Chemical Composition, Antibacterial and Phytotoxic Activities of Peganum harmala Seed Essential Oils from Five Different Localities in Northern Africa

Peganum harmala L., also known as Syrian rue or Pègano, is a herbaceous plant belonging to the Zygohpyllaceae family, and is widely used in traditional medicine. The chemical composition of essential oils of P. harmala seeds from five different regions of Northern Africa (Algeria, Egypt, Libya, Morocco and Tunisia) was studied by GC and GC-MS analyses. A total of 105 compounds were identified, the main components being oxygenated monoterpenes and oxygenated sesquiterpenes. Eugenol is the main component in all oils. The antimicrobial activity of the essential oils was assayed against some bacterial strains: Staphylococcus aureus (DSM 25693), Bacillus cereus (DSM 4313), Bacillus cereus (DSM4384), Escherichia coli (DMS 857) and Pseudomonas aeruginosa (ATCC 50071). All the oils showed different inhibitory activity. In the twentieth century this is an important result; we need possible new botanical drugs because the problem of resistance to antimicrobial drugs has become apparent. Moreover, the essential oils were evaluated for their possible in vitro phytotoxic activity against germination and initial radicle growth of Raphanus sativus L., Lepidium sativum L., and Ruta graveolens L. The results showed that both germination and radical elongation were sensitive to the oils.


Introduction
Peganum harmala L., also known as Syrian rue or Pègano, is a herbaceous plant belonging to the Zygohpyllaceae family. It is native to arid regions ranging from the eastern Mediterranean to northern India, Mongolia, and is present in South-East Europe, including southern Italy (Puglia and Sardinia) in arid steppes and sides of roads [1].
The seed oil is marketed in Egypt as an aphrodisiac. In ancient times, seeds and other parts of the plant were burned to produce a dense smoke during Zoroastrians rituals, and this use survives today. In Ladakh, India, the seeds of P. harmala are roasted and pulverized to obtain a fine powder, called techepakchiatzen, taken alone or smoked with tobacco to obtain narcotic effects [2]. In Iran, the smoke of its seeds is traditionally used as a disinfectant [3]. Different parts, including seeds, fruits, roots,

Chemical Composition of Essential Oils
Hydrodistillation yielded 0.005%, 0.003%, 0.002%, 0.001% and 0.01% of essential oil (on a dry mass basis) for P. harmala from Algeria (A), Egypt (E), Libya (L), Morocco (M) and Tunisia (T), respectively. Table 1 shows the chemical composition of the five P. harmala oils; compounds are listed according to their elution order on a HP-5MS column. The GC profile of five essential oils are present in Figure 1. Altogether, 105 compounds were identified, 45 for P. harmala from A, 38 for E, 20 for L, 38 for M and 37 for T, accounting for 89%, 93.3%, 95.4%, 75.72% and 95% of the total oil compositions.   a Retention time; b Kovats retention index determined relative to the t R of a series of n-alkanes (C10-C35) on HP-5MS column; c 1 = Kovats retention index, 2 = mass spectrum, 3 = co-injection with authentic compound; Tr = trace (<0.1%).

Antimicrobial Activity
Using the agar diffusion test, we evaluated the potential antimicrobial activity of the EOs obtained from P. harmala grown in the five countries of the Mediterranean area. All samples were capable of inhibiting the growth of the bacteria used as tester strains (Table 2). Escherichia coli is more sensitive to all oils, especially at a concentration of 15 µg/mL, with an inhibition area of 10.0 mm using the Egyptian oil. This value was higher than that shown by the control tetracycline against the same microorganism (6.0 ± 0.5 mm). The essential oil from Egypt showed quite similar effects against Bacillus cereus DSM 4384 at the same concentration. However, Staphylococcus aureus is more sensitive to essential oil from Morocco. Bacillus cereus DSM 4313 was the least sensitive to lower concentrations, but still shows a zone of inhibition of 9 ± 0 mm when the tests were effectuate with the essential oil from Libya at 15 µg/mL.

Phytotoxic Activity
The five essential oils were evaluated for their activity against germination and radicle elongation of radish (Raphanus sativus L.)-a species frequently utilized in biological assays-of garden cress (Lepidium sativum L.), and rue (Ruta graveolens L.). The five oils seem to be effective against germination of all these species (Table 3). In particular, treatment of seeds with concentrations of 100 µg/mL of the oil from Algeria is the most active against germination of radish. The concentration of 25 µg/mL of essential oils from Libya and concentrations of 50 µg/mL and of 25 µg/mL of essential oil from Tunisia inhibited significantly the germination of garden cress.
The essential oils from Algeria, Egypt and Libya, at all doses tested, significantly inhibited the radicle elongation of R. graveolens. However, concentrations of 25 µg/mL and 12.5 µg/mL of the oil from Tunisia are active against the germination of seeds oh the same species. The five essential oils affected significantly, at all doses tested, the radicle elongation in R. graveolens (Table 4).

Discussion
Oxygenated monoterpenes are highly predominant in P. harmala essential oils from A, L, M and T. Non-terpenic compounds are the main constituents of essential oils. In all oils, eugenol (13.2%-69.2%) was the most abundant component. Another component present in substantial quantity is thymol (6.9% for A, 5% for L, 5% for M and 1.8% in T). However this component is not found in essential oil from Egypt. Our results corroborate previous studies reporting the presence of eugenol but does not report the presence of thymol in the P. harmala essential oil from Egypt [12]. Tahrouch et al. [13] showed the presence of thymol in an essential oil from Morocco, but not of eugenol.
Chemical profiling is only basic, however DNA profiling will provide an additional safeguards on quality authentication. In fact, if the active components of a medicinal plant or essential oil become known and used as active treatment agent, uniformity will become mandatory and Good Agricultural Practice (GAP) should be practiced and enforced [27].
The composition of the essential oil of P. harmala in different countries is quite varied, suggesting that different factors can affect the oil composition. Optimization, standardization, and full control of growing conditions can guarantee quality-controlled production of plant-derived compounds [28]. In fact, the development stage is one of the determining factors of the yield and composition of essential oil [29]. In many cases an increase of the yield of oil from the bud stage to mature flower can change the chemical composition of the oil, and in some cases in the early stages this can vary more by than 10%. In Ocimum spp., the relative concentration of eugenol decreased with the development of the leaves, probably depending on the use of these compounds in the synthesis of lignin [30]. According to Manez et al. the alteration of the composition of the essential oil with maturation is closely related to the biosynthetic pathways leading to greater cyclization and dehydration of oil components [31].
Generally, the antimicrobial activity of P. harmala is attributed to specific components present in the hydroalcoholic or hexane portion of plant [32][33][34]. In our findings, few studies ascertained the antimicrobial potentialities of P. harmala essential oil [35]. Selim et al. [12] demonstrated the antimicrobial activity of P. harmala oil against B. cereus, S. aureus and E. coli, even if they used a higher concentration. We did not find an evident difference in sensitivity between the Gram+ and Gram− strains used in the experiments.
Taking into account the chemical composition of the five EOs, the difference in bioactivity could be attributable to the presence of nerol acetate [36], or to the different content of oxygenated sesquiterpene. The presence of eugenol, active on bacterial membranes is also of interest.
Eugenol, as isoeugenol, vanillin, and cinnamaldehyde are safrole phenylpropenes with antimicrobial activity [37], with isoeugenol more active than eugenol. Data show that the effectiveness of the oils is not strictly correlated to the percentage of eugenol, in fact the oil from M with a lower percentage of eugenol (13.2%) showed similar or higher antimicrobial activity than the others where eugenol was present at 17.8% or 69.2%. This shows that often the biological properties cannot be attributed to a single compound but to the synergy between the different components. In our study we evaluated the possibility of using the essential oil and not a singular component (eugenol) whose activity was already shown in previous studies. In our view, the use of P. harmala essential oil could impulse and improve the economy of these areas of the Mediterranean.
In all cases, the antimicrobial activity exhibited by the P. harmala oil against both Gram-positive (B. cereus and S. aureus) and Gram-negative (E. coli and P. aeruginosa) bacteria may indicate the presence of a broad spectrum of compounds with antibiotic activity. E. coli and S. aureus resulted generally more sensitive to the action of the 5 µg of the five P. harmala oils, in comparison to Pseudomonas aeruginosa, at their lowest concentration. We cannot attribute the different antimicrobial activity to a specific compound, present or absent in significant way in the two EOs from Libya and Tunisia, nor could this explain the slightly different behavior exhibited by the B. cereus 4384, which showed more sensitivity with low concentrations (5 and 10 µg) of EO used in the experiments. The antimicrobial compounds are commonly found in the essential oil fractions and many have a wide spectrum of antimicrobial activity [38]. Probably the method of extraction of essential oils influences their chemical composition and consequently their biological activities, in fact Shaverdi et al. have not observed antimicrobial activity of their n-exane smoke condensed extract derived from P. harmala seeds [3]. The main compounds of their smoke preparation are totally different from essential oil ones except for two compounds: hexadecane and heptadecane, this could explain the different antimicrobial activity.
The difference in phytotoxic activity of the oils could be attributed to their chemical composition. Oxygenated monoterpenes are predominant in essential oils from Algeria, Libya, Morocco and Tunisia. Our results agree with previous studies showing that monoterpenes and essential oils possess potent herbicidal effects on weed germination and seedling growth of various plant species [39][40][41][42][43]. The main monoterpene of five essential oils studied is eugenol. Vaid et al. [44] reported that eugenol suppresses some weedy species. Moreover, eugenol has been reported to damage plant tissue by damaging its cellular membranes [45,46].

Plant Material and Essential Oil Extraction
The seeds of P. harmala were collected from Tunisia (region of Kasserine-Bouzguem), Algeria (South of Algeria-Bousaada), Libya (region of El Hisha-South Derna), Morocco (Marrakech-Haouz) and Egypt (region of Marsa-Mattrouh). The main characteristics are reported in Table 5. The plant material was authenticated by Ms. Maroua Khadhr. The seeds were collected from individuals of the entire population to get an adequate representation of genetic diversity. Inflorescences were air-dried; seeds were separated from spikes and cleaned before surface. The seeds were dried at room temperature in the shade and weighed each day until the difference between two successive weights was less than 5%.
One hundred grams of dried seeds of each sample were ground in a Waring blender and then, subjected to hydrodistillation for 3 h according to the standard procedure described in the European Pharmacopoeia [47]. The oils were solubilized in n-hexane, filtered over anhydrous sodium sulphate and stored under N 2 at +4 • C in the dark, until tested and analyzed.

GC-FID Analysis
Analytical gas chromatography was carried out on a Perkin-Elmer Sigma-115 gas-chromatograph (Pelkin-Elmer, Waltham, MA, USA) equipped with a FID and a data handling processor. The separation was achieved using a HP-5 MS fused-silica capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness). Column temperature: 40 • C, with 5 min initial hold, and then to 270 • C at 2 • C/min, 270 • C (20 min); injection mode splitless (1 µL of a 1:1000 n-hexane solution). Injector and detector temperatures were 250 • C and 290 • C, respectively. Analysis was also run by using a fused silica HP Innowax polyethylene glycol capillary column (50 m × 0.20 mm i.d., 0.25 µm film thickness). In both cases, helium was used as carrier gas (1.0 mL/min).

Identification of the Essential Oil Components
Most constituents were identified using gas chromatography by comparison of their Kovats retention indices (R i ) (determined relative to the t R of n-alkanes (C 10 -C 35 )), with either those of the literature [48][49][50][51] and mass spectra on both columns or those of authentic compounds available in our laboratories by means of NIST 02 and Wiley 275 libraries [52]. The components' relative concentrations were obtained by peak area normalization. No response factors were calculated.

Antibacterial Activity
The antibacterial activity was evaluated in vitro, by means of the agar diffusion test on the plate. The activity of the essential oils was tested on five species of bacteria; S. aureus (DMS 25693), B. cereus (DSM 4313) and B. cereus (DSM 4384), representative of the Gram-positives; and E. coli (DMS 857) and P. aeruginosa (ATCC 50071) for Gram-negatives.
Microbial strains were previously grown in Nutrient Broth (Sigma, Milano, Italy), at 37 • C for 18 h. The microbial suspensions (1 × 10 7 cfu/mL) were uniformly distributed on Nutrient agar plates in sterile conditions. Different amounts of essential oils were spotted on the inoculated plates: 5 µg/mL, 10 µg/mL and 15 µg/mL. After 10 min, under sterile conditions, plates were then incubated at 37 • C for 24 h.
The antimicrobial activity performed with essential oils was evidenced by measuring the diameter (in mm) of the zone of inhibition. The diameter of the clear zone shown on plates was accurately measured using the "Extra steel caliper mod 0289", mm/inch reading scale, precision 0.05 mm (Mario De Maio, Milan, Italy). Dimethylsulfoxide (DMSO) was the negative control; on the other hand, tetracycline (7 µg) and gentamycin (8 µg) were used as positive standards. The value of MIC was evaluated as the lowest concentration of the sample that did not allow any visible growth of the microorganisms after incubation. Each oil was tested in triplicate.

Phytotoxic Activity
The phytotoxic activity was evaluated on germination and root elongation of three different plant species: R. sativus cv 'Saxa' (radish), L. sativum (garden cress) and R. graveolens (rue). These seeds are usually used in assays of phytotoxicity because they easily germinable and well known from the histological point of view. The seeds of R. sativus and R. graveolens were purchased from Blumen group srl (Emilia Romagna), the seeds of garden cress from Euroselect (Bari). The seeds were surface sterilized in 95% ethanol for 15 s and sown in petri dishes (Ø = 90 mm), containing five layers of Whatman filter paper, impregnated with distilled water (7 mL, control) or the tested solution of the essential oil (7 mL), at the different doses. The germination conditions were 20 ± 1 • C, with natural photoperiod. The essential oils, in water-acetone mixture (99.5:0.5), were assayed at the doses of 100, 50, 25 and 12.5 µg/mL Controls performed with water-acetone mixture alone showed no appreciable differences in comparison to controls in water alone. Seed germination was observed directly in petri dishes every 24 h. A seed was considered germinated when the protrusion of the root became evident [53]. After 120 h (on the fifth day), the effects on radicle elongation were measured in cm. Each determination was repeated three times, using petri dishes containing 10 seeds each. Data are expressed as the mean ± SD for both germination and radicle elongation.

Statistical Analysis
All experiments were carried out in triplicate. Data of each experiment were statistically analyzed using GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, CA, USA), followed by comparison of means (one-way ANOVA) using Dunnett's multiple comparisons test, at the significance level of p < 0.05.

Conclusions
We characterized the chemical composition of the essential oils of P.harmala grown in different Mediterranean countries. The composition of the essential oil of P. harmala in five countries is quite varied; in fact, the relative percentage of oxygenated monoterpenes, sesquiterpenes, and oxygenated sesquiterpenes are different. This could indicate a strong influence of the external environment on the metabolic pathway of the same plant. The studied samples showed antimicrobial activity against both Gram-positive and Gram-negative microorganisms, although it should be impossible to attribute such activity to a specific compound or group of biomolecules. However, the antimicrobial activity registered confirms some of the traditional uses of the plant. The essential oils studied showed different phytotoxicity, namely in the inhibition of radical growth of the three test species studied.