GC/MS Analyses of the Essential Oils Obtained from Different Jatropha Species, Their Discrimination Using Chemometric Analysis and Assessment of Their Antibacterial and Anti-Biofilm Activities

The essential oils of Jatropha intigrimma, J. roseae and J. gossypifolia (Euphorbiaceae) were analyzed employing GC/MS (Gas Chromatography coupled with Mass Spectrometry) analyses. A total of 95 volatile constituents were identified from J. intigrimma, J. gossypifolia and J. roseae essential oils, accounting for 91.61, 90.12, and 86.24%, respectively. Chemometric analysis using principal component analysis (PCA) based on the obtained GC data revealed the formation of three discriminant clusters due to the placement of the three Jatropha species in three different quadrants, highlighting the dissimilarity between them. Heneicosane, phytol, nonacosane, silphiperfol-6-ene, copaborneol, hexatriacontane, octadecamethyl-cyclononasiloxane, 9,12,15-Octadecatrienoic acid, methyl ester and methyl linoleate constitute the key markers for their differentiation. In vitro antibacterial activities of the essential oils were investigated at doses of 10 mg/mL against the Gram-negative anaerobe Escherichia coli using the agar well diffusion method and broth microdilution test. J. gossypifolia essential oil showed the most potent antimicrobial activity, demonstrating the largest inhibition zone (11.90 mm) and the least minimum inhibitory concentration (2.50 mg/mL), followed by the essential oil of J. intigrimma. The essential oils were evaluated for their anti-adhesion properties against the Gram-negative E. coli biofilm using a modified method of biofilm inhibition spectrophotometric assay. J. intigrimma essential oil showed the most potent biofilm inhibitory activity, demonstrating the least minimum biofilm inhibitory concentration (MBIC) of 31.25 µg/mL. In silico molecular docking performed within the active center of E. coli adhesion protein FimH showed that heneicosane, followed by cubebol and methyl linoleate, displayed the best fitting score. Thus, it can be concluded that the essential oils of J. gossypifolia and J. intigrimma leaves represent promising sources for antibacterial drugs with antibiofilm potential.


Introduction
Essential oils are natural, volatile components with a complex nature, possessing mostly fragrant odors manufactured by the plants as secondary metabolites. They are commonly prepared either by hydro-distillation or steam distillation; meanwhile, they are highly popular due to their observable biological potential, which is highly attributed to their different classes of compounds, particularly terpenoids. They are popular for their antimicrobial, antiviral, anti-inflammatory, analgesic, spasmolytic, anticancer, anti-aging and the literature retention indices, in an order of increasing retention indices (RIs) on the Rtx-5MS column, are summarized in Table 1.  Table 1. Essential oil compositions obtained from J. intigrimma, J. gossypifolia and J. roseae leaves using the Rtx-5MS column.

Discrimination of the Three Jatropha Species Using GC Data Coupled with Chemometrics
Chemometric analysis was adopted using an unsupervised pattern recognition technique represented by principal component analysis (PCA) based on the obtained GC data. Chemometric analysis constitutes an advanced approach for the better discrimination of closely related species, relying upon data gathered from different chromatographic and spectroscopic techniques. Principal component analysis (PCA) was initially performed to categorize data and to correlate between the examined samples and the used variables [13]. PCA based upon the number as well as the relative peak area of volatile constituents obtained from GC spectra for different Jatropha species, illustrated in Figure 3, revealed the formation of three discriminant clusters representing the three species.
PCA score plot for principal components (PCs), which are PC1 versus PC2, illustrated in Figure 3A accounts for 71% and 29% of the total variance, respectively. This perfectly results in the placement of the three Jatropha species in three different quadrants, which in turn highlights the evident dissimilarity between the three species. Both PC1 and PC2 effectively discriminate between J. gossypifolia and J. roseae, where the former lies in the left lower quadrant showing negative values for both PCs in contrast to the latter that is positioned in the upper right quadrant revealing positive values for both PCs. Regarding J. intigrimma that lies in the right lower quadrant in the PCA score plot, only PC1 could effectively discriminate between it and J. gossypifolia, as J. intigrimma showed positive values, while J. gossypifolia showed negative values for PC1. However, J. intigrimma and J. roseae could be discriminated only via PC2, where the former displayed negative values and the latter showed positive values. By careful analysis of the loading plot illustrated in Figure 3B, it was clearly obvious that heneicosane, phytol, nonacosane and silphiperfol-6-ene were the key markers for the discrimination of J. roseae from the other two species, while copaborneol constitutes the key marker for its differentiation from the other two species. Regarding J. intigrimma, hexatriacontane, octadecamethyl-cyclononasiloxane, 9,12,15-Octadecatrienoic acid, methyl ester and methyl linoleate represent the key markers. The results from chemometric analysis coupled with GC data allowed the clustering of samples, and this undoubtedly leads to better visualization of the differences among the essential oils obtained from different Jatropha species and in turn reflected the differences between their biological behaviors.   In vitro antibacterial activities of the essential oils obtained from the leaves of the three Jatropha species, J. intigrimma, J. gossypifolia and J. roseae, were investigated at doses of 10 mg/mL against the Gram-negative anaerobe Escherichia coli. The agar well diffusion method was adopted for calculating the mean diameter of inhibition zones produced by the three oil samples in comparison with the standard antimicrobial drug, Gentamicin. Furthermore, the minimum inhibition concentrations (MIC) values were estimated for the oil samples using the broth microdilution test. The essential oil obtained from J. gossypifolia leaves demonstrated the most potent antimicrobial activity against E. coli, demonstrating the largest inhibition zone (11.90 ± 0.46 mm) and the least minimum inhibitory concentration (2.50 mg/mL), followed by the essential oil obtained from J. intigrimma leaves. The latter exhibited an inhibition zone of 9.57 ± 0.40 mm and an MIC of 5.00 mg/mL. The oil of J. roseae demonstrated the least inhibition zone (8.93 ± 0.60 mm) but had MIC values equal to those of J. intigrimma oil of 5.00 mg/mL. The results were compared to the standard gentamycin that exhibited a mean inhibition zone of 27.09 ± 0.01 mm at a dose of 4 µg/mL and demonstrated an MIC of 2 µg/mL. This experiment was repeated in triplicate and data were represented as mean ± S.D.
2.3.2. Antibiofilm Activities of J. intigrimma, J. gossypifolia and J. roseae Essential Oils The essential oils of the different Jatropha species were further evaluated for their potential anti-adhesion properties against the Gram-negative E. coli biofilm. A modified method of biofilm inhibition spectrophotometric assay was adopted for the determination of the inhibitory activity of essential oils against the formation of E. coli biofilm and for the calculation of the minimum concentration required for complete inhibition of visible biofilm cell growth (MBIC). The essential oil of J. intigrimma demonstrated the most potent biofilm inhibitory activity, demonstrating the least minimum biofilm inhibitory concentration (MBIC) of 31.25 µg/mL. However, the essential oils of J. roseae and J. gossypifolia demonstrated less potent antibiofilm activities, demonstrating minimum biofilm inhibitory activities (MBIC) of 250 and above 1000 µg/mL, respectively, as displayed in Table 2. Data are presented as means ± S.D. n = 3.

Molecular Docking Studies of Adhesion Proteins with Major Constituents in Jatropha Essential Oils
In silico molecular docking of the major compounds identified from Jatropha essential oils was performed within the active site of the adhesion proteins associated with E. coli that enable the bacterium to attach to the surfaces and consequently form its invasive biofilm as FimH (PDB ID 1TR7; 2.10 Å) downloaded from the protein data bank. The docking experiments were carried out using Discovery Studio 4.5 (Accelrys Inc., San Diego, CA, USA) using the C-Docker protocol. The results displayed in Table 3 revealed that heneicosane, followed by cubebol and methyl linoleate, displayed the best fitting score within the active center of E. coli adhesion protein FimH with free binding energies equal to −30.68, −8.92 and −4.55 Kcal/mole, respectively. Heneicosane forms two alkyl and π-alkyl bonds with Ile52 and Tyr48, in addition to the formation of Van der Waals interactions with many amino acid residues at the active center ( Figure 4A). However, cubebol forms one conventional H-bond with Asp140, in addition to two alkyl and π-alkyl bonds with Ile13 and Phe142 together with Van der Waals bonds at the active site ( Figure 4B). Regarding methyl linoleate, it forms two conventional H-bonds with Asn135 and Phe1, one alkyl bond with Ile52, in addition to three C-H bonds with Asp54 and Asn46, together with many Van der Waals interactions, as shown in Figure 4C.

Discussion
This study represents the first report investigating the volatile constituents of J. rosea leaf oil. In addition, a comparative investigation of the volatile constituents of the three Jatropha species, J. intigrimma, J. gossypifolia and J. roseae, was conducted. A previous study on J. intigrimma leaves obtained from Nigeria reported β-ionone as one of the major volatile constituents of the oil, which was also identified in our study in a lower concentration. However, the other major constituents reported by Eshilokun et al., pentadecanal and 1,8-cineole, were absent in the present study [12]. Previous literature by Aboaba et al. on J. gossypifolia leaves grown in Nigeria reported the predominance of sesquiterpenes accounting for 74.3% of the oil content, which is almost relative to our study [14]. Meanwhile, fatty acids were reported by Ababa et al. in J. gossypifolia oil despite their scarcity in our J. gossypifolia oil. The major constituents reported by Aboaba et al., germacrene and hexahydrofarnesyl acetone, were identified in the present study but in negligible quantities. Another study in different regions of Nigeria [11] reported the predominance of phytol (33.4%) and linalool (9.81%) in J. gossypifolia oil, which were identified in the current study in different percentages.
The chemical composition variability among the essential oils of Jatropha species grown in Egypt and those in different regions of Nigeria or elsewhere are attributable to multiple exogenous and endogenous factors, such as seasonal variation, geographical region affecting soil, precipitation and light exposure, extraction method, and the age of the plant and its different chemotypes [15]. Additionally, chemometric analysis was adopted using an unsupervised pattern recognition technique represented by principal component analysis (PCA) based on the obtained GC data. PCA based upon the number, as well as the relative peak area, of volatile constituents obtained from GC spectra for different Jatropha species revealed the formation of three discriminant clusters due to the placement of the three Jatropha species in three different quadrants, which in turn highlights the evident dissimilarity between the three species evidenced by the score plot. By careful analysis of the loading plot, it was obvious that heneicosane, phytol, nonacosane and silphiperfol-6-ene, copaborneol, hexatriacontane, octadecamethyl-cyclononasiloxane, 9,12,15-Octadecatrienoic acid, methyl ester and methyl linoleate constituted the key markers for the differentiation of the three species.
Essential oils have long been known for their antimicrobial potential that made them crucial in different fields, including the food industry, preservation and medication [16]. To the best of our knowledge, few reports have addressed the antimicrobial activities of essential oils of different Jatropha species. Our current study presented the first report on the antimicrobial activities of J. intigrimma and J. roseae essential oils against E. coli Gram-negative bacterium and compared them with the activity of J. gossypifolia essential oil. The results of the in vitro antibacterial activities of the essential oils exhibited the superior potency of J. gossypifolia essential oil, followed by J. intigrimma essential oil, against the Gram-negative anaerobe E. coli. Results were in accordance with the previous literature reporting the bacteriostatic activity of J. gossypifolia leaf oil against the Gram-negative bacterium E. coli at a dose of 0.10 mg/mL [11]. It is worth mentioning that phytol, a major constituent of J. gossypifolia leaf oil constituting 10.33 % of the oil content, was previously reported for its potent antimicrobial activity against Escherichia coli, exhibiting growth inhibition at a minimum concentration (MIC) of 62.5 µg/mL [17,18]. Moreover, caryophylline oxide, identified in J. gossypifolia leaf oil, was reported to possess moderate inhibitory activity against Gram-negative E. coli with an estimated MIC of 60 ppm [19].
Bacterial biofilms are colonies of microorganisms lying in a matrix of polysaccharides attached to surfaces. They represent physical barriers that inhibit the penetration of antimicrobials to their target sites. Hence, bacterial biofilms represent rational biological risks in drinking water, food, and clinical and industrial environments [20]. Nowadays, increased interest has been directed toward investigating the different mechanisms of inhibiting bacterial biofilm formation and growth. Because attachment represents the initial step in almost all types of biofilm formation, the antiadhesive properties of natural products have recently become a prime interest of study in an aim for the early prevention of microbial biofilm and inhibiting the formation of micro colonies [21]. Furthermore, inhibiting the cell attachment of microbial biofilms has been found to be more readily achieved than preventing the growth of already established biofilms [22]. Hence, the three Jatropha oils were investigated for their inhibitory activities against E. coli adhesion. This study represents the first report of the antibiofilm activities of the essential oils of the three Jatropha species. The results demonstrated the superior antibiofilm activity of the essential oil of J. intigrimma leaves compared to the other two Jatropha oils. In addition, in silico molecular docking of the major compounds identified from Jatropha essential oils was performed within the active site of one of the adhesion proteins associated with E. coli that enables the bacterium to attach to the surfaces and consequently forms its invasive biofilm, FimH. The docking experiments revealed that heneicosane, followed by cubebol and methyl linoleate, displayed the best fitting score within the active center of E. coli adhesion protein FimH with free binding energies equal to −30.68, −8.92 and −4.55 Kcal/mole, respectively.
Previous literature has reported the anti-biofilm activities of different fatty acids and their methyl esters [23,24]. In addition, the monoterpene D-Limonene, an essential component of J. intigrimma essential oil, has been reported to possess strong anti-biofilm activity against the heterotrophic, Gram-negative, rod-shaped bacterium Aeromonas hydrophila isolated from fish [25]. The anti-adhesive properties and the capabilities of inhibiting the initial biofilm formation can be explained by the possibility of interference with the attraction forces that support the bacterial film with the affected surface or by interrupting the access of vital nutrients for bacterial growth and adhesion [26]. Thus, the antibiofilm of the essential oil could be attributed to the synergistic action of the existing compounds.

Plant Material
The fresh leaves of the three Jatropha species: J. intigrimma Jacq., J. gossypifolia L. and J. roseae

Chemicals, Reagents and Strains
Dimethyl sulfoxide (DMSO), crystal violet and trypan blue dye were obtained from Sigma (St. Louis, MO, USA). Crystal violet stain was prepared as 1% using 0.5% (w/v) crystal violet and 50% methanol that were adjusted to volume using distilled water and subsequently filtered through a Whatman No.1 filter paper. The studied bacterial strain was Escherichia coli, ATCC 25922, obtained from the American type culture collection (ATCC).

Preparation of Essential Oils
The fresh leaves of the three Jatropha species were separately hydro-distillated for 6 h utilizing a Clevenger-type apparatus. The obtained essential oils were dried over anhydrous sodium sulfate and gathered in separate and sealed vials that were maintained at −30 • C until further analyses. The yield was calculated as % v/w after being determined in triplicate, where calculation was performed based on the initial plant weight. and attached to a Shimadzu mass spectrometer. An initial set of temperature of the column at 50 • C for 3 min was done that was gradually elevated from 50 • C to 300 • C at a rate of 5 • C/min, followed by isothermal maintenance for 10 min at 300 • C. The injector temperature was maintained at 280 • C, while the interface and the ion source temperature were kept at 220 and 280 • C, respectively. The flow rate of helium, which was used as a carrier gas, was 1.37 mL/min. One microliter was injected from the diluted sample with a concentration of 1% v/v through a split mode using a split ratio of 15:1. The mass spectrum was recorded using an EI mode of 70 eV in the range of m/z 35 to 500. Compound quantitation relied upon the normalization method, taking the reading of three chromatographic runs. Identification of compounds was achieved depending on the retention indices of the detected compounds with regard to a homologous series of n-alkanes (C8-C28) that were injected under the same conditions and via comparison mass spectra of the detected compounds with those recorded in the Wiley library database as well as the National Institute of Standards and Technology (NIST) and together with the literature [1,[27][28][29].

Discrimination of the Three Jatropha Species Using GC Data Coupled with Chemometrics
Chemometric analysis using principal component analysis (PCA) as an unsupervised pattern recognition technique was done based on the obtained GC using CAMO's Unscrambler ® X 10.4 software (Computer-Aided Modeling, As, Norway) as previously described [3,5]. This was done in an effort to allow the clustering of samples, which undoubtedly leads to better visualization of the differences among the essential oils obtained from different Jatropha species. 4.6. Evaluation of Antibacterial and Anti-Biofilm Activity 4.6.1. Susceptibility Test Using the Agar Well Diffusion Method Susceptibility tests were performed according to NCCLS recommendations (National Committee for Clinical Laboratory Standards) [30]. Screening tests concerning the inhibition zone were performed employing the well diffusion assay previously conducted by Hindler et al. [31]. Preparation of the inoculum suspension was performed from cultures grown overnight on an agar plate that were concomitantly inoculated into Mueller-Hinton broth. A sterile swab was adopted for the inoculation of Mueller-Hinton agar plates (fungi using malt agar plates) after being immersed in the inoculum suspension. The examined samples at different concentrations (2.5, 5 and 10 mg/mL) were solubilized in dimethyl sulfoxide (DMSO) and the inhibition zones were determined around each well after 24 h at 37 • C where the control was prepared using DMSO.

Determination of the Minimum Inhibitory Concentration (MIC) Using the Broth Microdilution Method
The minimum inhibitory concentration (MIC) was determined as previously recommended by the Clinical and Laboratory Standards Institute (CLSI). Briefly, the dilution of a stock solution composed of 10% of the examined oil in the brain heart infusion broth (BHI) in two-fold serial dilutions was performed to obtain 0.02 to 25 mg/mL concentrations at a total volume of 100 mL per well in 96-well microtiter plates. One-hundred milliliters of each tested strain adopting a concentration of 1 × 106 CFU/mL were added to each well, followed by their incubation at 37 • C in appropriate conditions. The medium was used as the non-treated control, while 10% DMSO was employed as the negative control, whereas 0.1% (w/v) CHX was the positive control. MIC is the lowest concentration that completely prohibited growth when compared to the non-treated control. All experiments were repeated three times in duplicate.

Evaluation of Anti-Biofilm Activity
The volatile oil samples obtained from hydro-distillation of the three Jatropha species were evaluated for their inhibitory activity against biofilm formation of the Gram-negative anaerobe Escherichia coli. Biofilm inhibition assay was performed in 96-well plates adopting the modified method of biofilm inhibition spectrophotometric assay [32]. Briefly, 100 µL of an Escherichia coli cell suspension was added to a 96-well titer plate together with different concentrations of samples (1000, 500, 250, 125, 62.5, 31.25, 15.63 and 7.81 µg/mL); in addition, DMSO was added and incubated for 24 h at 37 • C. After incubation, the liquid suspension was removed, and 100 µL of 1% w/v aqueous solution of crystal violet was added. Removal of the excess crystal violet was achieved after 30 min of staining at room temperature followed by washing the wells thoroughly and the addition of 95% ethanol and incubation for 15 min. The reaction mixture was read spectrophotometrically at a wavelength of 570 nm using a microplate reader (TECAN, Inc.) after being shaken gently. The percent of inhibition of biofilm formation was determined according to the following equation: % inhibition = OD in control − OD in treatment × 100 OD in control.
The relation between biofilm formation inhibitory % and drug concentration is plotted to obtain the inhibitory curve after treatment with the specified compound. MBIC was the concentration required to completely inhibit biofilm formation.

Molecular Docking Studies of Adhesion Proteins with Major Constituents in Jatropha Essential Oils
Molecular docking analysis was performed on the major constituents existing in Jatropha essential oils regarding adhesion proteins associated with E. coli that enable the bacterium to attach to the surfaces and consequently form its invasive biofilm as FimH (PDB ID 1TR7; 2.10 Å) [33]. This protein was downloaded from the protein data bank and docking experiments were carried out using Discovery Studio 4.5 (Accelrys Inc., San Diego, CA, USA) using the C-Docker protocol as previously reported [5,[34][35][36], where binding energies (∆G) were calculated from the following equation: ∆G binding = E complex − (E protein + E ligand ) Where; ∆G binding : The ligand-protein interaction binding energy, E complex : The potential energy for the complex of protein bound with the ligand, E protein: The potential energy of protein alone and E ligand : The potential energy for the ligand alone.

Conclusions
In conclusion, the essential oil obtained from J. intigrimma, J. gossypifolia and J. roseae leaves revealed considerable variation, as revealed by GC analyses. This variation becomes clearly obvious when coupled with chemometric analysis that results in the placement of the three Jatropha species in three different quadrants, which in turn highlights the evident dissimilarity between the three species. Moreover, the essential oils of J. gossypifolia and J. intigrimma leaves represent promising sources for a new generation of antibacterial drugs. Their distinctive antibacterial and antibiofilm activities are probably attributed to their major bioactive chemical constituents, as well as the possible synergistic effect among them. To further confirm the obtained results, in silico molecular docking of the major compounds identified from Jatropha essential oils was performed within the active center of E. coli adhesion protein FimH and results showed that heneicosane followed by cubebol and methyl linoleate displayed the best fitting score. Thus, additional in vivo studies and bioavailability studies are highly recommended to ascertain the obtained results.