Essential oils compounds
As S. flavescens and P. harmala plants showed the best antimicrobial activities, they were selected for GC/MS analysis to identify the effective compounds. The results are shown below, separately.
S. flavescens
Thirty three constituents were recognized in the essential oil of S. flavescens aerial parts, representing 93.70% of the total essential oil. The essential oil combinations are listed in the order of their elution on the HP-5MS column as follows: Decane (0.44%), p-Cymene (0.31%), γ-Terpinene (0.39%), α-Terpinolene (0.26%), Terpinen-4-ol (0.35%), 4-isopropyl-2-cyclohexenone (0.46%), 1,6- cyclodecadiene (4.59%), Benzaldehyde, 4-(1-methylethyl)- (1.12%), Thymol (1.70%), Carvacrol (0.26%), β-Damascenone (0.91%), Caryophyllene (1.09%), Nerylacetone (0.44%), 2,6,10,14-Tetramethylheptadecane (0.49%), Alloaromadendrene (6.59%), α-curcumene (0.55%), β-Ionone (0.55%), 3,5-Di-tert-butylphenol (0.48%), Germacrene D (0.35%), Dodecanoic acid (3.37%), (+)-spathulenol (15.39%), Caryophyllene oxide (1.43%), Ledene (0.67%), Tetradecanoic acid (1.13%), 6,10,14-trimethylpentadecan-2-one (5.15%), Diisobutyl phthalate (0.65%), methyl 14-methylpentadecanoate (1.99%), n-Hexadecanoic acid (8.86%), Butyl 2-ethyl hexyl phthalate (1.20%), Squalene (8.87%), Ethyl linoleolate (4.99%), Neophytadiene (17.61%), and Linoleic acid (1.06%).
GC/MS analysis showed that the main components of the essential oil were Neophytadiene (17.61%), Spathulenol (15.39%), and Squalene (8.87%).
P. harmala
Eighteen components were identified in the essential oil of P. harmala fruits representing 91.76% of the total essential oil. The essential oil compounds are listed in the order of their elution on the HP-5MS column as follows: Decane (1.05%), m-Cymene (0.78%), γ-Terpinene (0.74%), 4- carvomenthenol (1.52%), 4-isopropyl-2-cyclohexenone (0.81%), Cuminaldehyde (2.58%), Thymol (2.46%), β-caryophyllene (1.44%), 6,10-dimethyl-5,9-undecadiene-2-one (0.88%), Alloaromadendrene (5.00%), (-)-Spathulenol (37.83%), (+)-Aromadendrene (1.07%), β-oplopenone (0.39%), Methyl palmitate (1.14%), n-Hexadecanoic acid (13.21%), Methyl linoleate (1.04%), Linoleic acid (11.08%), and Elaidic acid (8.72%).
GC/MS analysis showed that the main components of the essential oil were Spathulenol (37.83%), n-Hexadecanoic acid (13.21%), and Linoleic acid (11.08%).
Protein content and enzymes activity
Plants have evolved antioxidant pathways that are usually sufficient to protect them from oxidative injury during periods of natural growth and moderate stress. Both enzymatic and non-enzymatic systems protected tissue from the activated oxygen species, produced as the result of external environmental stresses, such as dryness, chilling and air pollution. Certain enzymatic antioxidant defense systems contain Super Oxide Dismutase (SOD), Catalase (CAT), and Guaiacol Peroxidase (GPX) [27]. In this research, the activity of 2 enzymes (CAT and GPX) was evaluated. Moreover, protein content was measured by bovine serum albumin as a standard. The results are exhibited in Figure 1. As shown, the maximum and the minimum activities of catalase were found in J. conglomeratus and S. flavescens plants, respectively. Besides, guaiacol peroxidase activity assay indicated that J. conglomeratus plant had the highest activity. Furthermore, the minimum guaiacol peroxidase activity was related to R. repens plant. Moreover, the maximum and the minimum protein contents were observed in M. azedarach fruit and J. conglomeratus plant, respectively.
DPPH radical scavenging effect
The effect of antioxidants on DPPH. was assumed to be because of their hydrogen donating capability [28]. Table 1 shows the DPPH radical scavenging effect of the tested plants. As presented, the highest free radical scavenging capacity of the plants was determined in P. harmala extract with an IC50 value of 0.46 ± 0.12 µg mL-1.
Total phenol and flavonoid content of the extracts
Plants have limitless ability to synthesize aromatic secondary metabolites, most of which are phenols or their oxygen-substituted derivatives. Important subclasses in this group of compounds include phenols, phenolic acids, quinones, flavones, flavonoids, flavonols, tannins and coumarins. These groups of compounds show antimicrobial effect and serves as plant defense mechanisms against pathogenic microorganisms. Phenolic toxicity to microorganisms is due to the site(s) and number of hydroxyl groups present in the phenolic compounds. Phenolic compounds cause cell membrane disruption, increase of ion permeability and leakage of vital intracellular constituents or impairment of bacterial enzyme systems in pathogenic microorganisms [34, 35].
It has been recognized that the antioxidant effect of the flavonoids and their effectiveness on human health and nutrition are considerable. Chelating or scavenging procedures are the action mechanism of flavonoids [29]. The evaluation of total flavonoid content was based on the determining the absorbance amount of tested plant solutions reacting with aluminum chloride reagent, and comparing with the standard solution of quercetin equivalents. The standard curve of quercetin was performed utilizing quercetin concentration ranging from 12.5 to 100 µg mL-1. The following equation stated the absorbance of the standard solution of quercetin as a function of concentration:
Y= 0.0056x + 0.1764, R2 = 0.9878
where, x is the absorbance and Y is the quercetin equivalent (mg g-1). The flavonoid content of samples is shown in Table 1. As shown, the highest phenol content was determined in A. maurorum, P. harmala and S. flavescens extracts with a value of 45.43, 39.3 and 39.07 mg of quercetin equivalents g-1 of dry matter, respectively.
Phenolic compounds gained from plants are a class of secondary metabolites, acting as an antioxidant or free radical terminators. Therefore, it is necessary to evaluate the total content of phenols in the tested plants [30]. The designation of the total phenolic amount was based on the absorbance amount of sample solutions (100 µg mL-1) reacting with Folin-Ciocalteu reagent, and comparing with the standard solution of gallic acid equivalents. The standard curve of gallic acid was performed utilizing gallic acid concentration ranging from 12.5 to 100 µg mL-1. The following equation stated the absorbance of the gallic acid standard solution as a function of concentration:
Y= 0.0954x + 0.196, R2 = 0.9973
where, x is the absorbance and Y is the gallic acid equivalent (mg g-1). The phenol content of the samples is presented in Table 1. As shown, the highest phenol content was determined in P. harmala and A. maurorum extracts with a value of 155.29 ± 0.20 and 146.71 ± 0.02 mg Gallic Acid Equivalents (GAE) g-1 dry matters, respectively.
Antibacterial screening
The antibacterial activity of methanolic and chloroformic extracts including A. maurorum, S. flavescens, R. repens, M. azedarach, P. harmala and J. conglomeratus in different concentrations (0.01, 0.03, 0.06, 0.12, 0.25 and 0.5 ppm) were tested versus 3 gram-positive (B. subtilis, S. aureus, R. toxicus) and 5 gram-negative (P. aeruginosa, E. coli, X. campestris, P. viridiflava, P. syringae) bacteria. The results at 0.5 ppm are shown in Figures 2 and 3. In addition, as in other concentrations, similar results were observed, for simplifying the discussion we considered only 0.5 ppm concentration. As shown in Figure 2, methanolic extracts of S. flavescens, P. harmala fruit and J. conglomeratus and chloroformic extracts of P. harmala fruit, S. flavescens, and P. harmala showed the maximum antibacterial activity on P. aeruginosa, respectively. Furthermore, methanolic extract of J. conglomeratus fruits and chloroformic extracts of M. azedarach and J. conglomeratus fruit had no antibacterial effect on P. aeruginosa (Figure 2a). The methanolic extract of P. harmala and chloroformic extracts of P. harmala fruit, R. repens, and M. azedarach had the maximum antibacterial activity against B. subtilis, respectively. Besides, chloroformic extract of A. maurorum extract had no antibacterial activity on B. subtilis (Figure 2b). The methanolic extracts of P. harmala fruit, P. harmala, and J. conglomeratus and chloroformic extracts of M. azedarach and P. harmala fruit indicated the maximum antibacterial activity on E. coli, respectively (Figure 2c). Moreover, the methanolic extracts of P. harmala fruit, the aerial part and chloroformic extracts of S. flavescens and P. harmala fruit had the maximum antibacterial activity on S. aureus, respectively (Figure 2d). Moreover, the antibacterial activity of tested plants on plant bacteria strains is shown in Figure 3. As indicated, methanolic extracts of P. harmala fruit and S. flavescens and chloroformic extracts of R. repens and M. azedarach showed the maximum antibacterial activity against R. toxicus, respectively (Figure 3a). Furthermore, methanolic extracts of R. repens and P. harmala fruit and chloroformic extracts of P. harmala fruit, J. conglomeratus fruit and, A. maurorum presented the maximum antibacterial activity against X. campestris, respectively (Figure 3b). The methanolic extract of P. harmala fruit and chloroformic extracts of P. harmala and J. conglomeratus displayed the maximum antibacterial activity on P. viridiflava (Figure 3c). Besides, the methanolic extracts of S. flavescens, P. harmala fruit and R. repens and chloroformic extracts of R. repens represented the maximum antibacterial activity on P. syringae, respectively. However, the methanolic extract of J. conglomeratus fruit showed no antibacterial activity (Figure 3d).
In order to compare the antibacterial activities of methanolic and chloroform extracts, independent-sample t-test was used, indicated with asterisk in Figures 2 and 3. For example, in Figure 2a, methanolic and chloroform extracts of plants 1, 2, 3, 5, 7 and 8 showed significant differences on Pseudomonas bacteria. In Figure 2b, methanolic and chloroform extracts of plants 2, 3, 4, 5, 6 and 7 displayed significant differences on B. subtilis. In Figure 2c, methanolic and chloroform extracts of plants 1, 2, 3, 4, 5, 7 and 8 exhibited significant differences on E. coli. In Figure 2d, methanolic and chloroform extracts of plants 1, 2, 3, 4, 5, 7 and 8 exhibited significant differences on S. aureus. While in Figure 3a, methanolic and chloroform extracts of plants 1, 2, 4, 5, 7 and 8 presented significant differences on R. toxicu, in Figure 3b, methanolic and chloroform extracts of plants 1, 2, 3, 4, 5, 6, 7 and 8 presented significant differences on X. campestris. Besides, in Figure 3c, methanolic and chloroform extracts of plants 1, 2, 3, 4, 5, 6, 7 and 8 showed significant differences on P. viridiflava, whereas in Figure 3d, methanolic and chloroform extracts of plants 1, 2, 3, 4, 5, 6, 7 and 8 showed significant differences on P. syringae.
Furthermore, Tables 2 and 3 illustrate the MIC and MBC values of the methanolic and chloroformic extracts of the tested medicinal plants against bacteria, respectively. The methanolic extract of P. harmala fruits showed the maximum activity against S. aureus and E. coli with MIC = 1.56 µg mL-1. In addition, chloroformic extracts of S. flavescens and P. harmala fruit indicated maximum activity against S. aureus and P. aeruginosa with MIC = 1.56 µg mL-1, respectively.
Antifungal activity
The antifungal properties of the methanolic and chloroformic extracts were tested using the agar well diffusion method. The results of the experiments showed that none of the tested plants had antifungal activity.
The use of herbal extracts as antioxidant and antimicrobial agents has two separate advantages: the natural origin and the related low risk. This means that they cause fewer side effects for people and the environment [31]. Based on the results, methanolic and chloroformic extracts of P. harmala fruit showed the maximum antibacterial activity against most of the tested bacteria pathogens, attributable to higher content of phenolic and flavonoid compounds. In addition, our findings were in agreement with those of Hayet et al. [9] and Guergour et al. [32]. Methanolic and chloroformic extracts of S. flavescens indicated the maximum antibacterial activity against P. aeruginosa and S. aureus, respectively. Our findings were in according with Han and Guo [10] and Yang et al. [31]. Chloroformic extract of M. azedarach represented the maximum antibacterial activity on E. coli, in accordance with Sen and Batra [11]. methanolic and chloroformic extracts of A. maurorum indicated antibacterial activity against all tested bacteria pathogens, in agreement with the study of Ahmad et al. [12].