Effective antioxidant, antimicrobial and anticancer activities of essential oils of horticultural aromatic crops in northern Egypt

Background Identifying ornamental plants as new natural antioxidant and antimicrobial sources is always of great importance for the ornamental and horticultural industries. Methods The antimicrobial activities of leaves and fruits peel essential oils of twelve ornamental and horticultural crops were determined by screening against wide spectrum of fungi and bacteria, and their respective in vitro antioxidant capacity was evaluated. Furthermore, the anticancer activities against several cancer cells, and one normal human cell line (HEK-293) were examined. Results Origanum vulgare L. essential oil showed the best antioxidant, antibacterial and anticancer activities compared to screened crops by means of the DPPH and linoleic acid assays for antioxidants, MIC and MBC values for antibacterial activities and IC50 for antiproliferative activities. Such important activities in O. vulgare was attributed to high pulegone ratio (77.45%) as revealed by the GC/MS assay. Rosmarinus officinallis L. essential oil showed the highest antifungal activities by means of lowest MIC and MFC values which might be attributed to 1, 8-cineole (19.60%), camphor (17.01%) and α-pinene (15.12%). Conclusion We suggest that oxygenated monoterpenes (i.e. linalool, terpinen-4-ol and pulegone) and monoterpene hydrocarbons play an important role in the essential oil antioxidant, antibacterial, antifungal and anticancer activities of diverse Egyptian ornamental and horticultural crops. Some species showed bioactivities similar to standards compounds and might be suitable for pharmaceutical and food industries.


Background
In an attempt to preserve human health and avoid food losses in the face of global growing food insecurity, synthetic preservatives were introduced in the food industries [1,2]. Unfortunately, some of these preservatives were reported to cause undesirable health side effects to humans [1,2]. For example, lipid peroxidation in stored food may lead to rancidity and reduction of food quality [3][4][5].
The consumption of spoiled food can cause a wide spectrum of human diseases. Furthermore, the development of resistant microorganisms to synthetic preservatives is another threat facing the continuous use of these chemicals.
Essential oils might be used as anticancer agents as found before in Pinus koraiensis against HCT116 colorectal cancer cells [28], Myracrodruon urundeuva against HeLa, HEK-293, and Vero E6 cells [29] and Yemeni medicinal plants against A-427, 5637 and MCF-7 cancer cells [30]. Such activities might be attributed to major essential oil constitutes including cineol, capillin and others [31,32]. There are dozens of unstudied medicinal horticultural crops in Egypt with certain traditional uses such as skin, face and hair treatments [33] and urological, gastrointestinal, respiratory, neurological, cardiological and immunological diseases control [34]. This great diversity of alternative medicine application of essential oils and leaf extracts might be of pharmaceutical importance that need to be explored.
The study investigated the antioxidant and antimicrobial activities of essential oils obtained from important horticultural and economic ornamental medicinal plants. We also associate the main components of the essential oil with specific antioxidant and antimicrobial properties found to explore novel crop additive values.

Plant materials
The leaves or fruit peels of twelve plant species were collected from Alexandria, Behera and Matrouh in northern Egypt in the spring and summer of 2016. The leaves were obtained from mature non flowering herbal plants of Rosmarinus officinalis L., Artemisia judaica L., A. monosperma, Origanum vulgare L. and Pelargonium graveolens L'Her. Also the mature leaves of ornamental non trees of Rosmarinus officinalis Callistemon viminalis G. Don, Cupressus macrocarpa Gordon , Schinus molle L., and Thuja occidentalis L. The fruit peels were obtained from mature fruits of Citrus aurantifolia Swingle, C. limon (L.) and C. paradisi. The plant samples were collected during the months of April to August, 2016. Plants were identified by Prof. Samir Abdelgaleil and Assoc. Prof. Hosam Elansary. Specimens were vouchered in Department of Pesticide Chemistry and Technology, Alexandria University.

Essential oils analysis
The leaves were dried for five days (26 ± 1°C) while the fruit peels were used fresh. The essential oils were obtained by hydrodistillation in a Clevenger-type apparatus for 1 h. Gas chromatography/mass spectrometry (Hewlett Packard 5890) apparatus detected the the essential oil constitutes after dilution in diethyl ether. Gas chromatography column properties and injection conditions matches those described [3]. Oil compositions were identified by comparing retention time and indices of identified constitutes to n-alkanes (C 10 -C 36 , Sigma-Aldrich, Cairo) subjected to the same conditions. NIST Ver. 2.0 and Wiley libraries were used as well for the identificaton of the compounds.

Antioxidants
The antioxidant activity of the essential oils was estimated using the DPPH [35] and the β-carotene-linoleic acid methods [35]. Positive control (BHT) and negative control were treated as samples and a calibration curve was obtained. The amount of the samples that inhibited 50% of each antioxidant solution (IC 50 ) were considered as the antioxidant activities and experiments were repeated twice.

Antifungal activity assay
The antifungal activities of the essential oils were estimated using the microdilution method [36]. The minimum inhibitory concentration (MIC) was the lowest concentration inhibiting the fungal growth using a binocular. The minimum fungicidal concentration (MFC) was estimated by serial sub-cultivations (0.1-4.0 mg/ mL). The MFC was determined as the concentration causing no visible growth and killing 99.5% of the original inoculum. Fluconazole (FLZ) and ketoconazole (KLZ) were used as positive control and experiments were repeated twice.

Antibacterial activity assay
The antibacterial activities were determined by the micro-dilution method [36]. To determine the minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations the MIC was considered as the lowest concentrations causing no growth using the binocular. The MBC was quantified using serial sub-cultivation of each bacterium (0.1-2.0 mg/mL) into 100 μL of TSB and incubated for one day. MBC was the lowest concentration showed no growth and killed 99.5% of the original inoculum. Experiments were performed twice and negative control (5% DMSO) as well as positive (streptomycin and ampicillin, 0.01-10 mg/mL) were used.

Antiproliferative activity assay
Essential oils antiproliferative activities against MCF-7, HeLa, Jurkat, HT-29,T24 and HEK-293 followed the MTT method [37] with modifications. Five doses of leaf extracts were used to reach a final concentration of 50, 100, 200, 300, and 400 μg/mL culture media. Negative controls and positive (vinblastine sulfate and taxol) were used.
Furthermore, IC 50 values were obtained by plotting percentage of cell viability against extract concentration and expressed in μg/ml.
The results of chemical analysis indicated that components including limonene, α-pinene were found in more than one species, while others were species specific. Essential oils major constitutes could be divided into three groups including oxygenated monoterpenes (i.e. linalool and pulegone); monoterpene hydrocarbons (i.e. limonene and sabinene) and sesquiterpene hydrocarbons (i.e., σ-cadinene and σ-selinene).

Antioxidant activities
The essential oil of O. vulgare showed significantly the highest antioxidant activities with IC 50 values of 2.8 and 1.1 mg/L in the DPPH and linoleic acid assays, respectively compared to other essential oils ( Table 2). The oils of A. judaica, A. monosperma and C. viminalis followed O. vulgare oil in their antioxidant activities and their IC 50 values ranged between 4.7-5.3 and 2.7-3.3 mg/L in the DPPH and linoleic acid assays, respectively. The oil of S. molle showed the lowest antioxidant activity among all essential oils examined in the study. In addition, the oils of C. limon, C. macrocarpa, P. graveolens and R. officinallis showed no significant differences regarding their antioxidant activities.

Antifungal activities
The activities of the essential oils were expressed as MIC (Table 3) and MFC (Table 4). In general, all examined oils showed antifungal activities against A. flavus, A.

The antibacterial activities
There were large differences regarding essential oils antibacterial activities by means of MIC (Table 5) and MBC ( Table 6). The oils of O. vulgare, C. macrocarpa and C. paradisi showed the highest antibacterial activities with MIC values ranged between 0.11-0.76 mg/mL against examined bacteria. O. vulgare, C. macrocarpa and C. paradise were the three most active essential oils, the MBC was in the range of 0.21 to 1.59. The most resistant bacterium in this case of D. solani and L. monocytogenes. In general, the highest MIC values were recorded for the essential oils of A. monosperma, C. lemon, R. officinallis and S. molle. Most essential oils showed slightly higher MIC values compared to antibiotics, however, O. vulgare showed comparable values to antibiotics in some cases. The antibacterial activities were higher than antibiotics.

Anticancer activities
There were variations in essential oils anticancer activities against selected cancer cell lines as shown in Table 7. The inhibition (expressed as IC 50 ) of different types of cancer cells proliferation ranged between 8.1 and ˃300 μg/ml. The highest antiproliferation activities were found in O. vulgare, Citrus sp. and A. monosperma against MCF-7, HeLa, Jurkat, HT-29 and T24 cancer cells. The lowest antiproliferative activities were found in the essential oils of P. graveolens. No inhibition activity was found in any oil against HEK-293 (kidney epithelial).

Discussion
Essential oils showed high variation in term of composition even among closely related species (i.e. species of the same genera), which is in agreement with previous reports on several plants [39][40][41]. All essential oils examined showed antioxidant capacities; however, O. vulgare showed the highest antioxidant activities. This might be attributed to specific essential oil such as the pulegone (77.45%). The pulegone is a known compound found in the family Lamiaceae and has been associated with high antioxidant activities [42][43][44]. Although previous studies reported that pulegone is the major oil component of O. vulgare [44], environmental conditions of the plant may alter the chemical composition of the oil [41,43] leading to the variation of chemical composition that we found among closely related species. Similarly, many other species showed high antioxidant activities (e.g. A. judaica, A. monosperma and C. viminalis). cajor components and their synergetic effects. β α. Specifically, we found a high ratio of β -thujone (49.83%) and α-thujone (8.21%) in A. Judaica, supporting the previous report in Artemisia sieberi Besser in Italy and Iran [45]. The essential oil ratio found in Italy and Iran [45] is slightly higher than that found in our study. However, A. monosperma showed high ratios of α-pinene and terpinen-4-ol which are known to have noticeable antioxidant activities [46]. The high antioxidant activities that we found in relation with the presence of βthujone may indicate that this compound may play a major role in the antioxidant activities of different Artemisia species.  The essential oil in R. officinallis showed the highest antifungal activities by means of lowest MIC and MFC values which might be attributed to 1,8-cineole (19.60%), camphor (17.01%) and α-pinene (15.12%) as the major oil constituents as previously reported [47]. It was reported that 1,8-cineole, terpinen-4-ol and other compounds showed strong in-vitro antifungal activities against some plant pathogenic fungi including Fusarium cerealis, Aspergillus tubingensis and A. carbonarius [48]. In the current study, we report strong antifungal activities of the 1,8-cineole-rich R. officinallis against plant and human pathogenic fungi including A. flavus, A. ochraceus, A. niger, P. funiculosum, P. ochrochloron and C. albicans. The oil of O. vulgare followed R. officinallis in term of level of antifungal activities, and such interesting bioactivities of the essential oil might be attributed to the presence of pulegone (77.45%) and the menthone (4.86%).
In the current study, we document the highest antibacterial activity for the O. vulgare essential oil by means of MIC and MBC values compared to other species, potentially because of the high pulegone ratio. Previous investigations reported strong antibacterial activities of essential oils of the pulegone-rich Mentha longifolia L. and pulegone-rich O. vulgare against different bacteria such as Pantoea agglumerans and human pathogenic Acinetobacter baumannii, respectively [43,44]. The high antifungal as well as antibacterial activities of pulegone rich essential oils suggest that this compound might have a wide spectrum of antifungal and antibacterial activities. The oils of C. macrocarpa and C. paradise followed O. vulgare in their level of antibacterial activities, which might be attributed to terpinen-4-ol and  sabinene in C. macrocarpa as well as limonene and linalool in C. paradise. A strong antibacterial activity of five different terpenoids was reported [49] against Campylobacter spp. In addition, it was previously reported that the monoterpene terpinen-4-ol enhanced the antibacterial performance of Melaleuca alternifolia essential oils against S. aureus and E. coli [50]. In the same trend, limonene is the major component of citrus essential oils in most species and was proven to have specific bacterial inactivation mechanism in E. coli and others [51] thus justifying the noticeable antibacterial activities of C. paradisi. However, other species studied here (C. limon and C. aurantifolia) showed slightly lower antibacterial activities which could be partially explained by lower ratios of limonene and synergetic effects with other oil constituents such as the linalool in C. paradisi. Some essential oils in R. officinallis and O. vulgare showed comparable antibacterial and antifungal activities to antibiotics in some cases, which may indicate that these essential oils might be useful sources of natural products  for commercial applications in the pharmaceutical and food industries. Several studies indicated that Gram-positive bacteria are less resistant than negative ones against natural products such as essential oils [52]. This resistance had been attributed to the additional outer membrane (this is likely the case in our study) and that most resistant bacterium was D. solani. Finally, the essential oils compositions and percentages obtained from this study are comparable to studies on geranium [53], citrus [54], C. viminalis [55], R. officinallis [47], Artimisia [45], and O. vulgare [44]. However, the essential oils composition of the current collection of horticultural crops differed in some cases from those reported in the literature for S. molle (mainly composed of pinene [56], T. occidentalis (mainly composed of thujone and fenchone [57] and O. vulgare (mainly composed of pulegone). Interestingly, other report on S. molle found that the essential oil might be composed of cubenol (27.1%) and caryophyllene oxide (15.3%) [58]. Indeed, a great diversity is known in essential oil plants due to environmental as well as genetic factors.
Few species showed promising anticancer activities against the proliferation of cancer cells. The main effect of the essential oils is attributed to main constitutes such as the capillin in A. monosperama, limonene in the   [64] reported that α-Phellandrene found in Schinus molle influenced cell cycle and apoptosis in murine leukemia WEHI-3 cells. Chen et al. [65,66] found that α-pinene has antitumor activities through inducing cell cycle arrest. We aimed to highlight the biological activities in ornamental and horticultural aromatic plants against pathogenic microbes in the Mediterranean region. We demonstrated their antioxidant activities by two different methods and their promising role as natural antioxidant resources. One of the important factors influencing the decision of the farmer to grow specific horticultural crop is the availability of different ways of marketing such as the use of the whole crop as a fresh crop or the use of the essential oil for the pharmaceutical industries (Table  8). Here the bioactivity that we found for these crops is an added value that may encourage farmers to grow these crops and increase the marketing possibilities of their end crops. On the medical level, the pharmaceutical values of those medical crops are promising for pathogenic diseases and human cancer control.

Conclusion
O. vulgarehad the best antioxidant and antibacterial activities with a high and unique pulegone ratio (77.45%). The essential oil of R. officinallis essential oil showed the highest antifungal activities by means of lowest MIC and MFC values which might be attributed to 1,8-cineole, camphor and α-pinene. The essential oils of O. vulgare, Citrus sp. and A. monosperma showed the highest antiproliferation activities against different cancer cells. Oxygenated monoterpenes (i.e. linalool and pulegone) as well as monoterpene hydrocarbons including pinenes plays a pivotal role in the antioxidant, antibacterial, antifungal and anticancer activities of the essential oils of different horticultural aromatic plants. Some species showed antioxidant and antimicrobial activities comparable to Artemisia monosperma Delile Leaves and flowers Spice, soft drink and cosmetic uses [37] Callistemon viminalis weeping bottlebrush Leaves and flowers Ornamental and source of antioxidants, antifungal and antibacterial products [47] Citrus aurantifolia Key lime Fruits and leaves The fruits are eaten and the essential oils are extracted from the fruit coat. The leaves are used for medicinal puposes [46,58] Citrus limon lemon Fruits and leaves The fruits are eaten and the essential oils are extracted from the fruit coat. The leaves are used for medicinal puposes [46,58] Citrus paradisi grapefruit Fruits and leaves The fruits are eaten and the essential oils are extracted from the fruit coat. The leaves are used for medicinal puposes [46,58] Cupressus macrocarpa Monterey cypress Stem, leaves and cones Ornamental and source of antioxidants, antifungal and antibacterial products [38] Origanum vulgare wild marjoram Leaves and flowers Spice, soft drink and cosmetic uses [25] Pelargonium graveolens Geranium Leaves and flowers Spice, soft drink and cosmetic uses [45] Rosmarinus officinalis Rosemary Leaves and flowers Spice, soft drink and cosmetic uses [25] Schinus molle American pepper Leaves, flowers and fruits Ornamental and source of antioxidants, antifungal and antibacterial products [48] Thuja occidentalis northern white-cedar Stem, leaves and cones Ornamental and source of antioxidants, antifungal and antibacterial products [49] standard compounds, which may indicate that these crops are valuable resources of natural compounds useful for pharmaceutical and food industries.