All fluoroquinolones, ethidium bromide reserpine, piperine, fluorescein isothiocyanate (FITC) and 4′,6-diamidino- 2-phenylindole (DAPI) were purchased from Sigma Chemical Co. (St. Louis, MO, United States). Microbiological media were purchased BD Microbiology Products (Sparks, MD, United States). All other chemicals were of analytical grade.

Staphylococcus aureus ATCC 29213, SA-1199B, SA-1199, SA-K1758, MT23142, and MRSA15187 were cultivated on trypticase soya agar (TSA). Important characteristics are described in Table 1.

Authentic samples of B. diffusa roots were collected from Jammu region (Voucher Specimen No. 21713), dried and powdered followed by solvent extraction in dichloromethane: methanol mixture (1:1 v/v) using cold maceration method. Crude extract was concentrated and purified by repeated silica gel column chromatography using hexane: ethyl acetate (95: 5 to 75: 25 v/v).

Drug potentiation or synergistic action of compound was assessed by broth checkerboard microdilution technique, which is most known method (Eliopoulos and Wennersten, 2002). Combination of ciprofloxacin and boeravinone B was tested in Mueller Hinton Broth (MHB; pH 7.0) against all bacterial strains using 96 well microtitre plates at a concentration range of 0.06–32 μg/ml and 0.8–50 μM, respectively. Additionally, combination of ethidium bromide and boeravinone B was assessed in similar way using S. aureus SA-1199B. Cell suspension of a density of 0.5 McFarland (≈1.5 × 10⁸ cfu per ml of Escherichia coli) was used as inoculum after diluting 1:100, a volume of 0.1 ml (5 × 10⁵) was added to plates. The plates were incubated at 37°C for 18–20 h. Reserpine and piperine (known efflux pump blocker) were used as the control in this study. Minimum effective concentration (MEC) of boeravinone B was determined which is defined as concentration at which there is atleast fourfold reductions in MIC of ciprofloxacin. In a similar way various other fluoroquinolones and ethidium bromide were also assessed for synergistic potential of boeravinone B.

Time-kill study of ciprofloxacin alone and in combination with boeravinone B was performed in 50 ml volume conical flasks containing 25 ml of MHB using the previously described method (Eliopoulos and Moellering, 1996). Ciprofloxacin at 2 μg/ml (0.25 × MIC) was tested alone and in combination with boeravinone B at the MEC concentration (12.5 μM) as determined above. Ciprofloxacin was also tested alone at an MIC of 8 μg/ml. NorA overexpressing S. aureus SA-1199B was used as the test bacterium. Ten folds serial dilutions of these aliquots were spotted on TSA plates.

Frequency of resistance (FOR) or Mutation prevention concentration (MPC) of ciprofloxacin against S. aureus ATCC 29213 was determined by method described elsewhere (Drugeon et al., 1999). MHA plates were supplemented with MIC, 2×, 4×, and 8× MIC of ciprofloxacin’s concentrations with or without boeravinone B at MEC. A bacterial suspension of 0.1 mL (10⁹ cfu) was plated on to each ciprofloxacin containing plates without and with boeravinone B. MPC was determined as the ratio of colonies appearing on ciprofloxacin or ciprofloxacin with boeravinone B combination to total colonies plated on a drug free plate.

Fluorescence based method as described by Brenwald et al. (1998) was used to determine the ethidium bromide efflux inhibitory as well accumulation studies. For this purpose a freshly grown NorA overexpressing S. aureus SA-1199B was suspended to an optical density of 0.2 in uptake buffer (110 mM NaCl, 7 mM KCl, 50 mM NH₄Cl, 0.4 mM Na₂HPO₄, 52 mM Tris base and 0.2% glucose, adjusted to pH 7.5 with HCl). Cells were loaded with 10 μg/ml ethidium bromide for 30 min at 37°C. Extracellular ethidium bromide was removed by centrifugation and pellet was resuspended in same volume of uptake buffer with and without boeravinone B (12.5 μM). Reserpine at 25 μg/ml was used as a known efflux pump inhibitor. For accumulation studies bacterial cells loaded with ethidium bromide for 30 min after which EPIs (boeravinone B and reserpine) were added. Loss or gain of fluorescence in presence of compound and known EPI was deduced at 530 and 600 nm for excitation and emission wavelength, respectively, using multimode reader Infinite 200 Pro (Tecan Mannedorf, Switzerland).

Effect of boeravinone B on the biofilm inhibitory potential of ciprofloxacin was studied with drug combination on biofilm formation, as previously described (Schillaci et al., 2010). For biofilm formation, cultures (S. aureus SA-1199 and SA-1199B) were grown overnight using TSB supplemented with 2% glucose (TSBG) and diluted to an optical density (570 nm) of 0.015 after diluting 1:200 (1 × 10⁹ cells/mL). Microtitre plates for inhibitory studies were prepared as described for synergistic activity with ciprofloxacin by checkerboard microdilution method. After 24 h, wells were washed thrice with PBS followed by fixation with methanol for 15 min and finally air-dried in inverted position at 37°C. The wells of the dried plates were stained with 0.1% (w/v) crystal violet for 10 min and rinsed thoroughly with water until the negative control wells (without biofilms) appeared colorless. To quantify biofilm formation, 0.2 mL of 95% ethanol was added to wells of plate that were stained with crystal violet. Immediately absorbance read at 595 nm using microplate reader. In a separate plate the contents were decanted and washed with PBS to remove the planktonic cells and again filled with fresh TSB. After incubation for 30 min, 15 μl of 0.04% resazurin and 12.5 μl of 20% Tween80 were added in each well of the plate to access the viability of the cells within biofilm. The plates were incubated for 15 min and fluorescence was determined at an excitation at 560 nm and emission at 590 nm (Tecan Infinite M200 microplate reader) as described previously (Bauer et al., 2013). Minimal biofilm inhibitory concentration (MBIC) is defined as the concentration at which absorbance was reduced by ≥90% or was nearly equivalent to media control wells.

The potentiating effect of boeravinone B was further confirmed by staining the biofilms with FITC and DAPI which stains protein (amine reactive) and nucleic acid (DNA), respectively, by modified method as described by Yang (using DAPI in place of Hoechst) followed by visualization by confocal laser scanning microscopy (Yang et al., 2015). The biofilm of S. aureus ATCC 29213 was grown in six well poly styrene plate (Nunc) containing sterile 18 mm glass cover slips. Ciprofloxacin was used at 4 μg/ml which is MBIC and at 1 μg/ml which is subMBIC (0.25× MBIC) as determined above. Boeravinone B at 12.5 μM (MBIC) was added to sub-inhibitory concentration of ciprofloxacin (0.25× MBIC). The plates were incubated for 24 h at 37°C. PBS washes were given to eliminate planktonic cells followed by fixation of biofilm with 5% para-formaldehyde for 1 h at 50°C. Fixed biofilm was flooded with 0.001% (w/v) FITC and 1 μg/ml DAPI at kept at room temperature for 1 h. Images were seen and captured on 40× oil immersion lens using CLSM (Olympus flou View 1000).

P-glycoprotein inhibitory activity was assessed by the method described earlier using colorectal cell line LS180 (Jin et al., 2012; Joshi et al., 2014). Cell monolayer was prepared in 96 well plate using Dulbecco’s Modified Eagle’s medium (DMEM) media. The media was decanted and replaced with Hank’s buffer containing 10 mM of Rh123 as a P-gp substrate. Twofold serial dilutions of boeravinone B (80–5 μM) were added and plate was incubated at 37°C in CO₂ incubator for 90 min. A known P-gp inhibitor elacridar (10 μM) was used as positive control. The contents of the wells were decanted and washed four times with cold PBS followed by cell lysis for 1 h using 200 μl of lysis buffer (0.1% Triton X 100 and 0.2 N NaOH). A total of 100 μl of lysate was used for reading fluorescence of Rh123 at an excitation at 485 nm and emission at 590 nm. All samples were normalized by dividing fluorescence of each sample with total protein present in the lysate. Half minimal inhibitory concentration (IC₅₀) value of boeravinone B was calculated using Graphpad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, United States). Data is expressed as mean ± SD or representative of one of three similar experiments. Comparisons were made between control and treated groups or the entire intra group using one way ANOVA with post Bonferroni test through GraphPad Prism 5.00.288 statistical analysis software. ^∗p-values <0.05 were considered significant.

Due to unavailability of crystal structures of human P-gp and S. aureus NorA, molecular docking studies were carried out using the homology models. The homology models for human P-gp (Jin et al., 2012) was prepared from Caenorhabditis elegans P-gp (PDB ID: 4AZF) and S. aureus NorA efflux pump (Aller et al., 2009) was prepared from glycerol 3-phosphate transporter pump from E. coli (PDB ID: IPW4) using Glide Induced Fit Docking in default setting. For Nor A docking studies, ciprofloxacin/reserpine binding site was used to construct grid file whereas for P-gp docking, verapamil binding site was used (Aller et al., 2009).

To assess the impact of boeravinone B on invasion of S. aureus in macrophage, previously described method was used (Cheung and Bayles, 2007). Murine macrophage cell line J774 maintained on Roswell Park Memorial Institute medium (RPMI) supplemented with 10% (v/v) fetal calf serum was used as host. The macrophages were grown in monolayer to a density to 10⁵ cells/well in 24 well plate in physiological conditions, i.e., 37°C and 5% CO₂ for 24 h. 10⁶ cfu/well of overnight grown S. aureus strains SA-1199B, SA-1199, and SA-K1758 were used to infect macrophages (MOI of 10) in presence and absence of boeravinone B at its MEC (12.5 μM) for 2 h. Extracellular and adherent bacteria washed with PBS (pH 7.4) followed by treatment with 50 μg/ml gentamicin for 30 min. Intracellular bacteria were quantified by host cell lysis by brief treatment of 0.07% SDS which followed neutralization with 6% BSA. Viable bacteria were counted by plating on TSA. In a similar experiment RPMI devoid of FCS was used to assess the role of FCS.

By using the cold maceration method, 6,9,11-trihydroxy-10-methylchromeno[3,4-b]chromen-12(6H)-one (boeravinone B; Figure 1) was isolated from the roots of B. diffusa. It is a yellow solid compound, with a melting point of 201°C–203°C. The structure was confirmed through high-resolution mass spectroscopy (m/z 313.0708 [M + H⁺], as calculated for C₁₇H₁₂O₆ + H⁺: 313.0707) and comparison of the ¹H and ¹³C NMR data. Boeravinone B used in this study had 96% purity, as determined through high performance liquid chromatography. Data related to the purity and structure of the isolated boeravinone B was characterized by comparison of spectral data with described reported values (Kadota et al., 1989) (Supplementary Data S1).

The resistance reversal activity of boeravinone B was quantified by calculating the fold reduction in the MIC of ciprofloxacin. Boeravinone B lack anti-staphylococcal activity when assessed up to 100 μM against all tested strains. This MIC of ciprofloxacin alone and in combination with boeravinone B was determined (Table 2). Boeravinone B reduced the MIC of ciprofloxacin by twofold in S. aureus SA-1199 and ATCC 29213, whereas the effect was stronger in the NorA-overexpressing S. aureus strains SA-1199B and MT-23142, where the MIC was reduced by eightfold. Conversely, SA-K1758 (NorA knocked out) did not show any reduction in the MIC of ciprofloxacin. Boeravinone B was also active against a methicillin-resistant S. aureus (MRSA) strain, which showed a fourfold reduction in the ciprofloxacin MIC.

In addition to ciprofloxacin, spectrum of activity was also assessed using other fluoroquinolones like ofloxacin and norfloxacin which are well known substrates for NorA efflux pump and newer fluoroquinolones like gatifloxacin and moxifloxacin which are poor efflux substrates for NorA. Boeravinone B was used at 4× MEC for ethidium bromide (6.25 μM or 2 μg/ml). Potential of boeravinone B to inhibit the efflux of other fluoroquinolones was investigated using S. aureus strains SA-1199B, SA-1199, SA-K1758, and MT23142 (Table 3). MIC potentiation using ethidium bromide yielded similar effects of eightfold reduction in the MIC of ethidium bromide (2 μg/mL) compared with that of ethidium bromide alone, which exhibited an MIC of 16 μg/mL against SA-1199B. In NorA overexpressing strains (SA-1199B and MT23142), boeravinone B reduced the MIC of norfloxacin by eightfold while only twofold reduction in MIC of ofloxacin, moxifloxacin, and gatifloxacin was observed. It was observed that ciprofloxacin MIC reduction was relatively less (two–fourfold) in case of wild type S. aureus SA-1199 against these fluoroquinolones. There was no synergistic effect against S. aureus SA-K1758 due to absence of NorA efflux pump. Significant reduction in MICs against NorA overexpressing strains of S. aureus concludes that ciprofloxacin and norfloxacin have stronger affinity for NorA and efflux can be inhibited using boeravinone B.

The effect of boeravinone B on the time–kill kinetics of ciprofloxacin was assessed in S. aureus SA-1199B. Ciprofloxacin alone (MIC: 8 μg/mL) showed 3-log reduction in growth, whereas the same was accomplished at a subinhibitory concentration of the ciprofloxacin (0.25 × MIC) in combination with boeravinone B at a minimum effective concentration (MEC; 12.5 μM). The sub MIC of ciprofloxacin alone could not reduce the growth. Ciprofloxacin alone at an MIC effectively reduced the colony-forming units (CFU) in 6 h, but cell growth reoccurred after 24 h. The combination of ciprofloxacin (at subinhibitory concentration) and boeravinone B prevented the growth of resistant colonies (Figure 2).

For effective clinical applications, drugs should be able to suppress mutations and resistance mechanisms. A resistant mutant selection study was performed on wild-type S. aureus ATCC 29213 because this strain does not have any known mutations in the regulatory region of NorA and drug targets (DNA gyrase and topoisomerase IV). The minimum concentration of a drug at which no resistant mutant is selected is defined as its MPC. Ciprofloxacin alone showed an MPC of 4 μg/ml whereas, in combination with 12.5 and 25 μg/ml of boeravinone B, the MPC of ciprofloxacin reduced to 2 and 1 μg/ml, respectively (Table 4). The MPC of this combination was lower than the C_max of ciprofloxacin (3–4 μg/ml) in human plasma, indicating the clinical relevance of these combinations in restricting the selection of resistant mutants.

Ethidium bromide is a well-known substrate for the NorA MDR efflux pump. SA-1199B cells were allowed to be loaded with ethidium bromide with and without boeravinone B and placed in a fluorometer cuvette containing fresh medium. Ethidium bromide fluoresces only when bound to nucleic acids in cells; therefore, a rapid decrease occurred in the fluorescence because of the NorA-mediated efflux of ethidium bromide. As shown in Figure 3, only the control cells without boeravinone B maximally extruded ethidium bromide, resulting in significantly decreased fluorescence in the assay period. Conversely, in cells treated with boeravinone B, the efflux of ethidium bromide was prevented, resulting in the prolonged retention of fluorescence. In the accumulation studies, ethidium bromide uptake was allowed in bacterial cells for 30 min. A counterbalance effect occurred because of the simultaneous efflux of ethidium bromide, which effectively increased the fluorescence. The addition of boeravinone B at this stage resulted in a surge in the fluorescence because of the inhibition of efflux pumps, resulting in a higher retention of fluorescence (Figure 3). Reserpine, a known efflux pump inhibitor, was used as the positive control in both studies. When compared, ethidium bromide accumulation in SA-1199B, SA-1199, and SA-K1758, accumulation was maximum in knockout followed by wild type and overexpressing which is in correlation with the phenotypes of the tested strains. In presence of boeravinone B, accumulation was significantly increased in NorA overexpressing, while minor effect was seen for wild type. S. aureus SA-1199. There was no effect on the accumulation efficiency of SA-K1758 which is devoid of functional norA gene (Figure 4).

The minimum concentration of a drug for inhibiting a biofilm is defined and expressed as its MBIC. The MBIC of a drug is typically higher than its MIC because of the lesser permeability of the drug within the matrix of a biofilm. In this study, ciprofloxacin showed an MBIC of 4 and 16 μg/mL for S. aureus SA-1199 and SA-1199B, respectively. A concentration-dependent decrease occurred in the MBIC of ciprofloxacin against both strains in the presence of boeravinone B (6.25–25 μM; Table 5). To ascertain that the biofilm inhibition was caused by the killing of the bacteria embedded in the matrix of biofilm, the resazurin staining of a parallel set of plates was performed. The change in the color of this redox dye from blue to red indicated the viability of the cells in the well. The biofilm inhibition correlated well with the cell viability (Supplementary Figure S1).

Effect of boeravinone B plus ciprofloxacin was evaluated by microscopic studies of the S. aureus ATCC 29213-generated biofilm. Fluorescein isothiocyanate stains proteins and imparts green fluorescence, whereas 4′,6-diamidino-2-phenylindole binds to nucleic acids and yields blue fluorescence when observed in the whole image field. As shown in Figure 5, the untreated biofilms were associated with a large amount of proteins and DNA. The sub MBIC of ciprofloxacin did not inhibit the biofilms; however, in combination with the MEC of boeravinone B, ciprofloxacin inhibited the biofilm, as did the MBIC of ciprofloxacin, as evident by the reduced intensity of green and blue fluorescence.

Boeravinone B was observed to interact with P-gp at the verapamil binding site, particularly with the residues Met69, Phe72, Tyr307, Tyr310, Phe314, Leu332, Phe335, Phe336, Leu339, Phe728, Phe759, Phe957, Phe978, and Val982 through hydrophobic π–π interactions (Figure 6A). Consequently, it led to an increase in the intracellular accumulation of the substrate Rh123. Similarly, the NorA efflux pump is a transmembrane pump, in which ciprofloxacin or reserpine optimally bind to site 1 through strong hydrogen bonding of carboxyl functionality with the Arg98 cationic guanido group. The hydrophobic and hydrogen interactions of ciprofloxacin were mimicked by boeravinone B. Molecular modeling studies with the bacterial NorA efflux pump homology model revealed that boeravinone B binds to the Ile23 and Glu222 residues of the pump through strong hydrogen bonding formed by phenolic hydroxyl groups and thus block the interaction of substrate quinolones with the efflux binding cavity (Figure 6B).

The intracellular invasion of S. aureus SA-1199B was nearly 1.5 log₁₀ higher than that of S. aureus SA-1199 in the murine macrophage cell line J774. Boeravinone B at its MEC (12.5 μM) was not active when the experiment was performed in the presence of 10% fetal calf serum (FCS). When the experiment was repeated using RPMI media without FCS, the invasiveness of S. aureus had reduced, suggesting the binding of boeravinone B to FCS. The invasiveness of SA-1199B showed greater reduction (1.6 log₁₀) than did that of S. aureus SA-1199 (0.5 log₁₀). S. aureus SA-K1758 exhibited the least penetration into macrophages, and boeravinone B did not affect the invasiveness of this strain (Figure 7).