Phytochemical Characterization, Antioxidant Potential and Antibacterial Activity of Araucaria columnaris against Methicillin-Resistant Staphylococcus aureus (MRSA) and Streptococcus pyogenes

Objectives: In recent years, increasing rates of antibiotic resistant infectious pathogens together with the decline of novel antibiotics urged the need to discover powerful antimicrobial therapy. This is aimed to characterize A. columnaris chemically and investigate its antioxidant and antimicrobial activities. Methods : DPPH, ABTS, iron metal chelation, FRAP and ORAC assays were used to evaluate the antioxidant activity. Agar well diffusion and tube dilution methods were employed to detect the antimicrobial activity and the minimum inhibitory concentrations of the plant extracts. Results : A. columnaris exhibit antibacterial activity against Gram-positive pathogens, S. aureus , S. pyogenes and Methicillin Resistant Staphylococcus aureus (MRSA). As for both MRSA and S. pyogenes clinical isolates, the MIC of extract obtained corresponded to concentration 10.4 mg/ml. Moreover, total phenolic and flavonoid contents of non-flowering aerial partsextractwere 39.0161± 1.53 mg GAE and 49.2214 ± 3.47 mg RE per gram of dried extract, respectively. GC/MS analysis of the essential oil of A.columnaris resulted in the detection of eighty-one components (98.03%) with hibaene (23.35%) as a major constituent. Additionally, in-silico study was performed on the identified polyphenolic compounds of A. columnaris against both the PBP2a allosteric site and active site to explain the mechanism of action against MRSA. Conclusion : A. columnaris extract is a potential candidate of an alternative therapy to both infectious and non-infectious diseases and further studies are required to expand the use of A. columnaris as antibacterial for treating resistant Gram-positive pathogens circulating in hospital settings.


INTRODUCTION
The consumption of medicinal plants is increased as it contains several natural constituents with therapeutic properties [1].The literature shows that antioxidants, anticancer and antibacterial activities were proved for various ornamental plants like Catharanthus roseus and Hibiscus rosa which highlighted its value [2].
Araucaria columnaris (G.Forst.)Hook., has a symmetrical pattern appearance, and is known as Cupressus columnaris J. R. Forst [19],which is used as an attractive plant.This evergreen coniferous plant grows to an average height of 60 m and has a variety of short shoots, a clear trunk, grey bark, and a slender, spirelike crown.Cook pine is the common name for this ornamental plant.
The results of prior studies showed that the nonflowering aerial parts of methanol and dichloromethane extracts of A. columnaris revealed the presence of antifungal, cytotoxic, antibacterial, and phytotoxic properties [20].
However, there have been a few findings of the tested plant that have not yet been fully explored for its phytochemical analysis and biological potential.Thus, the present study was conducted to investigate the antimicrobial activity of A. columnaris beside the characterization of its chemical profile and antioxidant activity using the alcoholic extract as a tool of standardization.

Plant Material
Araucaria columnaris (G.Forst.)Hook.(Family: Araucariaceae) non-flowering aerial parts (branches and leaves) were collected in January 2019 from Prince Mohamed Ali palace garden in Egypt.Dr. Therese Labib, senior head of plant identification, confirmed the plant material's identity.A voucher specimen (A C-7 A. columnaris) was placed in the pharmacognosy department's herbarium at Ahram Canadian University's Faculty of Pharmacy.

Preparation of Plant Extracts and oil extraction
Fresh non-flowering aerial parts of Araucaria columnaris (200g) were suspended in twice their volumes of distilled water and subjected to steam distillation for 6-8 h using essential oil distillation apparatus (VWR Scientific, catalog no.26319-008).Prior to distillation, the samples were chopped into about 2 cm long pieces.The distillate was allowed to cool at room temperature, the essential oil was allowed to separate from water and each essential oil sample was weighed on an analytical scale (0.5 ml 0.25% v/w) from Araucaria columnaris the oils were collected, dried over anhydrous Na2SO4 and kept in a freezer at -5°C until the GC-MS analysis can be performed.
Fresh non-flowering aerial parts of Araucaria columnaris were air-dried in the shade and powdered.Cold maceration was applied to extract the powdered specimen (50 g) with absolute methanol and allowed to stand at room temperature with occasional shaking for fifteen days.The extraction process was repeated twice with the same solvent and the solvent was then evaporated under reduced pressure at temperatures not higher than 40 C using a rotatory evaporator (Buchi, G. Switzerland) to give green residue (5.2 g), and the dried methanol extract was stored in sealed containers at 4 C until use.

Determination of Total Phenolic Content
The total phenolic content of the methanolic extract of Araucaria columnaris non-flowering aerial parts was determined using the Folin-Ciocalteu method and the improved assay procedures mentioned by Attard [21].Utilizing standard calibration curve of gallic acid (50 -1000 g/mL), the total phenolic content was presented as mg gallic acid equivalent (GAE)/g of Araucaria columnaris extract.

Determination of Flavonoid Content
The total flavonoid content of the methanolic extract of Araucaria columnaris was evaluated by measuring the yellow colour generated when flavonoids were interacted with AlCl3 reagent following the improved assay conditions described by (Kiranmai, Kumar [22].The total flavonoid content was displayed in mg rutin equivalent (RE)/g extract using a rutin calibration curve (6.25 -1000 g/mL).

Gas chromatography/Mass spectrometry (GC/MS) GC/MS analysis
Shimadzu GCMS-QP2010 (Tokyo, Japan) equipped with Rtx-1MS fused bonded column (30 m x 0.25 mm i.d.x 0.25 m film thickness) (Restek, USA) and with a split-splitless injector was applied to evaluate mass spectra.The initial column temperature was maintained at 45 °C for 2 minutes (isothermal) before being conditioned to 300 °C at a rate of 5 °C/min and

Peak identification
By comparing mass spectra and retention indices with those stored in the NIST, WILEY library database and published in the literature, compounds were identified.Kovats Indices (KI) relative to n-alkanes (C6-C20) were also used to identify the compounds.The Kovats Index was obtained by using GC/MS solution program.

Molecular modeling
Docking simulations was performed using the structure preparation application in Molecular Operating Environment (MOE), 2014.10 [23][24][25].The x-ray crystallographic structure Crystal structure of penicillinbinding protein 2a (PBP2a) from methicillinresistant Staphylococcus aureus (MRSA) in complex with piperacillin and quinazolinone (PDB code: 6Q9N) was retrieved from Protein Data Bank of the Research Collaboration for Structural Bioinformatics from (RCSB) website [www.rcsb.org][26].

DPPH Assay
Using Trolox as a reference, the DPPH (2,2diphenyl-1-picryl-hydrazyl-hydrate) free radical test was performed (Boly, Lamkami [27].In a 96-well plate (n=6), 100 µL of newly made DPPH reagent (0.1% in methanol) was applied along with 100 µL of the sample.The reaction was then allowed to sit at room temperature for 30 minutes while it was dark.The subsequent decrease in DPPH colour intensity was recorded at 540 nm at the conclusion of the incubation period.Based on the given formula, data are shown as means ±SD:

ABTS Assay
With minor adjustments, distilled water was used to fill the remaining 50 mL of the volumetric flask after 192 mg of ABTS had been dissolved in it (Arnao, Cano [28].17 µL of 140 mM potassium persulphate were combined with 1 mL of the prior solution, and the combination was then kept in the dark for 24 hours.The final ABTS dilution for the test was completed by diluting 1 mL of the reaction mixture to 50 mL with methyl alcohol.In a 96-well plate (n=4), 190 µL of newly made ABTS reagent was mixed with 10 µL of the samples.The reaction was then allowed to sit at room temperature for 120 minutes while kept in the dark.At 734 nm, the intensity of the ABTS colour declined after the incubation period.Based on the given formulas, data are shown as means ±SD:

FRAP assay
With minor changes in the method Benzie and Strain [29], A newly made TPTZ reagent (300 mM acetate buffer (PH=3.6), 10 mM TPTZ in 40 mM HCl, and 20 mMFeCl3, in a ratio of 10:1:1 v/v/v, respectively) was used in the ferric reducing ability experiment.In a 96-well plate (n=3), 190 uL of newly made TPTZ reagent were mixed with 10 uL of the test.The reaction was then left to sit at room temperature for 30 minutes while kept in the dark.The last measurement of the blue hue after incubation was made at 593 nm.Data is displayed as means ± SD.

Ferrozine iron metal chelation assay
The assay was conducted with a few minor adjustments to the original method (Santos, Brizola [31], In a 96-well plate (n=6), 20 µL of newly made ferrous sulphate (0.3 mM) was combined with 50 µL of the sample/compound.Then, each well received 30 µL of ferrozine (0.8 mM).For 10 minutes, the reaction mixture was incubated at room temperature.The reduction in the intensity of the generated colour was assessed at 562 nm at the conclusion of the incubation period.Based on the given formulas, results are shown as means ± SD:

Bacterial isolates
The ATCC bacteria and clinical isolates that were collected from hospitalized patients were the microorganisms utilized in this investigation.The Muller-Hinton broth was used to cultivate the bacterial isolates, including Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Streptococcus pyogenes clinical isolate, and Methicillin-resistant Staphylococcus aureus (MRSA) clinical isolate.Both MRSA and S. pyogenes clinical isolates were acquired from wound infections and streaked on blood agar plates (Oxoid, Basingstoke, UK) before being cultured aerobically at 37°C for 24 hours to produce a single colony.Growing colonies were isolated and analyzed using microbiological best practices and VITEK II (bioMerieux, Marcy l'Etoile, France) for further confirmation.The Clinical and Laboratory Standards Institute (CLSI) [32] described disc diffusion method was used to assess the antibiotic susceptibility of MRSA and S. pyogenes isolates using the following drugs: ciprofloxacin, ofloxacin, clindamycin, gentamicin, vancomycin, doxycycline, erythromycin, rifampicin, levofloxacin, amikacin, linezolid, mup (Oxoid, Basingstoke, UK).Bacterial suspensions were turbidityadjusted to match the McFarland Standard's turbidity of 0.5, and then incubated overnight at 37°C in an aerobic environment.

Inoculum preparation
The density of the inoculums was standardized to be 106 colony-forming units (CFU)/ml.The test organism was inoculated into 5 ml of Mueller Hinton broth and left to incubate for 24 hours at 37 °C.To standardize the culture to 10 6 CFU/ml, 0.2 ml of the organism's 24-hour culture was poured into 10 ml of sterile Mueller Hinton broth and cultured for 3-5 h.(equivalent to 0.5 McFarland standard).Within 15 minutes of standardizing the inoculum, plates were infected.

Agar well diffusion assay
Two hundred mg of the dried plant methanolic extract were dissolved in 0.4 ml of 10% dimethylsulfoxide (Sigma, Hamburg, Germany) to make the plant extract solution 500mg/ml.The solution was then sterilized by filtering using a 0.22 m cellulose membrane filter (Indiamart, Vadodara, India).A volume of the microbial inoculum is distributed across the whole surface of the Muller-Hinton agar plate to inoculate it.Then, a 100-L volume of the plant methanolic extract solution at the necessary concentration is added to the well after an aseptic hole of 6 to 8 mm in diameter is punched with a sterile cork borer.The test microorganism is then cultured on agar plates under the proper conditions [33].Standard antibiotics like doxycycline and amikacin were administered as a control.Negative control (DMSO) which showed no inhibition was used.

Minimum Inhibitory Concentrations of the plant Extract
The Tube broth dilution method was done to detect the minimum inhibitory concentrations of the methanolic plant extract against MRSA and S. pyogens according to CLSI guidelines 2020 (CLSI 2020).A concentration of 250 mg/ml of the plant methanolic extract was prepared as the stock solution and two-fold serial dilutions in each tube were done to obtain the following concentrations 125 mg/ml,62.5mg/ml, 20.8 mg/ml, 10.4 mg/ml, 5.2mg/ml, 2.6mg/ml, 1.3mg/ml, 0.65mg/ml, 0.32mg/ml and 0.16 mg/ml in Muller Hinton Broth.The incubation was completed for 24 h at 37 °C and the MIC was set up as the lowest concentration showed no visible bacterial growth.The mean value of three replicates was obtained as the result.

Antifungal activity
The agar well diffusion technique was used to examine the extract's antifungal efficacy [34].The extract's final concentration matched the 0.5 McFarland standard after being adjusted spectrophotometrically at 530 nm and tested against C. albicans ATCC 10231.After the plates had been incubated at 28 °C for 24 hours, the inhibition zones that resulted were quantified.Amphotericin, a common antibiotic, served as the standard.The means of each experiment's three replications were noted.

Determination of total phenolic and total flavonoid contents
Total phenolic and total flavonoid contents of the methanolic extract of thenon-flowering aerial partsof Araucaria columnariswere 39.0161± 1.53 mg GAE and 49.2214 ± 3.47 mg RE per gram of the dried extract, respectively.

GC/MS analysis of the essential oil of Araucaria columnaris
The chemical composition of oil of A. columnaris which was analyzed by GC/MS gave rise to the recognition of 81 constituents representing 98.03% of the essential oil content, Table 1, Figure 1.

Molecular modeling studies
More than 200,000 different small molecules are produced by plants, many of which have antibacterial properties (such as phenolics) [35].The majority of the flavonoids appeared to have PBP2a as their most likely  .The tyrosine residue (TYR-466) that closes the active site of this modified protein serves as a gate to block the entry of the ligand (such as β-lactam antibiotics) [37].The allosteric site 60 Å away from the active site is responsible for opening up this closed state, and ligand binding to this allosteric site can cause a series of conformational changes throughout the entire protein, leading to an accessible active site that is easily inhibited by β-lactam antibiotics.
Flexible docking was performed on the identified polyphenolic compounds of Araucaria columnaris such as Chlorogenic acid, Gallic acid, Syringic acid, Quercetin, Catechin and Rutin catechin [38-43] against both the PBP2a allosteric site and active site using Molecular Operating Environment (MOE) in order to further explain the anti-MRSA mechanism.
MOE docking results of the proposed library against the active site shows that Piperacillin (cocrystalized ligand) has S-score of -15.6655 kcal/mol and exhibited 9 hydrogen bonds with the main interacting amino acids Lys 597 of distance (2.37, 2.37, 3.54 Å), Ser598 of distance (2.35 Å), Ser 403 of distance (2.7 Å), Thr 600 of distance (2.84 Å), Gln 521 of distance (2.61 Å), Asn 464 of distance (2.61 Å) and Asn 601 of distance (2.72 Å), As shown in Table 3.Furthermore, the proposed library was found to be able to interact with most of the main amino acids.(Figure 2, 4)

Antioxidant activity
The antioxidant ability of the methanolic extract of A. columnariswere measured by 5 different assays; DPPH, ABTS, iron metal chelation, FRAP and ORAC assays.The extract IC50 value using DPPH assay was 599.4 ± 0.58 µg/mL versus 24.42 ± 0.87 µM for Trolox.The total antioxidant capacity was 144.338 ± 3.64, 233.821 ± 14.98 and 352.775 ± 20.32 µM Trolox equivalent per mg of the extract using ABTS, FRAP and ORAC assays, respectively, and 68.281 ± 5.89 µM EDTA equivalent per mg of the extract using iron metal chelation assay, Figure 5.

Antimicrobial activity
The results of agar well diffusion method showed that the methanolic extract of A.columnaris nonfloweringaerial partshas antibacterial activity towards S.aureus ATCC 25923, S.pyogenes clinical isolate which was susceptible to all the tested antibiotics except erythromycin, clindamycin and vancomycin and MRSA which were resistant to all tested antibiotics except levofloxacin, rifampicin, amikacin, vancomycin, teicoplanin, mupirocin and linezolid, Table 4 and Figure 6.The highest antibacterial activity was shown towards S. aureus ATCC 25923 (Zone of inhibition: 30 mm) followed by MRSA (Zone of inhibition: 17mm) and then S. pyogenes (Zone of inhibition: 15 mm), Table 5.The plant extract has no antibacterial activity towards E. coli (Gram-negative bacteria) and does not possess antifungal activity as well, Table 5.For both MRSA and S. pyogenes clinical isolates the MIC obtained was corresponding to concentration 10.4 mg/ml of the methanolic plant extract.

DISCUSSION
Currently, medicinal plants are significant pool for the discovery of novel drug.The importance of ethnopharmacological data as a source for the identification of bioactive compounds from plants is clearly demonstrated in this study.Our results revealed that the methanolic extract of Araucaria columnaris has a significant antimicrobial activity and antioxidant activity.Many chronic diseases like cancers, atherosclerosis is related to the free radical existence, so as to explore this, the antioxidant potential of A. columnaris extract were determined by 5 different assays; DPPH, ABTS, iron metal chelation, FRAP and ORAC assays showing a moderate antioxidant activity using a potent radical scavenger of peroxyl and alkoxyl radicals, trolox, a hydrophilic homologue of vitamin E as standard or equivalent drug.The antioxidant effect can be credited to its high content of phenolics and flavonoids that has been proven by determination of the total phenolic and total flavonoid contents of the methanolic extract of non-flowering aerial parts of Araucaria columnaris as 39.0161± 1.53 mg GAE and 49.2214 ± 3.47 mg RE per gram of dried extract, respectively.The oil content of Araucaria columnaris which is obtained by hydro-distillation represent (0.25% v/w) was analyzed by (GC/MS), Table 1, Figure 1.Showing a total of 81 compounds representing a percentage of 98.03% of identified compounds.The major classes identified in the essential were sesquiterpene (46.17 %; 33 compounds) and diterpene (34.36 %; 11 compounds) hydrocarbon beside other classes such as oxygenated sesquiterpenes, monoterpene and terpene related compounds.Two major compounds were noticed hibaene (23.35 %) and β-Selinene (21.49%) as diterpene and sesquiterpenes, respectively.The data of the present study greatly differs from the previous published data on Araucaria columnaris essential oil composition which showed that the major constituent are γ-muurolene and α-copaene [44].
Because MRSA is a Gram-positive pathogen that spread and established in the hospital settings and cause severe infections as bloodstream infections, surgical site infections, pneumonia, and sepsis.Generally, Staph aureus has an extraordinary ability to develop resistance to any antibiotic which it has been subjected.Additionally, multidrug resistant pyogenic Streptococci have been reported to cause infectious diseases with a significant impact to public health.Therefore, it is crucial to find an alternative to antibiotics to help in therapy and overcome the antibiotic resistant problem.They were chosen after the extract was tested on standard S.aureus strain ATCC 25923 and showed good antibacterial activity, so we decided to assess the antibacterial activity of the extract on clinical isolates which are problematic and difficult to treat in critically ill patients for being multidrug resistant.
The antimicrobial properties of methanolic extract of A.columnarisnon-flowering aerial parts was investigated in the present study against S.aureus ATCC 25923 and multidrug resistant Gram-positivebacteria (MRSA and S.pyogens) recovered from clinical specimens and Candida albicans ATCC 10231.The extract exhibited no antifungal activity.The results of MIC of the methanolic plant extract using tube dilution method against both MRSA and S.pyogenes clinical isolates is equal to 10.4 mg/ml.While no effect was observed against Gram-negative E.coli standard strain.Differences in structures between Gram positive and Gram-negative bacteria makes the plant extracts and different antibiotics have different activity against Gram positive and Gram-negative bacteria.The structure of Gram-negative bacteria is more complex and make them more resistant to different antimicrobial agents (natural compounds as plant extracts or antibiotics).

Figure 1 .
Figure 1.Total Ion Chromatogram (TIC) of essential oil of the non-flowering aerial parts of A. columnaris.

Figure 2 .
Figure 2. I. 3D-overlay of Quinazolinone and the proposed library in PBP2a allosteric site.II.3D-overlay of Piperacillin and the proposed library in PBP2a active site.

Figure 3 .
Figure 3. Top ranked 2D-poses of Quinazolinone and the proposed library showing interactions in PBP2a allosteric site.

Figure 4 .
Figure 4. Top ranked 2D-poses of Piperacillin and the proposed library showing interactions in PBP2a active site.