Essential oils compounds
S. flavescens and P. harmala plants had shown the best antimicrobial activities. Therefore were selected for GC/MS analysis to identify the effective compounds. The results are shown below, separately.
S. flavescens
Thirty three constituents were known in essential oil of S. flavescens aerial parts, representing 93.70% of the total essential oil. The essential oil combinations are listed in order of their elution on the HP-5MS column. 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 essential oil of P. harmala fruits representing 91.76% of the total essential oil. The essential oil compounds are listed in order of their elution on the HP-5MS column. 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 common enough to maintain them from oxidative injury during times of natural growth and moderate stress. Both enzymatic and non-enzymatic systems protect tissue from activated oxygen species, produced as the consequence of exterior environmental stresses, such as dryness, chilling and air pollution. Certain of the 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 outcomes are exhibited in Figure 1. As shown, the maximum and minimum activities of catalase were found in J. conglomeratus and S. flavescens plants, respectively. Also, guaiacol peroxidase activity assay indicated that J. conglomeratus plant had the most activity. Furthermore, the minimum guaiacol peroxidase activity was related to R. repens plant. Moreover, the maximum and minimum protein content was seen in M. azedarach fruit and J. conglomeratus plant, respectively.
DPPH radical scavenging assess
The effect of antioxidants on DPPH.is assumed to be because of their hydrogen donating capability [28]. Table 2 shows the DPPH radical scavenging effect of tested plants. As presented, the most 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
It has been recognized that the flavonoids demonstration antioxidant effect and their effectiveness on human health and nutrition are considerable. Chelating or scavenging procedures are the mechanism of action of flavonoids [29]. The evaluation of total flavonoid content was based on the absorbance amount of tested plant solutions that react with aluminum chloride reagent, followed using comparing by 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 3. As shown, the most 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 which act as an antioxidant or free radical terminators. Therefore, it is needed 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) that react by Folin-Ciocalteu reagent, followed with comparing by 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 3. As shown, the most 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
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. Also, in other concentrations, similar results were observed that 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. Also, 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). Also, 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. Also, chloroformic extract of A. maurorum extract had no antibacterial activity on B. subtilis (Figure 2b). Furthermore, 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, methanolic extracts of P. harmala fruit and aerial part and chloroformic extracts of S. flavescens and P. harmala fruit had the maximum antibacterial activity on S. aureus, respectively (Figure 2d). Also, 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 indicated the maximum antibacterial activity against X. campestris, respectively (Figure 3b). Moreover, 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). Also, 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. But, methanolic extract of J. conglomeratus fruit showed no antibacterial activity (Figure 3d).
Antifungal activity
Antifungal property of the methanolic and chloroformic extracts was tested using the agar well diffusion method. The results of the experiments showed that none of the tested plants had antifungal activity.