Phytochemical Composition, Antioxidant and Antiproliferative Activities of Citrus hystrix, Citrus limon, Citrus pyriformis, and Citrus microcarpa Leaf Essential Oils against Human Cervical Cancer Cell Line

The essential oil derived from Citrus plants has long been used for medicinal purposes, due to its broad spectrum of therapeutic characteristics. To date, approximately 162 Citrus species have been identified, and many investigational studies have been conducted to explore the pharmacological potential of Citrus spp. oils. This study investigated the volatile constituents of essential oil distilled from the leaves of C. hystrix, C. limon, C. pyriformis, and C. microcarpa, using gas chromatography–quadrupole mass spectrometry. A total of 80 secondary compounds were tentatively identified, representing 84.88–97.99% of the total ion count and mainly comprising monoterpene (5.20–76.15%) and sesquiterpene (1.36–27.14%) hydrocarbons, oxygenated monoterpenes (3.91–89.52%) and sesquiterpenes (0.21–38.87%), and other minor chemical classes (0.10–0.52%). In particular, 27 compounds (1.19–39.06%) were detected across all Citrus species. Principal component analysis of the identified phytoconstituents and their relative quantities enabled differentiation of the Citrus leaf oils according to their species, with the loading variables contributing to these metabolic differences being identified. The Citrus leaf oils were tested for their antioxidant and antiproliferative activities using 2,2-diphenyl-1-picryl-hydrazylhydrate (DPPH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The results indicated that C. limon displayed the highest DPPH radical scavenging ability (IC50 value of 29.14 ± 1.97 mg/mL), while C. hystrix exhibited the lowest activity (IC50 value of 279.03 ± 10.37 mg/mL). On the other hand, all the Citrus oils exhibit potent antiproliferative activities against the HeLa cervical cancer cell line, with IC50 values of 11.66 μg/mL (C. limon), 20.41 μg/mL (C. microcarpa), 25.91 μg/mL (C. hystrix), and 87.17 μg/mL (C. pyriformis).


Introduction
The genus Citrus belongs to the Rutaceae family, which is comprised of about 160 genera and approximately 162 species (according to the Tanaka classification system), which are distributed throughout the world [1,2]. Apart from culinary uses, Citrus spp. plants have been cultivated and exploited for their nutritional and therapeutic value. In particular, the aromatic oil extracted from these plants is valued due to its medicinal properties and Plants 2023, 12, 134 2 of 15 economic significance, with potential applications in the food, perfume, cosmetic, and pharmaceutical industries [3,4]. Citrus spp. essential oils (EO) have reportedly exhibited various therapeutic properties and/or beneficial pharmacological effects, such as antibacterial [5,6], anticancer [7,8], anti-inflammatory [9,10], and antioxidant [11][12][13] activities. It is worth noting that these associated bioactivities and their organoleptic properties are attributed to the complex pool of phytoconstituents in Citrus EO, which vary according to genetic variability and diversity, origin, climate, seasonal factors, and others [14].
The phytochemistry of Citrus spp. EOs is comprised of monoterpenic and sesquiterpenic hydrocarbons, as well as their oxygenated derivatives, which include alcohols, aldehydes, ketones, esters, and others [15]. The characteristic EO profiles of Citrus spp. have been reported to identify species/cultivars, determine genetic diversity, and interpret sensory attributes [16]. Given the availability of Citrus plants around the world and their associated pharmacological potentials, numerous studies have targeted investigating the chemical composition of EOs derived from different Citrus species and/or varieties that confer distinct pharmacological properties [17]. Notably, cancer has become a focus of attention, as one of the most critical diseases and the second highest cause of death after heart disease [18]. There are many reported causes of cancer (e.g., physiological and biochemical factors), which include ultraviolet rays, smoke, poisoning by bacteria and fungi, and the presence of free radicals that cause oxidative damage to vital biomolecules [19]. Therefore, the search for cures from plant-derived products (e.g., essential oil) has become an important subject [20,21]. Considerable evidence has demonstrated that selected Citrus spp. EOs exhibit an antioxidant potential that can eliminate excess free radicals (e.g., hydroxyl radicals, peroxide radicals, and super oxygen radicals) in the human body [22]. Some recent studies have also highlighted the chemopreventive potential of some Citrus spp. EOs that displayed cytotoxicity effects against cervical (HeLa), breast (MCF-7), kidney (HEK-293), colon (HT- 29), and liver (HepG2, BEL-7402) cancer cell lines [23][24][25][26].
To our knowledge, reports that have comprehensively examined the chemical constituents and pharmacological potential of EOs derived from the leaf of Citrus spp. plants are still lacking. Considering these points, this study aimed to (i) profile steam-distilled leaf essential oil from four commercially cultivated Citrus species (C. hystrix, C. limon, C. pyriformis, and C. microcarpa) in Malaysia; (ii) determine the antioxidant activities of Citrus spp. EOs using the 2,2-diphenyl-1-picryl-hydrazylhydrate (DPPH) radical scavenging method; and (iii) evaluate the in vitro antiproliferative activities of these oils against human adenocarcinoma cervical cancer (HeLa) cell line. The phytocomplexity and variation of the secondary compounds within different Citrus spp. leaf oils that may contribute to different biological effects are discussed. Discriminations based on chemometrics analyses, to objectively compare the fundamental similarities and differences among the different Citrus spp. are presented.

Phytochemical Compositions
GC-MS analysis enabled the tentative identification of 80 secondary compounds (46 in C. hystrix, 52 in C. microcarpa, 54 in C. pyriformis, and 45 in C. limon), which represented between 84.88% and 97.99% of the total ion count within the four Citrus spp. leaf EOs. The chromatographic profiles of secondary compounds in the different Citrus spp. leaf oils are presented in Figure 1, highlighting distinctive chemical patterns. These tentatively identified compounds are distributed into 15 monoterpene hydrocarbons, 22 oxygenated monoterpenes, 16 sesquiterpene hydrocarbons, 19 oxygenated sesquiterpenes, 1 diterpenes, 1 oxygenated diterpene, and others (6). A total of 26 compounds were detected in common across all species, comprising mainly monoterpene hydrocarbons (0.01-70.40%). Table S1 (Supplementary Information) summarizes the identified secondary compounds and their relative abundance based on total ion counts. The degree of variation in the metabolic compositions ( Figure 1 and Table S1) corresponding to different Citrus spp.
identified compounds are distributed into 15 monoterpene hydrocarbons, 22 oxygenated monoterpenes, 16 sesquiterpene hydrocarbons, 19 oxygenated sesquiterpenes, 1 diterpenes, 1 oxygenated diterpene, and others (6). A total of 26 compounds were detected in common across all species, comprising mainly monoterpene hydrocarbons (0.01-70.40%). Table S1 (Supplementary Information) summarizes the identified secondary compounds and their relative abundance based on total ion counts. The degree of variation in the metabolic compositions ( Figure 1 and Table S1) corresponding to different Citrus spp. EOs is noteworthy, which could be translated into differences in the expression of their pharmacological potential.  Table S1 (Supplementary Information).
The identified major compounds (relative abundance >1%) for different species of Citrus leaf oils are detailed in Table 1, while the relative composition of the classified chemical groups is illustrated in Figure 2. Notably, monoterpenic compounds predominated in C. hystrix, C. limon, and C. pyriformis, with total ion count abundances of 94.7%, 91.4%, and 89.7%, respectively. Interestingly, the content of monoterpenes (18.4%) in C. microcarpa was much lower compared to the other species (≥89.7%). Monoterpenic hydrocarbons constituted the highest proportion in C. limon (48.6%) and C. pyriformis (76.1%), as compared to C. hystrix (5.2%) and C. microcarpa (14.5%). Limonene was the major compound in C. limon and C. pyriformis, accounting for 33.6% and 70.4% of the total ion count, but only constituting a relatively small amount in C. microcarpa (1.7%) and C. hystrix (0.2%). C. hystrix had the highest content of oxygenated monoterpenes (89.5%), with citronellal being the principal constituent for C. hystrix (77.7%), while C. microcarpa showed the lowest amount of oxygenated monoterpenes (3.9%). Apart from C. hystrix, Eucalyptus  Table S1 (Supplementary Information).

Discrimination via Principal Component Analysis
In order to examine the metabolic differences among the different species of Citrus leaf oils, principal component analysis (PCA) was applied to the chemical abundances of the 80 compounds identified in respective species. Figure 3A illustrates a score plot for the different Citrus spp. leaf oils, highlighting the potential for chemotaxonomic classification based on species. PC-1 produced the highest variation (48%) of data, followed by PC-2 (31%), which cumulatively explained 79% of the variance in the dataset. The analyzed leaf oils were segregated into four different groups, revealing discriminating secondary compounds according to the Citrus species, which could be explained by the loading plot ( Figure 3B). It is worth noting that PC-2 separated C. limon and C. pyriformis from C. hystrix, and the proximity of C. limon and C. pyriformis as situated in the same quadrant (negative PC-2 axis) suggests a certain degree of similarity with respect to their secondary compounds. The main explanatory variables were trans-linalool oxide, 4-thujanol, citronellal, and citronellol acetate for C. hystrix; cosmene, α-thujene, camphene, β-pinene, and humulene for C. microcarpa; geranyl propionate, β-bisabolene, 3-carene, terpinolene, trans-β-ocimene, geranyl acetate, and phytol for C. limon; and trans-sesquisabinene hydrate, p-vinylguaiacol, and α-farnesene for C. pyriformis.

Discrimination via Principal Component Analysis
In order to examine the metabolic differences among the different species of Citrus leaf oils, principal component analysis (PCA) was applied to the chemical abundances of the 80 compounds identified in respective species. Figure 3A illustrates a score plot for the different Citrus spp. leaf oils, highlighting the potential for chemotaxonomic classification based on species. PC−1 produced the highest variation (48%) of data, followed by PC−2 (31%), which cumulatively explained 79% of the variance in the dataset. The analyzed leaf oils were segregated into four different groups, revealing discriminating secondary compounds according to the Citrus species, which could be explained by the loading plot ( Figure 3B). It is worth noting that PC−2 separated C. limon and C. pyriformis from C. hystrix, and the proximity of C. limon and C. pyriformis as situated in the same quadrant (negative PC−2 axis) suggests a certain degree of similarity with respect to their secondary compounds. The main explanatory variables were trans-linalool oxide, 4-thujanol, citronellal, and citronellol acetate for C. hystrix; cosmene, α-thujene, camphene, β-pinene, and humulene for C. microcarpa; geranyl propionate, β-bisabolene, 3-carene, terpinolene, transβ-ocimene, geranyl acetate, and phytol for C. limon; and trans-sesquisabinene hydrate, pvinylguaiacol, and α-farnesene for C. pyriformis.

Antioxidant Activity
The antioxidant capacity of the Citrus leaf EOs was investigated by evaluating their DPPH free-radical scavenging ability. The citrus oils displayed a dose-dependent scavenging activity against DPPH. Based on the linear curve plotted between the scavenging activity inhibition percentage and the EO concentrations, C. limon exhibited the highest antioxidant activity (IC50 value of 29.14 ± 1.97 mg/mL), while C. hystrix had the lowest antioxidant activity (IC50 value of 279.03 ± 10.37 mg/mL). Additionally, C. limon and C. pyriformis leaf oils showed the highest DPPH radical scavenging ability, as compared to the positive control (ascorbic acid). The IC50 values of the antioxidant activity of Citrus oils and values reported from previous studies are shown in Table 2. The current results demonstrated that the antioxidant potential of Citrus oils might be attributed to the variations in their phytoconstituents [32]. A few studies have reported that antioxidant activity might be correlated to the level or proportion of limonene within Citrus EOs [11,33,34]. Overall, the current findings indicated that the antioxidant activity was higher in oils containing a higher proportion of limonene. However, the inter-relation of the limonene  Table S1 (Supplementary Information).

Antioxidant Activity
The antioxidant capacity of the Citrus leaf EOs was investigated by evaluating their DPPH free-radical scavenging ability. The citrus oils displayed a dose-dependent scavenging activity against DPPH. Based on the linear curve plotted between the scavenging activity inhibition percentage and the EO concentrations, C. limon exhibited the highest antioxidant activity (IC 50 value of 29.14 ± 1.97 mg/mL), while C. hystrix had the lowest antioxidant activity (IC 50 value of 279.03 ± 10.37 mg/mL). Additionally, C. limon and C. pyriformis leaf oils showed the highest DPPH radical scavenging ability, as compared to the positive control (ascorbic acid). The IC 50 values of the antioxidant activity of Citrus oils and values reported from previous studies are shown in Table 2. The current results demonstrated that the antioxidant potential of Citrus oils might be attributed to the variations in their phytoconstituents [32]. A few studies have reported that antioxidant activity might be correlated to the level or proportion of limonene within Citrus EOs [11,33,34]. Overall, the current findings indicated that the antioxidant activity was higher in oils containing a higher proportion of limonene. However, the inter-relation of the limonene content and antioxidant potential of Citrus oil necessitates further investigation, as conflicting reports have been made [35]. Thus, it is difficult to explain how Citrus oil functions as an antioxi-dant, due to the intricacy of their multicomponent mixes and insufficient studies on their molecular mechanisms.

Antiproliferative Evaluation
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the cytotoxicity of Citrus spp. EOs against a human adenocarcinoma cervical cancer (HeLa) cell line, with a concentration range of 2.3 to 75.0 µg/mL. Figure 4 indicates that all Citrus oils decreased the viability of the Hela cells in a dose-dependent manner (p < 0.01). After 24 h of treatment, C. limon oil (IC 50 = 11.7 µg/mL) exhibited the most significant effect, followed by C. microcarpa (IC 50 = 20.4 µg/mL) and C. hystrix (IC 50 = 25.9 µg/mL), while the least potent was C. pyriformis (IC 50 = 87.2 µg/mL). To our knowledge, this is the first study to report a comparative investigation of the antiproliferative activities in C. microcarpa, C. hystrix, and C. pyriformis leaf oils. Interestingly, the results indicated no correlation was observed between the antioxidant activity (C. hystrix < C. microcarpa < C. pyriformis < C. limon) and the antiproliferative effects (C. pyriformis < C. hystrix < C. microcarpa < C. limon) against HeLa cells. The cytotoxicity of C. limon oil was observed to be notably higher compared to previous studies concerning different plant parts and origins. For instance, the in vitro cytotoxicity activity of C. limon peel oil from Northern Egypt against the HeLa cell line resulted in an IC 50 value of 51.0 µg/mL, and Iranian C. limon peel oil displayed an IC 50 value of 17.0 µg/mL [23,40]. On the contrary, C. limon leaf oil from India presented a much higher cytotoxicity (IC 50 = 4.75 µg/mL) compared to the current study [41]. Interestingly, the antiproliferative activities of the analyzed Citrus leaf EOs were comparable to cytotoxic studies using Citrus spp. peel EOs with IC 50 values <100 µg/mL. For instance, C. hystrix peel EO displayed potent cytotoxic activity against human melanoma cells (WM793, A375, and HTB140) with IC 50 values of 59.2-88.4 µg/mL, while C. limon peel EO was found to be cytotoxic to HepG2 cells and HCT-116 colorectal carcinoma cells (IC 50 value of 48.2 and 72.6 µg/mL, respectively) [42,43]. It is known that Citrus spp. oils contain high quantities of terpenes, especially limonene, a wellestablished chemopreventive and therapeutic agent against numerous tumor cells [44][45][46][47]. Our findings indicated that C. limon oils that contained high relative amount of limonene (33.6%) exhibited the highest cytotoxicity against HeLa cells, which agreed with a previous finding, where limonene demonstrated potential cytotoxicity (22.1 µg/mL) against HeLa cells [48]. Surprisingly, C. pyriformis, which contained the highest proportion of limonene (70.4%), displayed the lowest antiproliferative activity. A few studies have highlighted that limonene might not be a primary contributor to the cytotoxicity of essential oils [49][50][51]. On the other hand, several other terpenes that are found in Citrus spp. oils were reported to exhibit a better cytotoxic potential against HeLa cells. For instance, citral demonstrate the ability to suppress cell proliferation, through the increment of intracellular reactive oxygen species and dissipation of mitochondrial membrane potential in HeLa cells [52]. Linalool can induce apoptosis in HeLa cells through cyclin-dependent kinase inhibitors and src kinases [53]. Other compounds with a cytotoxicity effect against various cancer cell lines include phytol, nerolidol, α-humulene, geraniol, β-caryophyllene, and β-elemene [54][55][56][57][58][59]. These findings suggest that different constituents in Citrus EOs may synergistically contribute to their antiproliferative activity against HeLa cells, instead of being the sole contribution of a single bioactive compound. elemene [54][55][56][57][58][59]. These findings suggest that different constituents in Citrus EOs may synergistically contribute to their antiproliferative activity against HeLa cells, instead of being the sole contribution of a single bioactive compound.

Plant Material and Isolation of Essential Oil
Citrus spp. leaf samples were collected from selected orchards located at Gemencheh, Negeri Sembilan-C. hystrix and C. limon; Batu Ferringhi, Penang-C. pyriformis; and Gelugor, Penang-C. microcarpa in March 2021. Approximately 400 g of fresh leaves was subjected to steam distillation ( Figure S1) for 3 h. The yields for the steam-distilled Citrus spp. leaf EOs ranged from 0.28 to 0.72% (w/w) on fresh weight basis (Table S2; Supplementary  Information). The essential oil layer was collected and stored at 4 °C in glass vials, until further being analyzed, and were then diluted in 2% v/v acetone prior to being injected into the GC system.

Gas Chromatography-Quadrupole Mass Spectrometry System
Chemical profiling of the Citrus spp. leaf EOs was conducted on an Agilent Technologies 7890B gas chromatography system (Agilent Technologies, Santa Clara, CA, USA) equipped with a 5977B GC/MSD quadrupole mass spectrometer, 7693A autosampler, and a split/splitless inlet. The separation was effected using a nonpolar (HP-5ms (5% phenylmethylpolysiloxane)) capillary column of dimensions 30 m × 0.25 mm I.D. × 0.25 μm df.

Plant Material and Isolation of Essential Oil
Citrus spp. leaf samples were collected from selected orchards located at Gemencheh, Negeri Sembilan-C. hystrix and C. limon; Batu Ferringhi, Penang-C. pyriformis; and Gelugor, Penang-C. microcarpa in March 2021. Approximately 400 g of fresh leaves was subjected to steam distillation ( Figure S1) for 3 h. The yields for the steam-distilled Citrus spp. leaf EOs ranged from 0.28 to 0.72% (w/w) on fresh weight basis (Table S2; Supplementary Information). The essential oil layer was collected and stored at 4 • C in glass vials, until further being analyzed, and were then diluted in 2% v/v acetone prior to being injected into the GC system.

Gas Chromatography-Quadrupole Mass Spectrometry System
Chemical profiling of the Citrus spp. leaf EOs was conducted on an Agilent Technologies 7890B gas chromatography system (Agilent Technologies, Santa Clara, CA, USA) equipped with a 5977B GC/MSD quadrupole mass spectrometer, 7693A autosampler, and a split/splitless inlet. The separation was effected using a nonpolar (HP-5ms (5% phenylmethylpolysiloxane)) capillary column of dimensions 30 m × 0.25 mm I.D. × 0.25 µm d f . The GC conditions used were oven temperature program, 40 • C (hold 2 min) at 3 • C min −1 to 220 • C, 2 • C min −1 to 300 • C; injector temperature of 250 • C; helium (purity of 99.999%) at a flow rate of 1.0 mL min −1 ; and injection volume of 1 µL, and using a split ratio of 5:1. The quadrupole MS was operated in 70 eV electron ionization mode, ion source temperature of 230 • C, solvent delay time of 3.0 min, deactivated fused-silica capillary as the transfer line (0.8 m × 0.1 mm I.D.) thermostated at 280 • C, and a mass scan range of 45-600 Da.

Data Handling
GC-QMS data acquisition and processing were performed using Agilent Mass Hunter Qualitative Analysis 10.0 (Agilent Technologies). The chemical constituents of the EOs were tentatively identified using their retention indices (RI) (relative to n-alkanes C 7 -C 30 ), which were calculated using the Van den Dool and Kratz equation and mass spectrum matching, according to the NIST 14.0 MS library database (National Institute of Standards and Technology; Gaithersburg, MD, USA), and compared with previously reported RI values [60,61]. A matching score ≥ 80 in conjunction with consistent RI values within ±10 when compared to reported RI, were employed as criteria for the tentative identification of the chromatographically separated compounds. The relative concentration of tentatively identified compounds was calculated based on the acquired TICs and presented as the mean ± standard deviation from three repeated samples injections. Principal component analysis (PCA) was performed using Unscrambler X 10.3 (CAMO Software, Oslo, Norway) to identify differences for the obtained chemical profiles. All data were presented using Excel software (Microsoft Corporation, Washington, DC, USA) and Origin 8 (Origin Lab Corporation, Northampton, MA, USA). One-way analysis of variance (ANOVA) was performed using MS Excel software (Version 2211 Build 16.0.15831.20098).

Antioxidant Activity by DPPH Assay
The scavenging activity of four Citrus spp. leaf essential oils was determined using the DPPH method, as reported by Gursoy et al. [62], with slight modifications. Briefly, C. limon, C. pyriformis, and C. microcarpa EOs were prepared in methanol at different concentrations (3.12, 6.25, 12.50, 25.00, 50.00, and 100 mg/mL), while C. hystrix was prepared in a concentration range of 3.12-400 mg/mL. Subsequently, 50 µL of oil was added to 150 µL of the methanolic DPPH solution (0.1 mM), incubated in 96-well microplate for 30 min, and protected from light. Ascorbic acid solutions in methanol were prepared and used as a positive reference standard; as a negative control, methanol solution was used. At the end of the incubation period, the absorbance was immediately measured at 517 nm using an Epoch microplate UV-Vis spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA). The assay was conducted in triplicate. The DPPH free-radical scavenging ability was calculated using the following equation: DPPH scavenging activity (%) = Abs control − Abs sample Abs control × 100 (1)

HeLa Cell Culture
The HeLa cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). The HeLa cells were cultured in Dulbecco's modified Eagle's medium-high glucose (DMEM; Sigma-Aldrich), with 10% fetal bovine serum and 1% penicillin or streptomycin at a temperature of 37 • C and with 5% CO 2 .
3.7. Antiproliferative Activity with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay Cells were seeded in 96 well plates at a density of 3000 cells/well supplemented DMEM (Dulbecco's modified Eagle's medium) and incubated for 24 h in a humified atmosphere (37 • C and 5% CO 2 ). Citrus leaf EOs were re-dissolved in ethanol at a final concentration of 50 mg/mL, and the medium was removed, and each well was supplemented with different concentrations of EOs (2.34 to 150.00 µg/mL) diluted with DMEM. The ethanol concentration in each well was ≤0.3%. An MTT assay was conducted after the 24 h incubation period, to determine cell viability via the measurement of color alterations, which gauge the activity of the enzyme that reduces MTT to formazan, giving a purple color [54]. The plates were measured for optical density at 540 nm using a SkanIT absorbance micro-plate reader (Thermo Scientific, St. Peters, MO, USA). Media containing cells were used as a positive control, while media containing no cells were used as a negative control. Cells and medium with 0.3% ethanol were used as the vehicle control. All the assays were conducted in triplicate. The percentage of cell viability was determined using the following equation: Cell viability (%) = Mean absorbance in test well Mean absorbance in vehicle control well × 100 (2)

Conclusions
The present study detailed the metabolic profiling of secondary compounds in leaf essential oils of C. hystrix, C. limon, C. pyriformis, and C. microcarpa sourced in Malaysia. The metabolic profiles revealed notable differences for phytoconstituents within the four Citrus spp. leaf oils. C. hystrix was dominated by oxygenated monoterpenes (89.5%), C. microcarpa was dominated by oxygenated sesquiterpenes (38.9%), and C. pyriformis and C. limon were dominated by monoterpene hydrocarbons (≥48.6%). PCA specifically revealed the discriminating metabolites and allowed chemotaxonomical classification according to Citrus species. All EOs showed DPPH radical scavenging ability, with C. limon and C. pyriformis exhibiting higher antioxidant activities (IC 50 < 40 mg/mL). Additionally, all the analyzed oils displayed potent antiproliferative activities against the HeLa cell line, with C. limon showing the highest antiproliferative activity, with an IC 50 value of 11.7 µg/mL. The discrepancies observed in the antioxidant and antiproliferative activities of the leaf oils extracted from different Citrus spp. suggest an inter-relation of metabolic diversities in effecting these bioactivities via specific molecular mechanisms, which will require further investigations. Nevertheless, this study can serve as an a priori reference for the development of bioactive foods and nutraceuticals incorporated with Citrus leaf oil that can provide health-promoting properties.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants12010134/s1, Figure S1. Steam distillation equipment used for the extraction of Citrus spp. leaf essential oils. Table S1. Secondary compounds identified in different Citrus spp. leaf oils using GC-MS. Table S2. Yields of four Citrus spp. leaf essential oils.