MsrA efflux pump inhibitory activity of Piper cubeba L.f. and its phytoconstituents against Staphylococcus aureus RN4220
Authors: Pallavi Ahirrao, Rushikesh Tambat, Nishtha Chandal, Nisha Mahey, Anju Kamboj, Upendra K Jain, Inder Pal Singh, Sanjay M. Jachak, and Hemraj S Nandanwar
This manuscript has been accepted after peer review and appears as an Accepted Article online prior to editing, proofing, and formal publication of the final Version of Record (VoR). This work is currently citable by using the Digital Object Identifier (DOI) given below. The VoR will be published online in Early View as soon as possible and may be different to this Accepted Article as a result of editing. Readers should obtain the VoR from the journal website shown below when it is published to ensure accuracy of information. The authors are responsible for the content of this Accepted Article.
To be cited as: Chem. Biodiversity 10.1002/cbdv.202000144
Link to VoR: https://doi.org/10.1002/cbdv.202000144
MsrA efflux pump inhibitory activity of Piper cubebaL.f. and its phytoconstituents against Staphylococcus aureus RN4220
Pallavi Ahirrao1*, RushikeshTambat2, Nishtha Chandal2, Nisha Mahey2, Anju Kamboj1, Upendra K Jain1, Inder Pal Singh3, Sanjay M. Jachak3, Hemraj S Nandanwar2**
1*Research scholar of IK Gujral Punjab Technical University, Kapurthala; Department of Pharmaceutical Chemistry, Chandigarh College of Pharmacy, CGC, Landran, Mohali-140307, Punjab, India; 1Department of Pharmaceutical Chemistry, Chandigarh College of Pharmacy, Landran-Mohali-140307, Punjab, India; 2Clinical Microbiology & Bioactive Screening Laboratory, CSIR-Institute of Microbial Technology, Sector 39-A, Chandigarh-160036, India; 3Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER), Sector-67, SAS Nagar, Mohali-160062, Punjab, India.
*Corresponding Author: Mrs. Pallavi Ahirrao, Department of Pharmaceutical Chemistry, Chandigarh College of Pharmacy, Landran-Mohali-140307, Punjab, India.
Email: [email protected], Telephone: +91 9876115171
**Co-Corresponding Author: Dr Hemraj Nandanwar, Clinical Microbiology & Bioactive Screening Laboratory, CSIR-Institute of Microbial Technology, Sector 39-A, Chandigarh- 160036, India.
Email: [email protected] Telephone: +91-172-6665338 Fax: +91-172-2690585/2690632
Methionine sulfoxide reductase A (MsrA), an efflux pump belonging to ATP-binding cassette (ABC) transporter family that conferred resistance to macrolides, was detected in Staphylococcus aureus strain. Herein we report isolation of phytoconstituents from Piper cubeba fruit methanol extract and investigated their efflux pump inhibitory potential against S. aureus MsrA pump. Four isolated compounds viz. pellitorine, sesamin, piperic acid and tetrahydropiperine when studied in combination with erythromycin in S. aureus RN4220, exhibited 2-8 fold reduction in minimum inhibitory concentration (MIC) of erythromycin. Pellitorine and sesamin decreased MIC of erythromycin by 8-fold. The real-time fluorometry-based efflux and accumulation studies of ethidium bromide (EtBr) on S. aureus RN4220 in the presence of these compounds showed reduced efflux and enhanced uptake, thus indicating inhibition of the efflux pump. Pellitorine showed significant post-antibiotic effect of erythromycin. The results revealed that the primary mechanism of action of these compounds involves steady ATP production impairment. Keywords: Piper cubeba; Methicillin-Resistant Staphylococcus aureus (MRSA); MsrA efflux pump; Pellitorine; Sesamin; Phytoconstituents
Methicillin-Resistant Staphylococcus aureus (MRSA) is responsible for causing severe fatal infections in humans worldwide and the fact that MRSA strains causing severe fatal infections have become resistant to almost all clinically used β-lactam antibiotics. A limited number of drugs are available to treat MRSA infection such as vancomycin and due to its overuse, MRSA developed resistance against vancomycin.  Methionine sulfoxide reductase A (MsrA) belongs to ABC transporter family, confers resistance to macrolides and streptogramins. MsrA is a trans- membrane protein composed of 488 amino acids with two ATP binding motifs and it functions independently when cloned in RN4220 strain.  As a part of our continuing efforts in search for novel efflux pump inhibitors (EPIs) against various pumps of MRSA, Piper cubeba L.f. (Piperaceae) fruit MeOH extract was studied for MsrA EPI activity. P. cubeba is mainly grown in Java and Sumatra islands of Indonesia and therefore also called as Java Pepper. It is also cultivated in South Indian states.  In traditional medicine it is used as stomachic, appetizer, expectorant and stimulant.  The fruits are reported to possess antifungal, antibacterial and bactericidal properties. [5, 6] Fruits of P. cubeba are reported to contain lignans such as (+)- cubebin, (-) dihydrocubebin, (-) hinokinin and (-)-sesamin and an alkaloid, piperine. [7, 8] In this article, we describe phytoconstituents of P. cubeba as MsrA EPIs using a synergistic assay in combination with erythromycin against S. aureus RN4220 strain. Additionally, a series of in vitro mechanistic experiments were carried out.
Results and Discussion
The compounds 1-5 and 7 were characterized by Mass and 1H- and 13C-NMR spectroscopic techniques and comparing observed spectral data with that reported in the literature as sesamin
(1), trans-cubebin (2), methyl trimethoxycinnamate (3), pellitorine (4), tetrahydropiperine (5) and piplartine (7). Compound 6 and 8 were identified as piperine and piperic acid, respectively, by co-TLC with reference standard available in our laboratory (Fig.1). The purities of the isolated compounds were ≥95% as determined by HPLC/UPLC. The purity of cubebin was determined to be 98% by HPLC. Further analysis of 1H- and 13C-NMR spectral data of cubebin revealed that it is trans-cubebin and consists of two epimers with the ratio of 3:2 (as per integration of C-9’ protons in the 1H-NMR spectrum). Therefore, extra peaks are seen in 1H- and 13C-NMR spectra of cubebin. The specific rotation value for trans-cubebin was observed as [α]24 -58.6 (c 0.1, CHCl ). 
Minimum inhibitory concentration (MIC) of erythromycin was determined alone as well as in the combination of methanol extract of P. cubeba fruits and fractions viz. 5%, 10%, 20%, 40% and 80% ethyl acetate (EtOAc) in hexanes obtained by performing vacuum liquid chromatography of MeOH extract and of all the eight isolated compounds from these fractions viz. pellitorine, piperic acid, sesamin, cubebin, piperine, piplartine, methyl trimethoxycinnamate
and tetrahydropiperine at sub-inhibitory concentrations. Bioassay-directed fractionation resulted in identification of the most active compounds viz. pellitorine, sesamin, piperic acid and tetrahydropiperine. These compounds were studied for their synergistic effect with erythromycin at1/4 × MIC, 1/8 × MIC, 1/16 × MIC and 1/32 × MIC. It was observed that the susceptibility of erythromycin increased at sub-inhibitory concentrations of these compounds. Pellitorine and sesamin decreased the MIC of erythromycin by 8-fold whereas piperic acid and tetrahydropiperine decreased it by 4-fold against S. aureus RN4220 strain at various concentrations (Table 1). Pellitorine and sesamin at 1/4 × MIC displayed the best fold reduction in MIC of erythromycin, rendering them more prominent EPIs of MsrA efflux pump.
Table 1 MIC and fractional inhibition concentration indices (FICI) of four compounds isolated from Piper cubeba fruits and their combination studies with erythromycin for S. aureus RN4220
Compound MIC (μg/mL) Concentration used as an inhibitor (μg/mL) Fold Reduction in MIC of Erythromycin FICI
Pellitorine 64 16 8 0.375
8 4 0.375
4 4 0.3125
2 2 0.531
Piperic acid 256 64 4 0.5
32 2 0.625
16 2 0.5625
8 1 1.031
Sesamin 256 64 8 0.375
32 4 0.375
16 2 0.5625
8 1 1.031
Tetrahydropiperine 256 64 4 0.5
32 2 0.625
16 1 1.062
8 1 1.031
Erythromycin 128 NA NA NA
These compounds were tested for EtBr efflux inhibition assay in S. aureus RN4220 strain. The bacterial cells were incubated with EtBr for 30 min with continuous shaking at 37˚C. EtBr fluoresces when it is bound to nucleic acid inside the bacterial cell. There was a rapid decrease in the fluorescence due to MsrA mediated EtBr efflux in control well. Pellitorine and sesamin at 1/4 × MIC, displayed higher EtBr efflux inhibition potential compared to piperic acid and tetrahydropiperine (Fig. 2A). In EtBr accumulation assay relative final fluorescence (RFF) value is a measure of how effective a compound is on the inhibition of the EtBr efflux (at a given concentration) by comparison of the final fluorescence at the last time point (45 minutes) of the treated cells with that of cells treated with EtBr. An index of activity above zero indicated that the cells accumulate more EtBr under the condition used than those of the control (non-treated cells taken as zero). In case of negative RFF values, these indicated that the treated cells accumulated less EtBr than those of the control. Values above 1 in the presence of the efflux inhibitors revealed enhanced accumulation of EtBr inside the cells. At 1/4× MIC, pellitorine and sesamin showed higher RFF values displaying their enhanced ability of efflux inhibition as compared to piperic acid and tetrahydropiperine (Table 2). Time-kill kinetics study was performed to measure the bacterial killing effect of pellitorine, the most active EPI in combination with erythromycin in S. aureus RN4220. The sub-inhibitory concentrations of pellitorine 16µg/mL (1/4 × MIC) and erythromycin 16µg/mL (1/8× MIC; synergistic MIC) tested alone, had no effect on the viability of S. aureus RN4220 even after 24 h. When both tested in combination, there is ≥3 log10 reduction below the initial log10 CFU at 0 h (Fig. 2B) in the viability of S. aureus RN4220 after 12h of exposure; while re-growth was observed at 24 h. In time-kill curves, synergy was defined as a ≥2 log10decrease in CFU/mL between the combination and it’s most active constituent after 24 h (at least one of the test compounds must be present at a
concentration that does not affect the growth curve of the test organism).  These results clearly demonstrated the synergy between pellitorine and erythromycin as a consequence of MsrA efflux inhibition.
[A] Fluorescence based determination of EtBr efflux by S. aureus RN4220 cells in the presence and absence of four compounds at concentrations mentioned above (n = 3) [B] Time- kill curves of S. aureus RN4220.*PEL-Pellitorine, SES-Sesamin, PA-Piperic acid, THP- Tetrahydropiperine, ERY-Erythromycin
Table 2 Relative final fluorescence (RFF) values based on the accumulation of EtBr for S. aureus RN4220 in the presence of four efflux inhibitors. [a]n=3, Results were considered significant when*p< 0.05 and highly significant when **p< 0.01 and ***p< 0.001.
Compounds Conc. used as an inhibitor (μg/mL) RFF ± SD[a]
Pellitorine 64 8.14 ± 0.5**
Piperic Acid 64 2.87 ± 0.15*
Sesamin 64 7.23 ± 0.71**
Tetrahydropiperine 64 2.76 ± 0.18*
The minimum concentration of a drug at which no mutant is selected is defined as its mutation prevention concentration (MPC). For the selection of resistant mutants, we performed experiments on S. aureus ATCC 25923, which is a drug-susceptible strain with no known mutation in domain of MsrA efflux pump and erythromycin target domain. Erythromycin, when used at concentration of 2 µg/mL (16 × MIC), had no mutants selected, is defined as its MPC. When used in combination with pellitorine (16 µg/mL), there was no mutant selection even at concentration 0.5 µg/mL (4 × MIC) of erythromycin (Table
3).Table 3 Mutation frequency of S. aureus ATCC 25923 in the presence of erythromycin alone and in combination with pellitorine (16 μg/mL).
Pellitorine (µg/mL) 2 × MIC
(0.25 µg/mL) 4 × MIC
(0.5 µg/mL) 8 × MIC
(1 µg/mL) 16 × MIC
0 8.98 × 10-7 6.99 × 10-7 9.74 × 10-7 <10-7
16 2.49 × 10-7 <10-7 <10-7 <10-7
The MPC of the combination was found to be four-fold lower than erythromycin (16 × MIC) alone, indicating the clinical significance of combination in limiting the selection of resistant mutants. The PAE is the phenomenon of continued suppression of bacterial growth after a short exposure of bacteria to antimicrobial agents.  The PAE of erythromycin alone and in combination with pellitorine was determined against S. aureus RN4220. Erythromycin alone exhibited a PAE of 0.45, 1.05 and 1.79 h at 0.25× MIC (32 µg/mL), 0.5 MIC (64 µg/mL) and MIC (128 µg/mL), respectively. The same concentrations of erythromycin when tested in combination with pellitorine at 16 µg/mLresulted in significantly prolonged PAEs of 1.1, 1.5 and
2.43 h, respectively.
The disturbed membrane functions can affect respiratory chain and hence curtail level of ATP in the cell.  To evaluate effect of pellitorine, piperic acid, tetrahydropiperine and sesamin on the intracellular ATP levels, RN4220 MsrA over-expressed strain was exposed to their 1 × MIC, ½ × MIC and ¼ × MIC at 0 h, 4 h and 24 h. At 0 h that is immediately after treatment, the ATP levels remain unchanged at all the three concentrations of the EPIs including positive control, carbonyl cyanide 3-chlorphenylhydrazone (CCCP) as compared to that of EPI free control. After 4 h and 24 h of exposure, the level of ATP of the EPI free control increased significantly while ATP levels in the wells containing EPIs remained almost unaffected until the end of the experiment, with the exception of the wells containing ½ x MIC and ¼ x MIC at 24 hr time point. Similar results were observed for CCCP (Fig. 3). This showed MsrA being an ABC type efflux pump uses ATP as the source of energy to efflux out the substrates of the pump.
Effect of pellitorine (PEL), piperic acid (PA), tetrahydropiperine (THP) and sesamin (SES) on S. aureus ATP levels. The ATP levels were quantified using a luciferin-luciferase bioluminescence detection assay.
Pellitorine, sesamin, piperic acid and tetrahydropiperine showed no cytotoxicity with IC50> 256 μg/mL. Within this concentration range all the four compounds, (256-16 μg/mL) showed viability of ≥ 80% in both human embryonic kidney cells (HEK293T) and murine macrophage cells (J774).
Bacteria have evolved sophisticated mechanisms of resistance, including efficient drug efflux pumps that incorporate a wide range of substrates; towards both antibiotics as well as some non-antibacterial compounds. Thus, the inefficiency of many antibiotics triggers the search for the EPIs as adjunctive therapies. P. cubeba fruit extracts have been reported to possess antimicrobial property. In our preliminary experiments, the extracts of P. cubeba fruits showed no intrinsic activity on bacterial growth inhibition but had synergistic effect when tested with antibiotics. Using bioassay-directed fractionation, eight compounds viz. cubebin, pellitorine, piperic acid, piplartine, piperine, sesamin, tetrahydropiperine and methyl trimethoxycinnamate were isolated from the methanol extract of P. cubeba fruits. All the eight compounds showed no bacterial growth inhibition (at a concentration used in combination studies) but some of them exhibited significant synergistic activity with erythromycin against S. aureus RN4220, which expresses macrolide specific MsrA efflux pump. There are no reports so far on the synergistic potential of pellitorine, piperic acid, sesamin and tetrahydropiperine against erythromycin- resistant S. aureus. The MIC values of the erythromycin combined with these compounds were several times less than that of the erythromycin alone. Considering that the strain S. aureus RN4220 over-expressing MsrA efflux pump, we propose that the modulation effects were as a result of MsrA efflux pump inhibition by these four compounds. The accumulation and efflux of EtBr are good indicators of the involvement of efflux pumps in the resistance mechanism,[15, 16] particularly in drug-resistant strains of S. aureus.
Till date nine natural products of plant origin have been reported to show MsrA efflux pump inhibition activity in S. auereus RN4220 strain. Totarol, a phenolic diterpene showed 8-fold reduction in the MIC of erythromycin against S. aureus RN4220 strain that over-expresses macrolide specific MsrA pump and was the first EPI reported against this pump.  Oleic acid and linoleic acid isolated from methanol extract of Portulaca oleracea leaves have been recently reported to exhibit EPI activity when combined with erythromycin against MsrA pump in S. aureus RN4220 strain. Linoleic acid at 16 µg/mL and oleic acid at 32 µg/mL reduced MIC of erythromycin by 8-fold and 4-fold, respectively.  It was reported that caﬀeic acid cause a reversal of the resistance phenotype and it inhibited the MsrA pump belonging to the RN-4220 strain. Caﬀeic acid showed greater eﬃcacy in the docking model, in agreement with the demonstrated experimental eﬃcacy.  A diterpene, (4S,9R,14S)-4α-acetoxy-9β,14α- dihydroxydolasta-1(15),7diene isolated from C. cervicornis showed modulatory activity by decreasing the MIC of erythromycin by 16 fold.  Tannic acid at 1/8th of its MIC exhibited significant reduction in MIC of erythromycin in S. aureus RN 4220 strain indicating EPI activity. It is proposed that this EPI activity of tannic acid may be due to its interaction with structures of membrane proteins involved in the efflux system of RN4220 strain.  Furanocoumarins viz. imperatorin and isopimpinellin isolated from Rutaceae family plants showed 4-fold reduction in MIC of erythromycin in S. aureus RN4220 strain. It is reported that lipophilicity of putative EPIs of medicinal plant origin plays an important role in their EPI activity.  In case of imperatorin and isopimpinellin it is also reported that these furanocoumarins showed better EPI activity due to their lipophilicity.
 Menadione (vitamin K3) when assayed with erythromycin at 1/8th of its MIC, reduced the MIC of erythromycin significantly in S. aureus RN4220 strain. The combination of lipid soluble vitamins like vitamin K3 with antibiotics is an attractive alternativeto enhance antibiotic activity of the drug (erythromycin) in humans. Being lipophilic in nature menadione can alter fluidity of bacterial membrane making it more susceptible to penetration by antibiotics such as erythromycin. 
Our results are in agreement with earlier literature reports of the natural products and we observed that pellitorine was the most active compound and it reduced the MIC of erythromycin by 8-fold at 16 µg/mL. The real-time fluorometry-based efflux and accumulation studies of EtBr-pre-loaded MsrA over-producing S. aureus cells in the presence of the four compounds showed reduced efflux and enhanced uptake, thus indicating inhibition of the efflux system.
Combination of erythromycin (16 μg/mL) and pellitorine (16 μg/mL) exhibited a bactericidal effect, whereas when tested alone both the compounds did not show bactericidal effect. The combination significantly reduced the emergence of erythromycin-resistant mutants. Furthermore, pellitorine enhanced the PAE of erythromycin in a concentration-dependent manner. The most comprehensive PAE of 2.43 h was observed with erythromycin at 128 μg/mL when tested in combination with pellitorine (16μg/mL). At lower concentrations, the EPIs inhibit the efflux systems by interrupting with the necessary energy to maintain the functioning of the pump, which shows that the EPIs act as an energy inhibitor like CCCP. The results reveal that the EPIs show an off-target effect and authenticate our results that indicate the impairment in ATP synthesis as the primary mechanism of action for the EPIs.
In this study, we identified pellitorine, sesamin, piperic acid and tetrahydropiperineas novel EPIs for the first time. These EPIs exhibited specific inhibitory effects against MsrA efflux pump of S. aureus that contributes to their synergistic effect with erythromycin against this strain.T hese compounds were devoid of any intrinsic bactericidal activity and ultimately reduced thpossibility of new drug-resistant mutants. Furthermore, exploration of pellitorine and sesamin with respect to their invivo efficacy and toxicity could lead us to obtain more potent EPIs.
General Experimental Procedures
The optical rotations were measured on a Rudolph Autopol IV automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). Mass spectra were recorded on LCQ spectrometer with APCI/ESI probe (Finnigan Mat, Thermo,USA), 1H and 13C NMR spectra were recorded in deuterated solvents on Bruker 400 Ultra ShieldTM NMR spectrophotometer (Bruker Biospin AG, Faellanden, Switzerland) with TMS as an internal standard. Purity of isolated compounds was checked by HPLC on a Waters 600 HPLC system equipped with a Delta 600 quaternary solvent pump, an in-line degasser, a 717 plus auto sampler, a temperature control module, a 2996 photodiode array (PDA) detector and Empower 2TM software (Waters, Milford, MA, USA) using C18Xbridge column (4.6 × 250 mm, 5.0 µm) (Waters, Milford, MA, USA) and Waters ACQUITY UPLC H-Class system, equipped with a binary pump, autosampler, photodiode array detector (PDA) and Empower™3 software (Waters, Milford, MA, USA) using a BEH Shield RP-18 column (2.1 × 100 mm, 1.7 μM) (Waters, Milford, MA, USA).Column chromatography was performed with Silica gel (60–120 and 100–200 mesh) (Qualigens Fine Chemicals, Mumbai, Maharashtra, India). Gel permeation chromatography was performed on Sephadex LH-20 (Sigma-Aldrich, St. Louis, MO, USA).TLC was performed on Silica gel 60 F254 plates (0.20 mm thickness, Merck, Darmstadt, Germany).
Fruit of P. cubeba L.f. was purchased from Hyderabad in November 2018, India and was authenticated by a Dr. AlokGoyal, Botanist, Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER), SAS Nagar, Punjab. The voucher specimen (CCP/HB/KPH/07) was deposited in the Herbarium of Chandigarh College of Pharmacy, Landran, Punjab.
Extraction and isolation of phytoconstituents from P. cubeba fruits
Dried powdered fruits (930 g) of P. cubeba were extracted with methanol by maceration (3 x 3 L). The combined methanol extracts were concentrated to dryness on rotary evaporator to yield
122.6 g of the extract. The methanolic extract (90 g)was subjected to vacuum liquid chromatography on silica gel (230-400 mesh) andeluted with the gradient wise with hexane-ethyl acetate starting with 5% EtOAc and subsequently in continuation with 10, 20, 40, 80 and 100% EtOAc. All these fractions were analyzed byTLC and concentrated to dryness on rotary evaporator to yield 1 g, 23.4 g, 3.1 g, 7.0 g, 2.4 g and 0.4 g of residues, respectively. The fraction eluted with 10% EtOAc (20 g), was subjected to repeated column chromatography on Silica gel 230-400 mesh, eluting with hexane-ethyl acetate gradient, resulted in isolation of compound 1 (45 mg) and compound 2 (780 mg).The fraction eluted with 20% EtOAc (2 g) was subjected to repeated column chromatography on Silica gel, 230-400 mesh eluting with hexane and ethyl acetate gradient to give compound 3 (16 mg).Fraction eluted with 40% EtOAc (6 g) was subjected to repeated column chromatography on silica gel (230-400 mesh) and Sephadex LH-20 resulted in isolation of compounds 4 (29 mg), 5 (21 mg), 6 (53 mg)and 7 (460 mg).Compound 8 (15 mg) was isolated from 80% EtOAc fraction (1.5 g) by repeated column chromatography on Sephadex LH-20.
Bacterial strains and Growth Conditions
S. aureus strain RN4220 that possesses MsrA efflux protein was provided by Prof. Simon Gibbons, UCL School of Pharmacy, London. S. aureus strain ATCC 25923 was purchased from Himedia®, India.Cation-adjusted Mueller-Hinton broth (MHB; Becton-Dickinson, USA) was used for combination studies as well as time-kill experiments. For mutation studies and bacterial colony counts, Mueller-Hinton agar (MHA; Himedia®) was used.
Determination of MIC
MIC values were determined following the guidelines of Clinical Laboratory Standards Institute (CLSI). 
Checkerboard synergy assay
The drug-resistant S. aureus strains described above wereused in broth micro-dilution antimicrobial checkerboard assays to assess for the presence of a synergistic interaction. MTT colorimetric assay was performed to determine the MIC values, which were used to evaluate the effects of the combination of compounds and antibiotics by calculating the FICI according to the formula:
FICI= MIC (antibiotic combined with compound)/MIC (antibiotic alone) + MIC (compound combined with antibiotic)/MIC (compound alone)
“Synergy” effects were indicated when the FICI was less than orequal to 0.5. When the FICI was greater than 0.5 and less than orequal to 4.0 this was regarded as an “indifference” effect; whilst “antagonistic” effects were observed when the FICI was greater than 4.0. 
Ethidium bromide efflux inhibition studies
The fluorometric determination of EtBr efflux from S. aureus RN4220 was performed as described previously. 
Ethidium bromide accumulation assay
The effect of inhibitors on EtBr accumulation was assessed by fluorometry as previously described. 
Time–kill studies of erythromycin (Sigma-Aldrich) in the presence of pellitorine were conducted in MHB and evaluated using a time-kill curve method, as described previously with modification. S. aureus RN4220 used as the test bacterium in this assay. Bacterial suspension in its logarithmic phase (106 CFU/mL) was used as the inoculum. Erythromycin at 16 μg/mL (0.125× MIC) was tested alone and in combination with pellitorine at the concentration (16 μg/mL; 0.125× MIC), as determined above. The CFU/mL was determined by a serial dilution method in triplicate on MHA at 0 (untreated control), 2, 4, 8, 12 and 24 h of incubation at 37ºC.
Selection of resistant mutants
The MPC of erythromycin against S. aureus ATCC 25923 was determined as described previously.  A bacterial suspension of 108 CFU (100 μL) was plated onto MHA containing erythromycin concentrations equal to 2×, 4×, 8× and 16× MIC. The same concentrations of erythromycin were also tested in the presence of pellitorine at 16 and 8 μg/mL respectively. The mutation frequency was calculatedby counting the total number of colonies appearing after 48 h
ofincubation at 37ºC on the drug-containing plate and then dividing thenumber by the total number of CFU plated.
Post-antibiotic effect (PAE)
The PAE was determined by the method described elsewhere.  A final inoculum of 2 × 106 CFU/mL of S. aureus RN4220 was achieved in each tube byadding 50 μL of an inoculum with a turbidity equivalent to that of a 0.5 McFarland standard (2 × 108 CFU/mL) into 5 mL fresh broth containing erythromycin at concentrations equivalent to MIC (128 μg/mL), 0.5 × MIC (64 μg/mL) and 0.25 × MIC (32 μg/mL) alone and in combination with pellitorine (16 μg/mL). After 2 h incubation at 37ºC, with shaking, samples (50 μL) from each tube were diluted 1:1000 with fresh broth (5 mL) to effectively remove antibiotic and pellitorine. Viability counts were determined on MHA from each tube before exposure and immediately after dilution (0 h), and then every 1 h until visual turbidity was observed in the control tube. The PAE calculated by the viable count method was the difference in the time for growth in the exposed culture (T) and the correspondingunexposed control (C) to increase by 1 log10CFU/ml immediately after drug removal and is represented by the formula, PAE = T – C.
Intracellular ATP levels determination
The level of ATP was measured using the ATP determination kit (Invitrogen, LifeTechnologies, Paisley, UK) in consonance with the manufacturer’s protocol. The RN4220 MsrA over- expressed strain was grown to achieve an OD600 of 0.3 at 37˚C with shaking. The culture was then diluted to a cell density of 1 x 105 CFU/mL in MHB. Then 100 µL of culture was added along each test tube containing pellitorine, piperic acid, tetrahydropiperine and sesamin at their
individual 1 × MIC, ½ × MIC and ¼ × MIC concentrations. CCCP was used as positive control and culture control was included to monitor the normal growth of the strain. The aliquots were collected at 0 h, 4 h and 24 hintervals and were given alternative cold and heat shock, followed by liquid sonication bath for 10 minutes. The cell lysate so formed was then transferred to black flat 96-well plate and ATP standard assay solution was added. The relative luminescence was measured using the synergy microplate reader (BioTek, Winooski, Vermont, USA). 
Supporting information for this article is provided separately.
Pallavi Ahirrao is thankful to IK Gujral Punjab Technical University, Kapurthala for providing research facilities and Chandigarh college of Pharmacy, Landran for providing necessary facilities to carry out the described research work in the manuscript.
Author Contribution Statement
Pallavi Ahirrao conceptualized the study and carried out detailed phytochemical investigation. Hemraj Nandanwar planned and designed the study. Rushikesh Tambat performed in vitroEtBr accumulation, efflux assays, PAE and also performed ex vivo toxicity assays. Nisha Mahey and Nishtha Chandal performed in vitro MIC determination, checkerboard synergy assay, time kill kinetics, MPC and intracellular ATP determination assays. Anju Kamboj and Upendra Jain are Ph.D. supervisor and co-supervisor, respectively for Pallavi Ahirrao. Inder Pal Singh had
provided phytochemical reference standards required for the study. Sanjay Jachak helped in structure elucidation of isolated compounds, editing and proof reading the manuscript.
Conflict of interest
We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
 M.Z. David, R.S.Daum,‘Community-associated methicillin-resistant Staphylococcus aureus: epidemiology and clinical consequences of an emerging epidemic’, Clin. Microbiol. Rev. 2010, 23 (3), 616-687.
 S.S. Costa, M. Viveiros, L. Amaral, I.Couto,‘Multidrug efflux pumps in Staphylococcus aureus: an update’, Open Microbiol. J. 2013, 7, 59-71.
 R. Chopra, S. Nayer, I. Chopra. ‘Glossary of indian medicinal plants, National Institute of science and Communication’, CSIR Publication, New Delhi, India, 1956.
 E.M. Choi, J.K. Hwang,‘Investigations of anti-inflammatory and antinociceptive activities of Piper cubeba, Physalisangulata and Rosa hybrida’, J.Ethnopharmacol.2003, 89 (1),171-175.
 M. Khan, M. Siddiqui,‘Antimicrobial activity of Piper fruits’, Nat. Prod. Radiance 2007,
6 (2), 111-113.
 Accepted Manuscript
1 S.F.H. Zaidi, K. Yamada, M. Kadowaki, K. Usmanghani, T. Sugiyama,‘Bactericidal activity of medicinal plants, employed for the treatment of gastrointestinal ailments, against Helicobacter pylori’, J. Ethnopharmacol. 2009,121 (2), 286-291.
 S. Koul, S. Taneja, K. Dhar, C.Atal,‘Lignans of Piper clusii’, Phytochemistry 1983, 22 (4), 999-1000.
 V.S. Parmar, S.C. Jain, K.S. Bisht, R. Jain, P. Taneja, A. Jha, O.D. Tyagi, A.K. Prasad, J. Wengel, C.E. Olsen, ‘Phytochemistry of the genus Piper’, Phytochemistry 1997,46, 597- 673.
 I.C. de Pascoli, I.R. Nascimento, L.M. Lopes, ‘Configurational analysis of cubebins and bicubebin from Aristolochia lagesiana and Aristolochia pubescens’, Phytochemistry 2006, 67,735-742.
 N.P. Kalia, P. Mahajan, R. Mehra, A. Nargotra, J.P. Sharma, S. Koul, I.A. Khan,‘Capsaicin, a novel inhibitor of the NorA efflux pump, reduces the intracellular invasion of Staphylococcus aureus’, J. Antimicrob. Chemother. 2012, 67, 2401-2408.
 A.Belley, E.Neesham-Grenon, F.F. Arhin, G.A. McKay, T.R. Parr, G. Moeck,‘Assessment by time-kill methodology of the synergistic effects of oritavancin in combination with other antimicrobial agents against Staphylococcus aureus’, Antimicrob Agents chemother. 2008, 52 (10), 3820-3822.
 J.M. Blondeau, X. Zhao, G . Hansen, K. Drlica,. ‘Mutant prevention concentrations of fluoroquinolones for clinical isolates of Streptococcus pneumoniae’, Antimicrob. Agents Chemother. 2001,45(2), 433-438.
 K.A. Brogden,‘Antimicrobial peptides: Pore formers or metabolic inhibitors in bacteria?’,
Nat. Rev. Microbiol. 2005, 3 (3), 238 – 250.
 Accepted Manuscript
1 X-Z. Li, H. Nikaido,‘Efflux-mediated drug resistance in bacteria’ Drugs 2009, 69 (12), 1555-1623.
 G.W. Kaatz, V.V. Moudgal, S.M. Seo, J.E. Kristiansen,‘Phenothiazines and thioxanthenes inhibit multidrug efflux pump activity in Staphylococcus aureus’, Antimicrob. Agents Chemother. 2003, 47 (2), 719-726.
 X-Z. Li, K. Poole, H. Nikaido,‘Contributions of MexAB-OprM and an EmrE homolog to intrinsic resistance of Pseudomonas aeruginosa to aminoglycosides and dyes’, Antimicrob. Agents Chemother. 2003, 47 (1), 27-33.
 E.C. Smith, G.W. Kaatz, S.M. Seo, N. Wareham, E.M. Williamson, S. Gibbons, ‘The phenolic diterpene totarol inhibits multidrug efflux pump activity in Staphylococcus aureus’ Antimicrob. Agents Chemother. 2007, 51, 4480-4483.
 K.P. Fung, Q.B. Han, M. Ip, X.S. Yang, C.B.S. Lau, B.C.L. Chan, ‘Synergists from Portulaca oleracea with macrolides against methicillin-resistant Staphylococcus aureus and related mechanism’, Hong Kong Med. J. 2017, 23 (Suppl. 5), S38-42.
 J. F.S. dos Santosa, S.R. Tintinob, T. S. de Freitasb, F. F. Campinab, I. R. de A. Menezesc, J.P. Siqueira-Júniord, H. D.M. Coutinho, F. A.B. Cunha, ‘In vitro e in silico evaluation of the inhibition of Staphylococcus aureus eﬄux pumps by caﬀeic and gallic acid’, Comparative Immunol. Microbiol. Infectious Diseases, 2018, 57, 22-28.
 C.S. de Figueiredo, S.M.P. de Menezes Silva, L. S. Abreu, E. F. da Silva, M. S. da Silva,
G. E. C. de Miranda, V.C. de O. Costa, M. Le Hyaric, J. P. de Siqueira Junior, J.M.B. Filho, J.F. Tavare, ‘Dolastane diterpenes from Canistrocarpus cervicornis and their effects in modulation of drug resistance in Staphylococcus aureus’, Nat. Prod. Res., 2019 33, 3231-3239.
 Accepted Manuscript
1 S.R. Tintino, C.D. Morais-Tintinom, F.F. Campina, M.S. Cosa, I.R.A. Menezes, Y.M. L.S.de Matos, J.T. Calixto-Junior, P.S. Pereira, J.P. Siqueira-Junior, T.C. Leal-Balbino,
H.D.M. Coutinho, V.Q. Balbino, ‘Tannic acid affects the phenotype of Staphylococcus aureus resistant to tetracycline and erythromycin by inhibition of efflux pumps, Bioorg. Chem. 2017, 74, 197-200.
 S. Gibbons, ‘ Anti-staphylococcus plant natural products’, Nat. Prod. Rep., 2004, 21, 263-277.
 S.A.L. Madeiro, N.H.P. b. Borges, A.L. Souto, P.T.R. de Figueiredo, J.P. Siqueira-Junior,
J.F. Tavares, ‘Modulation of the antibiotic activity against multidrug resistant strains of coumarins isolated from Rutaceae species, Microbiol. Path., 2017, 104, 151-154.
 S.R. Tintino, C.D.M. Oliveira-Tintino, F.F. Campina, P.W. Limaverde, P.S. Pereira, J.P. Siqueira-Junior, H.D.M. Coutinho, L.J. Quintans-Junior, T.G. da Silva, T.C. Leal- Balbino, V.Q. Balbino, ‘Vitamin K enhances the effect of antibiotics inhibiting the efflux pumps of Staphylococcus aureus strains’, Med. Chem. Res., 2018, 27, 261-267.
 P. Wayne. Clinical and laboratory standards institute. Performance Tetrahydropiperine standards for antimicrobial susceptibility testing, 2011.
 H.A. Junio, A.A. Sy-Cordero, K.A. Ettefagh, J.T. Burns, K.T. Micko, T.N. Graf, S.J. Richter, R.E. Cannon, N.H. Oberlies, N.B. Cech,‘Synergy-directed fractionation of botanical medicines: a case study with goldenseal (Hydrastis canadensis)’, J. Nat. Prod. 2011,74 (7), 1621-1629.
 L. Machado, G. Spengler, M. Evaristo, J. Handzlik, J. Molnar, M. Viveiros, K. Kiec- Kononowicz, L. Amaral, ‘ Biological activity of twenty-three hydantoin derivatives