MAE is clearly faster than HD extraction. The extraction using MAE is three times faster than HD extraction. In addition, only 10 min using MAE was enough to reach the extraction temperature, which is equal to the boiling temperature of water (100°C); however, 30 min or more was required by HD extraction to reach the boiling temperature of water. Moreover, an extraction time of 60 min using MAE offered better yield than obtained using HD extraction for 3 h, confirming significant saving of extraction time and energy consumed.
The yield of the essential oil extracted from C. cinerea by hydro‐distillation was 0.31% in the Red Sea location and 0.34 in the El Alamein location, while the EO yield was 0.35% in the Red Sea location and 0.37 in the El Alamein location by using MAE. The above results show that the EO extracted by MAE (0.36%) was higher than the EO yield extracted by HD (0.33%), and also the data indicate that the EO extracted from plants grown in the El‐Alamein (0.36%) location was higher than EO extracted from plants grown in the Red Sea (0.33%) location (Table 1, Figure 1).
TABLE 1.
Essential oil yield percentage of C. cinerea extracted using HD and MAE.
| The plant source | Method of extraction | Essential oil yield % |
|---|---|---|
| Red Sea | HD | 0.31 ± 0.01 |
| Red Sea | MAE | 0.35 ± 0.01 |
| El Alamein | HD | 0.34 ± 0.01 |
| El Alamein | MAE | 0.37 ± 0.01 |
FIGURE 1.
The composition and the percentage composition of C. cinerea essential oils extracted using HD and MAE.
The GC/MS analysis of the extracted essential oil resulted in the identification of 19 constituents and the determination of their percentages in all samples tested, and they accounted for 99.87%–99.99% of the total composition of the oil. Table 2 shows the identified compounds and their relative percentage in the essential oils of C. cinerea extracted by hydro‐distillation (HD) and microwave‐assisted extraction (MAE). The representative GC chromatograms of the essential oils of both geographical locations obtained by both HD and MAE are shown in Figure 2a–d. The chemical composition of the essential oils studied had the majority of monoterpenes, mainly oxygenated monoterpenes. Pinocarvone was identified as the predominant constituent in most samples, ranging from 43.88% to 62.05%, followed by α‐thujone (6.62%–31.67%), camphene (7.07%–13.49%), santolina triene (2.68%–8.08%), α‐pinene (2.35%–4.86%), β‐pinene (1.28%–2.71%), and sabinene (1.12%–2.31%). Monoterpene hydrocarbons (21.70%–26.26%) and oxygenated monoterpenes (73.73%–78.17%) were the major terpene classes found in the oils. The results that were obtained indicated significant differences in the relative abundance of the volatiles as a function of geographical origin and extraction method. In general, extraction of MAE led to a slight enrichment of oxygenated monoterpenes compared to conventional hydro‐distillation.
TABLE 2.
The composition and the percentage composition of C. cinerea essential oils extracted using HD and MAE.
| No. | Name | KILit a | KIcal b | El Alamein | Red sea | Structure | Class | ||
|---|---|---|---|---|---|---|---|---|---|
| HD | MW | HD | MW | ||||||
| Santolina triene | 906 | 907 | 8.08 ± 0.05 | 6.49 ± 0.04 | 3.2 ± 0.03 | 2.68 ± 0.02 | C10H16 | MH | |
| 1 | Tricyclene | 921 | 920 | 0.57 ± 0.01 | 0.66 ± 0.02 | 0.41 ± 0.02 | 0.91 ± 0.02 | C10H16 | MH |
| 2 | α‐Thujene | 924 | 922 | 0.04 ± 0.01 | 0 | 0 | 0 | C10H16 | MH |
| 3 | α‐Pinene | 932 | 933 | 2.35 ± 0.03 | 3.41 ± 0.03 | 4.86 ± 0.05 | 4.46 ± 0.04 | C10H16 | MH |
| 4 | Camphene | 946 | 950 | 7.07 ± 0.1 | 10.7 ± 0.2 | 13.49 ± 0.22 | 13.15 ± 0.32 | C10H16 | MH |
| 5 | 2,2‐Dimethyl‐3‐vinyl‐bicyclo[2.2.1]heptane | 949 | 952 | 0 | 0 | 0.11 ± 0.00 | 0.17 ± 0.01 | C10H16 | MH |
| 6 | Sabinene | 969 | 967 | 2.31 ± 0.02 | 1.12 ± 0.02 | 1.48 ± 0.02 | 1.63 ± 0.01 | C10H16 | MH |
| 7 | β‐Pinene | 974 | 976 | 1.28 ± 0.02 | 1.77 ± 0.01 | 2.71 ± 0.03 | 2.11 ± 0.02 | C10H16 | MH |
| 8 | Eucalyptol | 1031 | 1033 | 0.26 ± 0.01 | 0.95 ± 0.01 | 4.2 ± 0.02 | 2.72 ± 0.02 | C10H18O | OM |
| 9 | cis‐Sabinene hydrate | 1065 | 1066 | 0.68 ± 0.01 | 0.16 ± 0.01 | 0.21 ± 0.02 | 0.87 ± 0.02 | C10H18O | OM |
| 10 | α‐Thujone | 1110 | 1112 | 31.67 ± 0.21 | 13.24 ± 0.2 | 9.06 ± 0.3 | 6.62 ± 0.2 | C10H18O | OM |
| 11 | 3,7‐Octadiene‐2,6‐diol, 2,6‐dimethyl | 1118 | 1121 | 0.33 ± 0.01 | 0.14 ± 0.01 | 0 | 0.26 ± 0.01 | C10H18O 2 | OM |
| 12 | trans‐Sabinene hydrate | 1135 | 1138 | 0.2 ± 0.00 | 0.09 ± 0.01 | 0 | 0.45 ± 0.01 | C10H18O | OM |
| 13 | β‐Pinenoxide | 1137 | 1139 | 0.09 ± 0.00 | 0.04 ± 0.0 | 0 | 0.16 ± 0.01 | C10H16O | OM |
| 14 | cis‐p‐Mentha‐1(7),8‐dien‐2‐ol | 1142 | 1144 | 0.45 ± 0.01 | 0 | 0 | 0 | C10H16O | OM |
| 15 | Camphor | 1146 | 1148 | 0 | 0 | 0.17 ± 0.01 | 0.11 ± 0.01 | C10H16O | OM |
| 16 | Pinocarvone | 1164 | 1166 | 43.88 ± 0.05 | 60.51 ± 0.40 | 59.41 ± 0.04 | 62.05 ± 0.50 | C10H14O | OM |
| 17 | Terpinen‐4‐ol | 1174 | 1179 | 0.46 ± 0.01 | 0.22 ± 0.01 | 0.21 ± 0.01 | 0.36 ± 0.01 | C10H18O | OM |
| 18 | (–)‐Bornyl acetate | 1285 | 1293 | 0.15 ± 0.01 | 0.48 ± 0.01 | 0.47 ± 0.01 | 1.22 ± 0.01 | C12H20O2 | OM |
| Monoterpene hydrocarbons | 21.70 | 24.15 | 26.26 | 25.11 | |||||
| Oxygenated monoterpenes | 78.17 | 75.83 | 73.73 | 74.82 | |||||
| Total identified compounds | 99.87 | 99.98 | 99.99 | 99.93 | |||||
aKILit.: Kovats index reported in literature.
bKIcal: Experimentally calculated Kovats index. Identification was performed via the comparison of the component mass spectral data and retention indices with the (i) NIST Mass Spectral Library, (ii) Wiley Registry of Mass Spectral Data (eighth edition, 2), and (iii) literature [22].
FIGURE 2.
The chromatograms of essential oils of C. cinerea aerial parts collected (a) Wadi El Natron–El Alamein desert road, Beheira Governorate (HD), (b) Wadi El Natron–El Alamein desert road, Beheira Governorate (MAE), (c) El Sheikh Fadl–Ras Gharib Road, Red Sea Governorate (HD), (d) El Sheikh Fadl–Ras Gharib Road, Red Sea Governorate (HD).
2.2. Principal Component Analysis (PCA) and Hierarchical Clustering Analysis (HCA)
The multivariate distribution pattern among the investigated samples was clearly observed when C. cinerea essential oils were subjected to Principal Component Analysis (PCA) of the chemical composition. The first two principal components (PC1 and PC2) accounted for 88.74% of the total variance, suggesting that the majority of the chemical variation could be captured by considering these two components. The PCA score plot (Figure 3A) showed good separation between the essential oil samples, and this separation was observed by both geographical and extraction method. The samples were definitely differentiated from the Red Sea region samples, suggesting the biosynthesis of the volatile constituents to be influenced by strong environmental and geographical factors. The relative abundance of monoterpenes with oxo and monoterpene hydrocarbons, especially pinocarvone, α‐thujone, camphene, santolina triene and α‐pinene, was mainly responsible for the discrimination between the samples as indicated by the corresponding loading plot (Figure 3B). Moreover, the second principal component (PC2) indicated the effect of extraction method, as the microwave‐assisted extraction (MAE) samples differed slightly from the hydro‐distillation (HD) samples, particularly in terms of oxygenated monoterpenes content. The results of the PCA were also confirmed by the hierarchical clustering analysis (HCA) (Figure 3C), which classified the samples of essential oils in different clusters depending on the geographical origin and extraction method. The high similarity between PCA and HCA shows that extraction methods and environmental conditions have a considerable effect on the chemical composition of the essential oils of C. cinerea [23, 24, 25, 26].
FIGURE 3.
Principal component analysis (A) score plot, loading plot (B), and hierarchical clustering analysis (C). The model explains 88.74% of the total variance prescribed by PC1 and PC2 for the essential oils extracted by two different methods from C. cinerea, which were collected from two different locations.
2.3. Antimicrobial Activity
The inhibitory effects of the essential oils on the tested bacteria, yeast, and fungi were carried out by the agar disc‐diffusion method. Results presented in Tables 3 and 4 showed that the oil obtained from C. cinerea collected from the Red Sea has antibacterial activity against all tested Gram‐positive and Gram‐negative bacteria without any inhibitory effect against the unicellular yeast fungi and filamentous fungi. There was similar antibacterial activity to the oil extracted by conventional hydro‐distillation and MW‐assisted hydro‐distillation against Gram‐positive S. aureus and L. cereus (IZD 9 mm) and (MIC, 1.25 µL). On the other hand, the oil extracted by MW‐assisted hydro‐distillation showed an inhibitory effect against the Gram‐negative bacteria E. coli (IZD, 11 mm & MIC, 0.625 µL) better than that extracted by hydro‐distillation (IZD, 9 mm & MIC, 1.25 µL). The obtained results are in compliance with the previously published data on biological properties of C. cinerea essential oil from the Sahara of Algeria [27].
TABLE 3.
The inhibitory activity of the essential oils extracted from C. cinerea against the pathogenic bacteria, yeast and fungi.
| Plant source | Methods of extraction | Inhibition zone diameter (IZD) (mm) | ||||||
|---|---|---|---|---|---|---|---|---|
| Bacteria | Yeast | Fungi | ||||||
| S. aureus ATCC29213 | L. cereus ATCC14579 | E. coli ATCC25922 | C. albicans ATCC 10321 | C. tropicalis ATCC750 | A. niger NRC53 | A. alternata NRC43 | ||
| Red Sea | HD | 9 ± 0.00 | 9 ± 1.41 | 9 ± 0.00 | N.A. | N.A. | N.A. | N.A. |
| MW | 9 ± 0.71 | 9 ± 0.70 | 11 ± 1.41 | N.A. | N.A. | N.A. | N.A. | |
| El Alamein | HD | 8 ± 0.00 | 8 ± 0.70 | 7 ± 0.00 | 13 ± 0.71 | 7 ± 0.700 | 15 ± 2.12 | 9 ± 0.00 |
| MW | 14 ± 1.41 | 15 ± 2.11 | 10 ± 0.71 | 15 ± 2.12 | 12 ± 0.00 | 25 ± 1.41 | 10 ± 0.70 | |
| Reference drug | Thiophenicol (antibacterial) | 25 ± 0.71 | 23 ± 0.00 | 16 ± 0.70 | N.A. | N.A. | N.A. | N.A. |
| Treflucan (antifungal) | N.A. | N.A. | N.A. | 26 ± 1.41 | 25 ± 0.71 | 13 ± 0.14 | 16 ± 0.00 | |
Note: HD: hydro‐distillation, MW: MW‐assisted hydro‐distillation. N.A.: no activity.
TABLE 4.
Minimal inhibitory concentration of the essential oils extracted from C. cinerea against the pathogenic strains.
| Plant source | Method of extraction | Minimal inhibitory concentration (MIC) (µL/disc) | ||||||
|---|---|---|---|---|---|---|---|---|
| Bacteria | Yeast | Fungi | ||||||
| S. aureus ATCC29213 | L. cereus ATCC14579 | E. coli ATCC25922 | C. albicans ATCC 10321 | C. tropicalis ATCC750 | A. niger NRC53 | A. alternata NRC43 | ||
| Red Sea | HD | 1.25 | 1.25 | 1.25 | — | — | — | — |
| MW | 1.25 | 1.25 | 0.625 | — | — | — | — | |
| El Alamein | HD | 2.5 | 2.5 | 5 | 5 | 5 | 2.5 | 5 |
| MW | 1.25 | 2.5 | 1.25 | 5 | 5 | 1.25 | 5 | |
| Reference drug | Thiophenicol (antibacterial) | 3.13 | 3.13 | 6.25 | — | — | — | — |
| Treflucan (antifungal) | — | — | — | 12.5 | 25 |
50‐ |
25 | |
Note: HD: hydro‐distillation, MW: MW‐assisted hydro‐distillation. N.A.: no activity.
Results emphasized that the oil obtained from C. cinerea collected from El Alamein showed a wide range of antimicrobial activity against bacteria, yeast, and fungi. The oil showed different inhibitory activities depending on the extraction method, where the oil extracted by MW‐assisted hydro‐distillation has good antibacterial activity (IZD) in the range from 10 to 15 mm and (MIC) in the range from 1.25 to 2.5 µL. However, that extracted by conventional hydro‐distillation has a low inhibitory effect against bacteria in the range from 7 to 8 mm and (MIC) in the range from 2.5 to 5 µL. The effectiveness of the oil extracted by MW‐assisted hydro‐distillation against yeast and fungi, in a range of 10–25 mm (MIC, 1.25–5 µL), was also better than that extracted by conventional hydro‐distillation (IZD, 7–15 mm & MIC, 2.5–5). From the experiments, the oil obtained from the C. cinerea collected from El Alamein and extracted by MW‐assisted hydro‐distillation showed the highest antimicrobial activities, especially against the fungus A. niger, with a larger growth inhibition zone diameter (25 mm) in comparison to synthetic Treflucan (13 mm). The detailed mechanistic investigations will be the subject of future studies to further elucidate the antimicrobial mode of action of the most active essential oil samples.
3. Conclusions
This study demonstrates that both geographical origin and extraction technique are decisive factors shaping the chemical composition and bioactivity of C. cinerea essential oil. Microwave‐assisted extraction proved superior to conventional methods, providing higher oil yields and enhanced antimicrobial activity, particularly for samples collected from the El Alamein region. Multivariate chemometric analyses (PCA and HCA) clearly discriminated the essential oils according to origin and extraction method, confirming their combined influence on essential oil quality. Collectively, these results highlight the necessity of controlled sourcing and the adoption of MAE as a standardized extraction approach to ensure reproducible chemical profiles and optimized bioactivity, thereby supporting the potential pharmaceutical and nutraceutical applications of C. cinerea essential oil.
4. Experimental Section
4.1. General Materials and Experimental Procedures
4.1.1. Plant Material
The aerial parts of C. cinerea were collected during the flowering stage in February 2017 from two ecologically distinct regions in Egypt: Wadi El Natron–El Alamein Desert Road, Beheira Governorate (30°29.113′ N, 30°09.923′ E; 18 m above sea level) and El Sheikh Fadl–Ras Gharib Road, Red Sea Governorate (28°57.423′ N, 32°03.272′ E; 227 m above sea level). Plant identification was carried out by a plant taxonomist at the National Research Centre (NRC), Giza, Egypt. Voucher specimens were authenticated and deposited in the Herbarium of the National Research Centre, Giza, Egypt, under assigned voucher numbers for future reference.
4.1.2. Extraction of Essential Oil
4.1.2.1. Conventional Hydro‐Distillation
The fresh aerial parts (100 g) were washed and extracted using a Clevenger‐type apparatus. Hydro‐distillation was performed for 3 h. The essential oil was dried with anhydrous sodium sulfate, then the essential oil was recovered, and its volume was determined by micropipette (calculated as percentage, volume of recovered oil per weight of sample) and stored under refrigeration at 4°C–6°C in dark glass tubes for further use [28]
4.1.2.2. Microwave‐Assisted Hydro‐Distillation
The fresh aerial parts (100 gm) were washed and placed in a 5000 mL round‐bottomed flask and were then extracted using focused microwave apparatus (CEM Corporation, Matthews, NC, USA), model (MARS 240/50, No. 907511, frequency 2450 MHz) operating at 2450 MHz with maximum power at 1600 W, connected to Clevenger‐type apparatus outside of the microwave oven. Temperature was adjusted at 100°C; extraction time of 45 min and 800 W microwave irradiation power were applied to the sample matrix. After MW‐assisted extraction, the same post‐extraction procedures for hydro‐distillation were applied to the recovered oil [28].
4.1.2.3. GC/MS Analysis
The essential oil compounds of the C. cinerea oils were analyzed and identified depending upon GC/MS analysis. The GC‐MS analysis of the essential oil samples was carried out using a gas chromatography‐mass spectrometry instrument at the Department of Medicinal and Aromatic Plants Research, National Research Center, Egypt, according to the reported protocols [29], under the same techniques and conditions previously reported [30, 31]. A TRACE GC Ultra Gas Chromatography, coupled with a Thermo ISQ single quadrupole mass spectrometer detector (Thermo Scientific Corporate, Waltham, MA, USA), was used. The GC/MS system was equipped with a TR‐5 MS column (30 m × 0.32 mm inner diameter, 0.25 µm film thickness, Thermo Scientific Corp). Helium was used as the carrier gas at a flow rate of 1.0 mL/min and a split ratio of 1:10. The column temperature was held at 60°C for 1 min, then raised at 4.0°C/min to 240°C and held for 1 min. The temperature of the injector and detector was maintained at 210°C. Each sample was injected into the injector in a small volume (1 µL in n‐hexane) with a concentration of 1:10 (v/v). The mass spectral data were collected at 70 eV by electron ionization with a spectrum spanning m/z 40 and 450.
4.1.2.4. Identification of Essential Oil Constituents
The identification of the chemical constituents of the essential oil was deconvoluted using AMDIS software (www.amdis.net) and identified by their retention indices (relative to n‐alkanes C8‐C22), mass spectrum matching to authentic standards (when available), and the Wiley spectral library collection and NSIT library database.
4.1.2.5. GC‐MS Multivariate Data Analyses
Based on the EOs composition, the correlation between four C. cinera species (four sources of EO) was assessed by agglomerative hierarchical cluster (AHC) as well as principal component analysis (PCA). We constructed a data matrix from the percentage of various classes of the EOs and then subjected them to PCA. However, we performed the AHC based on a data matrix of a total of 19 major identified compounds from the EOs. We performed the AHC based on the similarity using Pearson's coefficient of correlation and with the agglomeration method of un‐weighted pair‐group average. The AHC and the PCA analyses were performed using XLSTAT statistical computer software package, version 14 (Addinsoft, New York, USA).
4.1.2.6. Detection of Antimicrobial Activity
The agar disc diffusion method was used for screening of antimicrobial activity of each essential oil obtained from C. cinerea extracted by two different methods (hydro‐distillation and MW‐assisted extraction). The essential oils were tested against Gram‐positive bacteria (Staphylococcus aureus ATCC29213, Lactobacillus cereus ATCC14579), Gram‐negative bacteria (Escherichia coli ATCC 25922), yeast (Candida albicans ATCC 10321, Candida tropicalis ATCC750), and fungi (Aspergillus niger NRC53, Alternaria alternate NRC43) based on the documents [32, 33].
Sterile paper discs (6 mm in diameter) were saturated with 5 µL of pure essential oil and placed on plates inoculated with 1×106 spores‐ml of fungi (potato dextrose agar medium) and1×108 spores‐mlof bacteria (nutrient agar medium). Inoculated agar plates were left for 30 min at 4°C for oil diffusion; after that, the plates were incubated for 24 h at 30°C for bacteria and 72 h at 28°C for fungi. Commercial Thiophenicol (Thiamphenicol, Sanofi Aventis, France) and Treflucan (Fluconazole, Egyptian International Pharmaceutical Industries Company, EIPICO) were used as reference antibacterial and antifungal drugs, respectively (50 µg /disc). The diameter of inhibition zones, including the disc diameter, was measured in millimeters at three different points, and the average values are reported as Mean ± SD using MS Excel.
4.1.2.7. Minimal Inhibitory Concentration (MIC)
The MIC of the essential oils against seven micro‐organisms was determined. The essential oils were tested at the final volumes of 2.5, 1.25, 0.625, and 0.3125 µLdisc‐1. The lowest concentration showing an inhibition zone around the disc was taken as the MIC.
Author Contributions
Sherin K. Ali: conceptualization, methodology, software, data curation, investigation, validation, formal analysis, visualization, Writing – original draft, resources. Ibrahim A. Saleh: conceptualization, methodology, software, data curation, investigation, validation, formal analysis, supervision, visualization, writing – original draft, writing – review and editing, resources. Abd El–Nasser G. El‐gendy: conceptualization, investigation, writing – original draft, methodology, validation, visualization, writing – review and editing, software, formal analysis, data curation, supervision, resources. Abeer A. Abd El Aty: methodology, software, data curation, investigation, validation, formal analysis, visualization, resources, writing – original draft, writing – review and editing. Tarik A. Mohamed: conceptualization, methodology, software, data curation, investigation, validation, formal analysis, supervision, visualization, project administration, resources, writing – review and editing. Mohamed–Elamir F. Hegazy: conceptualization, methodology, software, data curation, investigation, validation, formal analysis, supervision, visualization, project administration, writing – review and editing. All authors have read and agreed to the published version of the manuscript.
Conflicts of Interest
The authors declare no conflicts of interest.
Contributor Information
Tarik A. Mohamed, Email: tarik.nrc83@yahoo.com.
Mohamed‐Elamir F. Hegazy, Email: elamir77@live.com.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.