Chemical Composition of Kickxia aegyptiaca Essential Oil and Its Potential Antioxidant and Antimicrobial Activities.

The exploration of new bioactive compounds from natural resources as alternatives to synthetic chemicals has recently attracted the attention of scientists and researchers. To our knowledge, the essential oil (EO) of Kickxia aegyptiaca has not yet been explored. Thus, the present study was designed to explore the EO chemical profile of K. aegyptiaca for the first time, as well as evaluate its antioxidant and antibacterial activities, particularly the extracts of this plant that have been reported to possess various biological activities. The EO was extracted from the aerial parts via hydrodistillation and then characterized by gas chromatography-mass spectrometry (GC-MS). The extracted EO was tested for its antioxidant activity via the reduction in the free radicals, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). In addition, the EO was tested as an antibacterial mediator against eight Gram-negative and Gram-positive bacterial isolates. Forty-three compounds were identified in the EO of K. aegyptiaca, with a predominance of terpenoids (75.46%). Oxygenated compounds were the main class, with oxygenated sesquiterpenes attaining 40.42% of the EO total mass, while the oxygenated monoterpenes comprised 29.82%. The major compounds were cuminic aldehyde (21.99%), caryophyllene oxide (17.34%), hexahydrofarnesyl acetone (11.74%), ar-turmerone (8.51%), aromadendrene oxide (3.74%), and humulene epoxide (2.70%). According to the IC50 data, the K. aegyptiaca EO revealed considerable antioxidant activity, with IC50 values of 30.48 mg L-1 and 35.01 mg L-1 for DPPH and ABTS, respectively. In addition, the EO of K. aegyptiaca showed more substantial antibacterial activity against Gram-positive bacterial isolates compared to Gram-negative. Based on the minimum inhibitory concentration (MIC), the EO showed the highest activity against Escherichia coli and Bacillus cereus, with an MIC value of 0.031 mg mL-1. The present study showed, for the first time, that the EO of K. aegyptiaca has more oxygenated compounds with substantial antioxidant and antibacterial activities. This activity could be attributed to the effect of the main compounds, either singular or synergistic. Thus, further studies are recommended to characterize the major compounds, either alone or in combination as antioxidants or antimicrobial agents, and evaluate their biosafety.

1. Introduction

Human beings are putting increased pressure on the planet’s various resources. Scientists and researchers are doing their best to explore new, green, eco-friendly natural bioactive compounds that can be used in various treatments in agriculture, pharmaceuticals, and industry [1]. Plants are the main source of bioactive compounds (phytochemicals). Essential oils (EOs) are considered promising bioactive compounds due to their various biological activities and their chemical diversity [2,3,4]. Kickxia genus includes 47 species worldwide including Africa, Europe, Asia, and Macaronesia [5]. The Kickxia genus is one of the largest genera of the family Plantaginaceae in the flora of Egypt, where it is represented by 11 species [6]. The plants belonging to Kickxia genus are well known for the presence of several metabolites such as flavonoids [7,8], alkaloids [9], terpenoids [10], and iridoids [11]. Several traditional uses of Kickxia species around the world have been documented, such as laxatives, diuretics, tonics, anti-diabetic, and antiscorbutic, alongside the treatment of disorders such as hemorrhoids, wounds, and vascular treatments [12].

Kickxia aegyptiaca (L.) Nábělek is a wild perennial herbal plant of the family Plantaginaceae. It is widely distributed in Egyptian sandy plains, wadis, deserts, Sinai Peninsula, oases, and the Mediterranean coastal areas [13]. Its synonyms include Antirrhinum aegyptiacum, Linaria micromerioides, and Linaria aegyptiaca [6]. The plant grows up to 50 cm, with a dense and woody base. Leaves are ovate or lanceolate, with an entire or dentate margin. The plant has yellow flowers, and flowering time extends from February to June [13]. Kickxia species, including K. aegyptiaca, were documented as significant traditional plants in the treatment of vascular diseases, haemorrhoids, and wounds, along with their uses as laxatives, anti-diabetics, anti-scorbutics, diuretics, and, tonics agents [12,14]. The phytochemical characterization of this plant revealed that it is rich in flavonoids, phenolic acids, glycosides, and iridoids [7,15,16]. The different K. aegyptiaca extracts and their isolated metabolites were documented to have antioxidant activity [17,18], larvicidal activity [18], and cytotoxic activity [15,19]. Flavonoids, pectolinarigenin, tangeretin, and gardenin were also isolated from K. aegyptiaca, which exhibited potential antiviral activity against SARS-CoV-2 [16].

To our knowledge, and according to the literature review, only K. spuria EO chemical profile was described by Morteza-Semnani, Saeedi and Akbarzadeh [10]. However, its EO K. aegyptiaca has not been studied to date to the best of our knowledge. Therefore, the present document (i) describes, for the first time, the chemical characterization of EO of the aerial parts of K. aegyptiaca, and (ii) evaluates the antioxidant and antibacterial potential of its EO.

2. Results and Discussion

2.1. Chemical Characterization of K. aegyptiaca EO

The hydrodistillation of K. aegyptiaca aerial parts produced a golden–yellow oil of 0.51% (v/w). This amount of EO was found to be comparable to that obtained from K. spuria, 0.40% [10]. The extracted EO was analyzed by GC-MS and the ion chromatogram is presented in Figure 1. The chemical characterization led to the identification of 43 components, which are comparable in number with those identified in the EO of K. spuria [10]. The identified compounds represented 97.36% of the extracted EO.

Figure 1

Chromatogram of K. aegyptiaca EO compounds derived from GC-MS analysis. The peaks in major compounds are numbered (1–6).

The identified chemical compounds are listed in detail, along with their retention times (Rt), and Kovats indexes (KI) in Table 1. These compounds can be categorized into seven classes of components, including monoterpenes (oxygenated and hydrocarbons), sesquiterpenes (oxygenated and hydrocarbons), diterpenes (oxygenated only), carotenoid derived components, and other hydrocarbons (Figure 2). These data revealed that this oil is very rich in terpenoid compounds, which represented 75.46% of the total EO mass. From overall terpenoids, a relative concentration of 70.57% was found in oxygenated forms. The abundance of terpenoids, particularly the oxygenated ones, was in harmony with the published data for K. spuria EO [10].

Table 1

Chemical profile characterization of the essential oil extracted from the aerial parts of K. aegyptiaca.

No	Rt a	Conc.% b	Compound	Formula	KI c
	Oxygenated monoterpenes
1	6.39	1.31 ± 0.03	α-Terpineol	C10H18O	1186
2	9.82	0.55 ± 0.01	α-Linalool	C10H18O	1095
3	11.92	21.99 ± 0.21	Cuminic aldehyde	C10H12O	1239
4	13.42	2.17 ± 0.06	p-Cymen-7-ol	C10H14O	1287
5	13.87	1.45 ± 0.04	Carvacrol	C10H14O	1298
6	16.07	1.51 ± 0.04	Eugenol	C10H12O2	1356
7	17.26	0.84 ± 0.02	10-(acetylmethyl)-3-Carene	C13H20O	1380
	Monoterpene hydrocarbons
8	10.82	0.63 ± 0.03	2-Bornene	C10H16	1165
	Oxygenated sesquiterpenes
9	19.68	0.65 ± 0.03	Neryl acetone	C13H22O	1436
10	21.81	0.25 ± 0.01	trans-Nerolidol	C15H26O	1561
11	24.02	0.43 ± 0.01	trans-Sesquisabinene hydrate	C15H26O	1577
12	24.16	0.97 ± 0.02	Spathulenol	C15H24O	1577
13	25.38	0.95 ± 0.02	Isoaromadendrene epoxide	C15H24O	1579
14	24.59	17.34 ± 0.11	Caryophyllene oxide	C15H24O	1582
15	25.19	0.42 ± 0.01	Carotol	C15H26O	1594
16	25.58	1.24 ± 0.03	Widdrol	C15H26O	1599
17	26.8	2.70 ± 0.06	Humulene epoxide	C15H24O	1608
18	27.05	0.43 ± 0.03	Clov-2-ene-9α-ol	C15H24O	1616
19	27.53	3.84 ± 0.07	Aromadendrene oxide-(2)	C15H24O	1631
20	28.14	8.51 ± 0.20	ar-Turmerone	C15H20O	1669
22	34.21	1.85 ± 0.04	trans-Z-α-Bisabolene epoxide	C15H24O	1675
22	36.28	0.53 ± 0.02	(E,E)-Farnesyl acetone	C18H30O	1915
	Sesquiterpene hydrocarbons
23	17.72	1.07 ± 0.03	Longicyclene	C15H24	1374
24	18.24	1.97 ± 0.04	Isocaryophillene	C15H24	1408
25	19.42	0.39 ± 0.02	trans-Caryophyllene	C15H24	1417
26	20.32	0.57 ± 0.01	β-Farnesene	C15H24	1442
27	20.53	0.31 ± 0.01	ar-Curcumene	C15H22	1480
28	22.85	0.26 ± 0.01	α-Calacorene	C15H20	1544
	Oxygenated diterpenes
29	29.86	0.33 ± 0.02	trans-Geranylgeraniol	C20H34O	2201
	Carotenoid derived compounds
30	12.77	1.21 ± 0.04	dihydroedulan II	C13H22O	1284
31	13.13	0.56 ± 0.02	Theaspirane A	C13H22O	1298
32	16.84	0.72 ± 0.03	β-Damascenone	C13H18O	1384
33	34.02	11.74 ± 0.13	Hexahydrofarnesyl acetone	C18H36O	1845
	Others
34	28.24	2.32 ± 0.06	Benzyl acetylacetate	C11H12O3	1486
35	32.11	0.30 ± 0.01	n-Octadecyl chloride	C18H57Cl	1399
36	35.48	0.27 ± 0.01	n-Nonadecane	C17H34O2	1900
37	36.77	0.78 ± 0.02	Methyl palmitate	C21H44	1921
38	41.77	0.93 ± 0.03	n-Heneicosane	C17H34O2	2100
39	43.03	0.50 ± 0.02	9,12-Octadecadienoic acid	C18H32O2	2085
40	44.69	0.26 ± 0.01	2-Nonadecanone	C19H38O	2106
41	47.52	0.78 ± 0.02	n-Docosane	C22H46	2200
42	48.14	0.54 ± 0.01	n-Tetracosane	C24H50	2400
43	57.76	0.99 ± 0.04	n-Octacosane	C28H58	2800
Total		97.36

a retention time, b average concentration of three replications ± standard deviation, c Kovats retention index.

Figure 2

Percentage of the various classes of the recognized chemical compounds (a) and the total oxygenated and non-oxygenated compounds (b) in the essential oil of the K. aegyptiaca.

Sesquiterpenes were identified with a relative concentration of 44.68% of the overall oil mass, including both the oxygenated sesquiterpenes (40.42%) and sesquiterpenes hydrocarbon (4.26%) forms. The dominance of the sesquiterpenes is also described in the EO of K. spuria [10]. Out of the 14 identified oxygenated sesquiterpenes, caryophyllene oxide (17.34%), ar-turmerone (8.51%), aromadendrene oxide (3.84%), and humulene epoxide (2.70%) represented the major ones (Figure 3), while trans-nerolidol (0.25%) represented the minor.

Figure 3

Chemical structure of the major identified compounds in the EO of K. aegyptiaca.

Caryophyllene oxide was determined in a high concentration (8.90%) in the EO of K. spuria [10]. Some of the identified compounds were also described in the constituents of K. spuria EO, such as ar-curcumene, spathulenol, as well as the cis isomer of sesquisabinene hydrate. Caryophyllene oxide, ar-turmerone, aromadendrene oxide, and humulene epoxide are widely distributed compounds in the EOs of several plants, such as Centaurea species [20], Artemisia campestris [21], Cullen plicata [2], Chromolaena odorata [22], and Heliotropium curassavicum [23]. On the other side, five sesquiterpene hydrocarbons were assigned, including isocaryophillene (1.97%) and longicyclene (1.07%) as the main compounds.

Monoterpenes represented the second class of identified compounds (30.45%), which encompass oxygenated monoterpenes as the main compounds, with a relative concentration of 29.82%, along with 0.63% of monoterpene hydrocarbons. Seven compounds were assigned as oxygenated monoterpenes, in which cuminic aldehyde (21.99%) and p-cymen-7-ol (2.17%) were determined as major compounds. The profile of the monoterpenes was totally different compared to that reported in K. spuria EO, except for eugenol [10]. This variation could be ascribed to the genetic differences in both species [24], and the environmental and climatic conditions have also been reported to affect the composition of the EO [23,25,26,27]. Cuminic aldehyde is basically the main compound of Cuminum cyminum, with a concentration of 22.4–41.5% [28,29,30]. On the other hand, p-cymen-7-ol was described as a major compound in the EOs of several plants, for instance, Curcuma cf. xanthorrhiza [31], Eucalyptus largiflorens [32].

Diterpenes have been known as rarely described compounds in EOs of the aromatic plants; nevertheless, they were reported as a major compound in the EO of Lactuca serriola [33], Euphorbia mauritanica [34], Araucaria bidiwillii [35], Araucaria heterophylla [3,36]. The results of the current study agreed with the scarcity of diterpenes, identifying only one oxygenated diterpenoid, trans-geranylgeraniol (0.33%), with a complete absence of diterpene hydrocarbons.

In addition to terpenoid components, four carotenoid-derived compounds were determined in the EO of K. aegyptiaca (Table 1). Hexahydrofarnesyl acetone attained a remarkable concentration (14.23%) in the K. aegyptiaca (Figure 3). This compound was assessed as an abundant constituent of EOs of Launaea mucronata and Launaea nudicaulis [37], H. curassavicum [23], and Bassia muricata [38]. The other hydrocarbons were represented with a relative concentration of 7.67% and ten compounds were represented as a mixture of oxygenated and non-oxygenated compounds. Benzyl acetylacetate with a concentration of 2.32% represented the main non-terpenoids, while n-nonadecane (0.27%) represented the minor one. The presence of hydrocarbons in the EO of K. aegyptiaca is consistent with the published data of Iranian K. spuria [10].

2.2. Antioxidant Activity of K. aegyptiaca EO

The EO of K aegyptiaca showed a substantial antioxidant activity based on both DPPD and ABTS methods compared to the ascorbic acid as a standard synthetic antioxidant (Figure 4). The scavenging activity increased with the increment of EO concentration. At a concentration of 20 mg mL−1 of K. aegyptiaca EO, the DPPH and ABTS colors were reduced by 39.85% and 33.16%, respectively, while ascorbic acid showed a reduction of 83.48% and 71.33%, respectively, at the same concentration (Figure 4).

Figure 4

Antioxidant activity of various concentrations and IC50 of the essential oil of K. aegyptiaca (a) and a standard antioxidant, ascorbic acid (b) based on the scavenging of DPPH and ABTS. Values are means (n = 3) ± standard deviation. Different letters inside each graph reveal values significant variation at p ≤ 0.05 (Duncan’s test).

According to the IC50 data, the K. aegyptiaca EO revealed IC50 values of 30.48 mg L−1 and 35.01 mg L−1 for DPPH and ABTS, respectively. The standard antioxidant, ascorbic acid, showed IC50 values of 9.45 mg L−1 and 12.61 mg L−1, regarding DPPH and ABTS, respectively. The considerable antioxidant activity of K. aegyptiaca EO in the present study could be attributed to the effect of key compounds, such as cuminic aldehyde, caryophyllene oxide, hexahydrofarnesyl acetone, ar-turmerone, aromadendrene oxide, and humulene epoxide. These compounds could act in either singular or in synergistic ways [39,40]. The major compound (cuminic aldehyde) has been reported in a high concentration (52.56%) in C. cyminum, showing strong antioxidant activity [29,41]. On the other hand, the second major compound in the present study (caryophyllene oxide) has been reported to possess substantial antioxidant activity [2,42]. The carotenoid-derived compound, hexahydrofarnesyl acetone, has been reported in the EOs of various plants that showed strong antioxidant activity, such as Launaea species [37], H. curassavicum [23], and B. muricata [38]. The aromadendrene oxide-rich EO of Cleome amblyocarpa has been described to have allelopathic, antioxidant, and anti-inflammatory activities [43]. The K. aegyptiaca EO showed a higher antioxidant activity than the EOs of other reported plants, such as Persicaria lapathifolia [25], Cleome droserifolia [44], and Deverra tortuosa [45], while it showed a lower antioxidant activity than those reported for the EOs of E. mauritanica [34].

2.3. Antibacterial Activity of K. aegyptiaca EO

The EO extracted from K. aegyptiaca aerial parts displayed considerable antibacterial activity against Gram-positive and Gram-negative bacterial isolates (Table 2). The EO showed varying inhibitory activity on various bacterial strains, but it did not reveal antibacterial activity against Streptococcus epidermis (Gram-negative strain). The antibacterial effect can be ordered as follows: Salmonella typhimurium > Bacillus cereus > Escherichia coli > Staphylococcus aureus > Pseudomonas aeruginosa > Staphylococcus xylosus > Staphylococcus haemolyticus (Table 2).

Table 2

Antibacterial activity of the essential oil extracted from K. aegyptiaca aerial parts, expressed by the diameter of the inhibition zone (mm) and minimum inhibitory concentration (MIC), as well as some selected reference antibiotics at a concentration of 10 mg mL−1.

Microbes	K. aegyptiaca(10 mg mL−1)	MIC(10 mg mL−1)	Cephradin	Tetracycline	Azithromycin	Ampicillin
Gram-negative bacteria
Escherichia coli	22.04 ± 0.74 C,#	0.031	15.67 ± 0.42 E	20.11 ± 0.55 B	18.08 ± 0.44 C	20.97 ± 0.75 C
Pseudomonas aeruginosa	13.67 ± 0.91 E	0.044	0.00 G	0.00 E	12.57 ± 0.31 D	0.00 F
Salmonella typhimurium	26.08 ± 1.02 A	0.038	0.00 G	9.47 ± 0.37 D	0.00 E	0.00 F
Streptococcus epidermis	0.00 H	0.00	11.05 ± 0.81 F	21.07 ± 0.98 A	20.36 ± 0.77 A	10.57 ± 0.57 D
Gram-positive bacteria
Bacillus cereus	23.11 ± 0.58 B	0.031	19.6 ± 0.43 C	9.68 ± 0.27 D	20.15 ± 0.33 A	6.45 ± 0.36 E
Staphylococcus aureus	16.17 ± 0.51 D	0.052	20.17 ± 0.79 B	18.51 ± 0.65 C	20.48 ± 0.49 A	29.14 ± 1.20 A
Staphylococcus haemolyticus	6.24 ± 0.11 G	0.562	24.17 ± 0.66 A	20.30 ± 1.01 B	19.19 ± 0.61 B	20.95 ± 0.94 C
Staphylococcus xylosus	11.61 ± 0.32 F	0.092	18.34 ± 0.77 D	18.48 ± 0.88 C	18.75 ± 0.73 B	24.66 ± 0.68 B
LSD 0.05	0.51 ***		0.52 ***	0.49 ***	0.45 ***	0.44 ***

# values are average (n = 3) ± standard error. Dissimilar superscript letters in each treatment express significant variation at a probability level of 0.05 (Duncan’s test). LSD: least significant difference. *** p < 0.001.

The selected antibiotics showed varied activity against the bacterial strains, with a general trend that Gram-negative bacteria were more resistant than Gram-positive strains. This observation is consistent with most research [26,45,46,47,48,49], where it is ascribed to the structure of the bacterial cells [46]. Cephradin showed the highest activity against S. haemolyticus, while it was inactive against P. aeruginosa and S. typhimurium at a dose of 10 mg mL−1. The tetracycline exhibited maximum inhibition on S. epidermis, but did not show activity against P. aeruginosa. On the other hand, the antibiotic azithromycin showed maximum activity against S. aureus, S. epidermis, and B. cereus at a concentration of 10 mg mL−1, while it did not show any activity against S. typhimurium. At a concentration of 10 mg mL−1, ampicillin was detected as a powerful antibacterial agent against S. aureus, while it did not have any activity against P. aeruginosa and S. typhimurium (Table 2). Based on the data of the minimum inhibitory concentration (MIC), the EO activity was highest (0.031 mg mL−1) againt E. coli and B. cereus, while the activity against the other bacterial isolates can be sequenced as follows: S. typhimurium, P. aeruginosa, S. aureus, S. xylosus, and S. haemolyticus. However, S. epidermis was comopletely resistant to the EO of K. aegyptiaca. The antibacterial activity of the K. aegyptiaca EO in the present study was higher than those reported for the EO of D. tortuosa [45] and Teucrium polium [48], while it was lower than others, such as Thymus decussatus [48], Achillea fragrantissima, Artemisia Judaica, and Tanacetum sinaicum [47].

The observed antibacterial activity of K. aegyptiaca could be attributed to the activity of the major compounds (cuminic aldehyde, caryophyllene oxide, hexahydrofarnesyl acetone, ar-turmerone, aromadendrene oxide, and humulene epoxide), either singly or synergistically. The insecticidal activity of Rosmarinus officinalis has been attributed to the synergistic interaction between camphor and 1,8-cineole [40]. A similar study by de Sousa, et al. [50] indicated that the combination of carvacrol and 1,8-cineole maximizes the inhibitory activity against bacterial strains associated with vegetable processing. In addition, cuminic aldehyde has been reported to have antibacterial effects [29,30]. In contrast to our results, the EO of C. cyminum, rich with cuminic aldehyde, did not show antibacterial activity against Pseudomonas species [29].

Caryophyllene oxide has been reported as a strong antimicrobial agent against a wide range of microbes [51]. In addition, the EOs that were rich in caryophyllene oxide were reported to have a considerable antimicrobial activity, such as Satureja coerulea [52], Psidium guajava [53], and Pinus eldarica [54]. Moreover, EOs rich in hexahydrofarnesyl acetone have been described as possessing considerable antimicrobial activity [55,56]. In addition, ar-turmerone has been reported as antimicrobial agent in Artemisia integrifolia [57]. Our findings supported the potential uses of the EO of K. aegyptiaca in the food industries, as well as in the manufacturing of cosmetics and aromas, due to its potent antioxidant and/or antimicrobial significance.

3. Materials and Methods

3.1. Plant Materials

The K. aegyptiaca aerial parts were collected during the flowering season (April 2019) from different populations growing in Wadi Araba, Eastern Desert, Egypt (28.9781482N, 32.2019523E). The collected samples were healthy and flowering. Plant authentication was carried out following Boulos [13] and Tackholm [58]. From the collected sample, a voucher specimen was prepared and deposited in the Botany Department Herbarium at College of Science, Mansoura University, Egypt, with the code Mans.191101007 (Figure 5).

Figure 5

Kickxia aegyptiaca (L.) Nábělek. (A) overview of the plant in sandy habitat showing dense branching, (B) close view showing the flowering branches, and (C) close view of the yellow flower.

3.2. Extraction of EO and GC-MS Analysis

The EO was extracted via the subjection of ~180 g of the air-dried K. aegyptiaca aerial parts to hydro-distillation for 3 h over the Clevenger apparatus. The separation of the EO layer was performed by n-hexane, dried by anhydrous Na2SO4 (0.5 g), and then saved at 4 °C in glass vials until further chemical and biological analyses. The extracted EO was chemically analyzed via gas chromatography-mass spectrometry (GC-MS). The characterization and identification of chemical constituents were performed with the same conditions and protocol as described previously [25,59]. The GC-MS apparatus was made up of TRACE GC Ultra-Gas Chromatographs (THERMO Scientific™ Corporate, Waltham, MA, USA) with a quadrupole mass spectrometer (Thermo Scientific ISQ™ EC, Waltham, MA, USA). The GC-MS column dimension was 30 m × 0.32 mm and i.d. 0.25 µm film thickness. At a flow rate of 1.0 mL per min, helium was used as a transporter gas, with a split ratio of 1 to 10. The temperature program was accustomed as follows: 60 °C for 1 min., raised to 240 °C with 4 °C/min. The diluted sample in n-hexane (1 µL) at a ratio of 1:10 (v/v) was injected into the instrument, where the injector and detector were adjusted at 210 °C. The mass spectra of compounds were charted by electron ionization (EI) at 70 eV, using a spectral range of m/z 40–450. The authentication and identification of the chemical compounds were performed using the Automated Mass spectral Deconvolution and Identification (AMDIS) software, NIST library database, Wiley spectral library collection, retention indices relative to n-alkanes (C8–C22), or assessment of the mass spectrum with authentic standards compounds. The relative concentrations of the compounds were performed based on Tentatively Identified Compounds (TICs) of the EO.

3.3. Antioxidant Activity of the EO

To test the antioxidant activity K. aegyptiaca EO, two protocols were considered: (a) reduction in the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma-Aldrich, Darmstadt, Germany) and (b) reduction in the radical 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, Sigma-Aldrich, Darmstadt, Germany). In the DPPH assay, the EO was prepared in a concentration range of 5–50 mg L−1, using methanol as a solvent. This range was selected based on the scavenging activity that enabled us to determine the IC50 (EO amount necessary to reduce the radical by 50%) [45]. According to Miguel [60], equal volumes of each concentration and DPPH (0.3 mM) were shaken vigorously and kept in dark conditions for 30 min. The absorbance was assessed at 517 nm via spectrophotometer, model Spectronic 21D, Milton Roy, CA, USA. On the other side, the ABTS assay was conducted according to Re, et al. [61]. In brief, about 0.2 mL of each concentration was mixed with 2 mL of freshly prepared ABTS and incubated in a dark condition for 6 min. The range of the EO concentration was similar to those of DPPH (5–50 mg L−1). The color absorbance was measured at 734 nm by Spectronic 21D spectrophotometer, Milton Roy, CA, USA. In addition, to refer the antioxidant activity to standard antioxidant, various concentrations (1–20 mg L−1) of ascorbic acid were prepared and their antioxidant activity was determined as previously described for the EO. The scavenging activity was calculated based on the following formula:

3.4. Antibacterial Activity of the EO

The EO extracted from K. aegyptiaca aerial parts was tested for its antibacterial activity against four Gram-negative bacterial strains (E. coli (ATCC 10536), P. aeruginosa (ATCC 9027), S. typhimurium (ATCC 25566), and S. epidermis (ATCC 12228)) and four Gram-positive bacterial strains (B. cereus (EMCC number), S. aureus (ATCC 6538), S. haemolyticus (ATCC 29970), and S. xylosus (NCCP 10937)). The bacterial isolates were obtained from the Cairo Microbiological Resources Centre (Cairo MIRCEN), College of Agriculture, Ain Shams University, Egypt. The bioassay was performed using the agar diffusion method [62]. In brief, filter paper discs (Whatman no.1, 5 mm) were saturated with the EO of K. aegyptiaca at a concentration of 10 mg mL−1 in dimethyl sulfoxide. Petri dishes (90 mm) were prepared and filled with sterilized nutrient agar medium and inoculated with 106 colony-forming units (CFU)/mL of each bacterial strain. The filter paper disc was adjusted above the medium in the center of the Petri dish, and the 1080 plates were immediately sealed with Parafilm® tape (Sigma, St. Louis, MO, USA) and incubated for 24 h at 37 °C. After incubation, the diameter of the inhibition zone (clear zone around disc without growth) was measured in mm at three random positions. The MIC was determined based on the dimensions of the inhibition zone for different EO concentrations. To compare the antibacterial activity of the EO with reference antibiotics, cephradin, tetracycline, azithromycin, and ampicillin were subjected to the same procedures.

3.5. Statistical Analysis

The experiments of both antioxidant activity and antibacterial activity were achieved three times with three replications for each treatment. The data were subjected to one-way analysis of variance (ANOVA), after Duncan’s test using CoStat software program (version 6.311, CoHort Software, Monterey, CA, USA). The data were expressed as mean values with standard error. The IC50 value for antioxidant assays was graphically calculated using MS-Excel 2016.

4. Conclusions

A GC-MS analysis of the extracted EO from the aerial parts of K. aegyptiaca revealed, for the first time, the presence of 43 compounds, mainly terpenes. Oxygenated compounds were predominant, particularly sesquiterpenes and monoterpenes. Cuminic aldehyde, caryophyllene oxide, hexahydrofarnesyl acetone, ar-turmerone, aromadendrene oxide, and humulene epoxide were identified as major compounds with a concentration of 66.02% of the total mass. The extracted EO showed considerable antioxidant activity as well as antibacterial activity. The major compounds have been reported to possess various biological activities, including antioxidant and antimicrobial activities. Therefore, further studies are recommended to evaluate the various biological activities of the major identified compounds, either alone or in combination, as well as to assess their biosafety.