Chemical Composition Related to Antimicrobial Activity of Moroccan Nigella sativa L. Extracts and Isolated Fractions

Background Nigella sativa L. (NS) is an aromatic and medicinal plant commonly used in Mediterranean cuisine. Its grains contain a large amount of fixed oil and have many therapeutic virtues and medicinal properties (antioxidant, antidiabetic, antimicrobial, and anticancer). Aim The aim of this work is to study the antimicrobial activity of Nigella sativa L. extracts and separated fractions on various pathogenic strains and to correlate that with its chemical composition. Methods Extracts from Moroccan Nigella sativa seeds were extracted using successive organic solvents, and their hexane and acetone extracts were separated by column chromatography. The chemical composition of extracts, fractions, and essential oil was determined by GC-MS and HPLC-DAD. Extracts and fractions were evaluated for antimicrobial activity through disk diffusion against Gram-positive bacteria (Staphylococcus aureus, Bacillus cereus, and Listeria innocua), Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), and yeast (Candida pelliculosa) for 1 mg/mL concentration. Bacterial strains were followed to study their behaviors over time in different concentrations. The minimum inhibitory concentration of Nigella sativa essential oil was determined against Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Candida albicans. Results and Conclusion. Although hexane extract was active against both types of bacteria (Gram+ and Gram−), some of its fractions were specifically active against only one type. Fraction (SH4) had the highest activity (15 mm inhibitory diameter). Acetone extract was nonactive but surprisingly resulted in specific active fractions, and the most interesting one was (SA7) that had an inhibitory diameter of 13 mm. This antibacterial effect was related to fatty acids (linoleic and palmitic acids) in (SH4) and 17 pentatriacontene in (SA7). Moreover, the antifungal activity of hexane fractions (10–13 mm) was higher than hexane extract (8 mm), but for acetone, it was the opposite. Acetone extract had a higher activity (18 mm) than its fractions (8–12 mm), except for (SA7) (19 mm). Those inhibitions were attributed to gallic acid, cysteine, and apigenin in acetone extract and cysteine with ascorbic acid in fraction (SA7). Antifungal activity of the essential oil was more pronounced than the antibacterial one. Indeed, determined MICs in the first case were on the microgram scale (MIC = 8 μg/mL, Candida albicans), while in the second case, they were on the milligram scale (MIC = 0.96 mg/mL for Staphylococcus aureus, 0.5 mg/mL for Bacillus cereus, and 0.68 mg/mL for Escherichia coli). This antifungal activity was attributed to three major compounds beta-cymene, alpha-thujene, origanene, and thymoquinone. Results of strains behavior over time at different concentrations of the fractions showed all the curves went through a maximum around 20 hours and had a delay of expression of 5 hours at the start. Taking all results into count, Nigella sativa L. extracts and/or derived principles could form promising antimicrobial agents for therapeutical and industrial uses.


Introduction
Commonly called black cumin, Nigella sativa (NS) is a yearly cool temperature resistant plant in the Ranunculaceae family. NS seeds are used in traditional medicine and as a culinary spice throughout its range from the Mediterranean basin to India. In Morocco, the plant is cultivated in coastal areas in July and traditionally used to treat asthma, diabetes, and other inflammatory diseases [1]. Indeed, research has confirmed an abundance of secondary metabolites [2]. e seeds are gaining popularity for their medicinal and dietary uses, and many cooperatives view the plant as an exploitable source of oil as the argan tree [3]. In fact, Moroccan Nigella sativa produces high quality oil even when compared to other variants [4]. Consequently, large-scale cultivation is being considered, despite considerable variation in yield [5].
Many studies have explored the biological properties of NS [6][7][8]. e plant has antibacterial effects against both Gram-positive and Gram-negative bacteria [9]. In addition, the seeds have an important activity against multidrug resistant bacterial strains [10]. NS seeds also have antifungal properties because of thymoquinone [11].
Previous studies of NS focused mostly on seed extracts or essential oils [12,13]. e diversity of chemical compounds in the extracts makes attribution of antimicrobial activity to specific compounds difficult, especially when considering toxicity. Hepatic damage can occur with an aqueous extract [14] and metabolic changes with a lipid extract [15]. Consequently, the specific chemical(s) that imparts antimicrobial properties to NS must be separated and purified for exploitation.
In this study, we investigate the antimicrobial activity of Moroccan NS fractions separated from hexane and acetone extracts and the essential oil. e level of inhibition by the fractions was evaluated against Gram-positive and Gramnegative bacteria and fungi strains and the impact of the separation process was evaluated.

Plant and Material.
Nigella sativa seeds (NS) were purchased from a local market in Oujda, Morocco. Organic analytical grade np solvents (98.9%), cycloheximide, and tetracycline were supplied by Sigma-Aldrich. Silica gel was from Fluka. Mueller-Hinton medium (BIOKAR, France) and potato dextrose (BIOKAR, France) were used for microbial cultures of bacteria and yeast, respectively. e microbial strains used as targets were Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Listeria innocua, Candida pelliculosa, and Candida albicans.
2.2. Extractions. Nigella sativa (NS) seeds were cleaned and reduced by a blender into a powder of 5 μm. e powder was added to warm organic solvents (50°C) with a Soxhlet apparatus and a liquid-seed ratio of 7:1. Hexane and acetone were successively used for the extraction, and organic extracts obtained were concentrated under a vacuum rotatory at 40°C and conserved at a low temperature (−4°C). e essential oil was extracted by hydrodistillation using hexane extract produced at 50°C. Hexane extract was mixed with water and then distillated for three hours at 40°C. e essential oil was collected through Clevenger and stored at −4°C.

Separation of Extracts.
Hexane and acetone extracts were fractioned using column chromatography. Hexane extract was separated using a silica gel column with 20% hexane/80% dichloromethane eluents. Acetone extract was separated using silica gel column chromatography and a 50% cyclohexane/50% dichloromethane eluent system [7]. e separation of both extracts was at ambient temperature and pressure.  [17]. e injection volume of samples was 20 μl, and the column temperature was set to 25°C. e components were observed by diode array detector (DAD) at 280-365 nm. [17]. Sample chromatograms were used to compare standard referring to retention time and λ max. e chromatographic characterization was carried out twice and results were median.  [18]. Agar cultures were prepared with overnight bacterial cultures of 10 8 cells/mL. Extracts and fractions at 10 mg/mL were aseptically positioned on sterile filter paper disks (6 mm) and on Petri plates. Papers were treated with gentamicin at 40 mg/mL as a standard antibiotic against bacteria. Dimethyl sulfoxide (DMSO) was used as the negative control. Plates with extracts and fractions were maintained at ambient temperature for prediffusion for two hours, and then the disk plates were incubated at 37°C for 24 hours [19,20]. After incubation, inhibition diameters were measured in millimeters. e antibacterial activity of the essential oil of NS was determined when using Hayes and Markovic's microdilution method [21]. 400 μL of the essential oil was diluted in 1 mL DMSO. Serial concentration (500 μg/mL to 1000 μg/mL) dilutions were prepared and mixed with 2% of MHB (Mueller-Hinton Broth) medium and then inoculated with overnight culture (10 7 CFU/mL) of targets [22]. e cultures were then incubated at 37°C for 24 h. e minimum inhibitory concentration (MIC) was defined using absorbance at 625 nm, which refers to the turbidity caused by microorganism's growth.

Antibacterial
e MIC was obtained from samples showing total inhibition of bacterial growth. DMSO, used as negative control, could not inhibit bacterial strains [23].

Disk Diffusion Test.
e antifungal activity of extracts and fractions of NS was determined using Candida pelliculosa as a target, according to the Barros et al.'s [24] method. Sterile paper disks were positioned on potato dextrose agar (PDA) plates and 1% of strain suspension. Extracts and fractions at 10 μg/mL were prepared and incubated at 28°C five days. Cycloheximide 0.01% (w/v) (mg/ L) was the antifungal standard and the negative control was DMSO. Fungal activity results were evaluated for five days and were expressed by diameter of inhibition halo.

Microdilution Test: Determination of Antifungal MIC of Essential Oil.
e antifungal activity of NS essential oil was performed using the Chebaibi et al.'s microdilution method [25]. Serial concentrations (1 μg/mL to 32 μg/mL) samples were prepared and added to the Candida albicans suspension and 2% MHB. Samples were incubated at 28°C for 48 hours. e MIC was determined using the concentration of completely inhibited microorganism growth.

3.1.
Extraction. Nigella sativa (NS) extractions were successively performed by hexane, chloroform, ethyl acetate, and acetone organic solvents using Soxhlet apparatus at 50°C. e solvents were evaporated under vacuum rotary at 40°C. Hexane extract gave a yield of 34.2% and acetone extract was represented by 2.03%. Nigella sativa essential oil constituted 1.12% of hexane extract and 0.28% of NS seeds.

Separation of Hexane and Acetone Extracts.
Hexane and acetone extracts were fractioned according to our previous work [7]. Hexane extract gave eight fractions (SH (1-8)) and acetone extract specified eleven fractions (SA (1-11)). ose specified fractions were the results of successive separations of the extract tell having single compounds.

HPLC-DAD.
e identification of the acetone extract and its fractions revealed interesting compounds. Major compounds included polyphenol derivatives. Before the column chromatography, separation, the acetone extract contained gallic acid as the most abundant constituent. Components such as thymoquinone, apigenin, naringenin, ascorbic acid, cysteine, rutin, quercetin, and kaempferol were also found in acetone extract with different proportionalities. After the separation, the compounds were purified and were concentrated in the fractions (Table 4). (SA1) had exclusively gallic acid while (SA2) had gallic acid, thymoquinone, and apigenin. Fractions (SA3) and (SA4) had mostly catechol and naringenin, but (SA7) had cysteine and rutin while (SA11) had quercetin and histidine. Overall, Evidence-Based Complementary and Alternative Medicine acetone fractions were mostly well separated and presented one, two, or three compounds in each fraction.

Determination of Antibacterial Activity by Disk Diffusion
Assay.

Determination of Antibacterial Activity by Quantitative
Assay. Antibacterial activity of fractions (FH), (SH1), (SH2), (SA7), and (SA8) of Nigella sativa (NS) were selected for their inhibitory effect previously determined and on the quantity limit. e fractions were tested for their inhibition of biomass growth of pathogenic bacteria in MH Broth, and the results obtained are reported in Figure 3. ese results showed that the higher the concentration of the fraction, the greater the inhibition in biomass growth of the targets (E. coli, P. aeruginosa, and L. innocua).
Every curve was labelled at the end of its evolution. On the third step, E. coli was illustrated with the dispersion of the concentration curves (Figures 3(a), 3(c), 3(f), and 3(i)), while P. aeruginosa grouped and converged to a precise point (Figures 3(b), 3(d), 3(e), and 3(g)). L. innocua curves evolved in similar fashion (Figure 3(h)). e gap between the control and the fractions against E. coli widened over time. As time increased, the gap widened and activity increased. is traduces the positive evolution of activity on E. coli strain. For P. aeruginosa, the opposite trend was observed. All curves of both the lowest and highest activity converged to a precise point at an average time. In the case of Listeria strain, the activities of fractions were maintained constant for 45 minutes.
If we compare the evolution of the fractions' activities based on time, the optical density decreased when the concentrations of fractions increased for all bacterial strains. At the end of the third step (t � 45 min), optical density varied from two to three times for E. coli, two to five times for P. aeruginosa, and four times for Listeria. e antibacterial activity sequences were arranged as follows:   6 Evidence-Based Complementary and Alternative Medicine  e NS essential oil was tested on three pathogenic strains (Staphylococcus aureus (SA), Bacillus cereus (BC), and Escherichia coli (EC)) with concentrations from 500 to 1000 μg/mL (Table 6). e minimum inhibitory concentration (MIC) values obtained were 960 μg/mL for SA, 500 μg/ mL for BC, and 680 μg/mL for EC. ose values of antibacterial activity of Nigella sativa essential oil were average.

Antifungal
Activity. Hexane and acetone extracts and their fractions were tested on Candida pelliculosa (Table 7). e compounds presented low, medium, and high activity. Acetone extract (FA) and fraction (SA7) had high activity but not comparable to the reference cycloheximide 0.01%. e fractions (SH1), (SH2), (SH3), and (SA3) presented a high antifungal activity. For the other hexane fractions, the antifungal effects were medium or low. Hexane extract gave low activity, but generated fractions were more active. Acetone extract generated fraction (SA7) that showed the same high activity as the extract; however, the other fractions were less active than their source (FA).

Essential Oil Antifungal Activity: Microdilution Test.
NS essential oil had a minimum fungal concentration equal to 8 μg/mL against Candida albicans strains ( Table 8).
e MIC was the lowest that was found in our fungal study. In general, Nigella sativa had high antifungal effect that makes the essential oil a specific active antifungal.  Evidence-Based Complementary and Alternative Medicine

Discussion
Solvent polarity had an impact on the extracted compounds. Successive extraction was performed in order to separate the extracted compounds based on polarity. We only used hexane and acetone extracts in this study, partly due to their polarity difference. Hexane solvent is responsible for the extraction of lipophilic components such as terpenes and sterols, and acetone extracted polar compounds such as flavonoids and polyphenols. With a yield value of 34.2%, NS seeds are high in fat. Many studies reported hexane extract was the most abundant extract from NS [26][27][28], while acetone extract had a yield of only 2.5% [29]. e essential oil had a low yield when it is directly extracted from NS seeds (0.18%), that is the reason why hydrodistillation was necessary for the hexane extract. Indeed, Singh et al. had a similar yield of 1.2% [30]. Volatile oil yields of 0.4% to 0.44% [31] were reported from hexane extracts by hydrodistillation and with supercritical CO 2 method, a yield of 0.1% to 0.3% [32] based on NS seeds origin.
On the esterified hexane extract, the major components were fatty acids [32,33], both unsaturated fatty acids such as linoleic and oleic acids and saturated fatty acids such as palmitic acid [26].
Essential oil composition depends directly on its geographical origin, climate factors, extraction method, and the temperature conditions. As a result, the composition of NS essential oil could vary from a study to another. In the literature, Burits and Bucar [31] reported alphapinene and beta.
In fact, even differences in the composition of essential oils harvested from the same geographical origin with the same extraction method are possible [36] because oxygen can cause transformation of p-cymene into many derivative compounds.
In hexane extract fractions, many compounds were found in multiple fractions. Repetitive acquisition of constituents is a result of not only the abundance of fatty acids but also the difficulties associated with their migration through the chromatography column. Some less common compounds in hexane extracts are reported in Table 3 such as eicosadien acid, 9.12-octadecanoic acid, and pentanone dimethyl [37,38]. e characterization of acetone extract was in line with the chemical composition reported by Mechraoui et al. [17]. In addition, the identification of acetone extract fractions was consistent with our previous studies [7] and the fractions contained compounds with interesting biological activities [39][40][41].
Acetone extract (FA) had low activity towards Staphylococcus aureus and E. coli [17]. However, chicks fed NS acetone extract subproducts showed high activity towards E. coli in the feces [46]. Apigenin had a low antibacterial effect [47], with thymoquinone being the active part of the fraction (SA2) [48]. (SA3) was identified as catechol, a phenolic compound with interesting antibacterial activity towards Gram-positive bacteria [49] and (SA7) activity was a result of ascorbic acid and cysteine which are well known for their antibacterial properties [50,51]. Also, acetone fraction (SA10) was active because it contained essentially rutin that resulted in antibacterial activity towards Gram-positive and Gram-negative bacteria [52][53][54]. e best antibacterial activity was attributed to the fraction (SA7) towards L. innocua and fraction (SH1) towards P. aeruginosa. Hexane extract had antibacterial activity against Gram-positive bacteria [55,56] because of thymoquinone, p-cymene, and carvacrol, all good antimicrobials [57,58]. In many studies, the antibacterial activity was attributed to the presence of fatty acids such as oleic, linoleic, and palmitic acids in the hexane extract [59][60][61]. After separation, the antibacterial activity of fraction (SH1) was noticed because it contained fatty acid components such as palmitic and heptadecanoic acids [44]. Fraction (SH2) activity is almost entirely due to palmitic acid (50%), linoleic acid (17%), and heptadecanoic acid (16.35%). ese compounds were reported as antimicrobials [44,62]. e activity of acetone fraction (SA7) is explained by the presence of cysteine and ascorbic acid. In fact, ascorbic acid inhibits E. coli [50] and cysteine inhibits L. innocua and S. aureus [51,63]. e inhibitory activity of strains by fraction (SA7) could then be caused by the intervention of one or both constituents or the result of a synergic relationship between cysteine and ascorbic acid [64].
Our essential oil MIC values for S. aureus and E. coli were similar to those obtained by Ainane et al. [65], with a MIC value of 964 μg/mL towards S. aureus and a MIC equal to 676 μg/mL towards E. coli. Our results for Bacillus cereus were similar to Landa et al. [66] (MIC � 512 μg/mL) but different from S. aureus (MIC � 512 μg/mL) and E. coli (MIC >1024 μg/mL). e constituents of the hexane extract are hydrophobic allowing for strong interactions with the microbial membrane. As a result, alterations to the microbial membrane increases permeability and allows high consumption of the active agent by microbial cells [67].
(SA3) caused high antifungal activity. Fungal strains are sensitive to catechol; in consequence, its antifungal activity was elevated [49,71]. Moreover, (SA7) was the most active fraction that even exceeded the acetone extract. (SA7) contained cysteine which changes the morphology of fungal colonies by creating a structural modification that generates nuclear degradation [72,73]. e chemical mechanism for the essential oil is difficult to explain due to its complexity. It is possible that every single component has its own mechanism of action. e most abundant compounds describe the essential oil efficacy. For example, terpenes and hydrocarbons have an inhibitory effect on bacteria strains. In fact, the lipophilic propriety of skeletal hydrocarbons and hydrophilic properties of alcohol, aldehydes, and ketones have a positive impact on antimicrobial activity [74,75]. ymoquinone and carvacrol in the essential oil had minimal antimicrobial activity [58,65]. Hydrocarbons such as lilac aldehyde D in NS essential oil had high antimicrobial activity likely because conjugated aldehyde groups with double carbon-carbon bonds are highly electronegative. Suggesting that increasing electronegativity would increase antimicrobial activity [76,77]. e presence of thymol in essential oil could show a medium activity against bacterial strains [78]. Terpenes such as α-pinene, β-pinene, and c-terpinene also present high antibacterial activity towards Gram-positive bacteria and medium activity towards Gram-negative bacteria [30,79,80].

Conclusion
Column chromatography was used to separate hexane and acetone extracts into their purified fractions. Characterization based on GC-MS and HPLC-DAD showed the nature of the extracts and fractions and exposed links between antimicrobial activity and chemical composition.
Hexane and acetone extracts and their fractions had antibacterial and antifungal activities. Most fractions were more active against Gram-positive than Gram-negative bacteria. However, the (SH4) was active towards Gramnegative bacterial strains, and (SA7) had the highest antifungal activity.
e microdilution method was proceeded on NS essential oil for the determination of antibacterial and antifungal activities through quantification by MIC. Our results show that antifungal activity is more important than antibacterial activity.
Antimicrobial activity was a consequence of separated compounds as witnessed by comparing activity before and after separation. is may be a result of a unique isolated compound in the fraction or several compounds that react simultaneously or synergistically, meaning two compounds would individually have low activities, but when combined within the same fraction, their activity would increase. In summary, Nigella sativa L. extracts and fractions are interesting antimicrobials that could be exploitable in therapy and industry. e next step is to test the active antimicrobial fractions against "sensitive" foods to determine if Nigella sativa can be used for food preservation. Gas chromatography-mass spectroscopy HPLC-DAD:

Abbreviations
High performance liquid chromatography coupled with diode array detector MIC: Minimal inhibition concentration.

Data Availability
All data used to support the finding of this study are available online and from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.