Yield, Phytochemical Constituents, and Antibacterial Activity of Essential Oils from the Leaves/Twigs, Branches, Branch Wood, and Branch Bark of Sour Orange (Citrus aurantium L.)

In the present work, essential oils (EOs) extracted from different parts of sour orange Citrus aurantium (green leaves/twigs, small branches, wooden branches, and branch bark) were studied through gas chromatography coupled with mass spectrometry (GC/MS). Furthermore, the EOs in the amounts of 5, 10, 15, 20, and 25 μL were studied for their antibacterial activity against three pathogenic bacteria, Agrobacterium tumefaciens, Dickeya solani, and Erwinia amylovora. The main EO compounds in the leaves/twigs were 4-terpineol (22.59%), D-limonene (16.67%), 4-carvomenthenol (12.84%), and linalool (7.82%). In small green branches, they were D-limonene (71.57%), dodecane (4.80%), oleic acid (2.72%), and trans-palmitoleic acid (2.62%), while in branch bark were D-limonene (54.61%), γ-terpinene (6.68%), dodecane (5.73%), and dimethyl anthranilate (3.13%), and in branch wood were D-limonene (38.13%), dimethyl anthranilate (8.13%), (-)-β-fenchol (6.83%), and dodecane (5.31%). At 25 μL, the EO from branches showed the highest activity against A. tumefaciens (IZ value of 17.66 mm), and leaves/twigs EO against D. solani and E. amylovora had an IZ value of 17.33 mm. It could be concluded for the first time that the wood and branch bark of C. aurantium are a source of phytochemicals, with D-limonene being the predominant compound in the EO, with potential antibacterial activities. The compounds identified in all the studied parts might be appropriate for many applications, such as antimicrobial agents, cosmetics, and pharmaceuticals.

from leaves/twigs (Petitgrain), branches (2-4 cm in diameter), the wood of branches, and branch bark were kept dry in sealed Eppendorf tubes and stored at 4 • C prior to chemical analyses.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
The chemical composition of the essential oils was determined using a Trace GC Ultra-ISQ mass spectrometer (Thermo Scientific, Austin, TX, USA) with a direct capillary column TG-5MS (30 m × 0.25 mm × 0.25 µm film thickness). Initially, the column oven temperature was held at 45 • C, then increased by 5 • C/min to 250 • C and held for 2 min, then increased to 280 • C by 10 • C/min. The injector and MS transfer line temperatures were kept at 250 • C. Helium was used as a carrier gas at a constant flow rate of 1 mL/min. The solvent delay was 2 min and diluted samples of 1 µL were injected automatically using an Autosampler AS1310 coupled with the GC in the split mode. EI mass spectra were collected at 70 eV ionization voltages over a range of m/z of 40-600 in full scan mode. The ion source was set at 200 • C. Identification of the constituents was performed on the basis of their retention times and by comparing the mass spectra with those found in the library search (NIST and Wiley) [39]. Type threshold values contained in Xcalibur 3.0 data system of GC/MS were used as match factors and to confirm that all mass spectra are appended to the library with measuring the Standard Index (SI) and Reverse Standard Index (RSI), where the value ≥650 is acceptable to confirm the compounds [40].

Antibacterial Activity
Antibacterial evaluation of the EOs was assayed against three phytopathogenic bacteria, Agrobacterium tumefaciens, Dickeya solani, and Erwinia amylovora (Microbiology Laboratory, Agricultural Botany Department, Faculty of Agriculture (Saba Basha), Alexandria University, Egypt). The antibacterial evaluation test of the studied four EOs was performed by measuring the inhibition zones (IZs) in millimeters around the loaded filter papers with different amounts of oils (5,10,15,20, and 25 µL) using disc diffusion method [40,41]. Sterile filter paper discs (Whatman filter paper no. 1) with a diameter of 4 mm loaded with different amounts of the studied EOs were placed on the surface of prepared agar plates. All the plates were incubated in incubator at 30 • C for 24 h. Negative control discs were left without any EO. All of the tests were performed in triplicate and the values of the IZs (the clear zones with no bacterial growth around the loaded discs) were reported including the diameter of the disc.

Antibacterial Activity of the EOs
From the main effects of the extracted oils from different parts of C. aurantium (Figure 2a), oil from leaves/twigs showed the highest activity against all the studied three phytopathogenic bacteria. The main effects of oil amount from all the studied plant parts (Figure 2b) showed that increasing the amount of oil (µL) also increased the antibacterial activity, as measured by the inhibition zone (IZ). Table 6 presents the antibacterial activity of the studied EOs from different parts of C. aurantium. The highest activity against the growth of A. tumefaciens was observed by the application of EO from branches at 25 µL (IZ value of 17.66 mm), followed by oil from leaves/twigs at 20 and 25 µL with IZ value of 15.66 mm. On the other hand, EOs from bark and branch wood did not show any activity

Antibacterial Activity of the EOs
From the main effects of the extracted oils from different parts of C. aurantium (Figure 2a), oil from leaves/twigs showed the highest activity against all the studied three phytopathogenic bacteria. The main effects of oil amount from all the studied plant parts (Figure 2b) showed that increasing the amount of oil (µL) also increased the antibacterial activity, as measured by the inhibition zone (IZ). Table 6 presents the antibacterial activity of the studied EOs from different parts of C. aurantium. The highest activity against the growth of A. tumefaciens was observed by the application of EO from branches at 25 µL (IZ value of 17.66 mm), followed by oil from leaves/twigs at 20 and 25 µL with IZ value of 15.66 mm. On the other hand, EOs from bark and branch wood did not show any activity against A. tumefaciens. At 25 µL of leaves/twigs EO, the highest activity (17.33 mm) against D. solani was reported, followed by the application of branch EO at 25 µL (16.66 mm) and 20 µL (16.66 mm). For the antibacterial activity of EOs against the growth of E. amylovora at oil amount of 20 and 25 µL from leaves/twigs, the highest IZ value was observed (17.33 mm), followed by branch EO at 25 µL with IZ value of 15.33 mm. Also, the EO from leaves/twigs at 10 and 15 µL showed good activity against E. amylovora with IZ value of 15.00 mm.
against A. tumefaciens. At 25 µL of leaves/twigs EO, the highest activity (17.33 mm) against D. solani was reported, followed by the application of branch EO at 25 µL (16.66 mm) and 20 µL (16.66 mm). For the antibacterial activity of EOs against the growth of E. amylovora at oil amount of 20 and 25 µL from leaves/twigs, the highest IZ value was observed (17.33 mm), followed by branch EO at 25 µL with IZ value of 15.33 mm. Also, the EO from leaves/twigs at 10 and 15 µL showed good activity against E. amylovora with IZ value of 15.00 mm.

Discussion
The results of the present work showed the variation in the chemical composition of the EOs from different parts of C. aurantium. Most previous studies have focused on the identification of chemical composition of EOs from the peels, pericarp, blossoms, and leaves, and no core results have been reported from branches, wood, or bark. Additionally, the trials of antimicrobial activities of the EOs were measured against human pathogenic bacteria and plant pathogenic fungi, with no results about the activity against plant bacterial pathogens. 4-terpineol (22.59%) and D-limonene (16.67%) were the most predominate components abundant in green leaves/twigs of C. aurantium, while D-limonene with percentages of 71.57%, 54.61%, and 38.13% was found in small green branches, branch bark, and branch wood, respectively. Results from Wolffenbuttel et al. [29] showed that limonene (39.5-92.7%) and linalool (14.2-24.8%) are the main components of the pericarp and leaves, respectively, of citrus oils obtained by steam distillation, hydrodistillation, or cold press extraction. Linalyl acetate, linalool, α-terpineol, geranyl acetate, geraniol, and geranial as oxygenated monoterpene hydrocarbons were primarily identified in petitgrain oil of C. aurantium var. amara [12], whereas limonene was present only at a concentration of 1.4%.
Linalyl acetate was present in Sicilian petitgrain oil with a lower amount of linalool [49]. Linalyl acetate and linalool were the main components in petitgrain oil from Turkey [50]. EOs of the peels, flowers, and leaves from C. aurantium, collected from northern Greece, exhibited the primary compounds linalool (29.14%), β-pinene (19.08%), trans-β-ocimene (6.06%), and trans-farnesol (5.14%) [51]. The EOs from blossoms of C. aurantium growing in the Darab region in Fars Province, Iran, showed that geraniol, α-terpineol, linalool, and benzene acetaldehyde were the main compounds [52]. Myrcene was found in low percentage of the present work and previously it was reported that myrcene, which found in the EO, is known to possess cytotoxic activity [53,54]. Dl-limonene with 94.81% is the main compound identified in peel EO from C. aurantium with promising larvicide against Anopheles stephensi [9]. Limonene, (E)-nerolidol, α-terpineol, α-terpinyl acetate, and (E,E)-farnesol were the main compounds in the flower EO of C. aurantium with good antibacterial activity against Pseudomonas aeruginosa [10]. α-terpineol and terpinene-4-ol, found in the leaf EO from C. hystrix, were more active against Acinetobacter baumannii, Streptococcus spp., and Haemophilus influenzae than crude oil, while limonene, the most abundant component of C. hystrix oil, had lower antibacterial activity [55].
Zest EO had limonene (85.22%), β-myrcene (4.3%), and α-pinene (1.29%) as the main components, and the EO showed higher antioxidant activity than did limonene alone with a potential for antibacterial activity against Staphylococcus aureus, Salmonella sp., Pseudomonas aeruginosa, Bacillus subtilis, and Escherichia coli [13]. Among 34 kinds of citrus EOs, four EOs from C. aurantium zest presented good antioxidant activities, as measured by a DPPH assay [16]. Strong fungicidal activity was exhibited by limonene and (E)-nerolidol present in the EO of the flowers of C. aurantium L. var. amara [56].
Limonene, linalool, citronellal, and citronellol were the main constituents of EO from C. aurantifolia leaves and fruit peels and exhibited promising antibacterial activity against oral pathogenic bacteria Streptococcus mutans and Lactobacillus casei [64].
Leaves EO of C. aurantium grown in Shiraz (south of Iran) showed the presence of limonene, linalool, and trans-β-ocimene as major components and exhibited strong antioxidant activity [65]. EO obtained by cold pressing of C. aurantium fruits with high percentage of limonene (77.90%) and minor percentages of β-pinene (3.40%) and myrcene (1.81%) was inactive against Escherichia coli and Pseudomonas, while moderately active against Stapylococcus aureus [66]. Limonene from linalool-rich essential oil inhibits S. aureus [67].

Conclusions
In the present study, variations in essential oils composition from different parts of C. aurantium were reported. 4-terpineol, followed by D-limonene, were the main constituents in EO from the leaves/twigs, while D-limonene was the main constituent in small green branches, the branch wood, and the branch bark. EOs from leaves and small branches promised to be potential antibacterial activates against Agrobacterium tumefaciens, Dickeya solani, and Erwinia amylovora. The EOs obtained from different parts of C. aurantium displayed bioactive compounds, which have the potential for application as biopreservative agents, antioxidants, antimicrobial compounds, cosmetics, and pharmaceuticals.