Evaluation of antioxidant and antimicrobial activities of chloroformic and methanolic extracts of 6 important medicinal plants collected from North of Iran

Background: as possible sources of natural bioactive molecules, the plant essential oils and extracts have been used globally and to treat infectious disease. Methods: In this research, the antimicrobial activities of chloroformic and methanolic extracts of Sophora flavescens , Rhaponticum repens , Alhagi maurorum , Melia azedarach , Peganum harmala , and Juncus conglomeratus were evaluated against 8 bacteria ( S. aureus , B. subtilis , R. toxicus , P. aeruginosa , E. coli , P. syringae , X. campestris , P. viridiflava ) and 3 fungi ( Pyricularia oryzae , Fusarium oxysporum and Botrytis cinerea ), through disc diffusion method. Furthermore, the essential oils of plants with the highest antibacterial activity were analyzed utilizing GC/MS. Moreover, the tested plants were exposed to screening for possible antioxidant effect utilizing DPPH test, guaiacol peroxidas, and catalase enzymes. Besides, the amount of total phenol and flavonoid of these plants was measured. Results: Among the tested plants, methanolic and chloroformic extracts of P. harmala fruits showed the highest antibacterial activity against the tested bacteria. Besides, the investigation of free radical scavenging effects of the tested plants indicated the highest DPPH, protein, guaiacol peroxidase, and catalase in P. harmala , M. azedarach , J. conglomeratus fruits, and J. conglomeratus fruits, respectively. In addition, the phytochemical analysis demonstrated the greatest amounts of total phenolic and flavonoid compositions in J. conglomeratus and P. harmala , respectively. Conclusion: The results indicated that these plants could act as a promising antimicrobial agent, due to their short killing time.


Introduction
The plant essential oils and extracts, considered as possible sources of natural bioactive molecules, have been utilized globally in new antimicrobial compounds, food preservatives, and alternatives to treat infectious disease [1]. There are many researches about the antibacterial and antifungal activities of plant extracts and essential oils [2][3][4][5][6].
For example, Srinivasan et al. [7]  typhi, Bacillus subtilis, and Staphylococcus aureus bacteria and did not show any antifungal activity on the tested fungus. Nagata et al. [8] investigated the antimicrobial activity of macrocarpals, phloroglucinol derivatives contained in Eucalyptus leaves, versus a diversity of bacteria containing oral bacteria. Among the tested bacteria, P. gingivalis presented the maximum sensitivity to macrocarpals. Furthermore, its trypsin-like proteinase activity and binding to saliva-coated hydroxyapatite beads were inhibited by macrocarpals. Hayet et al. [9] evaluated the antibacterial activities of ethyl acetate, chloroform, butanol and methanol extracts of peganum harmala leaves against some pathogens containing 11 gram-positive and 6 gram-negative bacteria, among which methanol and chloroform extracts exhibited a higher antibacterial activity versus grampositive than gram-negative bacteria. Han and Guo [10]  Chalmers. Besides, SE was moderately active to E. coli. Moreover, Sen and Batra [11] examined the antimicrobial activity of ethanol, methanol, petroleum ether and water extracts of Melia azedarach L. leaves versus 8 human pathogens including Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli, Aspergillus flavus, Aspergillus niger, Fusarium oxisporum, and Rhizopus stolonifera. All the extracts indicated considerable activity versus all pathogens; however, the alcoholic extract exhibited the maximum inhibitory concentration versus all the microorganisms. Ahmad et al. [12] studied the antibacterial effect of Alhagi maurorum leaves extract and showed that the crude extract, chloroform, and ethyl acetate fractions had prominent effects, giving over 80% inhibition versus Bacillus anthrax. The crude extract displayed 80% inhibition versus Shigella dysenteriae. Similarly, the ethyl acetate and crude extract acted well versus Salmonella typhe by 78.35% and 76.50% inhibition respectively. Furthermore, antioxidants helped to prevent cancer or heart diseases, as they could act as scavengers of free radicals and neutralized the damaging reactive free radicals in body cells before they could cause protein and lipid oxidation and decrease potential mutation [13]. Generally, plants include considerable extents of phytochemical antioxidants such as flavonoids, phenolics, carotenoids, and tannins, which can be utilized to scavenge the extra free radicals existing in the body [14]. Many researches have reported the antioxidant effect of essential oils and plant extracts. For example, Hayet et al. [9] examined the antioxidant activity of ethyl acetate, chloroform, butanol and methanol extracts of Peganum harmala leaves, demonstrating that methanol extract had the highest antioxidant activity. Nesrin and Tolan [15] proved the antioxidant effect of Hyssopus officinalis; however, it was lower than butylated hydroxytoluene and ascorbic acid. Ahmad et al. [12] indicated that extracts/fractions from Alhagi maurorum leaves displayed powerful radical scavenging activity, probably because of the existence of phenolic compounds in the plant.
The main aim of the present work was to study the chemical composition, antioxidant effects, and antimicrobial activities, while doing the phytochemical analysis of some important medicinal plants.

Plant materials
The plants studied in this research are displayed in Table 1. All plants were collected from the research field of Sari Agricultural and Natural Resources University (SANRU), located at 53º 04̍ E and 36º 39̍ N (Iran), and identified from flora resources. A botanist authenticated the samples (different parts of the mentioned plants) and the voucher specimen deposited in the laboratory.

Plant extracts preparation
The collection of plant materials complied with institutional guidelines, and whole plant materials were wild type requiring no licenses for the application. The fresh selected parts of each plant were washed by the distilled water, shade-dried and then powdered in a mechanical mill. Afterward, 10 g of powdered materials was soaked into 170 mL methanol and chloroform, separately. The plugged flasks of samples solution were placed at room temperature for 48 h by persistent shaking. The crude solutions were filtered through glass funnel and then dried via a rotary vacuum evaporator at 40 ºC temperature. Finally, the extracts were filter sterilized by a 0.22 µm Ministart (Sartorius) and stored at 4 ºC before utilization [16].

Essential oils separation
The powdered samples (75 g) were exposed to hydro-distillation for 4 h, using a Clevenger-type apparatus. The essential oils were dehydrated by sodium sulfate anhydrous and stored at 4 ºC before GC/MS analysis [17][18][19].

Gas Chromatography coupled to Mass Spectrometry (GC/MS) analysis
GC/MS analysis was performed on an Agilent Technologies 7890A (GC) coupled with Agilent Technologies 5975C, equipped with a fused silica capillary HP-5MS column (30 m × 0.25 mm iD, film thickness 0.25 µm). The oven temperature was increased from 50 ºC to 220 ºC at a speed of 15 ºC min -1 , retained at 220 ºC for 7 min; and then incremented to 260 ºC at a speed of 15 ºC min -1 . Transfer line temperature was 250 ºC. Helium was used as the carrier gas, at a flow speed of 1 mL min -1 . The inlet temperature was 280 ºC.
Afterwards, the homogenates were centrifuged (Eppendorf centrifuge 5430R) at 10000 g for 15 min. Finally, the supernatant fractions were utilized for the measurement of protein content and enzyme activities [20].

Measurement of catalase (CAT)
Catalase was examined via evaluating the primary rate of disappearance of H 2 O 2 , according to the Chance and Meahly [21] method. The reaction mixture, including phosphate buffer (2.5 mL, 50 mM, pH = 7), H 2 O 2 (0.1 mL, 1%) and enzyme extracts (50 µL), was diluted in order to keep the measurements within the linear range of the analysis. The absorbance of the reaction mixtures was recorded at 240 nm via spectrophotometer (Biochrom WPA Biowave II UV/Visible), in which the reduction in the absorbance at 240 nm was because of the reduction of H 2 O 2 . The activity was stated as µmole activity mg -1 protein.
The activity was stated as mmole activity mg -1 protein.

Measurement of protein
Protein concentrations were specified based on the Bradford [23] method, by Bovine Serum Albumin (BSA), as standard protein.

2, 2-Di-Phenyl-1-Picryl Hydrazyl (DPPH) scavenging
The antiradical activity of the methanol extract of samples was evaluated using a spectrophotometer, via Liyana-Pathirana and Shahidi [24]  where, Abs control is the absorbance of DPPH radical + methanol; and Abs sam ple is the absorbance of DPPH radical + samples methanol extract. The radical scavenger activity was stated as the extent of antioxidants required to reduce the primary DPPH absorbance by 50% (IC 50 ). The IC 50 amount for any sample was calculated graphically through plotting the percentage of disappearance of DPPH as a function of the sample concentration.

Phytochemical analysis
Total Phenolic Content (TPC) of the test samples was assayed using Yu et al. [25] Folin -Ciocalteu method, utilizing gallic acid as the standard. Briefly, double distilled water (900 µL) was added to the methanolic solution of test samples (100 µL, 100 µg mL -1 ). Then, Folin -Ciocalteu reagent (500 µL) was added, followed by the addition of sodium carbonate (1.5 mL, 20%). The volume of the mixture was reached to 10 mL by the distilled water. The mixture was afterward incubated at room temperature for 2 h. After that, the absorbance was assayed via spectrophotometer (Biochrom WPA Biowave II UV/Visible) at 725 nm. The same method was used for the standard solutions of gallic acid. Based on the evaluated absorbance, the concentration of phenolic content was determined from the calibration line. Finally, the total phenolic content of methanol extracts was stated as mg Gallic Acid Equivalents (GAE) g -1 dry matter.
In order to determine the flavonoid content, the colorimetric aluminum chloride method was utilized [26]. Each sample in methanol (0.5 mL, 1:10 g mL -1 ) was blended with methanol (1.5 mL), potassium acetate (0.1 mL, 1 M), aluminum chloride (0.1 mL, 10%), and the distilled water (2.8 mL). Then, the extracts were placed at room temperature for 30 min. Afterwards, the absorbance of the reactions was recorded using spectrophotometer (Biochrom WPA Biowave II UV/Visible) at 415 nm. The calibration curve was plotted through making quercetin solutions (12.5 to 100 µg mL -1 ) in methanol. Finally, the total flavonoid content was stated as mg of quercetin equivalents g -1 of dry sample.
The antifungal property of the methanol and chloroform extracts was examined with the agar-well diffusion method [16]. Potato Dextrose Agar (PDA) was seeded by tested fungus.
Sterile paper discs of 6 mm diameter (Padtan, Iran) were impregnated by 25 µL of the methanol and chloroform extracts of samples, separately. The sterile impregnated discs were put on the level of the seeded agar plate. The incubation conditions utilized were 28 ºC and 70% RH for 12-14 days for Pyricularia oryzae and 7-9 days for Botrytis cinerea, and Fusarium oxysporum. The antifungal activity was visualized as a zone of inhibition of fungal growth around the paper disc and the results were stated as mean ± SD after three repetitions. Pathogen grown on PDA without plant extract was utilized as control.

Statistical analysis
Methanol and chloroform extracts tested in triplicate for chemical analysis and bioassays.
The obtained data were exposed to Analysis of Variance (ANOVA), following a completely randomized design to determine the Least Significant Difference (LSD) at P < 0.05 by SPSS statistical software package (SPSS v. 11.5, IBM Corporation, Armonk, NY, USA). All results were stated as mean ± SD. Independent-sample t-test was used for selected comparisons between samples. Alpha value was set a priori at P < 0.05.

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 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 2 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 IC 50 value of 0.46 ± 0.12 µg mL -1 . 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 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.

Total phenol and flavonoid content of the extracts
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: 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 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

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.

Conclusion
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]. In this work, the antimicrobial and antioxidant activities of extracts of some plants used in Iranian folklore medicine were reported [33]. 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].
According to the obtained results, a high resolution GC/MS method reported for the evaluation of the constituents of P. harmala and S. flavescens plants, while in both plants, Spathulenol was the main component of the essential oil.
Following and confirming the work of other researchers on the effects of medicinal plants extracts on various pathogens [7-12, 15, 31-33], in this study, the antibacterial and antifungal activities of medicinal plants extracts on plant bacteria and fungi strains were evaluated for the first time. Furthermore, antioxidant assays including measurement of catalase, guaiacol peroxidase and protein were reported for the first time in this study.
In conclusion, the results confirmed the traditional use of the herb against antimicrobial diseases. These plants could act as a potential antimicrobial agent; however, further studies are required for them to be safely used in the control of disease and pests.
Declarations of Tabarestan (GABIT) is gratefully acknowledged. b Not specified  Figure 1 Enzymes activity and protein content strains. Data were exposed to Analysis of Variance (ANOVA), following a completely randomized design to determine the Least Significant Difference (LSD) at P < 0.05 by SPSS statistical software package (SPSS v. 11.5, IBM Corporation, Armonk, NY, USA). All consequences were stated as mean ± SD. strains. Data were exposed to Analysis of Variance (ANOVA), following a completely randomized design to determine the Least Significant Difference (LSD) at P < 0.05 by SPSS statistical software package (SPSS v. 11.5, IBM Corporation, Armonk, NY, USA). All consequences were stated as mean ± SD.