All media and medium components were procured from Hi-Media Laboratories, Mumbai. All organic solvents and other chemicals were purchased from Qualigens Fine Chemicals, India.
Bacterial strains and culture conditions:Pseudomonas aeruginosa PAO1 and Acinetobacter baumannii MTCC1425 were used as reference strains. Escherichia coli MG4/pKDT17 and Chomobacterium violaceum ATCC12472 were used as AHL reporter strain. Staphylococcus aureus MTCC737 strain was used for LasA protease assay. Luria Bertani (LB) medium was used for the bacterial growth. E. coli MG4/pKDT17 was grown in LB medium containing 100 μg ampicillin.
Tests and controls: CEE dissolved in 100 per cent dimethyl sulphoxide (DMSO), was used as the test in all assays. Azithromycin dihydrate (Sisco Research Laboratories, India), dissolved in sterile phosphate buffered saline (PBS), pH 6.0, was used as positive control for QS inhibition, and inhibition of QS regulated virulence and biofilm development8 (link). Azithromycin was used at a standardized concentration of 4.0 μg/ml. LB broth, with or without plant extract, was used as blank. DMSO and PBS were maintained as negative controls. Same volumes of the test and controls were used in all the assays.
Plant material collection and authentication: Dried stem of Tinospora cordifolia (Willd.) Miers was purchased from local vendor, authenticated and voucher deposited (Number: S/B-122) at Agharkar Research Institute, Pune, Maharashtra, India.
Preparation of CEE: Dried and powdered, stem (100 g) was macerated in 500 ml of ethyl acetate at ambient temperature (27°C) with intermittent shaking for 48 h. The extract was filtered using Whatman filter paper no. 1, dried in hot air oven at 45°C, and stored at 4°C in amber bottle.
Anti-QS activity at different concentrations: C. violaceum pigment production assay and long acyl-HSL production assay7 were performed to determine the anti-QS activity of plant extract (CEE) at the concentration range of 0.2 to 10.0 mg/ml (0.2, 0.5, 1, 2, 4, 6, 8, 10). Further, IC50 was calculated using regression analysis. The IC50 value was used as a basis to decide the test concentration for further assays.
C. violaceum pigment production assay:C. violaceum produces a short acyl-HSL (C6-HSL) which regulates production of dark purple pigment, violacein. Thus, inhibition of short acyl-HSL in C. violaceum is indicated by inhibition of violacein production. Agar well diffusion assay was performed to evaluate inhibition of short acyl-HSLs by CEE7 .
Long acyl-HSL production assay: The ability of CEE to inhibit long acyl-HSLs in P. aeruginosa and A. baumannii was tested using the reporter strain, E. coli MG4/pKDT177 . This strain expresses β-galactosidase gene in response to the exogenous addition of long acyl-HSLs. The enzyme activity thus corresponds to the amount of long acyl-HSLs added, and decrease in enzyme activity indicates long acyl-HSL inhibition. Overnight bacterial cultures of P. aeruginosa and A. baumannii were diluted with fresh LB broth, up to an OD600 of 0.2. Then, 0.1 ml of CEE (final concentration of 0.2 to 10.0 mg/ml)/controls was added to 4.9 ml of the diluted culture and incubated at 37 °C for
24 h. Acyl-HSL obtained in the culture supernatant was extracted with ethyl acetate, twice; ethyl acetate layers were pooled and evaporated to dryness under nitrogen. The dried extract was reconstituted in LB broth and added to 2.0 ml of overnight grown reporter strain culture and incubated for five hours at 37° C to induce the expression of β-galactosidase. The enzyme activity was determined as described by Miller using ortho-nitrophenyl-ß-galactopyranoside (ONPG) as substrate9 and expressed in terms of Miller units.
Collection of clinical isolates: Clinical isolates of P. aeruginosa and A. baumannii, obtained from wound infections, respiratory infections, urinary tract infections and bloodstream infections, were obtained from local government hospitals. The isolates were maintained as 20 per cent glycerol stocks at - 80°C.
Qualitative AHL detection: A modified protocol of cross feeding assay10 (link) was followed. The bacterial isolates were cross streaked in concentric circles rather than parallel straight lines. The test strain (clinical isolates of P. aeruginosa and A. baumannii) was spot inoculated and the reporter strain (E. coli MG4/pKDT17) was streaked around it, one cm apart, on LB agar, previously spread with 40 μl of 20 mg/ml X-gal solution (Sigma-Aldrich, USA). Reference strains were used as positive control, and reporter strain as negative control. Blue colouration in the growth of reporter strain suggested AHL production.
Qualitative protease detection: Clinical isolates of P. aeruginosa were spot inoculated on skim milk agar plates and incubated at 37°C. After overnight incubation, halo around growth indicated protease activity. Reference strain (PAO1) was used as positive control.
Typing of clinical isolates of P. aeruginosa: The procedure of pyocin typing given by Gillies and Govan11 (link) was modified. Due to unavailability of the Wahba indicator strains, the clinical isolates were cross streaked among themselves to check for growth inhibition of each isolate by the remaining isolates. Thus, depending on the growth inhibition pattern, the isolates were grouped.
Effect of CEE on virulence of P. aeruginosa: CEE, dissolved in DMSO, was used at a final concentration of 5 mg/ml (rounded off IC50 value) to evaluate its activity against the production of extracellular virulence factors in reference (PAO1) and clinical isolates (K04 and C03) of P. aeruginosa. The clinical strains, K04 and C03, formed the representatives for group I and group II isolates (classified on the basis of pyocin typing), respectively. Reduced enzyme activity or pyocyanin concentration in comparison to negative control suggested virulence inhibition. Per cent inhibition was calculated as [(enzyme activity/pyocyanin concentration for negative control - enzyme activity/pyocyanin concentration for CEE/azithromycin)×100] ÷ [enzyme activity/pyocyanin concentration for negative control].
Las A staphylolytic protease assay: The method of Kessler et al12 (link) was followed, with modifications. The reaction volume was maintained at 2.0 ml instead of 1.0 ml, and the reaction temperature was optimized at 37 °C. P. aeruginosa was inoculated in 5 ml LB broth containing controls/CEE, up to an OD600 value of 0.2, and incubated for 24 h, at 37°C. Culture was centrifuged at 8,000×g, 4°C. 0.5 ml of culture supernatant was mixed with 1.5 ml of boiled (15 min) S. aureus suspension (OD600 value of ~0.6) in 0.02 M Tris-Cl buffer (pH 8.5). OD600 was measured at 0 and 3 h post incubation at 37°C, using Perkin-Elmer UV-Vis Spectrophotometer Lambda 25 model. Enzyme activity was expressed as units/ml; 1 unit corresponding to decrease in OD by 0.01.
Las B elastase assay: Enzyme activity was studied as per the method of Pearson et al13 (link) with modifications. The bacterial cultures were grown in LB broth instead of peptone tryptic soy broth (PTSB), the substrate concentration was decreased from 20 to 10 mg Elastin-Congo Red (ECR) per ml of buffer and the incubation time was optimized to 24 h. P. aeruginosa was inoculated in 5 ml LB broth containing controls/CEE, up to an OD600 value of 0.2, and incubated for 24 h, at 37°C. Culture was centrifuged at 8,000×g, 4°C. One ml of buffer (Tris-Cl 0.1 M, CaCl2 1 mM, pH 7.5) containing 10 mg ECR substrate (Sigma-Aldrich, USA) was mixed with 0.5 ml of culture supernatant, incubated at 37°C, 24 h. The tubes were centrifuged at 8,000×g, 4°C and OD495 of the supernatant was measured. Enzyme activity was expressed as units/ml, where 1 unit represents increase in OD by 0.01.
Pyocyanin production assay: The procedure given by Huerta et al14 was followed, with modifications. The bacterial strains were grown in King's medium B for 48 h instead of culturing in LB medium with 24 h incubation. P. aeruginosa was inoculated in King's medium B broth containing controls/CEE up to OD600 value of 0.2 and incubated for 48 h, at 37°C. Pyocyanin was extracted from the culture supernatant using chloroform and measured spectrophotometrically at 690 nm. Pyocyanin concentration was calculated as mg/ml using the formula, OD690 ÷ 1615 (link).
Effect of CEE on biofilm formation of P. aeruginosa and A. baumannii: Biofilm formation of the reference strains and clinical isolates of the organisms was evaluated using 96-well microtiter plate assay, as per the method of Mathur et al16 (link), with modifications. LB broth supplemented with glucose was used for biofilm growth, biofilm fixation was done using methanol, and the bound dye was dissolved in ethanol before measuring its absorbance at 490 nm. An aliquots (200 μl) of overnight cultures diluted in LB broth (supplemented with 0.5% glucose) was added to the microtiter wells. CEE, dissolved in DMSO, was added at the concentration of 5.0 mg/ml. For positive control, azithromycin dihydrate, dissolved in PBS, pH 6.0, was used at a 4 μg/ml concentration and DMSO/PBS was used as negative control. The plates were incubated for 24, 48 and 72 h, at 37 °C. Following incubation, the contents in the wells were removed, washed with distilled water, and the adherent biofilm was fixed with methanol for 15 min. This was followed by staining with 1 per cent crystal violet dye for 15 min. Excess stain was removed, the wells were washed with distilled water and the remaining bound dye was resolubilized in 95 per cent ethanol for five min. OD490 was measured using BioRad Microplate reader Model 680 (Hercules, CA, USA). Per cent inhibition was calculated as [(biofilm density for negative control - biofilm density for CEE/azithromycin)×100] ÷ [biofilm density for negative control].
Phytochemical class detection of CEE: Standard chemical tests17 were performed for detection of carbohydrates, proteins, alkaloids, tannins, flavonoids, cardiac glycosides, anthroquinone glycosides, coumarin glycosides, saponin glycosides and steroids.
Activity guided fractionation of CEE: CEE (5 g) was subjected to silica gel (60-120 mesh) (Merck Co., India) column chromatography. A glass column of 600 mm length, 35 mm internal diameter and volume capacity of 600 ml, was packed with a silica bed of about 270 mm length. CEE was separately adsorbed on silica gel, dried, and loaded over the silica bed. Gradient elution was carried out using 100 per cent petroleum ether, followed by increasing concentration of ethyl acetate, up to 100 per cent ethyl acetate, and then increasing polarity with methanol, upto 80 per cent methanol. About 50 ml fraction volume was collected, and based on thin layer chromatography (TLC) profile, the fractions were pooled. In total, 11 fractions were obtained and further evaluated for anti-QS, anti-virulence, anti-biofilm and anti-bacterial activities. The fraction showing significant (P<0.05) anti-QS, anti-virulence, anti-biofilm activities and absence of antibacterial activity, was subjected to GC-MS analysis.
Identification of components in active fraction: One mg of the fraction (fraction 5) was derivatized by silylation reaction using 100 μl N-methyl-trimethylsilyltrifluoroacetamide (MSTFA) (Sigma-Aldrich, USA) and 50 μl pyridine, at 60°C, for 30 min. The mixture was dried, redissolved in ethyl acetate and analyzed using GC-MS18 . Shimadzu GCMS-QP2010 ultra equipped with a quadrapole mass spectrometer (Tokyo, Japan) was used. Chromatographic separation was performed on Rtx®-5MS capillary column (Restek, PA, USA), coated with 0.25 μm film of cross bond® 5 per cent diphenyl/ 95 per cent dimethyl polysiloxane; dimensions 30 m length × 0.25 mm internal diameter, 0.25 μm film thickness. Helium was used as the carrier gas at a flow rate of 44.5 cm/sec. The oven temperature programme was 80°C for 2 min, 80 to 250°C at 15°C/min and 10 min at 250°C. Two μl of sample was injected in 1:75 split ratio. The injector temperature and the interface temperature was 250°C. The mass spectrometer was operated in electron positive-mode ionization, with 70 eV ionization voltage. Ion source temperature was 250°C. The data were obtained in full scan mode (total ion current-TIC); mass range 35 - 500 amu. Identification of the compounds was done by comparing their mass spectra with the reference spectra in National Institute of Standards and Technology (NIST) library (http://chemdata.nist.gov/ ).
Molecular docking: Schrödinger Small-Molecule Drug Discovery Suite Release 2013-1 (Schrödinger 2013, New York, USA) and the products included therein were used for performing various molecular modelling studies. Glide version 5.9 was used to dock the compounds isolated from the active fraction, native ligand and known LasR inhibitors19 (link) with two target proteins, LasI and LasR. A single structure file of AHL synthase, LasI from P. aeruginosa, is available in the Protein Data Bank (PDB;www.rcsb.org/ ) (PDB ID 1RO5). For LasR of P. aeruginosa, the structure showing LasR bound to its autoinducer (PDB ID 2UV0) was used. The protein structures were prepared using Protein Preparation wizard and subjected to Receptor Grid Generation using Glide 5.9. The generated grid was used for docking the compounds in the active site. Similarly, the structures of the compounds identified in fraction 5 were built in Maestro. All the structures were then subjected to LigPrep2.6, using the default settings. This set of molecules was subjected to docking in the active site of target proteins. For docking studies, Extra Precision (XP) mode was used. The docked poses were minimized and root mean square deviation (RMSD) to input molecular geometries were calculated.
High performance (HP) TLC fingerprinting: HPTLC was performed for fingerprinting of the crude extract (CEE) and the column fractions using two mobile phases; n-hexane:ethyl acetate::17:3 which is known to separate diterpenoids20 and a general separation system, toluene:ethylacetate:methanol:formic acid::8:2:1:1.HPTLC was performed on 10×10 cm aluminium plates coated with 0.25 mm layer of silica gel 60 F 254 (Merck, Germany); 20 μl of the extract (10 mg/ml in ethyl acetate) was applied as 8 mm wide and 8.0 mm apart, at a rate of 5 μl/sec, using a DESAGA Applicator AS 30 sample applicator (Sarstedt, Nümbrecht, Germany) equipped with a 100-μl Hamilton (USA) syringe. The plates were air dried, allowed to develop up to 80 mm in a pre-saturated (30 min) glass twin trough chamber containing 20 ml of the respective mobile phase. After development, plates were dried and visualized under a UV cabinet at 254 and 366 nm. The plates were scanned at 254 and 366 nm, using a HPTLC Densitometer CD 60, 230V, equipped with ProQuant software (Sarstedt, Nümbrecht, Germany). The plates were derivatized using Anisaldehyde sulphuric acid reagent21 and again scanned at 366 nm and 540 nm.
Statistical analysis: The experimental data were analyzed using paired t test, Wilcoxon signed rank test, one way analysis of variance (ANOVA) with the post hoc test, Dunnett's test, considering the level of significance (P<0.05). The statistical analysis software, SPSS 17.0 (Chicago, USA) was used.
Bacterial strains and culture conditions:Pseudomonas aeruginosa PAO1 and Acinetobacter baumannii MTCC1425 were used as reference strains. Escherichia coli MG4/pKDT17 and Chomobacterium violaceum ATCC12472 were used as AHL reporter strain. Staphylococcus aureus MTCC737 strain was used for LasA protease assay. Luria Bertani (LB) medium was used for the bacterial growth. E. coli MG4/pKDT17 was grown in LB medium containing 100 μg ampicillin.
Tests and controls: CEE dissolved in 100 per cent dimethyl sulphoxide (DMSO), was used as the test in all assays. Azithromycin dihydrate (Sisco Research Laboratories, India), dissolved in sterile phosphate buffered saline (PBS), pH 6.0, was used as positive control for QS inhibition, and inhibition of QS regulated virulence and biofilm development8 (link). Azithromycin was used at a standardized concentration of 4.0 μg/ml. LB broth, with or without plant extract, was used as blank. DMSO and PBS were maintained as negative controls. Same volumes of the test and controls were used in all the assays.
Plant material collection and authentication: Dried stem of Tinospora cordifolia (Willd.) Miers was purchased from local vendor, authenticated and voucher deposited (Number: S/B-122) at Agharkar Research Institute, Pune, Maharashtra, India.
Preparation of CEE: Dried and powdered, stem (100 g) was macerated in 500 ml of ethyl acetate at ambient temperature (27°C) with intermittent shaking for 48 h. The extract was filtered using Whatman filter paper no. 1, dried in hot air oven at 45°C, and stored at 4°C in amber bottle.
Anti-QS activity at different concentrations: C. violaceum pigment production assay and long acyl-HSL production assay7 were performed to determine the anti-QS activity of plant extract (CEE) at the concentration range of 0.2 to 10.0 mg/ml (0.2, 0.5, 1, 2, 4, 6, 8, 10). Further, IC50 was calculated using regression analysis. The IC50 value was used as a basis to decide the test concentration for further assays.
C. violaceum pigment production assay:C. violaceum produces a short acyl-HSL (C6-HSL) which regulates production of dark purple pigment, violacein. Thus, inhibition of short acyl-HSL in C. violaceum is indicated by inhibition of violacein production. Agar well diffusion assay was performed to evaluate inhibition of short acyl-HSLs by CEE7 .
Long acyl-HSL production assay: The ability of CEE to inhibit long acyl-HSLs in P. aeruginosa and A. baumannii was tested using the reporter strain, E. coli MG4/pKDT177 . This strain expresses β-galactosidase gene in response to the exogenous addition of long acyl-HSLs. The enzyme activity thus corresponds to the amount of long acyl-HSLs added, and decrease in enzyme activity indicates long acyl-HSL inhibition. Overnight bacterial cultures of P. aeruginosa and A. baumannii were diluted with fresh LB broth, up to an OD600 of 0.2. Then, 0.1 ml of CEE (final concentration of 0.2 to 10.0 mg/ml)/controls was added to 4.9 ml of the diluted culture and incubated at 37 °C for
24 h. Acyl-HSL obtained in the culture supernatant was extracted with ethyl acetate, twice; ethyl acetate layers were pooled and evaporated to dryness under nitrogen. The dried extract was reconstituted in LB broth and added to 2.0 ml of overnight grown reporter strain culture and incubated for five hours at 37° C to induce the expression of β-galactosidase. The enzyme activity was determined as described by Miller using ortho-nitrophenyl-ß-galactopyranoside (ONPG) as substrate9 and expressed in terms of Miller units.
Collection of clinical isolates: Clinical isolates of P. aeruginosa and A. baumannii, obtained from wound infections, respiratory infections, urinary tract infections and bloodstream infections, were obtained from local government hospitals. The isolates were maintained as 20 per cent glycerol stocks at - 80°C.
Qualitative AHL detection: A modified protocol of cross feeding assay10 (link) was followed. The bacterial isolates were cross streaked in concentric circles rather than parallel straight lines. The test strain (clinical isolates of P. aeruginosa and A. baumannii) was spot inoculated and the reporter strain (E. coli MG4/pKDT17) was streaked around it, one cm apart, on LB agar, previously spread with 40 μl of 20 mg/ml X-gal solution (Sigma-Aldrich, USA). Reference strains were used as positive control, and reporter strain as negative control. Blue colouration in the growth of reporter strain suggested AHL production.
Qualitative protease detection: Clinical isolates of P. aeruginosa were spot inoculated on skim milk agar plates and incubated at 37°C. After overnight incubation, halo around growth indicated protease activity. Reference strain (PAO1) was used as positive control.
Typing of clinical isolates of P. aeruginosa: The procedure of pyocin typing given by Gillies and Govan11 (link) was modified. Due to unavailability of the Wahba indicator strains, the clinical isolates were cross streaked among themselves to check for growth inhibition of each isolate by the remaining isolates. Thus, depending on the growth inhibition pattern, the isolates were grouped.
Effect of CEE on virulence of P. aeruginosa: CEE, dissolved in DMSO, was used at a final concentration of 5 mg/ml (rounded off IC50 value) to evaluate its activity against the production of extracellular virulence factors in reference (PAO1) and clinical isolates (K04 and C03) of P. aeruginosa. The clinical strains, K04 and C03, formed the representatives for group I and group II isolates (classified on the basis of pyocin typing), respectively. Reduced enzyme activity or pyocyanin concentration in comparison to negative control suggested virulence inhibition. Per cent inhibition was calculated as [(enzyme activity/pyocyanin concentration for negative control - enzyme activity/pyocyanin concentration for CEE/azithromycin)×100] ÷ [enzyme activity/pyocyanin concentration for negative control].
Las A staphylolytic protease assay: The method of Kessler et al12 (link) was followed, with modifications. The reaction volume was maintained at 2.0 ml instead of 1.0 ml, and the reaction temperature was optimized at 37 °C. P. aeruginosa was inoculated in 5 ml LB broth containing controls/CEE, up to an OD600 value of 0.2, and incubated for 24 h, at 37°C. Culture was centrifuged at 8,000×g, 4°C. 0.5 ml of culture supernatant was mixed with 1.5 ml of boiled (15 min) S. aureus suspension (OD600 value of ~0.6) in 0.02 M Tris-Cl buffer (pH 8.5). OD600 was measured at 0 and 3 h post incubation at 37°C, using Perkin-Elmer UV-Vis Spectrophotometer Lambda 25 model. Enzyme activity was expressed as units/ml; 1 unit corresponding to decrease in OD by 0.01.
Las B elastase assay: Enzyme activity was studied as per the method of Pearson et al13 (link) with modifications. The bacterial cultures were grown in LB broth instead of peptone tryptic soy broth (PTSB), the substrate concentration was decreased from 20 to 10 mg Elastin-Congo Red (ECR) per ml of buffer and the incubation time was optimized to 24 h. P. aeruginosa was inoculated in 5 ml LB broth containing controls/CEE, up to an OD600 value of 0.2, and incubated for 24 h, at 37°C. Culture was centrifuged at 8,000×g, 4°C. One ml of buffer (Tris-Cl 0.1 M, CaCl2 1 mM, pH 7.5) containing 10 mg ECR substrate (Sigma-Aldrich, USA) was mixed with 0.5 ml of culture supernatant, incubated at 37°C, 24 h. The tubes were centrifuged at 8,000×g, 4°C and OD495 of the supernatant was measured. Enzyme activity was expressed as units/ml, where 1 unit represents increase in OD by 0.01.
Pyocyanin production assay: The procedure given by Huerta et al14 was followed, with modifications. The bacterial strains were grown in King's medium B for 48 h instead of culturing in LB medium with 24 h incubation. P. aeruginosa was inoculated in King's medium B broth containing controls/CEE up to OD600 value of 0.2 and incubated for 48 h, at 37°C. Pyocyanin was extracted from the culture supernatant using chloroform and measured spectrophotometrically at 690 nm. Pyocyanin concentration was calculated as mg/ml using the formula, OD690 ÷ 1615 (link).
Effect of CEE on biofilm formation of P. aeruginosa and A. baumannii: Biofilm formation of the reference strains and clinical isolates of the organisms was evaluated using 96-well microtiter plate assay, as per the method of Mathur et al16 (link), with modifications. LB broth supplemented with glucose was used for biofilm growth, biofilm fixation was done using methanol, and the bound dye was dissolved in ethanol before measuring its absorbance at 490 nm. An aliquots (200 μl) of overnight cultures diluted in LB broth (supplemented with 0.5% glucose) was added to the microtiter wells. CEE, dissolved in DMSO, was added at the concentration of 5.0 mg/ml. For positive control, azithromycin dihydrate, dissolved in PBS, pH 6.0, was used at a 4 μg/ml concentration and DMSO/PBS was used as negative control. The plates were incubated for 24, 48 and 72 h, at 37 °C. Following incubation, the contents in the wells were removed, washed with distilled water, and the adherent biofilm was fixed with methanol for 15 min. This was followed by staining with 1 per cent crystal violet dye for 15 min. Excess stain was removed, the wells were washed with distilled water and the remaining bound dye was resolubilized in 95 per cent ethanol for five min. OD490 was measured using BioRad Microplate reader Model 680 (Hercules, CA, USA). Per cent inhibition was calculated as [(biofilm density for negative control - biofilm density for CEE/azithromycin)×100] ÷ [biofilm density for negative control].
Phytochemical class detection of CEE: Standard chemical tests17 were performed for detection of carbohydrates, proteins, alkaloids, tannins, flavonoids, cardiac glycosides, anthroquinone glycosides, coumarin glycosides, saponin glycosides and steroids.
Activity guided fractionation of CEE: CEE (5 g) was subjected to silica gel (60-120 mesh) (Merck Co., India) column chromatography. A glass column of 600 mm length, 35 mm internal diameter and volume capacity of 600 ml, was packed with a silica bed of about 270 mm length. CEE was separately adsorbed on silica gel, dried, and loaded over the silica bed. Gradient elution was carried out using 100 per cent petroleum ether, followed by increasing concentration of ethyl acetate, up to 100 per cent ethyl acetate, and then increasing polarity with methanol, upto 80 per cent methanol. About 50 ml fraction volume was collected, and based on thin layer chromatography (TLC) profile, the fractions were pooled. In total, 11 fractions were obtained and further evaluated for anti-QS, anti-virulence, anti-biofilm and anti-bacterial activities. The fraction showing significant (P<0.05) anti-QS, anti-virulence, anti-biofilm activities and absence of antibacterial activity, was subjected to GC-MS analysis.
Identification of components in active fraction: One mg of the fraction (fraction 5) was derivatized by silylation reaction using 100 μl N-methyl-trimethylsilyltrifluoroacetamide (MSTFA) (Sigma-Aldrich, USA) and 50 μl pyridine, at 60°C, for 30 min. The mixture was dried, redissolved in ethyl acetate and analyzed using GC-MS18 . Shimadzu GCMS-QP2010 ultra equipped with a quadrapole mass spectrometer (Tokyo, Japan) was used. Chromatographic separation was performed on Rtx®-5MS capillary column (Restek, PA, USA), coated with 0.25 μm film of cross bond® 5 per cent diphenyl/ 95 per cent dimethyl polysiloxane; dimensions 30 m length × 0.25 mm internal diameter, 0.25 μm film thickness. Helium was used as the carrier gas at a flow rate of 44.5 cm/sec. The oven temperature programme was 80°C for 2 min, 80 to 250°C at 15°C/min and 10 min at 250°C. Two μl of sample was injected in 1:75 split ratio. The injector temperature and the interface temperature was 250°C. The mass spectrometer was operated in electron positive-mode ionization, with 70 eV ionization voltage. Ion source temperature was 250°C. The data were obtained in full scan mode (total ion current-TIC); mass range 35 - 500 amu. Identification of the compounds was done by comparing their mass spectra with the reference spectra in National Institute of Standards and Technology (NIST) library (
Molecular docking: Schrödinger Small-Molecule Drug Discovery Suite Release 2013-1 (Schrödinger 2013, New York, USA) and the products included therein were used for performing various molecular modelling studies. Glide version 5.9 was used to dock the compounds isolated from the active fraction, native ligand and known LasR inhibitors19 (link) with two target proteins, LasI and LasR. A single structure file of AHL synthase, LasI from P. aeruginosa, is available in the Protein Data Bank (PDB;
High performance (HP) TLC fingerprinting: HPTLC was performed for fingerprinting of the crude extract (CEE) and the column fractions using two mobile phases; n-hexane:ethyl acetate::17:3 which is known to separate diterpenoids20 and a general separation system, toluene:ethylacetate:methanol:formic acid::8:2:1:1.HPTLC was performed on 10×10 cm aluminium plates coated with 0.25 mm layer of silica gel 60 F 254 (Merck, Germany); 20 μl of the extract (10 mg/ml in ethyl acetate) was applied as 8 mm wide and 8.0 mm apart, at a rate of 5 μl/sec, using a DESAGA Applicator AS 30 sample applicator (Sarstedt, Nümbrecht, Germany) equipped with a 100-μl Hamilton (USA) syringe. The plates were air dried, allowed to develop up to 80 mm in a pre-saturated (30 min) glass twin trough chamber containing 20 ml of the respective mobile phase. After development, plates were dried and visualized under a UV cabinet at 254 and 366 nm. The plates were scanned at 254 and 366 nm, using a HPTLC Densitometer CD 60, 230V, equipped with ProQuant software (Sarstedt, Nümbrecht, Germany). The plates were derivatized using Anisaldehyde sulphuric acid reagent21 and again scanned at 366 nm and 540 nm.
Statistical analysis: The experimental data were analyzed using paired t test, Wilcoxon signed rank test, one way analysis of variance (ANOVA) with the post hoc test, Dunnett's test, considering the level of significance (P<0.05). The statistical analysis software, SPSS 17.0 (Chicago, USA) was used.