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

Background: The plant essential oils and extracts are possible sources of natural bioactive molecules and have been selected globally for new antimicrobial compounds, food preservatives, and alternatives to treat infectious disease. Methods: In this research, 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 oil of plants with the highest antibacterial activity was analyzed utilizing GC/MS. Moreover, 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 tested plants, methanolic and chloroformic extracts of P. harmala fruits showed the highest antibacterial activity against tested bacteria. Also, 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 . Conclusion: The results indicated that these plants can act as a promising antimicrobial agent due to its short killing time.


Introduction
The plant essential oils and extracts are possible sources of natural bioactive molecules and have been selected globally for new antimicrobial compounds, food preservatives, and alternatives to treat infectious disease [1]. There are many researches about the antibacterial and antifungal activity of plant extracts and essential oils [2][3][4][5][6]. For example, Srinivasan et al. [7]  Staphylococcus aureus bacteria and didn't show any antifungal activity on the tested fungus. Nagata et al. [8] investigated the antimicrobial activity of macrocarpals, which are phloroglucinol derivatives contained in Eucalyptus leaves versus a diversity of bacteria containing oral bacteria. Among 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 with macrocarpals. Hayet et al. [9] evaluated 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 and between tested extracts, methanol, and chloroform extracts exhibited a higher antibacterial activity versus gram-positive than gram-negative bacteria.
Han and Guo [10] investigated the antibacterial activity of Angelica sinensis extract (AE), Sophora flavescens extract (SE), and herb pair A. sinensis and S. flavescens extract (HPE) and displayed HPE had strong antibacterial activity on Escherichia 4 coli, Staphylococcus aureus, Shigella castellani, and Chalmers. Also, SE was moderately active to E. coli. Moreover, Sen and Batra [11]  Furthermore, antioxidants can act as scavengers of free radicals and can neutralize potentially damaging reactive free radicals in body cells before they cause protein and lipid oxidation and might decrease potential mutation and so, help prevent cancer or heart diseases [13]. Plants include considerable extents of phytochemical antioxidants like flavonoids, phenolics, carotenoids, and tannins which can be utilized to scavenge the extra free radicals existing in the body [14]. Many researches have reported 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 and demonstrated methanol extract had the highest antioxidant activity. Nesrin and Tolan [15] proved the antioxidant effect of Hyssopus officinalis. However, it was lesser than butylated hydroxytoluene and ascorbic acid. Ahmad et al. [12] indicated that extracts/fractions from Alhagi maurorum leaves displayed powerful radical scavenging activity that it might be because of the existence of phenolic compounds in the plant.
This research studies the chemical composition, antioxidant effects, antimicrobial activities and phytochemical analysis of some important medicinal plants.

Plant materials
The studied plants 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, 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
Collection of plant materials comply with institutional guidelines, and whole plant materials were wild type including no necessary getting any licenses for application.
The fresh selected parts of each plant were washed by distilled water, shade-dried and then powdered in a mechanical mill. Afterward, 10 g of powdered materials were soaked into 170 mL methanol and chloroform, separately. The plugged flasks of samples solution were placed for 48 h at room temperature by persistent shaking. The crude solutions 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
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 to 220 ºC at a speed of 15 ºC min -1 , and then retained at 220 ºC for 7 min; In the fallow, 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.

Measurement of catalase (CAT)
Catalase was examined using evaluating the primary rate of disappearance of H 2 O 2 , according to the Chance and Meahly [21] method. The reaction mixture included phosphate buffer (2.5 mL, 50 mM, pH = 7), H 2 O 2 (0.1 mL, 1%) and enzyme extracts (50 µL), diluted until retain the evaluations in the linear range of the analysis. The absorbance of the reaction mixtures was investigated at 240 nm via spectrophotometer (Biochrom WPA Biowave II UV/Visible), and the reduction of H 2 O 2 was because of the reduction in absorbance at 240 nm. The activity was stated as µmole 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 evaluate
The antiradical activity of the methanol extract of samples was evaluated through a spectrophotometer, using 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 essential to reduce the primary DPPH absorbance by 50% (IC 50 ). The IC 50 amount for any sample was investigated graphically through plotting the percent 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 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 using the adding sodium carbonate (1.5 mL, 20%). The volume of the mix was reached to 10 mL by distilled water. The mixture was afterward incubated at room temperature for 2 h. Thereafter, the absorbance was assayed via spectrophotometer (Biochrom WPA Biowave II UV/Visible) at 725 nm. The same method was repeated 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.
For the evaluation of flavonoid, 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 distilled water (2.8 mL). Then, extracts were placed for 30 min at room temperature; afterward, the absorbance of the reactions was evaluated using spectrophotometer (Biochrom WPA Biowave II UV/Visible) at 415 nm. The calibration curve was created 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. Antibacterial effect of the methanol and chloroform extracts of samples was assessed with the disk diffusion method utilizing Mueller-Hinton agar [17], and investigation of inhibition zones of the extracts. The filter paper discs of 6 mm diameter (Padtan, Iran) were sterilized then impregnated with 25 µL of methanol and chloroform extracts, separately. The sterile impregnated discs were put on the agar surface by flamed forceps and softly compressed down to ensure perfect contact of the discs with the agar surface. The incubation condition was 37 ºC for quality control strains and 27 ºC for plant bacteria for 24 h. Whole trials were performed in triplicate and the consequences were stated as mean ± SD.
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 effect was imagined as a zone of inhibition of fungal growth around the paper disc and the consequences 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 examined in a triple for chemical analysis and bioassays. 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.

Results and discussion
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, 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 IC 50 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 13 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, R 2 = 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 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.

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.

Conclusion
In conclusion, the consequences validate the traditional use of the herb against antimicrobial diseases. Also, the results indicated that these plants can act as a potential antimicrobial agent and deserves further studies to be safely used in the control of disease and pests. 16 Declarations