EXPERIMENTAL INVESTIGATIONS ON CAMELLIA KISSI WALL. FOR ANTIOXIDANT, ANTI-QUORUM SENSING AND ANTI-BIOFILM ACTIVITIES

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February – March 2020, vol. 9, no. 4
pages: 736-741
Article type: Microbiology of Microbiology
DOI: 10.15414/jmbfs.2020.9.4.736-741
Abstract: Plants are known for their widespread biological activities with special reference to the use in folkloric medicines for the treatment of several diseases and metabolic disorders from ancient times. The presence of bioactive phytochemicals especially phenolic compounds, tocopherol, phytol etc. are responsible for the potential bioactivities of plants. In the present study, the radical scavenging potential of ethanolic extract of Camellia kissi wall. was evaluated. In addition, the effect of C. kissi wall. on quorum sensing (QS) associated virulence and biofilm formation in Pseudomonas aeruginosa PAO1 was also investigated. The crude extract of C. kissi wall exhibited a significant antioxidant activity against DPPH and hydroxyl radicals with a scavenging percentage of 73.77 ± 3.58 and 75.3 ± 4.45 % respectively. The plant extract also significantly inhibited the QS regulated pyocyanin production, bacterial motility and recalcitrant biofilm formation in P. aeruginosa PAO1. The anti-biofilm activity was confirmed by confocal laser scanning microscopic (CLSM) analysis. The in vitro anti QS activity of C. kissi wall. was further confirmed by molecular docking studies specifically targeting the QS transcriptional regulatory protein, LasR. The present result will provide ample avenues to exploit medicinal plants in attenuating the QS regulated microbial infections and oxidative stress in the post-antibiotic era.
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EXPERIMENTAL INVESTIGATIONS ON CAMELLIA KISSI WALL. FOR ANTIOXIDANT, ANTI-QUORUM SENSING AND ANTI-BIOFILM ACTIVITIES


AUTHORS

Nagaraju Jalli, Santhi Sri KV, Sairengpuii Hnamte, Subhaswaraj Pattnaik, Parasuraman Paramanantham, Busi Siddhardha

ABSTRACT

Plants are known for their widespread biological activities with special reference to the use in folkloric medicines for the treatment of several diseases and metabolic disorders from ancient times. The presence of bioactive phytochemicals especially phenolic compounds, tocopherol, phytol etc. are responsible for the potential bioactivities of plants. In the present study, the radical scavenging potential of ethanolic extract of Camellia kissi wall. was evaluated. In addition, the effect of C. kissi wall. on quorum sensing (QS) associated virulence and biofilm formation in Pseudomonas aeruginosa PAO1 was also investigated. The crude extract of C. kissi wall exhibited a significant antioxidant activity against DPPH and hydroxyl radicals with a scavenging percentage of 73.77 ± 3.58 and 75.3 ± 4.45 % respectively. The plant extract also significantly inhibited the QS regulated pyocyanin production, bacterial motility and recalcitrant biofilm formation in P. aeruginosa PAO1. The anti-biofilm activity was confirmed by confocal laser scanning microscopic (CLSM) analysis. The in vitro anti QS activity of C. kissi wall. was further confirmed by molecular docking studies specifically targeting the QS transcriptional regulatory protein, LasR. The present result will provide ample avenues to exploit medicinal plants in attenuating the QS regulated microbial infections and oxidative stress in the post-antibiotic era.


KEYWORDS

Antioxidant, ROS, Biofilm, Quorum sensing, Molecular docking, CLSM

INTRODUCTION

In the metabolic process of living organisms, oxidation reactions represent an intermediate step and produce free radicals. However, imbalance in the production and subsequent discharge of these free radicals during the oxidation steps leads to the production of highly reactive oxygen species (ROS) which have the authority to damage the biological macromolecules such as nucleic acids, carbohydrates, proteins and lipids (Meena et al., 2012; Subhaswaraj et al., 2017a; Rajkumari et al., 2018). The generation of free radicals or ROS is not only dute to the metabolic imbalance but also elevate during environmental stress and chronic bacterial infections (Cap et al., 2012). The chronic bacterial infections are generally associated with highly complex, cell-density dependent cellular communication phenomenon called quorum sensing (QS). The QS network constitutes the production of specific signaling molecules with respect to cellular density, their interaction with specific cognate receptors and triggering the expression of virulence phenotypes and biofilm formation (Singh et al., 2009b). P. aeruginosa PAO1 is an opportunistic nosocomial Gram negative pathogenic bacterium cause cystic fibrosis, severe pulmonary infections and majority of hospital-acquired infections where QS network play an important role in pathogenesis. (Vasavi et al., 2016). The QS regulatory network also controls the adverse effect of ROS can be neutralized by in-built antioxidant machinery including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase, glutathione reductase, β-carotene and vitamin A, C and E (Rath et al., 2011). However, when the generation of ROS exceeds certain limit, the integral antioxidant machinery fails to scavenge the highly reactive free radicals wher exogenous supply of antioxidant is necessary to neutralize the ROS mediated oxidative stress. In this context, plant derived phytochemicals especially polyphenolic group of compounds reported for radical scavenging activity and neutralization of oxidative stress (Luis et al., 2016). C. kissi wall. belongs to the genus Camellia, has received considerable attention due to its application in beverages industries, pharmacological properties and antioxidant potential (Bashir et al., 2014).  In the present study, the ethanolic extract of C. kissi wall. was evaluated for its antioxidant ability to scavenge free radicals. The anti QS and anti-biofilm potential of the crude plant extract against P. aeruginosa PAO1 was also determined. The in vitro anti QS activity was further corroborated by molecular docking studies which provided an insight into the mechanism of QS inhibition.

MATERIALS AND METHODS

Collection of plant material and preparation of crude extract

The leaf samples of C. kissi wall. plant were collected from Mizoram, India. The leaf samples were thoroughly washed and dried. The dried plant materials were then homogenized into fine powder. Five grams of powdered plant material was soaked in ethanol (50 mL) for 2-3 days. The plant infusions were allowed to pass through Whattmann filter paper and the residues were collected and concentrated under vacuum on a rotary evaporator at 40 °C. The obtained crude extract was stored at 4 °C for further use.

Chemicals and reagents

The chemicals used in the present study are 2, 2-Diphenyl-1-picrylhydrazyl (DPPH), 2-deoxy ribose, ethylene diamine tetraacetic acid (EDTA), thiobarbituric acid (TBA) and acridine orange. The reagents used in the study are trichloroacetic acid (TCA), sulphuric acid (H2SO4), hydrochloric acid (HCl), ferric chloride (FeCl3), potassium ferricyanide [K3Fe(CN)6] and sodium hydroxide (NaOH). All the chemicals and reagents were procured from HiMedia laboratories Pvt Ltd, Mumbai, India.

Determination of antioxidant activity

DPPH free radical scavenging assay

The ability of ethanolic leaf extract of C. kissi wall. in scavenging DPPH free radicals was evaluated by the method described by Udayaprakash et al. (2015) with slight modifications. Briefly, 0.2 mM DPPH (in methanol) was mixed with different concentration of the sample (100–500 µg/mL) and incubated in the dark for 30 min at room temperature. After the incubation, the absorbance was measured at 517 nm. A control experiment was also set up without the plant extract. The DPPH radical scavenging activity (%) was calculated as per the following equation. The IC50 value was also determined.

Scavenging (%) = (Absorbance of the control – Absorbance of the treated sample) × 100
Absorbance of the control

Determination of reducing power

The reducing power assay was performed to determine the ability of C. kissi wall. ethanolic extract in reducing the ferric (Fe3+) to ferrous (Fe2+) form by forming Prussian blue complex at Briefly, a series of increasing concentrations of plant extract (100–500 µg/mL) were mixed with phosphate buffer (0.2 M, pH 6.6) and 1% [K3Fe(CN)6] in a ratio of 1:5:5. The reaction mixture was incubated at 50 °C for 20 min in a water bath. After incubation, the reaction was terminated by adding 10% w\v of TCA and the solution was centrifuged at 10,000 rpm for 10 min. The supernatant was diluted with deionized water and freshly prepared FeCl3 (0.1% w/v) in a ratio of 5:5:1. The absorbance of the reaction mixture was measured at 700 nm (Do et al., 2014).

Hydroxyl radical scavenging activity

The effect of ethanolic leaf extract of C. kissi wall. on scavenging of highly reactive free hydroxyl radicals was determined according to the method described by Tounkara et al. (2014) with slight modifications. Briefly, two sets of reactions were prepared by addition of sodium phosphate buffer (0.2 M, pH 7.0), 2-deoxyribose (10 mM), FeSO4-EDTA (10 mM), H2O2 (10 mM) with presence or absence of different concentration of sample (100–500 µg/mL). The reaction mixture was incubated at 37 °C for 4 h. After incubation, TCA (2.8% w/v) and TBA (1% w/v in NaOH) were added to the reaction mixture and boiled for 10 min followed by cooling to room temperature. The absorbance of the solution was then measured at 532 nm and hydroxyl radical scavenging was determined using the following equation,

Scavenging (%) = (Absorbance of the control – Absorbance of the treated sample) × 100
Absorbance of the control

Total antioxidant activities

The total antioxidant activity of ethanolic leaf extract of C. kissi wall. was determined by standard phosphomolybdate method. Briefly, different concentrations of leaf extract (100–500 µg/mL) were mixed with reagent solution (comprising of 0.6 M H2SO4, 28 mM sodium phosphate and 4 mM ammonium molybdate) in 1:9 ratios. The reaction mixture was incubated in a water bath at 95 °C for 90 min. After incubation, the reaction mixture was cooled down to room temperature and the absorbance was measured at 695 nm. The total antioxidant activity was expressed as ascorbic acid equivalents (Do et al., 2014).

Determination of anti QS and anti-biofilm activity

Bacterial strains and maintenance of culture

The anti QS activity of C. kissi wall. extract was determined against biomarker strain, C. violaceum (MTCC 2656) and test microorganism, P. aeruginosa PAO1. The anti-biofilm activity was evaluated against P. aeruginosa PAO1. Both the bacterial cultures were obtained from Microbial type culture collection (MTCC), IMTECH, Chandigarh, India.

Determination of minimum inhibitory concentration (MIC)

The MIC of C. kissi wall. extract against P. aeruginosa PAO1 was determined as per the recommendation of Clinical & Laboratory Standards Institute (CLSI, 2014) using broth macrodilutions method. All the anti QS and anti-biofilm activities were performed at sub-MIC concentration (El-Shaer et al., 2016).

Anti QS activity against biomarker strain, C. violaceum

Violacein inhibition activity

The effect of C. kissi wall. entrant on the production of violacein by C. violaceum was evaluated according to Husain et al., (2017) with slight modifications. Briefly, C. violaceum was grown in presence or absence of sub-MIC of C. kissi wall. at 30 °C for 24 h. After incubation period, the culture was centrifuged (10,000 rpm, 10 min) to precipitate the insoluble violacein and DMSO was added to the pellet to solubilize the violacein. The reaction mixture was recentrifuged (10,000 rpm, 10 min) and the absorbance of the resulting supernatant was measured at 585 nm.

Anti QS and anti-biofilm activities against P. aeruginosa PAO1

Pyocynanin inhibition activity

Pyocyanin is an important QS regulated virulence factor produced by P. aeruginosa PAO1. The effect on production of pyocyanin in presence of sub-MIC of C. kissi wall. was determined according to the method described by Husain et al. (2017) with slight modifications. Briefly, pyocyanin pigment was extracted from the cell-free supernatant of C. kissi wall. treated P. aeruginosa PAO1 by mixing the supernatant in chloroform in a 5:3 ratios. The reaction mixture was vortexed and the pyocyanin containing organic phase was re-extracted with HCl (0.2 M). The optical density of the aqueous phase was measured at 520 nm. A control experiment was performed without the addition of crude extract.

Effect on bacterial motility

The bacterial motility such as swimming and swarming is regulated by QS network and plays a critical role during biofilm formation and development. The effect of C. kissi wall. extract on swimming and swarming motility of P. aeruginosa PAO1 was determined according to Packiavathy et al. (2014) with slight modifications. Briefly, the plant extract treated P. aeruginosa PAO1 was point inoculated into specific swimming medium (composed of 1% tryptone, 0.5% NaCl and 0.3% agar agar) and swarming medium (composed of 1% bacteriological peptone, 05% NaCl, 0.5% filter sterilized glucose and 0.5% agar agar) and incubated at 37 °C.

Anti-biofilm activity using microscopic observation

The anti-biofilm activity of C. kissi wall. extract against P. aeruginosa PAO1 biofilm was determined according to the method described by Lewis Oscar et al. (2018) with slight modifications. Briefly, C. kissi wall. extract treated P. aeruginosa PAO1 was allowed to grow on the glass coverslips at 37 °C for 24 h. After the incubation, the adhered biofilms on the coverslip were stained with acridine orange (0.1% w/v) for 10 min under dark condition. After incubation, the excess stain was removed from the coverslip and observed under CLSM (LSM 710, Carl Zeiss, Germany). A control experiment was also performed for P. aeruginosa PAO1 without the plant extract.

Gas Chromatography-Mass Spectrometric (GC-MS) analysis

The phytochemical profile of ethanolic leaf extract of C. kissi wall. was analyzed by GC-MS (Thermo GC-Trace Ultra Version: 5.0, Thermo MS DSQ II equipped with a DB 35 – MS Capillary Standard non-polar column with dimensions of 30 mm × 0.25 mm ID × 0.25 μm film). Helium was used as a carrier gas with a constant flow rate of 1.0 mL/min. The injector was operated at 250 °C and the oven temperature was programmed as follows: 60 °C for 15 min, then gradually increased to 280 °C at 3 min. The phytochemicals present in the extract were identified based on the obtained spectrum, retention indices and NIST libraries (Gomathi et al., 2015).

Molecular docking studies

The docking studies were carried out in Schrodinger maestro software version 9.2 and the binding affinity of identified phytochemicals from GC-MS analysis and natural autoinducer to transcriptional receptor, LasR were analyzed. The ligand binding domain of LasR protein’s 3D-structure file (PDB ID: 2UV0) was retrieved from Protein Data Bank. The LasR protein was then subjected to preparation in protein preparation wizard of Schrodinger maestro software version 9.2. Grid generation was performed in Glide, version 5.7 in Schrodinger maestro software. For LasR protein grid were defined around the active site residues (Tyr-56, Trp-60, Asp-73, Thr-75 and Ser-129) where autoinducer C12-homoserine lactone (3-Oxo-C12-HSL) interacts with LasR protein (Bottomley et al., 2007). The above prepared grid was used for docking. The ligand compounds were obtained from PubChem database and submitted for preparation in Ligprep module 2.5 in Schrodinger suite and the prepared protein and ligand were subjected for docking.

Statistical analysis

All the experiments were performed in triplicates and the data was presented as mean ± standard deviation (SD). For each assay, a control experiment was performed without treating the test bacterium with plan extract.

RESULTS AND DISCUSSION

Determination of antioxidant activity

DPPH free radical scavenging activity

From the DPPH assay, C. kissi wall. extract exhibited significant DPPH radical scavenging in a concentration dependent manner with a scavenging potential of 24.0 ± 1.04 and 73.77 ± 3.58 % at a concentration of 100 and 500 µg/mL respectively. The plant extract showed an IC50 of 238.95 µg/mL (Figure 1).

Figure 1 DPPH free radical scavenging activity of different concentrations (100-500 µg/mL) of ethanolic leaf extract of C. kissi wall.

Reducing power activity

From reducing power assay experiment, it was observed that with the increase in concentration of C. kissi wall. extract from 100 to 500 µg/mL, a concomitant increase in the optical density from 0.3142 to 0.4935 was observed suggesting an increase in the reducing power (Figure 2). The ethanolic extract of C. kissi wall. showed an IC50 of 502.97 µg/mL for reducing power assay.

Figure 2 Determination of reducing power of different concentrations (100-500 µg/mL) of ethanolic leaf extract of C. kissi wall

Hydroxyl radical scavenging activity

A concentration dependent increase in hydroxyl radical scavenging was observed with concomitant increase in concentrations (100-500 µg/mL) of ethanolic leaf extract of C. kissi wall. with an IC50 of 256.97 µg/mL (Figure 3).

Figure 3 Hydroxyl radical scavenging activity of different concentrations (100-500 µg/mL) of ethanolic leaf extract of C. kissi wall.

Total antioxidant activity

Ethanolic leaf extract of C. kissi wall. Showed a concomitant increase in the absorbance from 1.46 ± 0.064 to 3.90 ± 0.126 with the concentration of 100 and 500 µg/mL respectively. The ethanolic leaf extract of C. kissi wall. showed an ascorbic acid equivalent of 137.12 µg/mL at 500 µg/mL (Figure 4).

Figure 4 Total antioxidant activity of different concentrations (100-500 µg/mL) of ethanolic leaf extract of C. kissi wall.

Anti QS and anti-biofilm activities of plant extract

Minimum Inhibitory concentration

The MIC of C. kissi wall. extract against P. aeruginosa PAO1 was observed to be 1000 µg/mL and sub-MIC was fixed at 250 and 500 µg/mL. All the anti QS and anti-biofilm activities were performed at both the sub-MIC concentrations.

Violacein inhibition activity against C. violaceum

On treatment with 250 and 500 µg/mL of C. kissi wall. extract, violacein production in biomarker strain, C. violaceum was inhibited by 56.25 ± 3.47 and 62.16 ± 3.21 % respectively (Figure 5).

Pyocyanin inhibition activity against P. aeruginosa PAO1

A concentration dependent increase in the inhibition of pyocyanin production was observed when P. aeruginosa PAO1 was treated with 250 and 500 µg/mL of plant extract with an inhibition of 50.27 ± 2.69 and 85.87 ± 4.40 % respectively (Figure 5).

Figure 5 Effect of sub-MIC concentrations of C. kissi wall. (250, 500 µg/mL) on production of violacein in biomarker strain, C. violaceum and pyocyanin in test microorganism, P. aeruginosa PAO1

Inhibition of bacterial motility

On treatment with sub-MIC (500 µg/mL) of ethanolic leaf extract of C. kissi wall., a significant decrease in swimming and swarming motility was observed as compared to untreated control (Figure 6).

Figure 6 Anti-swimming and anti-swarming activities of ethanolic leaf extract of C. kissi wall. (A) Swimming motility of P. aeruginosa PAO1 (untreated control), (B) Swimming motility of C. kissi wall. treated P. aeruginosa PAO1, (C) Swarming motility of P. aeruginosa PAO1 (untreated control), (D) Swarming motility of C kissi wall. treated P. aeruginosa PAO1.

Anti-biofilm activity using CLSM studies

C. kissi wall. extract exhibited a significant anti-biofilm activity against the 24 h biofilms of P. aeruginosa PAO1 as observed from CLSM analysis when compared to untreated control with comparatively thick and highly compact biofilm architecture (Figure 7).

Figure 7 Anti-biofilm activity of ethanolic leaf extract of C. kissi wall. against P. aeruginosa PAO1 biofilm (A) Biofilm formation in P. aeruginosa PAO1 (untreated control), (B) Inhibition of biofilm formation in P. aeruginosa PAO1 on treatment with C. kissi wall.

GC-MS analysis

From the GC-MS analysis and subsequent NIST library search, α-amyrin and β-amyrin were identified with 60.715 and 27.50 peak area (%) which were relatively higher than other identified phytoconstituents. The GC-MS spectrum and the identified compounds were presented in Table 1.

Table 1 GC-MS analysis of extract of C. kissi wall. List of phytochemicals identified from GC-MS analysis and their reported biological activities.

Sl no. Compound  name Peak area (%) Retention time

(min.)

Biological Activities References
1 phytol 2.06 16.68 Antioxidant, Anti Qs Santos et al 2013.Pejin et al 2015.
2 Olean-12-ene 3.658 29.09 Anti-inflammatory Hussien et al, 2006.
3 β-amyrin 27.5 29.45 Anti-bacterial Ogwuche et al,2008
4 α-amyrin 5.025 29.65 Anti-bacterial Fermandes et al,2013
5 2R-Acetoxymethyl-1,3,3-trimethyl-4t-(3-methyl-2buten-1-yl) -1T-cycloh 55.69 30.09 Anti-inflammatory Anupama et al , 2014
6 2,4,4-trimethyl-3 hydroxymethyl-5A-(3Methyl-but-2enyl) cyclohexen

 

6.068 31.17 Anti-bacterial Sivakumar And Gayathri, 2015.

Figure 8 GC-MS analysis of extract of C. kissi wall. GC-MS spectrum of C. kissi wall. extract,

Molecular Docking studies

Molecular docking studies of C. kissi wall. revealed that, phytol exhibited a docking score of – 6.891 kcal/mol for LasR which was relatively close to the binding energy of LasR with its natural ligand (-7.293 kcal/mol). Besides, 2R-Acetoxymethyl-1,3,3-trimethyl-4t-(3-methyl-2-buten-1-yl)-1T-cycloh and 2,4,4-Trimethyl-3-hydroxymethyl-5A-(3-methyl-but-2-enyl) cyclohexen also exhibited a docking score of -5.675 and -5.81 kcal/mol respectively (Table 2, Figure 9).

Table 2 Interaction of bioactive phytochemicals of C. kissi wall. with QS transcriptional regulatory protein, LasR of P. aeruginosa PAO1 expressed in terms of docking energy (kcal.mol), hydrogen bonds and hydrophobic residues

Sl no. Compounds/Ligands LasR
Docking score (kcal/mol) Hydrogen bond Hydrophobic residues
1. C12-HSL (Natural ligand) -7.293 Arg 61, Thr 75 Leu 36, Tyr 47, Trp 60, Tyr 64, Val 76, Tyr 93, Thr 115, Ser 129
2. Phytol -6.891 Leu 110 Tyr 47, Ala 50, Ile 52, Tyr 56, Arg 61, Tyr 64, Ala 70, Tyr 93, Phe 102
3. 2R-Acetoxymethyl-1,3,3-trimethyl-4t-(3-methyl-2-buten-1-yl)-1T-cycloh

 

-5.675 Tyr 47, Arg 61 Leu 36, Ile 52, Ala 70, Asp 73, Val 76, Phe 101
4. 2,4,4-Trimethyl-3-hydroxymethyl-5A-(3-methyl-but-2-enyl) cyclohexen -5.81 Tyr 93 Leu 36, Tyr 56, Trp 60, Thr 75, Phe 102, Ser 129

Figure 9 Molecular docking studies of the phytochemicals identified from the ethanolic leaf extract of C. kissi wall. and their interactions with the QS transcriptional regulator protein, LasR as compared to the interaction of natural ligand (C12-homoserine lactone). (A). 2D docked conformation of C12-HSL into the active pocket of LasR, (B) 2D docked conformation of phytol into the active site of LasR, (C) 2D docked conformation of 2R-Acetoxymethyl-1,3,3-trimethyl-4t-(3-methyl-2-buten-1-yl)-1T-cycloh into the active site of LasR, (D) 2D docked conformation of 2,4,4-Trimethyl-3-hydroxymethyl-5A-(3-methyl-but-2-enyl) cyclohexen into the active site of LasR.

DISCUSSION

From ancient times, India constitutes a global hotspot of diversified medicinal plants with tremendous ethnomedicinal values, being used as folkloric medicines for the treatment of diseases, disorders and microbial infections.  In the present study, the ethanolic extract of C. kissi wall. was evaluated for its antioxidant potential and ability to attenuate QS regulated virulence and biofilm formation. DPPH free radical assay is a standard method to evaluate the antioxidant potential of plant extract in determining the free radicals scavenge potential. C. kissi wall. showed significant DPPH scavenging with an IC50 of 238.95 µg/mL which was significantly higher than the Croton caudatus which showed an IC50 of 305.39 µg/mL (Subhaswaraj et al. 2017b). The reducing power assay provide the ability of plant extract in reducing Fe3+ to Fe2+, which corresponds to the radical scavenging potential. In the present study, C. kissi wall showed increase in reducing power with concomitant increase in the concentrations of extract, which was in accordance to the earlier report (Baba et al., 2015). Hydroxyl radicals are one of the important members of ROS causing severe damage to biological macromolecules. Plant extracts based on their phytochemicals composition showed differential rate of scavenging to highly reactive hydroxyl radicals. In the present study, C. kissi wall. exhibited an increase in the hydroxyl radical scavenging with subsequent increase in concentrations of crude extract with an IC50 of 256.97 µg/mL. This result was comparatively very high as compared to methanol and chloroform extract of Kedrostis foetidissima with an IC50 of 2.0 and 2.8 mg/mL (Pavithra and Vadivukkarasi, 2015). The total antioxidant activity was determined as per the formation of phosphomolybdate complex and expressed in terms of ascorbic acid equivalents. C. kissi wall. showed an ascorbic acid equivalent of 137.12 µg/mL which was nearly equal to the total antioxidant potential of Acacia nilotica leaf extract reported with an ascorbic acid equivalent of 152.79 µg/mL (Subhaswaraj et al., 2017a).

The QS regulatory network provides an attractive target for the development of novel anti-infectives from natural resources as it regulates the production of pathogenic determinants, biofilm formation, ROS generation and resistance to antibiotics.  In this regard, QS inhibition has emerged as a promising target to combat bacterial infections and associated health risks in the form of antibiotic resistance. At sub-MIC level, the ethanolic extract of C. kissi wall. significantly inhibited the production of violacein pigment as compared to untreated control in the biomarker strain, C. violaceum suggesting the efficacy of C. kissi wall. extract as potent QS inhibitor. P. aeruginosa utilizes a highly complex cascade of QS network for the production of an array of virulence phenotypes, siderophores, and most importantly several cytotoxic phenazine compounds, which play a critical role during host infection process. Pyocyanin is a highly toxic, ROS inducing phenazine compound and is an important biomarker during P. aeruginosa infection process and also involved in cytotoxicity (Chong et al., 2011). In the present study, C. kissi wall. significantly down-regulated the production of pycoyanin suggesting the ability of plant extract in minimizing the bacterial infection and cytotoxicity. The pyocyanin inhibition of 85.87 ± 4.40% was significantly higher than the earlier report where Terminalia bellerica showed 67.99 % at 500 µg/mL (Ganesh and Rai, 2018). The swarming motility in P. aeruginosa is regulated by flagellar synthesis and alteration in flagellar synthesis could lead to reduction in swarming motility which corresponds to reduction in biofilm formation (Husain et al., 2017). C. kissi wall. significantly inhibited the swarming motility of P. aeruginosa PAO1 as compared to untreated control suggesting its ability to combat flagellar-driven biofilm formation. The biofilm disruption ability of C. kissi wall. was further corroborated by CLSM analysis, which showed comparatively less thick and uncompact biofilm architecture (Packiavathy et al., 2014). The presence of phytol in the ethanolic leaf extract of C. kissi wall., as identified from GC-MS suggested the efficacy of plant extract in scavenging free radicals and also combating QS regulated virulence and biofilm formation in P. aeruginosa PAO1 (Santos et al., 2013; Pejin et al., 2015). From the molecular docking studies, it was observed that phytol exhibited promising docking affinity with LasR which is relatively close to that of natural ligand suggesting the efficacy of phytol in competitive binding with LasR and altering the LasR mediated bacterial virulence.

CONCLUSION

The present study demonstrated the widespread potential of C. kissi wall. in scavenging highly reactive free radicals thereby suggesting its use as antioxidant for exogenous supplementation for maintaining the balance in the living system. In addition, the plant extract also act as potent inhibitor of QS regulated virulence and biofilm formation in P. aeruginosa PAO1 by down-regulating the production of pyocyanin, inhibiting the bacterial motility and disrupting the recalcitrant biofilm architecture. The antioxidant and anti QS activity of C. kissi wall. could be attributed due to the presence of diverse group of phytochemicals including phytol, which has already been reported for antioxidant and anti QS properties. The present study will provide a lead in the antimicrobial drug discovery for the development of novel anti-infectives by recognizing the widespread potential of medicinal plants.

Acknowledgement: The authors are thankful to Bharathidasan University, Tiruchirappalli, Tamilnadu, India for providing the CLSM facility. The authors would also like to thank Sophisticated Instrumentation Facility (SIF), VIT University, Vellore, Tamilnadu, India for GC-MS analysis. The authors acknowledge the Centre for Bioinformatics, Pondicherry University for providing the molecular docking facility.

Conflict of interests: The authors declare no conflict of interests.

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